Bone-Marrow - IAEA

Bone-Marrow - Conservation, *, Culture and Transplantation

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BONE-MARROW CONSERVATION, CULTURE AND TRANSPLANTATION

The following States are Members of the International Atomic Energy Agency:

AFGHANISTAN GHANA PAKISTANALBANIA GREECE PANAMAALGERIA GUATEMALA PARAGUAYARGENTINA HAITI PERUAUSTRALIA HOLY SEE PHILIPPINESAUSTRIA HUNGARY POLANDBELGIUM ICELAND PORTUGALBOLIVIA INDIA ROMANIABRAZIL INDONESIA SAUDI ARABIABULGARIA IRAN SENEGALBURMA IRAQ SIERRA LEONEBYELORUSSIAN SOVIET ISRAEL SINGAPORE

SOCIALIST REPUBLIC ITALY SOUTH AFRICACAMBODIA IVORY COAST SPAIN .CAMEROON JAMAICA SUDANCANADA JAPAN SWEDEN ■CEYLON JORDAN , . SWITZERLANDCHILE KENYA SYRIAN ARAB REPUBLICCHINA KOREA, REPUBLIC OF THAILANDCOLOMBIA KUWAIT TUNISIACONGO, DEMOCRATIC LEBANON TURKEY

REPUBLIC OF LIBERIA UGANDACOSTA RICA LIBYA UKRAINIAN SOVIET SOCIALISTCUBA LIECHTENSTEIN REPUBLICCYPRUS LUXEMBOURG UNION OF SOVIET SOCIALISTCZECHOSLOVAK SOCIALIST MADAGASCAR REPUBLICS

REPUBLIC MALAYSIA UNITED ARAB REPUBLICDENMARK MALI UNITED KINGDOM OF GREATDOMINICAN REPUBLIC MEXICO BRITAIN AND NORTHERNECUADOR MONACO IRELANDEL SALVADOR MOROCCO UNITED STATES OF AMERICAETHIOPIA NETHERLANDS URUGUAYFINLAND NEW ZEALAND VENEZUELAFRANCE NICARAGUA VIET-NAMGABON NIGER YUGOSLAVIAGERMANY, FEDERAL REPUBLIC OF NIGERIA ZAMBIA

NORWAY

The Agency’s Statute was approved on 23 October 1956 by the Conference on the Statute of the IAEA held at United Nations Headquarters, New York; it entered into force on 29 July 1957. The Headquarters of the Agency are situated in V ienna. Its principal ob jectiv e is "to accelera te and enlarge the contribution of atom ic energy to peace, health and prosperity throughout the w orld".

Printed by the IAEA in Austria July 1969

PANEL PROCEEDINGS SERIES


PROCEEDINGS OF A PANEL ON CURRENT PROBLEMS OF

BONE-MARROW CELL TRANSPLANTATION WITH SPECIAL EMPHASIS ON CONSERVATION AND CULTURE

HELD IN MOSCOW, 22 - 26 JULY 1968

INTERNATIONAL ATOMIC ENERGY AGENCY VIENNA, 1969

BONE-MARROW CONSERVATION, CULTURE AND TRANSPLANTATION,(Panel Proceedings Series) ' *

ABSTRACT. Proceedings of a panel convened by the IAEA and held in Moscow, from 22 to 26 July 1968. The meeting was attended by 25 scientists from 13 countries and one international and one national organization.

Contents; Tissue culture of bone-marrow cells (2 papers) ; H istocompatibility and avoidance of secondary disease (9 papers) ; Conservation and storage of bone-marrow ce lls , white cells and thrombocytes (8 papers) ; Scien tific and organizational problems of bone-marrow ce ll banks (2 papers) ; Summary and recom mendations of the panel; List of participants.

Each'papet is in English with an abstract. One paper is presented by abstract only.

(195 p p ., 16 x 2 4 cm , paper-bound, 52 figures ; 1969) Price: US$ 5 . 00; £ 2 .1 .8 .


IAEA, VIENNA, 1969 STI/PUB/ 219

FOREWORD

A Panel on the current problems of bone-marrow cell transplantation with special emphasis on cell conservation and culture was organized by the International Atomic Energy Agency and held at the Central Institute of Haematology and Blood Transfusion in Moscow from 22 to 26 July 1968. Twenty-three scientists from 13 Member States and representatives of international and national organizations attended.

Many of the participants had done notable work on this subject. The following topics were discussed:

Tissue culture of bone-marrow cells;Histocompatibility and how to avoid secondary diseases;Conservation and storage of bone-marrow cells, white cells andthrombocytes;Scientific and organizational problems of bone-marrow cell banks.

The meeting was opened by Professor A. E. Kiselev, of the Central Institute, who welcomed the participants on behalf of the Agency's Director General and the host country. In his address he pointed out that bone- marrow cell transplantation deserved a great deal of attention because of its importance as a powerful tool in human therapy, including radiation disease. He further stressed that despite the remarkable achievements in this field, specifically with regard to auto- and homologous bone-marrow transplantation,, many problems remained ill-defined and unsolved — particularly on homologous bone-marrow transplantation. He considered that, to clarify these problems, broad international collaboration was needed among specialists in Member States as well as with international bodies such as the IAEA and WHO, both of which organizations should be centres for collecting and disseminating information from Member States and for encouraging and stimulating research.

In presenting much interesting work, the Panel clearly established the usefulness of bone-marrow transplantation for human therapy in specific conditions, and identified more clearly the practical problems still to be solved.

The recommendations together with the reports presented at the Panel are published in this volume.

CONTENTS

A. TISSUE CULTURE OF BONE-MARROW CELLS •

Agar technique for the cultivation in vitro of bone-marrow colonies. . . . 3D. M e t c a l f '

Differentiation of haemopoietic tissue in organ1 cultures . . . . . . . . .............. 13A . Y a . F r i e d e n s ht e in

B. HISTOCOMPATIBILITY AND AVOIDANCE OF SECONDARY DISEASE

Secondary disease: in radiation ch im eras.................. ...................... ................. 21C . C o n g d o n

Clinical questions of tissue incompatibility after allogenic bone-marrow transplantation..................................................................................................... 29F . E . F a i n : s h t e i n and E . A . Z o t i k o v

Prevention and control of secondary disease following allogenic :b o n e-m arro w tra n sp la n ta tio n ........................................................ ........................... 35D . W . van B e k k u m

Effects1 and-complications of bone-marrow transplantation in man(abstract only) ............................. ........................................................... ......... 47G. M a t h e / L . S c h w a r z e n b e r g , J . L . A m i e l ,M. Sc h n e i d e r . , A. C 'a ttan and J . R . S c h l u m b e r g e r

Are haemopoietic stem cells precursor cells in secondary disease?. . . 49J . L . C h e r t k o v

Lymphoid tissue grafts in m an ............................................................................... 51H. E . M.. K ay . • . •

Suppression of immunogenesis when using gangleron and prednisolone . 55K . A . An t o n y a n • .

Stem-cell inactivation on transplantation of haemopoietic cell suspensions from genetically different donors............................................... 59R . V . P e t r o v

Effect of massive blood transfusion on the therapeutic efficiency ofhomogenic bone marrow in acute radiation illn ess............................... 67V . S e r a p hi m o v - D i m i t r o v , Z. D e c h e v a and M . N e d y a l k o v a

C. CONSERVATION AND STO RA G E O F BO N E-M A RRO W C E L L S , W HITE C E L L S AND TH R O M BO C Y TES

C ytotoxic and agglutinating p ro p e rtie s of s e r a fro m m u lti-tra n sfu se dand n o rm al p e r s o n s ....................................................................................................... 77M a r t h a M . E i b l a n d H . E i b l

Sub-microscopic organization and functional properties of cellsstored in a bank for frozen leucocytes and platelets............................... 85F . R . V i no gr ad - F i nk e 1, E . I . T e r e n t i e v a ,V . A . L e o n t o v i c h , S . B . S k o p i n a , N . N . A b e z g a u z and A . A . T o t s k a y a

Viability tests for fresh and stored haemopoietic cells ................................... 95T . M . F l i e d n e r

Preservation of bone-marrow cells, leucocytes and platelets at lowtem peratures: a rev iew ..................................................... ........................ 107M . J . A s h w o o d - S mi t h

Posthumous bone marrow and its significance for transplantation . . . . . 139 N . G . K a r t a s h e v s k y and Т . К . M a m y s h e v a

Bone-marrow storage and transplantation . ' . . . . .................................... .. 1430 . C o s t à c h e l , I. C o r n e c i , T. A n d r i a n ,1. K i t z u l e s c u , N. P o p e s c u , D. P a s c u , E . B u z i and N. V o i c u l e t z

Preservation of bone marrow for clinical use ....................... ................. .. 163A . G . F e d o t e n k o v

Preservation of bone marrow by deep freezing with polyvinylpyrrolidone (PVP).................................................................. .■.............■............. 173S . S . L a v r i к

D. SCIENTIFIC AND ORGANIZATIONAL PROBLEMS OF. BONE-MARROW CELL BANKS . .

Scientific and organizational problems connected with the establishmentof bone-marrow and blood-component banks............................................ 181A . E . K i s e l e v

B a s ic co n sid era tio n s fo r the e stab lish m en t of a b o n e -m a rro w bank . . . . 187• R. . Ki e n

Summary and Recommendations of the Panel.................................... 191

L is t of P a rtic ip a n ts and S e c r e ta r ia t 193

A

TISSUE CULTURE OF BONE-MARROW CELLS

AGAR TECHNIQUE FOR THE CULTIVATION IN VITRO OF BONE-MARROW COLONIES*

D. METCALFWalter and Eliza Hall Institute, Royal Melbourne Hospital,Melbourne, V ictoria, Australia

Abstract

AGAR TECHNIQUE FOR THE CULTIVATION IN VITRO OF BONE-MARROW COLONIES. In solid-state agar cultures certain haem opoietic cells proliferate and form discrete colonies of 200 - 4000 ce lls. Colony formation is dependent on stimulation by the colony-stim ulating factor, and this is achieved by (1) the use o f a ce ll feeder layer, (2) the addition o f conditioned medium, or (3) the addition of human or mouse serum or urine containing the factor. AU colonies initially contain granulocytic cells which differentiate from myeloblasts to polymorphs as colony growth proceeds. Later colonies develop a second population of phagocytic mononuclear ce lls (macrophages). The colony-forming-system is sim ple, readily quantitated and highly reproducible. Linear dose responses occur between the dose of colony-stim ulating factor and the number and size o f colonies developing from a standard number of bone-marrow ce lls. In-vitro colony formation has been achieved with haem opoietic cells o f the following species: mouse, rat, hamster, guinea pig, rabbit and human. In the adult mouse, colony-form ing cells are located in the bone marrow, spleen and blood and in the embryo, in the yolk sac, liver and spleen. The colony-form ing ce ll appears to be an early member of the granulocytic series. The colony-forming system has been used as a quantitative assay system: (1) to assay levels o f colony-stim ulating factor in serum and urine and in the chem ical- characterization and purification o f the factor; and (2) to enumerate the number o f colony-forming cells in haem opoietic tissues in response to a variety of experim ental procedures and disease states. Since the system is applicable to human bone-marrow ce lls, it should prove of value in the quantitative assay of(1) survival o f human bone marrow on storage, and (2) bone-marrow content of granulocytic precursor cells in various disease states and following various types of therapy. The system is not suitable for the mass production in vitro o f haem opoietic cells for therapeutic use.

1. INTRODUCTION

The ability to cultivate haemopoietic cells in' large quantities in vitro would have many obvious applications, among which would be the therapeutic use of such cells in the management of persons exposed to large doses of ionizing irradiation and of patients with neoplasms or aplastic diseases of the haemopoietic tissues.

• From the point of view of both the research worker studying the biology of haemopoietic tissues and the clinician, it is highly desirable that any tissue culture system used should achieve the twin goals of allowing both proliferation and differentiation of the haemopoietic cells being cultured. It is to be regretted that conventional liquid-state culture systems have failed so far to achieve these goals when applied to haemopoietic cells. In most cases survival of such cultures has been short-term with little evidence of cell proliferation, or, if long-term survival with cell proliferation has been achieved, virtually no differentiation has been observed in the cultures. In some cases where continuous cell lines have been obtained from haemopoietic tissues, it is obvious that the cell lines

- successfully established have been fibroblasts rather than haemopoietic cells.

* This work was supported by the Carden Fellowship Fund of the Anti-Cancer Council o f V ictoria.

3

4 METCALF

The solid-state agar culture system was developed independently in 1965 by Bradley and Metcalf [ 1] and Pluznik and Sachs [ 2] . With this system, clonal proliferation of individual haemopoietic cells can be achieved in primary cultures with the formation of large haemopoietic colonies. A striking feature of such colony development is the capacity of the colony cells to exhibit normal differentiation and functional activity.

It is the purpose of the present paper to review briefly the technique of agar culture and the information so far derived from its use and to consider the potential applications of the technique to the clinical problems of the preservation, cultivation and therapeutic use of haemopoietic tissues.

2. THE AGAR CULTURE SYSTEM

In its present form the agar culture technique involves the short-term cultivation (for periods up to 10 - 14 days) of haemopoietic cells taken directly from the animal or patient (primary cultures). The culture re agents and techniques have been described in full elsewhere [ 1 , 3 ] .

Bone-marrow plugs or other haemopoietic cell suspensions are collected in bone-marrow collecting fluid (double strength modified Eagle's medium, 40 ml, 3% trypticase soy broth, 10 ml, distilled water, 50 ml). Single cell suspensions are prepared by aspirating the cell clumps up and down with a pipette. Cell counts for viable cells (usually greater than 90%) are performed in the routine fashion with eosin or nigrosin. Cultures are made in either glass or plastic petri dishes (50 mm for feeder layer cultures; 35-mm plastic dishes (Falcon Plastics, Los Angeles) for serum-conditioned medium or urine-stimulated cultures).

The reagents used in the culture system have the following composition:

E2020 medium: Eagle's balanced salt solution, 100ml; sodium bicarbonate 7.5%, 30 ml; MEM vitamins, 20 ml; MEM amino acids, 20 ml; MEM glutamine, 10 ml; sodium pyruvate, 10 ml; L-serine, 10 ml; phenol red 0.5%, 4 ml; penicillin/streptomycin (5000 units of each per ml), 2ml; distilled water , 94 ml; foetal calf serum, 100 ml; total volume, 400 ml. This mixture is filtered through a 0.45-/um Millipore membrane and stored at 4° С .Trypticase soy broth: 6 g trypticase soy broth in 200 ml distilled water,autoclaved 20 min and stored at 4° С for no more than 2 weeks.Bacto agar (Difco, Detroit : 0 .6 g agar in 100 ml distilled water, boiledfor 2 min, thoroughly dissolved and held in a 40° С water bath. Agar prepared immediately before use.

Four parts of E2020 are mixed with one part of trypticase soy broth and L-asparagine and DEAE dextran solutions are added to give a final concentration per ml of agar-medium of 20 ¡jtg and 75 /ug, respectively. Equal volumes of this medium are mixed with the 0. 6% agar solution held at 40° С and sufficient haemopoietic cells added to give a final cell concentration of usually 50 000 or 75 000 cells per ml of medium-agar.

One- or two-ml aliquots of these cell suspensions in medium-agar are pipetted into the culture dishes and allowed to gel at room temperature

AGAR TECHNIQUE 5

for 20 minutes. The culture dishes are then incubated at 37° С in a humidified atmosphere of 10% CO2 in air.

Fo r colony formation to occur, the haemopoietic cells in the above . agar gel must be stimulated by the colony-stimulating factor. This is achieved by one of four methods:

(a) The use of an underlying feeder layer of 0. 5% agar containing a trypsinized single-cell suspension of a variety of tissues, e. g. neonatal kidney or embryo cells. Such underlayers of feeder cells can be prepared before the addition of the overlying agar layer containing the haemopoietic cells [1, 2, 4, 5] .

(b) The addition to the system of 'conditioned medium' , prepared by incubating various cells in conventional liquid tissue cultures and harvesting the fluid after several days. Conditioned medium can be incorporated in an underlayer of agar as in the feeder layer technique or it can be placed directly in the empty culture dish and mixed with the liquid medium-agar mixture containing the haemopoietic cells, before gelling occurs [6, 7 ] .

(c) The addition of certain mouse or human sera to the culture dish before pipetting in the mixture of medium-agar and cells, and mixing thoroughly before gelling occurs [ 3, 8, 9] .

(d) The addition of dialysed and filtered human urine to the culture dishes as in (c) [ 10] .

In the presence of a feeder layer or colony-stimulating factor, proliferation of certain haemopoietic cells commences within 24 hours, and by 2 days small developing colonies of up to 20 cells may be observed. Colony growth is progressive for 7 - 1 0 days without further media change.

Certain technical comments should be made regarding the above culture technique.

(1) Initially, 5% CO2 in air was recommended, but it has since been realized that with many incubators this results in borderline conditions in which the pH often cannot be satisfactorily kept in a sufficiently acid state. Ten per cent CO2 in air results in a more uniformly satisfactory culture system.

(2) Wide variations occur in the effectiveness of different batches of foetal calf serum. Some sera are frankly cytotoxic and with others poor colony formation occurs. The basis for these variations is unknown and at present satisfactory serum pools can only be selected by trial and erro r.

(3) The addition of 5% horse serum to the E2020 commonly improves colony growth.

(4) Removal of culture dishes from the incubator for inspection should be kept to a minimum, as repeated exposure to room temperature and low CO2 concentrations for periods greater than a few minutes inhibits colony formation.

(5) The concentration of agar (0.3%) is critical for colony formation [ 1] . If the agar gel dries slightly, due to inadequate humidification of the incubator, colony formation is inhibited.

(6) Purified agar or agarase is not so satisfactory as bacto-agar.

6 METCALF

(7) The culture system appears to be remarkably free from contamination problems. Agar cultures can be set up.routinely in an open general laboratory with a contamination rate of less than 1% of the dishes. Obviously, a tissue culture room or cubicle is preferable for the preparation of dishes but is not essential.

Developing colonies are available for study at any time after the first day of incubation and colony counts are performed routinely at day 7 or 10 of incubation by means or á dissecting microscope. Colonies are normally removed for cytological examination with a fine Pasteur, pipette.They may be smeared on microscope slides and stained with a variety of stains. Routinely, 0. 4% orcein in 60% acetic acid is used for cytological classification of colony cells.

The above culture medium has been used routinely for the cultivation of mouse and rat haemopoietic colonies and has been used successfully for the growth of rabbit, hamster and guinea pig colonies. Growth of chicken haemopoietic colonies has not been achieved. We have not been successful with the cultivation of human haemopoietic cells with the above system, but Senn, IVlcCulloch and Till [11] have adapted the mouse kidney feeder layer technique to the growth of colonies from human bone- marrow cells. The colonies achieved were poor compared with those derived from cells of other species and it seems likely that certain,probably minor modifications to the above technique are required before routinely successful colony growth will be achieved with human cells.

3. COLONY FORMATION

Colonies arise by the proliferation of single colony-forming cells [ 2] and are initially loose clusters of cells. As. colony growth proceeds, the colonies enlarge and become roughly globular aggregates of cells which may either retain their loose structure or develop a dense central core of cells. Colonies reach 2000 - 4000 cells by day 10 of incubation with feeder-layer stimulation, but only 200 - 500 cells with serum or urine stimulation [ 12] .

Excessive colony crowding inhibits colony growth and colony cells have been shown to elaborate a diffusible factor inhibiting colony growth [ 13] . Conversely, a limited degree of colony crowding potentiates colony growth and cell breakdown products from degenerating non-colony forming cells in the medium are reutilized by colony cells and potentiate colony growth (Metcalf, unpublished data).

The earliest colony cells are identifiable, as primitive granulocytes (myeloblasts and myelocytes) and all early colonies are granulocytic in nature [ 12] . After 3 - 4 days, a second population of phagocytic mononuclear cells (macrophages) develops in the colonies and commences active proliferation. These cells ingest the metachromatic agar of the medium and can be mistaken for mast cells in Giemsa-stained preparations. With feeder-layer stimulation, colonies retain their mixed granulocytic/mononuclear composition for at least 14 days, but with conditioned medium, serum or urine stimulation; virtually all colonies become completely mononuclear in composition by day 7 of incubation.

AGAR TECHNIQUE 7

The granulocytic cells.in developing colonies differentiate through a metamyelo-cyte stage and form polymorphs, which appear to disintegrate after 1 - 2 days in the culture system. The ultimate disappearance of granulocytic elements from serum- or urine-stimulated colonies is due to the progressive differentiation of the initial granulocytic cells to metamyelocytes and polymorphs with subsequent breakdown of these cells. This sequence of loss by differentiation is less prominent in feeder-layer- stimulated colonies and in such colonies the whole spectrum of granulocytic cells from myeloblasts to polymorph is present throughout the incubation period. ■

Colonies may be sub-passaged to fresh agar plates by transferring intact colonies or dispersed colony cells [-14] . However, in our experience such colonies do not contain .genuine colony-forming cells and dispersed 3- or 4-day colony cells do not initiate granulocytic .colony.formation.The cell aggregates produced by the growth of dispersed colony cells represent the continued proliferation of thè mononuclear cells of the colonies. . . .

Regardless of the type, of colony stimulation used and the source of the colony-forming cells, all colonies obtained to the present time with this technique have been of the granulocytic-mononuclear cell variety-. Erythropoietic colonies have never been obtained and erythropoietin will not stimulate colony formation in the agar system. . Lymphoid cells have never been shown to exhibit colony formation, even after the addition of phytohaemagglutinin or various antigens. . ,

4. THE NATURE OF THE COLONY-FORMING CELL

In the mouse embryo, colony-forming cells have been detected first in the 8-day yolk sac and later in the liver and spleen. In the adult mouse, approximately-1 in 500 bone-marrow cells and 1 in 25 000 spleen cells are capable of colony formation in vitro. The incidence of colony-forming cells in the blood is lower still. These three tissues are the only tissues in the normal adult animal which appear to contain colony-forming cells.The figures quoted above are typical but are .probably underestimates of , the absolute incidence of colony-forming cells in the various, tissues. Maximum stimulation of colony formation has probably not yet been achieved in any of our current agar systems. In a myelo-monocytic leukaemia in BALB/c mice currently under study in this laboratory, as many as one in twenty of the leukaemic cells will form colonies of the above type.

Colony-forming cells are highly radiosensitive in vivo,and,in. the mouse a Dзт of 85 R has.been established for bonermarrow colony-forming cells [ 1 5 ] . ' i- . . .

Glass bead column separation of bone-marrow cells has been attempted but has not resulted in cell fractions of sufficient purity to allow identification of the colony-forming cell. •

The in-vitro colony-forming cell is.not a member of the erythropoietic series but may share a common ancestor with erythropoietic cells, since anaemia induction leads to a sharp fall in bone-marrow colony-forming cells and transfusion-induced polycythaemia to a sharp rise in colony- forming cells [ 16] . The in-vitro colony-forming cell does not appear to be self-sustaining in vivo by its own mitotic activity. It seems likely

8 METCALF

that m o st in -v itro co lo n y -fo rm in g c e lls a re e a r ly m em b ers of the gran u lo cy tic c e ll s e r ie s at a stag e o f d ifferen tia tio n in te rm e d ia te betw een the in -v iv o g ran u lo cy tic co lon y -form in g c e ll and the m y elo b last [ 17] .

5 . TH E CO LO N Y-STIM U LA TIN G FA C TO R

Colony fo rm ation is dependent on stim u lation by the co lo n y -stim u la tin g fa c to r . T h is fa c to r not only in itia te s colony fo rm ation but is requ ired continuously fo r p ro g re ss iv e growth of the colony [ 14] . L in e a r r e la tio n sh ip s can be d em onstrated in v itro betw een the dose o f colony- stim u latin g fa c to r and both the num ber and s iz e o f co lon ies a r is in g from a stand ard num ber o f b one-m arrow c e lls [ 3, 8] . Thus colony fo rm ation can be used as a p re c is e quantitative a ssa y sy stem fo r the m easu rem en t o f le v e ls of co lo n y -stim u latin g fa c to r .

T he fa c to r exh ib its som e sp e c ie s sp e c if ic ity but both human and m ouse co lo n y -stim u la tin g fa c to r w ill s tim u la te m ouse b o n e-m arro w c e l l s . Human u rin e fa c to r is an tigen ic for ra b b its and antibodies produced a fte r im m u nization with human u rin e can inhibit the in -v itro activ ity of the co lo n y -stim u la tin g fa c to r o f both m ouse and human o r ig in (M cN eill, unpublished d a ta ).

With a m ouse b o n e-m arro w cu ltu re sy stem , co lo n y -stim u la tin g fa c to r can be assayed in m ouse and human m a te r ia l . C o lo n y -stim u latin g fa c to r is d etectab le in the seru m of m o st n orm al m ice but not in seru m fro m n o rm al hum ans. L e v e ls of co lo n y -stim u la tin g fa c to r a re elevated in m ice with leu k aem ia of a ll m o rp h olog ical typ es [ 3, 8, 18] and in resp o n se to som e v iru s in fectio n s [1 9 ] . In hum ans, seru m colony- stim u latin g fa c to r le v e ls a re elevated in 15 - 70% o f patients with: (1) neop lasm s o f the re ticu lo en d o th e lia l sy stem , p a rticu la rly in the advanced, and c lin ic a lly activ e , s ta g e s , (2) c e r ta in n o n -n eo p lastic p ro life ra tiv e d is o rd e rs of h aem o p o iesis , and (3) in the acute s tag es of c e r ta in non- b a c te r ia l in fectio n s and m ononu cleosis [2 0 , 21] .

C o lo n y-stim u latin g fa c to r can be d em onstrated in the unconcentrated u rin e o f m o st n orm al hum ans, although le v e ls v e ry w idely from one p erson to another and from day to day in individ uals. The fa c to r is p resen t in elevated am ounts in som e c a s e s o f leu k aem ia [ 10] , although lev e ls flu ctu ate widely during the co u rse of the d is e a s e in individual p atien ts (M etca lf, unpublished d ata).

C h em ica l a n a ly sis o f the co lo n y -stim u la tin g fa c to r is incom p lete but su g g ests that th e re is a c lo se s im ila r ity betw een the conditioned m edium , m ouse seru m and human urine co lo n y -stim u la tin g fa c to r s . The fa c to r is f i lte ra b le , n o n -d ia ly sab le and re s is ta n t to e th e r and u.v. ir ra d ia tio n . It is h e a t-la b ile and is d estroyed by try p sin . It w ithstands s to ra g e at -20° C, rep eated freeze-th aw in g and tre a tm e n t with p eriod ate, D N A -ase and RNA- a s e . It is p recip ita ted by ethanol 40 - 48% and am m onium sulphate 40 - 60%. It m oves e le c tro p h o ré tic a lly as a s in g le peak in the p o st-a lb u m in reg ion and has a sed im en tation constan t o f 3 - 4 S . It se p a ra te s as a s in g le peak on Sephadex and DEAE c e llu lo se ch rom atograp hy. The data so fa r a re co n sis te n t with the in te rp re ta tio n that the co lo n y -stim u la tin g fa c to r is the p ro te in o f m o le cu la r weight 50 000 - 60 000 (Stan ley , M etca lf and B rad ley , unpublished d a ta ).

AGAR TECHNIQUE 9

T he co lo n y -stim u la tin g fa c to r m ay re p re se n t a n orm al hum oral reg u la to r o f g ra n u lo p o ies is . P re lim in a ry in -v ivo te s ts in m ice of human urine fa c to r and conditioned m edium fa c to r (derived from syngenic em bryo c e lls ) have indicated that the fa c to r e le v a te s the num ber of b o n e-m arro w , sp leen and blood co lo n y -fo rm in g c e lls and produces a polym orph leu co - cy to sis in which the k in e tics of granu locyte p ro life ra tio n and r e le a s e to the blood appear to be ap p roxim ately n orm al (B ra d ley et a l . , unpublished d a ta ).

H ow ever, it is obvious that the regulating sy stem fo r gran u lo p oiesis in the body is com plex and m ust involve the op eration o f a num ber o f o th er regulating in flu e n c e s . F o r exam p le, in the reg en era tio n of g ran u lo cytic c e lls a fte r w hole-body irra d ia tio n , th e re has been no evidence so fa r of an e levation in seru m le v e ls o f co lo n y -stim u la tin g fa c to r . F u r th e rm o re , d esp ite good g en era l c o r re la tio n s betw een seru m le v e ls o f co lo n y -stim u latin g fa c to r and le v e ls of g ran u lo p oiesis in v ario u s d ise a se s ta te s in m ice , c o r re la tio n at the individual m ouse le v e l betw een seru m fa c to r le v e ls and the num ber o f b o n e-m arro w co lo n y -fo rm in g c e lls o r the le v e l of blood polym orphs has been e x tre m e ly p oor. In part th is m ay be due to flu ctu ation s in seru m co lo n y -stim u latin g fa c to r lev e ls from day to day — a phenom enon known to o ccu r in hum ans with acu te leu kaem ia (F o s te r and M etcalf, unpublished data) — but it is lik e ly that o th er fa c to rs a re op erating in such m ice to m odify the o b serv ed le v e ls o f co lo n y -fo rm in g c e lls and polym orphs.

6. FR E Q U E N C Y O F CO LO N Y-FO RM IN G C E L L S IN ABNORM ALSITUATIONS

P o ten tia lly , the a g a r colony a ssa y sy stem fo r co lo n y -fo rm in g c e lls has its m ost obvious ap p lications in the enum eration of co lon y -form in g c e lls in v ario u s abnorm al s itu a tio n s . With a constant dose o f colony- stim u latin g fa c to r and titra tio n s o f the haem op oietic c e ll suspension under in v estigation , the sy stem has proved to be highly rep ro d u cib le in enum era tin g the absolu te num ber of co lo n y -fo rm in g c e lls p resen t and the num ber o f c e lls req u ired fo r such an a s s a y can be v e ry s m a ll (as few as 12 500 p er p la te ). In ou r exp erim en ta l work with m ouse b on e-m arrow c e lls , it has been p o ssib le fo r a s in g le in v e stig a to r in h alf a day to p re p a re and c a r r y out a ssa y titra tio n s on ten to twenty d ifferen t bone- m arro w c e ll su sp en sio n s.

We have used th is method o f assay in g co lon y -form in g c e lls fo r a wide v a r ie ty o f purposes in ou r la b o ra to ry . F o r exam p le, the incid ence of co lo n y -fo rm in g c e lls in m ouse bone m arrow has been d eterm ined a lread y in the follow ing s itu a tio n s: (1) aging, (2) g e rm -fre e s ta te , (3) p o s t - ir r a - d iation , (4) g en etic an om alies of e ry th ro p o ies is [ 2 2 ] , (5) spontaneous and v ira l-in d u ced leu kaem ia , (6) a fte r an ti-ly m p h ocyte seru m tre a tm e n t and/ o r thym ectom y, (7) a f te r bleeding o r h y p ertran sfu sio n , (8) a fte r antigenic s tim u lation , (9) a fte r sp lenectom y, and (10) a fte r co rtiso n e ad m in istra tio n . Many o f the data from th e se o b serv a tio n s a re in a p re lim in a ry form and w ill be rep orted in fu ll e lse w h e re .

P rovided the p resen t cu ltu re conditions fo r human haem op oietic ce ll colony form ation can be im proved slig h tly , th e re a re s e v e r a l obvious ap p lications fo r th is technique in c l in ic a l m ed icin e . The p resen t assay

10 METCALF-

sy stem would be of value in any situ ation w here it is d e s ira b le to a s s e s s the num ber o f g ran u lo cytic p re c u rs o r c e lls o r w here su rv iv al o f granulo c y tic p re c u rs o rs can be used as an index o f su rv iv a l of o th er haem op oietic c e ll ty p e s . Som e ap p lications would be (a) the a s s e s s m e n t of the e ffic ie n cy o f v ario u s m ethods fo r the s to ra g e of h aem op oietic c e lls , (b) the a s s e s s - : m ent of bone-m arro.w dam age a fte r irra d ia tio n o r high dose th erap y of cy to tox ic d rugs, (c) the a s s e s s m e n t of su rv iv a l of tran sfu sed haem op oietic c e l ls , (d) the a s s e s s m e n t of h aem op oietic c e ll r e s e r v e s in v ario u s d is e a s e s , e .g . leu k aem ia , and (e) the a s s e s s m e n t o f haem op oietic dam age resu ltin g fro m th erap eu tic p ro ced u res designed to su p p ress hom ograft re je c tio n .

7 . A P P LIC A TIO N OF TH É AGAR C U L TU R E TECHNIQUE TO O TH ERP R O B L E M S IN TH E STO R A G E AND CU LTIVA TIO N O F HAEM OP O IE T IC „CELLS .

In its p resen t fo rm , the a g a r cu ltu re technique does not ap p ear to be su itab le fo r m a ss production in v itro o f haem op oietic c e l ls : (a) Only g ran u lo cy tic and m ononu clear c e lls w ill p ro life ra te in the p re sen t sy stem and not e ry th ro p o ie t ic ,o r lym phop oietic c e l l s , (b) It is doubtful w hether a net gain in haem op oietic c e lls re s u lts in the p resen t cu ltu re s y s te m . C olonies do not g en erate co lo n y -fo rm in g c e l l s . F u r th e r , even with strong s tim u la tio n w here 100 co lo n ies develop from 50 000 b o n e-m arro w c e lls , it is com m on fo r each colony only to contain up to 500 c e l l s , which m eans th e re has been no in c re a s e in the to ta l num ber o f h aem op oietic c e lls achieved in the cu ltu re s y s te m , (c) Due to the p e cu lia r p ro p e rtie s of a g a r , it is e x trem ely d ifficu lt to fre e c e lls o f surrounding a g a r . T h is has fo re s ta lle d m any of o u r attem p ts to in je c t colony c e lls in vivo and to a s s e s s th e ir in -v ivo functional ca p a c ity . T h is m ight be o v erco m e by the u se o f a ltern a tiv e g ellin g agen ts, e .g . m ethyl ce llu lo se o r s ta r c h , (d) Colony growth cannot be m aintained fo r lo n g er than 14 days even a fte r re feed in g with fre s h o v e r la y e rs o f m edium and co lo n y -stim u la tin g fa c to r .

H ow ever, the p resen t a g a r sy stem points the way to p o ssib le new ap p roach es to the cu ltivation o f h aem op oietic c e lls and with su itab le m od ifica tio n an ag ar cu ltu re sy stem m ight be able to s e rv e as a continuous gen eratin g sy stem fo r at le a s t one type of haem op oietic c e ll .

8 . SUM M ARY

T he ag ar cu ltu re sy stem d escrib e d above has opened up new v is ta s in e x p erim en ta l haem ato logy . F o r the f i r s t tim e , .a re la tiv e ly sim p le , qu an tita tive , and highly rep ro d u cib le sy stem is av a ilab le fo r the cu ltu re in v itro o f one type o f h aem op oietic c e ll in such a.w ay that c e ll p r o li fe r a tion and d ifferen tia tio n a re read ily m e a su ra b le .

T h is sy stem has a lread y g re a tly in cre a se d ou r knowledge of the o r ig in s o f g ran u lo cy tic .p re cu rso r c e l ls , th e ir b iology and th e ir exact mode o f p ro life ra tio n and d iffe ren tia tio n .

In its p resen t fo rm , the a g a r cu ltu re sy stem has two ap p licatio n s which should be of value at the c l in ic a l le v e l : (Í ) It has d em onstrated the e x is te n ce of a pow erful hu m oral fa c to r regu lating g ran u lo p o iesis and

AGAR TECHNIQUE 11

can a c c u ra te ly a s s a y seru m o r u r in e -le v e ls o f th is fa c to r . The fa c to r is p resen t in h u m an u rin e in 'la rg e am ounts and the cu ltu re sy stem is being used as an a ssa y sy stem in the la r g e - s c a le p u rifica tio n o f th is fa c to r . T h is fa c to r m ay have a num ber o f ap p licatio n s in the th erap y o f d iso rd e rs o f h a e m o p o ie s is . ¿(2) The a g a r cu ltu re sy stem provides a quantitative sy stem fo r enum erating g ran u lo cy tic co lo n y -fo rm in g c e l l s . B ein g ap p lica b le .to human, c e lls , it can be used in the a s s e s s m e n t o f the e ffic ie n cy o f s to rag e p ro ced u res o f human haem op oietic c e lls and the a ss e s s m e n t o f h aem op oietic c e ll r e s e r v e s in v ario u s types o f patient.

U n less the sy stem can be m odified su b stan tia lly it does not appear to o ffe r a s a tis fa c to ry method fo r the m a ss production o f haem op oietic c e lls fo r th erap eu tic u se .

R E F E R E N C E S

[1] BRADLEY, T .R ., METCALF, D ., The growth of mouse bone marrow cells in vitro, Aust. J. exp. Biol. med. Sci.44 (1966) 287.

[2] PLUZNIK, D .H ., SACHS, l . t The cloning of normal "mast" cells in tissue culture, J. cell. Physiol. 66 (1965) 319.

[3] METCALF, D ., FOSTER, R . , Bone marrow colony stimulating activity of serum from mice with viral-induced leukemia, J. natn. Cancer Inst. 39 (1967) 1235.

[4] ICHIKAWA, Y . , PLUZNIK, D. H ., SACHS, L ., In vitro control of the development of macrophageand granulocyte colonies, Proc. natn. Acad. Se i.USA 56 (1966) 488,

[5] BRADLEY, T .R ., Aspects of stimulation of bone marrow colony growth in vitro, Aust. J. exp. Biol,med. Sei.(in Press).

[6] PLUZNIK, D .H ., SACHS, L ., The induction of clones of normal "m ast*'cells by a substance from conditioned medium, Expl cell. Res. 43 (1966) 553.

[7] BRADLEY, T .R ., SUMNER, M. A ., Stimulation of mouse bone marrow colony growth in vitro by conditioned medium, Aust. J. exp. Biol. med. Sei.(in Press).

[8] ROBINSON, W., METCALF, D ., BRADLEY, T , R .,. Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells, J. cell. Physiol. 69 (1967) 83.

[9] BRADLEY, T .R . , SIEMIENOWICZ, R ., Colony growth of rat bone marrow cells in vitro, Aust. J . exp. Biol. med. Sei.(in Press).

[10 ] ROBINSON, W. A ., STANLEY, E .R ., METCALF, D ., Stim ulation of bone marrow colony growth in vitro by human urine. Blood (in Press).

[11] SENN, J .S . , McCULLOCH, E. A ., TILL, J . E . , Comparison of colony-forming ability of normal and leukaemic human marrow in tissue culture, Lancet 2 (1967) 597.

[12] METCALF, D ., BRADLEY, T .R ., ROBINSON, W ., Analysis of colonies developing in vitro from mouse bone marrow cells stimulated by kidney feeder layers or leukemic serum, J. cell. Physiol. 69 (1967) 93.

[13] ICHIKAWA, Y ., PLUZNIK, D .H ., SACHS, L ., Feedback inhibition of the development of macrophage and granulocyte colonies. I Inhibition by macrophages, Proc. natn. Acad. Se i.USA 58 (1967) 1480.

[14] METCALF, D ., FOSTER, R ., Behavior on transfer of serum stimulated bone marrow colonies,Proc. Soc. exp. Biol. Med. 126 (1967) 758.

[15] ROBINSON, W. A ., BRADLEY, T .R ., METCALF, D ., Effect of whole body irradiation on colony production by bone marrow cells in vitro, Proc. Soc. exp. Biol. Med. 125 (1967) 388.

[16] BRADLEY, T .R ., ROBINSON, W., METCALF, D ., Colony production in vitro by normal, poly- cythaemic and anaemic bone marrow, Nature (Lond. ) 213 (1967) 511.

[17] WU, A .M ., SIMINÖVITCH, L ., TILL, J. E ., McCULLOCH, E .A ., Evidence for a relationship between mouse hemopoietic stem cells and cells forming colonies in culture, Proc. natn. Acad. Se i.USA (in Press).

[18] METCALF, D ., FOSTER, R ., POLLARD, M ., Colony stimulating activity of serum from germfree normal and leukemic mice, J. cell. Physiol. 70^(1967) 131.

12 METCALF

[19] FOSTER, R ., METCALF, D ., KÏRCHMYER, R ., Induction of bone marrow colony stimulating activity by a filterable agent in leukemic and normal mouse serum, J. exp. Med. 127 (1968) 853.

[20] FOSTER, R ., METCALF, D ., ROBINSON, W .A ., BRADLEY, T .R ., Bone marrow colony stimulating activity in human sera. Results of two independent surveys in Buffalo and Melbourne, Br. J. Haematol. 15 (1968) 147.

[21] METCALF, D ., WAHREN, В ., Bone marrow colony stimulating activity of sera in mononucleosis,Br. med. J. (in Press).

[22] BENNETT, M ., CUDKOWICZ, G.'f FOSTER, R .S ., METCALF, D ., Hemopoietic progenitor cells of W anemic mice studied in vivo and in vitro, J. cell. Physiol, (in Press).

DIFFERENTIATION OF HAEMOPOIETIC TISSUE IN ORGAN CULTURES

A . Y a . FRIEDENSHTEIN

N .F . G a m a le y a In stitu te for E p id em iology and M icro b io lo gy ,

USSR A ca d e m y o f M e d ica l S c ie n ce s ,

M oscow , USSR

Abstract

, DIFFERENTIATION OF HAEMOPOIETIC TISSUE IN ORGAN CULTURES. . As is well known, it is not possible to maintain over lengthy periods the normal differentiation of haemopoietic tissue in cultures. It seems probable that the lack of success attending such attempts is due to the fact that cultures do not present the necessary conditions for local interactions of haemopoietic matter with the stroma of the organs concerned.

In order to test this possibility, recourse was had to the method of organ culturing on millipore filters.It was found that when fragments of embryonic liver or embryonic bone of mouse are cultured on filters, hepatic parenchymatous matter on bone stroma develops. In the former case haemopoiesis is also maintained in the cultures for a duration of 20 days, the foci of myeloid and less frequently erythroid cells being visible. In such cultures colony-forming cells are also maintained, their number amounting to between 20 and 40 per 10s cells by day 8 to day 12. In the second case, where embryonic bone is cultured, the addition of adult bone-marrow cells results in haemopoiesis being maintained in the explant over a period of 16 days (without bone tissue haemopoiesis in organ cultures stops within 5 days).

The paper discusses the part played by such local interactions between haemopoietic cells and bone tissue or embryonic liver parenchyma in maintaining haemopoiesis.

1. INTRODUCTION.

The cu ltu re of h aem op oietic tis su e without a ffectin g its d iffe ren tia tio n in v itro and without lo s s of the p re c u rs o r c e l ls cap able of en su rin g h aem op o ie s is when tran sp lan ted in v itro is a p roblem that has not yet been solved .In s in g le -la y e r cu ltu re s in a liquid nu trien t m edium and in p lasm a cu ltu re s th e re is a rapid lo s s of d iffe ren tia tio n of the h aem op oietic c e lls and the f ib r o - b la s ta ce o u s c e lls w hich grow in such cu ltu re s do not d iffe ren tia te into h aem op oietic c e lls when subsequently tran sp lan ted .

The m ost su itab le m eans of m aintain ing c e ll d iffe ren tia tio n in v itro a re the organ cu ltu re m ethods, which a re b ased on the p rin cip le of p lacin g the tis su e at the boundary betw een a liquid and a gaseous m edium . In such cu ltu re s the n atu ra l in te r c e llu la r in te ra c tio n s c h a r a c te r is t ic of the tis s u e in question a re re ta in ed . T h e se in te ra c tio n s , as is w ell known, a re of the u tm o st im p ortan ce in m ain ta in in g the d ifferen tia tio n of h aem op oietic t is s u e in the o rg an ism its e l f . In th is conn ection , alongside such g e n era l env ironm ental fa c to rs as the horm one and v itam in b a s e s , e tc . , s p e c ia l im p ortan ce a ttach es to lo c a l in te ra c tio n s o ccasio n ed by tis su e in d ire c t co n tact with haem op oietic tis su e .

The question acco rd in g ly a r is e s w hether it would not be d e s ira b le , in attem pts to m ain tain the d iffe ren tia tio n of h aem op oietic t is su e in v itro , to a rra n g e fo r it to be in co n tact with the t is s u e s con cern ed in organ cu ltu re cond itions. T h is is the type of approach w hich has been follow ed in the w ork h e re rep o rted . We have sought to a rra n g e fo r h aem op oietic t is s u e to in te r -

13

1 4 FRIEDENSHTE1N

act in 'v itro with its two n atu ra l neigh b ou rs, em bryon ic l iv e r tis su e and bone tis su e . ' ■ ■ .

2. E X P E R IM E N T

The l iv e r o f l 6 - t o 2.0-day C B A m ou se em bryos was w ashed tw ice in 199-m ed iu m , then s l ic e s into frag m en ts 2 - 3 mm in d ia m ete r and explanted in v itro with the organ cu ltu re m ethod d escrib e d p rev iou sly (E . A. L u riy a ,I .E . Pyanchenko). C ulture w as c a r r ie d out on ANFS m illip o re f i l t e r s (pore s iz e 0. 6 - 0. 9 jum) p laced at the boundary betw een two p h ases — a liquid n u trien t m edium phase and a gaseous phase co n sistin g of 5% carb on dioxide in a ir . At the sam e tim e 12 - 13 explants w ere p laced in one cu ltu re v e s s e l . The com p osition of the m edium w as 70% 199-m ed iu m , 20% bovine seru m ,10% ch icken em bryo e x tr a c t ; to each 100 m l of p rep ared m edium w ere added7 m g v itam in C, ■ 4 0 0 m g g lu co se , 20 m g N aB -g ly cero -p h o sp h áte , 20 mg 1-g lutam in and 5000 un its each of p e n ic illin and strep to m y cin . The cu ltu re m édium w as changed ev ery 48 - 72 h o ú rs . ,

, The cu ltu re s w ere fixed in v itro a fte r 3,. 6, 8, .9, 12, 13,. 18, 21 and .24 days with F o rm o l s p ir it o r 96% a lcoh ol. B etw een 2 and 8 cu ltu re s w ere fixed a fte r each p eriod . A lto geth er 108 cu ltu re s w ere studied. Som e of the cu ltu re s w ere steeped in p ara ffin and cut into a s e r ie s o f s l i c e s 7 ßm th ick , which w e re 's ta in e d w ith a lu m haem atö xy len e. O ther cu ltu re s , w hich had been stained with haem atöxylene in to to , a fte r dehydration and c le a r in g in xylene w ere used to m ake to ta l p re p a ra tio n s. S till o th ers w ere su b jected to try p sin iza tio n to m ake c e ll su sp en sio n s which w ere then studied by G ie m sa - sta in ed s m e a rs o r ad m in istered in trav en o u sly to syngenic m ice to d eterm in e the num ber of co lo n y -fo rm in g u n its, by m eans of the method of T i l l and M acC u lloch . F ra g m e n ts of thigh bone fro m 17-d ay m ice em bryos w ere e x planted into th ese cu ltu re s and a fte r 10 - 14 days frag m en ts of'borie m arrow taken fro m the thigh bones of adult m ice w ere a lso explanted onto the f i l t e r s . O n e-th ird of the th igh-bone content was explanted . The m edium was changed e v e ry 48 - 72 h ou rs. ’ The cu ltu re s w ere fixed in 96% alcohol b e fo re t r a n s plantation of the bonè m arrow and follow ing tran sp lan ta tio n (a fte r 3, 6, 7, 8,9, 10, 11, 14, 16 and 18 days), betw een 3 and 5 cu ltu res being fixed a fte r each p eriod . The bone frag m en ts w ere rem oved fro m the f i l t e r s , c a re being taken to avoid dam age to the growth zone, and cirt into a s e r ie s of s l ic e s which w ere stained with alum h aem atö xy len e. On the f i l t e r s w hich had been fixed b efo re tran sp lan ta tio n of bone m arro w we induced G ô m ô ry 's re a c tio n fo r a lk alin e phosp hatase. The o th er f i l t e r s w ere stained with haem atöxylene and, á fte r dehydration and c le a r in g , en clo sed in Canada b a lsam in the fo rm of p erm anent p re p a ra tio n s.

. с

3. R E SU L T S

In 3-d ay cu ltu res of l iv e r frag m en ts one cán o b serv e tis su e d egen eration in the ce n tre of the explant and re te n tio n of the ep ith e lia l la y e r s , m yeloid c e lls and m e g ak ary o cy tes in the p e rip h e ra l re g io n s . Around the frag m en t on the f i l t e r 'th e r e is a growth of ep ith e lia l m em b ran es with the e je c te d h aem op oietic c e l l s . On the low er su r fa ce of the f i l t e r th e re is a growth of c e lls of the type

DIFFERENTIATION OF HAEMOPOIETIC TISSUE 15

of d en d ritic h is tio cy te s — probably s tro m a l m a tter that has p erm eated through the f i l t e r p o re s . •

At 6 days the frag m en ts a re surrounded by ep ith elia l m em b ran es with num erous c e l ls in p ro c e s s of m ito s is . The p e rip h e ra l reg io n s of the explant a re taken up by la rg e haem op oietic a re a s containing m yeloid and ery th ro id c e lls and m eg ak ary o cy tes d isp ersed am ong the ep ith e lia l c e lls .

A fter 8 - 9 days the p ro life ra tin g e p ith elia l m em b ran es on the upper s u r fa ce of the f i l t e r b eco m e m u lti- la y e r in c h a ra c te r and the ep ith elia contained in them acq u ire the c h a r a c te r is t ic m orphology of polygonal hepatic c e lls co lle c te d in tra b e c u la e . On the m em brane one finds a la rg e num ber of e ry th ro id and m yeloid c e l ls , w hich fo rm la rg e fo c i of h aem o p o iesis . T h ese fo ci co m p rise m atu re and im m atu re haem op oietic c e lls and m a tter in p ro c e s s of m ito s is . R eg en era tio n of the hepatic c e lls is under way in the c e n tra l p arts o f the frag m en t. H ow ever, h aem op oietic tis su e is found only in the p erip h era l re g io n s , w here it is a rran g ed alongside h ep atic c e lls .

B y 12 - 13 days the explant b eco m es flattened and its conn ective tissu e grow s m o re and m o re into the p o res of the f i l t e r . A m u lti- la y e r growth of hepatic tis su e co v e rs a co n sid erab le a re a of the f i l t e r and p re se n ts a un iform p ic tu re , both in the ce n tre and in the p e rip h e ra l a r e a s . The la y e r of hepatic epithelium , made up of la rg e c e lls with sh arp bound aries and a polygonal shape, s tre tc h e s o v er a netw ork of s m a lle r co n n e c tiv e -t is su e d en d ritic c e lls which have p erm eated into the f i l t e r p o res and a re grow ing on its low er s u r fa c e . In c e r ta in a re a s it i s now p o ss ib le to see the fo rm atio n of sp e c ific m u lti- la y e r s tru c tu re s m ade up of o rien ted ep ith elia l c e l ls co lle c te d into re n ifo rm s tru c tu re s and surrounded by elongated co n n e c tiv e -t is su e m a tte r , fo rm in g -as it w ere a cap su le around the e p ith elia l group. In th ese s tru c tu re s th e re is in ten se accu m ulation of yellow pigm ent p a r t ic le s . One can a lso see the fo rm ation of o rb ic u la r lacu n ae , the sid ew alls of which a re form ed of s im ila r elongated m a tte r w hile the flo o r i s paved with e p ith e lia l c e l ls . In cu ltu res of th is age in ten siv e h aem o p o iesis is to be seen . • M yeloid c e lls at vario u s stag es of d ifferen tia tio n , e ry th ro id fo rm s and m eg ak ary ocy tes are d isp ersed over wide a re a s above the e p ith e lia l la y e r and fo rm , as it w ere, the upper la y e r of ;the expiant.- It is im portant to note the groups of m eg ak ary o cy tes co n sistin g of 4 - 10 c e lls c lo s e ly ad joining each o th er. The haèm op oietic a re a s have n o 'sh arp b ou n d aries, one a re a m erg in g in another.It is n e v e rth e less p o ssib le to note w ithin each a re a s m a lle r fo c i of-m yeloid , ery th ro id and m eg ak ary o cy tic h aem o p o iesis , which in re s p e c t of m orphology and arran g em en t can be divided into s u b -s tru c tu r e s . It is a lso p o ssib le to see groups of m yeloid c e lls w hich, to judge fro m the shape of the n u clei, are a ll at one stage of h is to g e n e s is : "■

B y th e '18 th day of cu ltu re th e re is fu rth er m atu ration of the epithelium .As p rev iou sly , one can see la rg e a re a s of h aem op oiesis which a lso co m p r is e haem op bietic m a tte r in the p ro c e s s of m ito s is . In addition to th is th e re a re a co n sid erab le num ber of sm a ll fo c i of haem op oietic c e lls , containing som e ten s of c e lls each .

By the 21st day of cu ltu re the num ber of h a e m o p o ie tic -ce lls has fa llen by com p ariso n with p reviou s p erio d s. The fo c i of haem op oietic c e lls are to be found betw een the ep ith elia l tra b e cu la e , surrounded by a la y e r of con n ectiv e t is s u e . The num ber of c e lls in the fo c i v a r ie s fro m ten s to hundreds. Above the ep ith e lia l la y e r th e re a re a re a s of m atu ring m yeloid c e lls (m y elo c y te s ). One can now see the ap p earance of a la rg e num ber of m onocytic c e lls which w ere not to be ob served p rev iou sly .

16 FR1EDENSHTEIN

Of the th re e cu ltu re s fixed fo r 24 days in v itro two showed no h aem op o ie s is . Above and betw een the e p ith e lia l s tru c tu re s th e re a re la rg e groups o f m yeloid c e lls co m p risin g s e v e r a l hundred c e lls each and at v ario u s stag es of d ifferen tia tio n . One a lso n otes individual m eg ak ary o cy tes or groups of m e g ak ary o cy tes , so m e tim e s lo cated in lacu n ae .

S m e a rs taken fro m 3 - to 24-d ay cu ltu re s showed num erous h aem op oietic c e lls with m ito s e s . The num ber of co lo n y -fo rm in g units in the em bryonic l iv e r c e ll population on the 8th and 12th days of cu ltu re was d eterm ined in con ju n ction with D r. J . L . C hertkov (M oscow In stitu te of H aem otology and B lood T ra n sfu sio n ). T h e se r e s u lts a re given in T ab le I, which shows that the cu ltu re s re ta in a re la t iv e ly la rg e num ber of co lo n y -fo rm in g u n its, i. e. that the ca teg o ry of co lo n y -fo rm in g , in o th er w ords patently s tem c e lls of h aem op oietic c h a ra c te r is m aintained and p o ssib ly even grow s. T h e ir d if fe r en tiatio n potential is obviously unchanged in cu ltu re , and th is w as a lso to be expected fro m the g e n era l ap p earance of the co lo n ies and fro m the ra tio of co lo n ies contain ing h aem op oietic c e lls of d iffe ren t typ es.

T A B L E I . CO LO N Y-FO RM IN G UNITS (C FU ) IN EM BRYO N IC L IV E R C U L T U R E S

Duration of culture (d)

Number of available nucleated cells introduced

(X 10)

Number of colonies per spleen .

Number of CFU per 105 cells

7 66 8 13 16 20 21 22.8 ± 5 .2

7 132 25 28 31 32 32

12 54 17 20 21 21 22 22 23 41.7 ± 3 .9

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 (control)

In exp erim en ts on the cu ltu re of bone m arrow fro m adult m ice on f i l t e r s with p recu ltu red bone, it was found that on th o se f i l t e r s w here em bryonic bone was planted o ste o g e n e s is was to be found by day 10 - 14 in v itro , the growth zone of the f i l t e r co n sis tin g m ainly of osteog en ic tis su e w hich gave a p o sitiv e re a c tio n to a lkalin e phosp hatase; the growth zone did not at th is s tag e show any m o rp h olog ically m atu re bone tra b e cu la e .

By day 6 - 8 of com bined cu ltu re of bone m arrow and em bryonic m ouse bone, the growth zone ad joining the bone explant shows a re a s of co m pleted o ste o g e n e s is in the fo rm of bone tra b e cu la e with ca lc iu m dep osition . The p e rip h e ra l re g io n s of the growth zone show no c h a r a c te r is t ic bone tis su e m orphology, but a re made up of orien ted c e ll s tran d s without d ep osition of p rim a ry su b stan ce . The h aem op oietic m a tte r is m ainly con cen tra ted round the explanted bone fragm en t and fo rm s com p act c lu s te r s above the c e lls of the s tro m a .

B y the 6th day in v itro , the bulk of the explanted b o n e-m arro w frag m en t has died. In the bone frag m en t i t s e l f th e re is o s te o g e n e s is on the p erip h ery . In not one of the c a s e s o b serv ed w as any repopulation of h aem op oietic c e lls see n in the in te r io r .

DIFFERENTIATION OF HAEMOPOIETIC TISSUE 17

The 9th and, m o re e sp e c ia lly , the 11th day of exp lantation a re r e m a r k able fo r the in te n sifica tio n of o s te o g e n e s is and m yeloid h aem o p o iesis in the cu ltu re s . In the m u lti- la y e r growth zone, the bone explant is surrounded by num erous b ran ch in g bone tra b e cu la e with lo ca liz e d ca lc iu m d ep osition .In the fo c i of o s te o g e n e s is i t is p o ss ib le to o b serv e m atu re bone s tru c tu r e s , a p e rip h e ra l l a y e r ‘of o s te o b la s ts and en clo sed o s te o cy te s . In co n tact with the bone tra b e cu la e th e re a re m u lti-la y e r fo c i of m yeloid h aem o p o iesis which contain s e v e r a l hundreds of c e l ls . In the su r fa ce la y e r of the growth zone, w here th e re is no c le a r m o rp h olog ical evidence of o s te o g e n e s is , th e re a re a lso la rg e a re a s of in ten siv e m yeloid h aem o p o iesis ; the m yeloid m a tter co n ta in s a num ber of c e lls in p ro c e s s of m ito tic d iv ision . The m yeloid c e lls co v er a wide a re a extending to the p e r ip h e rie s of the growth zone.

In 1 4 - to 16-d ay cu ltu re s , the num ber of h aem op oietic c e lls is som ew hat le s s than at previou s s ta g e s . The m yeloid c e l ls , w hich a re at vario u s s tag es of d ifferen tia tio n , fo rm fo c i of d ifferen t s iz e s around the explant and on the p erip h ery of the su r fa ce la y e r of the growth zone. B y the 18th day a fte r explantation of the bone m arro w , the cu ltu res show only v e ry few v iable m y eloid c e lls .

Thus an organ cu ltu re of m ouse bone m arrow explanted onto p recu ltu red osteog en ic tis su e shows a prolonged in ten siv e h aem o p o iesis m aintained for 16 days in v itro . As is w ell known, when bone m arrow is explanted d ire c tly onto m illip o re f i l t e r s , h aem o p o iesis la s ts for 5 days.

T h e se re s u lts show that in organ cu ltu re conditions haem op oietic t is su e of em bryonic and adult bone m arrow can continue to p ro life ra te and d if fe r en tia te over long p erio d s. F o r th is purpose it is n e c e s s a ry fo r the tis su e to be in con tact with bone o r em bryonic hepatic t is su e , i . e. th o se t is s u e s which a re the n atu ral neighbours of haem op oietic tis su e in the o rg an ism and which e x e r t 't a x i s ' on i t . T h e re is re a so n to suppose that th e se t is s u e s e x e rt s p e c if ic h isto g en etic s tim u li on the h aem op oietic c e l ls , though no data is yet availab le on the natu re of the stim u li o r on the m ech an ism s by which they ac t.

вHISTOCOMPATIBILITY AND AVOIDANCE OF

SECONDARY DISEASE

SECONDARY DISEASE IN RADIATION CHIMERAS *

C . C . CÓNGDON

B io lo g y D ivision , O ak R idge N a tio n a l L a b o ra to ry ,

O ak R idgè, T e n n . , U nited S ta tes o f A m e ric a

Abstract

SECONDARY DISEASE IN RADIATION CHIMERAS. A review of research dealing directly or indirectly with the development o f bidirectional tolerance in radiation chim eras has been m ade, emphasizing some of the contemporary research on this subject in Oak Ridge and Knoxville. By controlling such factors as ce ll dose, age of donor anim al and day of ce ll in jection , it was possible to achieve bidirectional tolerance. Attempts to reduce bid irectional to lerance in favour of increasing the graft-versus-host reaction were less successful. Hypoxic caging demonstrated a new approach to achieving bidirectional tolerance through physiological com petition for growth. Graft-versus-host reactions have a lower growth priority than marrow regeneration or erythropoietic hyperplasia.

Study of pathologic processes, im munologic capability and the-biochem ical lesions in radiation .chimeras all lead to new ideas that involve bidirectional tolerance.

The investigations on dose rate in radiation suppression o f the immune response and on LD^ (30- to 9Cbday)values after in jection o f different numbers of marrow cells all have a bearing on control o f the host-versus-graft response and therefore are important in understanding bidirectional to lerance.

1. INTRODUCTION

It is freq u en tly ob serv ed that su p ra le th a lly irra d ia te d m ice tre a te d with a llo g en ic o r ra t bone m arrow develop sustained to le ra n c e in the h o s t-v e r s u s -g r a ft re a c tio n , and equally sustained to le ra n c e in the g ra ft- v e rs u s-h o s t re a c tio n [ 1 ,2 ] . The spontaneous developm ent o r ap p earance of the b id irec tio n a l to le ra n c e is noted m ost often in ro d en ts . It is o cca s io n a lly noted in dog e x p erim en ts [3] but apparently is n ever see n in m onkey [4] or m an [5 ]. B e ca u s e of th e se find ings, acq u isitio n of to le ra n c e in fo re ig n b o n e-m arro w rad ia tio n c h im e ra s is probably the sin g le m ost im portant top ic under in v estig atio n in m arrow tran sp lan t r e s e a r c h .

The fa c t that spontaneous tran sp lan ta tio n to le ra n c e o c c u rs in irra d ia te d rod ents is fo rtu n ate and m ak es it p o ss ib le to study the phenom enon in a g re a t v a r ie ty of w ays. T h e p re se n t w ork i s a rev iew of som e of our stu d ies in Oak R idge and K noxv ille o n a cq u isitio n of to le ra n c e in the m ouse rad ia tio n ch im e ra .

2. E X P E R IM E N T A L DESIGN STU D Y OF M O R T A L IT Y

In th is w ork, the m e a su re of to le ra n ce o r la c k of to le ra n c e is 90-d ay m o rta lity in le th a lly (950 R ) irra d ia te d m ice g iven a llo g en ic o r h etero logou s b o n e-m arro w c e l ls in trav en o u sly . The v a r ia b le s ch osen fo r study w ere age of b o n e-m arro w donor (3 -9 0 d ays), sex of donor and re c ip ie n t, tim e of m arrow in je c tio n with r e fe r e n c e to the day of irra d ia tio n (0 -4 ) , and num bers

* Research sponsored by the US Atom ic Energy Commission under contract with Union Carbide Corporation.

21

2 2 CONGDON

of b o n e-m arro w c e lls in je cte d (10X 1 0 6 - 60X 106 ). A n im als surviv ing 90 days w ere te s ted fo r c h im e ris m .

S ix designed exp erim en ts were com p leted . F iv e w ere a llo g e n ic , of which fou r w ere c a rr ie d out to ach ieve m inim um 90-d ay deaths and one was designed fo r m axim um 90-d ay m o rta lity . The six th exp erim en t was a h etero logou s ra t b o n e-m arro w donor type c a rr ie d out to ach iev e m inim um m o rta lity . A seventh exp erim en t is in p ro g re s s , continuing the s e a rc h fo r m inim um m o rta lity in m ice a fte r ra t b o n e-m arro w tran sp lan ta tio n .

T he g e n era l findings of th is w ork indicated that m axim um to le ra n c e , o r m inim um m o rta lity , with p e rs is te n c e of ch im e rism could be achieved in the a llog en ic situation ch osen . F o r ty m illio n c e lls fro m 3-d ay -o ld d on o rs, given at day 1 a fte r rad iatio n exp o su re , gave le a s t m o rta lity (15% deaths in 90 days [2] ). R eg ion s of high m o rta lity , or la ck of to le ra n c e , w ere not observed in the a llo g en ic d esign chosen to find h ighest 90-d ay d eaths, but th e re w ere in d ication s that m o rta lity in cre a se d sh arp ly when fa c to r le v e ls a sso c ia ted with le a s t m o rta lity w ere a lte re d .

In the h etero log ou s exp erim en t, day 1 in je c tio n of m arrow was im portant fo r re c ip ie n ts of both se x e s ; fu rth e r red uction in m ale m o rta lity was ob serv ed with 2 0 -d ay -o ld donors and a dose of 30X 1 0 6 c e lls [6 ,7 ] .The m inim um m o rta lity noted at 90 days was about 25%. Som e lo s s of c h im e ris m was a lso ob serv ed .

A few of the an im als dying fro m seco n d ary d ise a se w ere checked fo r p re se n ce of the com m on m u rine v iru se s , with e ss e n tia lly negative r e s u lts .

The exp erim en ta l d esign approach has substantiated the e a r l ie r indica tio n s that age of donor m arrow and day of in je ctio n a re im portant v a r ia b le s in ach iev in g m axim um to le ra n c e in the fo re ig n b o n e-m arro w rad ia tio n c h im e ra . T h e re a re a lso continued in d ication s (unpublished data on f i l t e r top caging) that environm ent of the rad ia tio n ch im era is of co n sid erab le im p o rtan ce . M ore v igorous control of p a ra s ite s and pathogens in m ice in c r e a s e s the b id ire c tio n a lto le ra n c e ; the an im al with seco n d ary d ise a se is not n e c e s s a r i ly lik e ly to die fro m the d ise a se i ts e lf but is m o re s u s cep tib le to a fa ta l in fectio n during the period that the seco n d ary d ise a se re a c tio n is activ e [8].

3. H YPO XIC CAGING AND TO LER A N C E

An e n tire ly d ifferen t approach to the ach iev em en t of b id irec tio n a l to le ra n c e is the use of hypoxic caging [9] to produce a physiologic .com p etitio n , within the irra d ia te d h ost, that w ill be disadvantageous to the growth of im m u nologically com petent c e l ls . Under conditions of e x trem e hypoxia, the le th a lly irra d ia te d m ouse given a fo re ig n m arrow tran sp lan t m u st, in o rd e r to su rv iv e , not only repopulate h is b o n e-m arro w s ite s but a lso a- ch iev e ery th ro p o ietic h y p erp lasia . T h ese two p ro g re ss iv e tis su e changes m ust take p lace even under c ircu m sta n ce s of re la tiv e s ta rv a tio n , b ecau se the hypoxic an im al does not eat well during the period of a cc lim a tiz a tio n .T he p re lim in a ry exp erim en ts rev ea led very lit t le g r a ft -v e r s u s -h o s t re a c tio n and high p e r cent su rv iv al at 90 days with p e rs is te n c e of the g ra ft [9 ]. E v i dently th e re is a h ie ra rch y of growth, with h aem o p o iesis and e ry th ro p o ies is taking p re ce d e n ce ov er the g r a ft -v e r s u s -h o s t p ro life ra tio n in lym phatic t i s s u e s ,

One can now su m m arize the d ifferen t ap p roach es to d ire c t co n tro l of b id ire c tio n a l to le ra n ce in the rad iatio n ch im e ra lis te d in T a b le I. A s shown

TABLE I. APPROACHES TO CONTROL OF BIDIRECTIONAL TOLERANCE

Technique or conditionMost likely site of action

Host-versus-graft reaction Graft-versus-host reaction Bidirectional

W hole-body irradiation of recipient +

In-vitro manipulation of donor cells (incubation, irradiation, e tc .) +

G enetic relationship of donor and host +

Antim etabolites and anti lymphocyte serum +

Controlled environment for irradiated recipient +

Physiologic com petition for ce ll growth (hypoxic caging) +

Manipulations of other variables (ce ll dose, age of donor, day o f ce ll in jec tio n , sex, e tc .) +

2 4 CONGDON

in the ta b le , an ex ten siv e and v aried s e r ie s of exp erim en ta l m anipulations has been in vestigated ; co n tro l of seco n d ary d ise a se in man w ill p resu m ably com e fro m one o r m o re of th e se m ethods.

4 . PA TH O LO G Y OF SECONDARY D ISEA SE AND TO LER A N C E

In sp ite of the d iv erse and g en era lized natu re of p ath o log ica l p r o c e s s e s seen in an im als and m en dying fro m seco n d ary d ise a se [4], it s t i l l see m s that th e re is a pathognom onic p ro g re ss iv e tis s u e change in lym phatic t is s u e s appearing within a few days a fte r the fo re ig n m arrow tran sp lan t [10 ]. T h e se p ro g re ss iv e tis su e changes a re a m an ifesta tio n of the c e llu la r im m une r e sponse and a re fu rth e rm o re the e a r lie s t m orphologic evidence of a g ra ft - v e rs u s -h o s t re a c tio n [10a], R e tro g re s s iv e changes with d estru ctio n of ly m phatic tis su e a re com m on seq u elae to the p ro life ra tiv e changes (se e R e f. [10] fo r l ite r a tu r e c ita tio n s ) . In the sam e an im al th e re m ay b e m arked d iffe re n ce s in the d eg ree of the g r a ft -v e r s u s -h o s t re a c tio n fr o m one lym phatic t is s u e s ite to an oth er. One lymph node can end up to ta lly d estroyed , although another is re g e n e ra te d . T h e se v a r ia tio n s in the outcom e o r even the extent of the g r a f t -v e r s u s -h o s t re a c tio n within a given lym phatic tis su e show the p o s s ib ility of to le ra n ce at the c e llu la r le v e l in lym phatic t is s u e s .

One can m ake the assum p tion — it s t i l l needs d etailed v e r if ic a tio n - that re g e n e ra tio n of lym phatic tis su e in the fo re ig n m arrow c h im e ra is the an a to m ica l e x p re ss io n of b id ire c tio n a l to le ra n c e .

5. IMM UNOLOGICAL C O M PE T E N C E IN THE RADIATION CHIM ERA

The v a r ia tio n s in the fa te of and seq u elae to g r a f t -v e r s u s -h o s t re a c tio n s in lym phatic t is s u e s a s studied m o rp h olog ica lly a re re fle c te d in the com p ete n ce of im m u nological re sp o n se to a planned antigenic, s tim u latio n . M ost in v e s tig a to rs , notably G engozian et a l . [ l l ] and A g a ro s s i and D o ria [12], find som e le s io n in the im m u nological re sp o n se of the surviv ing fo re ig n m arro w ch im e ra . M arked v a r ia tio n in im m u nocom petence, how ever, is noted [1 1 ]. In the work of G engozian et a l . , the a llog en ic rad ia tio n ch im e ra showed a d efect in ab ility to sh ift fro m 19S to 7S antibody production .

P re su m a b ly , absolu te b id ire c tio n a l to le ra n c e in the rad iatio n ch im e ra would be ' a s so c ia te d with n orm al lym phatic t is s u e s in both fu nctional and an ato m ica l m e a su re m e n ts .

6 . DOSE R A T E AND RADIATION SU P P R ESSIO N O F THE IMMUNER E SP O N SE . '■

C ou rtenay [13], G engozian [14] and G engozian et a l .[1 5 ] have' shown the im p o rtan ce of dose ra te of w hole-body X - and y - r a y s in su p p ressin g the im m une resp o n se ..' A Ílogen ic o r hetero log ou s r a t b o n e-m arro w tr a n s p lants did not take or p e r s is t when the éxp o su re ra te was le s s than 29 R/m in fo r 250-kV p X -r a y s and le s s than,40 R/m in fo r 7 - r a y s . U nder th e se c ir c u m s ta n c e s , fa ilu re to ach iev e m axim um su p p ressio n with.low dose ra te is p ro b ab ly cau sed by c e llu la r r e p a ir m e ch a n ism s.

In th e ir m o st re c e n t w ork, Gengozian et a l . [15] re p o rt that su p p ression of hu m oral antibody production i s g re a te s t fo r an in te g ra l dose of 600 R 7 -r a y s

SECONDARY DISEASE 25

when the dose ra te w as 72R/m in, a s com pared with low er r a te s of 40 and8 R/min. T he tim e .of antigen in je c tio n is a lso an e x tre m e ly im p ortan t v a r i ab le ; g re a te s t su p p ressio n o ccu rre d with in je c tio n 24 hours a fte r exp o su re . When the in te g ra l exp osu re was too low to give im m u nosu p p ression , a d o se ra te e ffe c t was ab sen t. At the h ighest in te g ra l ex p o su re s , the d o s e -ra te e ffe c t w as not m e a su ra b le by the m ethods of study.

When the quality of rad ia tio n was studied in re la tio n to dose r a te , it was found that X - r a y su p p ressio n of hum oral antibody production was d o sera te dependent in a re la t iv e ly n arrow in te g ra l exp osu re ra n g e . A d o s e -ra te e ffe c t on im m une su p p ressio n was absent fo r fis s io n -s p e c tru m n eu tron s.

E x ten sio n of the e a r l ie r w ork on dose ra te and fo re ig n m arrow tr a n s plantation again showed a d o s e -ra te dependence fo r m arrow tak e and p e r s is te n c e [1 5 ].. 'T id in g o v e r1 and su rv iv al fro m le th a l ir ra d ia tio n exposure w ere ob serv ed , in som e c irc u m s ta n c e s , with the te m p o ra ry g r a fts . In the te m p o ra ry 'ta k e ' a n im a ls , th e re was no evidence of seco n d ary d is e a s e .

7 . THE M E TA BO L IC LESIO N IN TH E RADIATION CHIM ERA

The idea that an ex ten siv e pathologic im m une re a c tio n , such as in seco n d ary d is e a s e , c r e a te s or is a s so c ia te d with m etab o lic le s io n s has been under in v estig ation fo r s e v e ra l y e a rs [16]. V a rio u s g e n e ra l p hysiolo g ica l stud ies have been c a rr ie d out on n itrogen b a lan ce [17], oxygen consum ption, and food in tak e , prom pted by the p a ra d o x ica l weight lo s s and the la ck of a re g u la r p attern of rad iatio n -in d u ced graying of the h a ir in fo re ig n b o n e-m arro w c h im e ra s . W hen a high d egree of b id ire c tio n a l to le ra n ce develops e a r ly a fte r tran sp lan ta tio n , the p a ra d o x ica l weight lo s s and fa ilu re to g ray a re ab sen t.

S p e c ific b io ch em ica l stu d ies on kidney lyso zym e [ 1 8 -2 1 ] , sk in and m u scle n itrogen [22], l iv e r RNA, and the fr e e am ino acid pools in the l iv e r w ere a lso p erfo rm ed [23 ]. The m arked l iv e r weight in c re a s e during the e a r ly in te rv a ls of the g r a ft -v e r s u s -h o s t re a c tio n continues to be a d ram atic yet n o n -sp e c ific finding, and it needs a d etailed m e ta b o lic explanation [24].

In the m ost re c e n t studies [ 2 4 a -2 7 ] , em phasis has cen tered on the pro b lem of s e r in e m e ta b o l ism in the foreign b o n e -m a rro w c h im e r a s .In a llog en ic rad ia tio n c h im e ra s , unlike syngenic an im a ls , r e s u lts ind ica te that in je c tio n of g lycin e and fo rm a te d e c re a s e the ra te of appearance of 14C fro m s e r in e into r e s p ir a to r y C O 2 . F u r th e r , th e se findings suggest that som e of the m etab o lic pathw ays involving fo lic acid a re se t to favou r production of s e r in e and te tra h y d ro fo lic ac id . M etab o lism of the 'o n e- carb on frag m en t' is ab n orm al in the fo re ig n m arrow c h im e ra .

8 . LD 50/30-90 STU D IES

An in te re s tin g and d etailed restu d y of the syngenic c e ll 'd o s e re q u ire m ents fo r su rv iv a l at 30 and 90 days a fte r X - ir ra d ia t io n has been c a rr ie d out by D oherty [28] in m ic e . He finds that the LD 50/30 and LD 50/90 values a re p a ra lle l and le s s than 75 R ap art ov er the dose range 5X 1 0 4 to 2 X 1 0 8 c e l l s . It .is quite su rp ris in g to se e that the L D 50/30 changes sh arp ly fro m 753± 6 R , in co n tro l m ic e , to 1220 ± 15 R a s in cre a s in g m arrow c e ll num bers

2 6 CONGDON

a re tested to a lev el of 2X 1 0 5 c e l l s . The change in LD 50/30 i s 467 R . F u r th e r in c r e a s e in syngenic c e l l num bers to 2X 108 c e l l s only i n c r e a s e s the value, at a much slow er ra te , to 1450 ± 35 R: the d if ference between the value for 2X 1 0 5 c e l l s and fo r 2X 1 0 s c e l l s is o n ly -230 R.

T h e se findings indicate that syngenic m a rro w ac ts th erap eu t ica l ly on haem opoietic death even with 5X 1 0 4 c e l l s (LD50/30 is 8 5 3 ± 1 1 R ) . In addition, with la rg e c e l l nu m bers , it red u ces death in the range of X - r a y exposure that i s usually a sso c ia ted with in test inal death. When the L D 50/7

value i s examined, it is seen that in co n tro ls the value was approxim ately 1200 R. With high m a rro w n u m b e rs , the value r o s e to 1425 R, indicating again that m arrow therapy has an e ffec t on the in test inal death syndrome and substantiating older studies, with m arro w and a n tib io t ics , that showed th erapeutic e ffec ts on the in test inal death syndrome a f te r 7 - and X - r a y s as well as whole-body neutron exposure .

Doherty a lso has extended L D 50/30-90 s tudies to include allogenic and and heterologous ra t bone m arrow , but not enough data have been accu m ulated to rep ort at this t im e . It s e e m s v ery d es ira b le to do th is work in view of the new b ase l ine fo r radiation e ffe c ts that must be estab l ished fo r pathogen-free m ice , s ince n ear ly all the older studies w ere c a r r ie d out in conventional an im als .

R E F E R E N C E S

[1 ] CONGDON, C .C . , KASTENBAUM, M .A ., GARDINER, D .A ., Factors affecting mortality from secondary disease in mouse radiation chim eras, JNCI 35 (1965) 227.

[2 ] CONGDON, C .C . , GARDINER, D .A ., KASTENBAUM, M .A ., Reduced secondary disease mortality in mouse radiation chimeras, JNCI 38 (1967} 541.

[3 ] EPSTEIN, R .B ., STORB, R ., RODGE, H ., THOMAS, E .D ., Histocompatibility typing and its application to allogeneic bone marrow transplantation in dogs, Expl Hem atol. 13 (1967) 35.

[ 4 ] VAN BEKKUM, D . W . , DE VRIES, M . J . , Radiation C him era s , A ca d e m ic Press (1 9 6 7 ) 9 2 - 9 4 .[5 ] MATHË, G ., "Secondary syndrome: a sturpbling block in the treatment of leukaem ia by whole-body

irradiation and transfusion of allogeneic haem opoietic ce lls" , in Diagnosis and Treatm ent of Acute Radiation Injury, Columbia University Press (1961) 191.

[6 ] MITCHELL, T . J . , GARDINER, D .A ., KASTENBAUM, M .A ., CONGDON, C .C . , Ninety daymortality in irradiated m ice treated with rat bone marrow, Fedn.Proc. 26 (1967) 571.

[ 7 ] MITCHELL, T . J . , CONGDON, C .C . , KASTENBAUM, M .A ., GARDINÍr , D .A .', Factorialdesign-response surface study of mortality from secondary disease in mouse radiation chimeras,Fedn.Proc. 27_(1968) 3Q7.

[8 ] WATSON, J .R . , HAMMOND, Carolyn W ., Incidence of endogenous bacterial infection in murine radiation chimeras with secondary disease, Radiat. Res. 32 (1967) 581,

[9 ] CONGDON, C .C . , SIMMONS, M .L ., TOYA, R .E ., "Reduced m ortality from secondary diseaseafter exposure to hypoxia", in Advance in Transplantation, (DAUSSET, J . , HAMBURGER, J . , MATHE, G. , Eds.) Munksgaard, Copenhagen (1968) 437.

[1 0 ] CONGDON, C .C . , The destructive effect of radiation on lymphatic tissue, Cancer Res. 26 (1966) 1211.[1 0 a ] WALBURG, H, E . , COSGROVE, G .E ., Severity of the parent to F 1graft-versus-host reaction in

irradiated germfree m ice, Transplantation, submitted for publication.[1 1 ] GENGOZIAN, N ., RABETTE, Barbara, CONGDON, C .C . , Abnormal immune mechanism in allogeneic

radiation chimeras, Science 149 (1965) 645.[1 2 ] AGAROSSI, G », DORIA» G ., Recovery of the hemolysis response in mouse radiation chim eras,

Transplantation 6 (1968) 419.[1 3 ] COURTENAY, (V .D ., Studies on the protective e ffe c to f allogeneic marrow grafts in the rat following

whole-body irradiation at different dose-rates, B r.J.R ad io l. 36 (1963) 440.[1 4 ] GENGOZIAN, N ., Transplantation of rat bone marrow in irradiated m ice: e ffe ct of exposure rate,

Science 146 (1964) 663.

SECONDARY DISEASE 2 7

[1 5 ] GENGOZIAN, N ., CARLSON, D .E ., GOTTLIEB, C .F . , "Radiation exposure rates: effects on the immune system”, in Proc. Symp. on Dose Rate in Mammalian Radiation Biology, co-sponsored by UT-AEC Agricultural Research Laboratory and US Atomic Energy Commission, Oak Ridge, Tennessee,29 April - 1 May, 1968.

[1 6 ] KRETCHMAR, A .L . , CONGDON, C .C . , "The m etabolic problem in secondary disease", in La Greffe des C ellules Hematopoietiques Allogeniques, C o ll.In t. du Centre National de la Recherche Scientifique,No. 147, Centre National de la Recherche Scientifique, Paris (1965) 347.

[1 7 ] McARTHUR, W .H ., CONGDON, C .C . , KRETCHMAR, A .L ., Nitrogen balance in radiation chimeras, Proc. So c .ex p .B io l. Med. 117 (1964) 171.

[1 8 ] SUU, V u-Thi, CONGDON, C .C . , KRETCHMAR, A .L ., Increase in lysozyme activity in kidneys of irradiation chim eras, Proc, Soc. exp .B iol. Med. 113 (1963) 481.

[1 9 ] SUU, V u-Thi, CONGDON, C .C . , KRETCHMAR, A .L ,, Lysozyme activ ity in radiation chimeras,Proc. Soc. exp. B io l. Med. 115 (1964) 825.

[2 0 ] TROUP, G .M ., WALFORD, R .L ., Transplantation disease, renal lysozyme and aging, Transplantation 5 (1967) 43 .

[2 1 ] ENDO, E ., Kidney lysozyme in hypoxic radiation chim eras, Expl.H em atol. 1Ъ (1967) 158,[2 2 ] KRETCHMAR, A .L ., McARTHUR, W .H ., CONGDON, C .C . , Shift in the relative distribution o f body

nitrogen between skin and carcass in mouse radiation chimeras, J.N utr. 87 (1965) 261.[2 3 ] KRETCHMAR, A .L . , CONGDON, C .C . , The levels of free aspartic acid , glycine and serine in

tissues of bone marrow chim eras, Transplantation _1 (1963) 298 .[2 4 ] CONGDON, C .C . , KRETCHMAR, A .L ., Increased liver weight in bone marrow chimeras, Expl m olec.

Pathol. 2 (1963) 277..[2 4 a ] KRETCHMAR, A .L ., PHIPPS, C .E . , " S e r in e -3 -MC metabolism in irradiation chim eras: respiration

pattern analysis” , in Research Report, M edical Division, Oak Ridge Associated U niversities(1966)48 .[2 5 ] KRETCHMAR, A .L . , Serine metabolism in radiation chim eras, Expl H em atol. 13 (1967) 33.[2 6 ] KRETCHMAR, A .L . , Serine metabolism in radiation chim eras, Expl H em atol. 14 (1967) 41 .[2 7 ] KRETCHMAR, A .L . , DOHERTY, D .G ., Carbon-14 activity in respiratory C 0 2 of m ice treated with

m ethotrexate and injected with 2 - 3 - ^ C -serin e , Expl H em atol. 14 (1967) 46 .[2 8 ] DOHERTY, D .G ., E ffecto f syngeneic bone marrow c e ll dose on the 30- and 90-day LD 50 of

X-irradiated m ice , Radiat.Res. (in Press).

CLINICAL QUESTIONS OF TISSUE INCOMPATIBILITY AFTER ALLOGENIC BONE-MARROW TRANSPLANTATION

F .E . FA IN SH TEIN , E . A . ZO TIKOV

C e n tra l In stitu te o f H a e m a to lo g y and Blood Transfusion,

M oscow , USSR

Abstract

CLINICAL QUESTIONS OF TISSUE INCOMPATIBILITY AFTER ALLOGENIC BONE-MARROW TRANSPLANTATION. This paper describes the results of studies concerning tissue incom patibility in the transplantation o f allogenic bone marrow into patients suffering from hypoplastic and aplastic anaem ia.Factual data are presented on the extent to which the immune activity and capacity for immunological response of recipients is preserved. Attention is mainly directed to the characteristics o f the spectrum showing serological activity o f the an ti-leu cocyte antibodies, which depends on the type of sensitization. These data point to the need for differential use of haemotherapeutic agents and are also o f some importance in the selection o f bone-marrow donors.

1. INTRODUCTION

Som e a s p e c ts of the e ffic ie n cy of a llo g en ic b o n e-m arro w tra n sp la n ta tio n with em p h asis on p ro b lem s of t is s u e in co m p atib ility fo rm the b a s is fo r th is p ap er. O ver a p eriod of ten y e a r s , tre a tm e n ts w ere p erfo rm ed on 145 p atien ts with hypo- and a p la s tic an aem ia who w ere g iven 210 t r a n s p lantations of a llo g en ic bone m arro w .

It would appear fro m th is e x p e rien ce that a llo g en ic b o n e-m arro w tran sp lan ta tio n is a u sefu l m eans of com p lex th erap y fo r su b -a cu te hypop la s tic an aem ia .

2. E X P E R IM E N T S AND R E S U L T S

A d istin ctiv e fe a tu re of su b -a cu te hyp op lastic anaem ia and a c h a r a c t e r i s t ic of panhaem ocytopenia and b o n e -m arro w hypoplasia (s o -c a lle d 'b lu e bone m arro w ' ) is the re la t iv e ly in c re a se d p ro life ra tio n of p ro e ry th ro b la s ts , b aso p h ilic e ry th ro b la s ts and p la sm a tic c e l l s .

Ju s t as in su b -a cu te hyp op lastic an aem ia , the tran sp lan ta tio n of a llog en ic bone m arrow in com bination with blood tra n sfu s io n , c o r tic o s te r o id h o rm o n es, group 'B ' v ita m in s , and a m ean s to in c r e a s e v a s c u la r -s tre n g th favou rably a l te r s the co u rse of the d is e a s e .

In one group of p atien ts su fferin g fro m hypoplastic an aem ia , by p r e venting h aem o rrh ag e and m aintain ing a n orm al o r high haem oglobin lev e l o v er a period of w eeks o r m onths a fte r tran sp lan ta tio n , a fav ou rable sh ift was ach iev ed slow ly in white c e ll fo rm u la and quantity and in th rom b ocyte nu m ber. H ow ever, im p ro v em en ts in the m o rp h olog ical content of the bone m arrow do not b eco m e apparent b e fo re 3 to 12 m onths have elap sed a fte r b o n e -m arro w tran sp lan ta tio n .

29

3 0 FAINSHTEIN and ZOTIKOV

In an oth er group of p atien ts with su b -acu te hyp op lastic anaem ia, the e ffic ie n cy of com plex th erap y , including b o n e -m arro w g ra ft , re su lte d in equ ilib riu m of the h aem op oietic index and p reven tion of b leed in g . Only when th is equ ilib riu m is ach iev ed , is a sp len ecto m y ju s tif ie d . U nder the in fluence of such a sy stem of th erap y , p a rtia l o r to ta l c lin ic o -h a e m a to lo g ic a l r e m iss io n should develop . M o reo v er, the e ffe c ts of s tro n g th erap y m ay only b eco m e apparent 5 - 6 m onths a fte r th erap y has begun.

Am ong 90 p atien ts with su b -acu te hypoplastic an aem ia , 53 had no r e m iss io n s fo r the f i r s t two y e a rs , but betw een two and sev en y e a rs th ese p atien ts did have re m is s io n s (T a b le I ) . Many of th o se who took m odern com plex th erap y a re c lo s e to p ra c t ic a l re c o v e ry .

T A B L E I. DURATION OF REM ISSIO N IN P A T IE N T S "WITH SU B -A C U T E H Y PO P L A STIC ANAEMIA

2 - 6 7 - 12

Remission in months

13 - 24 25 - 36 37 - 60 61 - 84

Number of patients 6 8 23 14 25 14

N .L . Sam oyllna and A .M . P o lan sk ay a have d eterm in ed that lym phocy tes of the p e rip h e ra l blood of p atien ts with hypo- and a p la s tic an aem ia in tis su e cu ltu re re m a in in good health when phytohaem agglutinins a re added. B la s t tra n sfo rm a tio n and m ito se s a lso take p lace (F ig . 1 ). P r o ceeding fro m r e s u lts of p reviou s in v e stig a tio n s, it can be concluded that the developm ent of im m une re sp o n se in hypo- and a p la s tic anaem ia p atien ts a fte r a b o n e-m arro w a llo g ra ft tran sp lan t is due to the functioning of the a llo g ra ft fo r a sh o rt p eriod .

FIG. 1. Short-lived lym phocyte culture of peripheral blood in the presence of phytohaemagglutinin (A - aplastic anaem ia patient, В - donor).

TISSUE INCOMPATIBILITY 31

B y u se of a phenotypic m a rk e r of the red c e lls of both donor and r e cip ien t as w ell as by u se of the sex ch ro m atin m a rk e r in leu co cy te s (in d ifferen t sex tra n sp la n ts ), it has been d eterm ined that donor b o n e -m arro w c e lls su rv iv e in d iseased bod ies of the re c ip ie n ts fo r two to fo u r w eeks (F ig s 2 and 3).

T he r e s u lts of th e se stu d ies do not enable the l ife - t im e of the t r a n s plant to be d eterm in ed .

F IG .2 . Survival in days o f bone-marrow transplant in four patients as determined by the donor red ce ll phenotypic marker.

FIG .3. Survival in days of fem ale bone-marrow transplant in four m ale patients as determined by the donor leucocyte sex chromatin m arker. .

A nother exam ple of the developm ent of im m une co n flic t is fo rm ation of antibodies in the re c ip ie n t a fte r a llog en ic b o n e-m arro w tran sp lan tatio n . T h e se data (T a b le II) suggest that developm ent of iso se n s it iv ity often o ccu rs with fo rm ation of a n ti-le u co cy te an tib o d ies. L eu coagglu tinating and cy to to x ic antibodies appeared in ap p roxim ately o n e-th ird of the p atien ts t r a n s fused with a llogen ic bone m arro w . The fo rm ation of anti throm bocyte antibodies was ob served m o re r a r e ly , and a n ti-re d c e ll antibody fo rm ation

32 FAINSHTEIN and ZOTIKOV

T A B L E II. : C H A R A C TER ISTIC S OF A N TIBO D IES R E C O V E R E D IN P A T IE N T S FO LLO W IN G A LLO G EN IC BO N E-M A RRO W TRA N SPLA N TA TIO N

Number of recipients Antibodies

Anti-red cells A nti-leucocytes Anti-throm bocytes

69 2 20 3

T A B L E III . P E C U L IA R IT IE S OF THE SP E C T R U M OF A N T I-L E U C O C Y T E A N TIBO D IES ARISING ON BLO O D TRA N SFU SIO N AND A LLO G EN IC BO N E-M A RRO W TRA N SPLA N TA TIO N

Form of sensitization Sera tested for antibodies

(N o.)

Leucocyte samples

Sam ple No. Samples reacting positively

A llogenic bone-marrow transplantations

(1 - 2) 11 12 1 - 3

Blood transfusion 3 - 5 10 12 2 - 5

10 - 20 33 12 4 - 7

50 - 100 8 12 8 - 10

o ccu rre d in only two c a s e s . The poor condition of p atien ts req u irin g e m e rg en cy g ra fts did not p erm it d onors to be se le c te d acco rd in g to a ll the v a r ie t ie s of R h -fa c to r s .

A n a ly sis of the s e ro lo g ic a l c h a r a c te r is t ic s of antibodies follow ing a l lo g en ic b o n e-m arro w tran sp lan ta tio n s and blood tra n sfu sio n s shows that the sp ectru m of antibodies depends on the kinds of se n sitiz a tio n that the patient d ev elop s. A n ti-le u co cy te antibodies form ed a fte r b o n e-m arro w g ra ft e x h ib it a co m p arab le sp ectru m of se ro lo g ic a l a c tiv ity (T ab le III). The g ra ft re a c te d with sam p les 1 - 3 fro m 12 sam p les of the panel of leu co cy te d onors. T he s e r a of p atien ts having m any blood tra n sfu s io n s showed p o sitiv e r e s u lts m o re often and th e ir s e ro lo g ic a l sp ectru m b eca m e w ider during the co u rse of in cre a s in g the num ber of blood tra n sfu s io n s .

F in a lly , we supposed that developm ent of im m une in com p eten ce a fte r ■ b o n e-m arro w tran sp lan ta tio n would re s u lt in in c re a se d p la sm a tic c e ll p r o life ra tio n .

T he above data on the phenom enon of im m une in co m p atib ility with g ra fts of a llog en ic bone m arrow m ay lead to m o re e ffe c tiv e use of tran sp lan ta tio n in th erap y .

We a re s t i l l of the opinion that in c a s e s w here m yelo therapy is req u ired the use of blood tra n sfu sio n and blood d e r iv a tes should be envisaged only when the p a tie n t's s ta tu s p e rm its . T h e se m e a s u re s would avoid a n ti-le u co cy te s and a n ti-th ro m b o cy te s , s in ce se n sitiz a tio n m ak es it d ifficu lt to s e le c t su itab le b o n e-m arro w th erap y .

A n ti-le u co cy te antibod ies that appear to be the re s u lt of a llog en ic bone- m arrow g ra ft in co m p ariso n with s im ila r ones appearing a fte r blood tr a n s -

TISSUE INCOMPATIBILITY 3 3

fu sion do not cau se a se r io u s o b sta c le to donor s e le c tio n , s in ce the s e r o lo g ica l a c tiv ity sp ectru m can be ch a ra c te r iz e d on the m arro w .

It h as th e re fo r e been concluded that tra n sfu s io n of washed red c e lls is the b e s t type of blood tra n sfu sio n to em ploy with a llo g en ic b o n e -m a rro w g ra ft .

A p o ss ib le m ethod of d ep ressin g the im m une re sp o n se of a re c ip ie n t a fte r a b o n e -m arro w g ra ft is to tre a t the patient with m a ss iv e d oses of a l lo gen ic antigen m a te r ia l. T h is has been attem pted by tran sp lan tin g stored bone m arrow fro m m any d on o rs. O ther w o rk e rs a re using m a s s iv e le u c o cy te tra n sfu s io n s to g eth e r with c o r t ic o -s te r o id h orm on es and ep silo am in o - c a p ro ic acid fo r b o n e -m arro w tra n sp la n ta tio n s .

3. CONCLUSIONS

In th is p ap er we have tr ie d to an a ly se the in form ation that has been accu m ulated in re g a rd to tis s u e in co m p atib ility in the tran sp lan ta tio n of a llo g en ic bone m arro w into p atien ts su fferin g fro m hypo- and a p la stic a n aem ia .

In p a r t ic u la r we have p resen ted data ind icating to what extent the host o rg an ism re ta in s im m u n olog ical fu nction .

We have a lso turned our a tten tion to sp e c ific data ind icating what c a p a c ity the org an ism of th e se p atien ts has fo r im m u nological re sp o n se . In th is con n ection , sp e c ia l in te r e s t p la in ly a tta ch es to the s h o r t - te r m functioning of in je c te d b o n e -m a rro w c e l l s and the sp e c ia l fe a tu re s of antibody fo rm ation in the host a fte r a llo g en ic b o n e-m arro w tran sp lan ta tio n .

It a lso seem ed d e s ira b le to us to dwell at g r e a te r length on the c h a r a c te r is t ic s of the sp ectru m of s e ro lo g ic a l a c tiv ity of a n ti-le u co cy te an tibo d ies, which depends on the type of s e n s itiz a tio n . T he data obtained a re without any doubt of g re a t im p ortan ce fo r the d ifferen tia ted use of haem otherapy in tran sp lan ta tio n and fo r a ra tio n a l ch o ice of b o n e-m arro w donor.

F in a lly , we d iscu sse d v ery g e n e ra lly som e a sp e c ts of p o ss ib le fu ture r e s e a r c h undertaken with a view to d isco v e rin g the b e s t m eans of su p p ressin g tis s u e in co m p atib ility phenom ena in the a llo g en ic tran sp lan ta tio n of bone m arrow into p atien ts su fferin g fro m hypo- and a p la s tic an aem ia .

PREVENTION AND CONTROL OF SECONDARY DISEASE FOLLOWING ALLOGENIC BONE-MARROW TRANSPLANTATION

D .W . VAN BEKKUM

R a d io b io lo g ica l In stitu te TN O ,

Rijsw ijk (Z H ), T h e N etherlands

Abstract

PREVENTION AND CONTROL OF SECONDARY DISEASE FOLLOWING ALLOGENIC BONE-MARROW TRANSPLANTATION. A review is presented of the various methods found to be effectiv e in preventing or am eliorating acute secondary disease in rodents and prim ates. Selectiv e physical elim ination of lymphoid ce lls by centrifugation over a discontinuous albumin gradient, post-transplantation administration o f cyclophosphamide, amethopterin or ALS and the selection of histocom patible bone-marrow donors are considered to be the most promising methods in primares. It is not known to what extent delayed secondary disease will occur in monkeys and man in cases where acute secondary disease has been successfully avoided.

1. INTRODUCTION

The tre a tm e n t o f rad ia tio n s ic k n e s s re su ltin g fro m w hole-body i r r a d i ation with le th a l d oses o f p en etratin g rad ia tio n s is s t i l l a s u b je c t o f cons id e ra b le in te re s t to m any rad ia tio n b io lo g is ts . Am ong the v ario u s m odes of tre a tm e n t that produce in c re a se d su rv iv a l, the tran sp lan ta tio n o f bone- m arro w c e lls has rem ain ed , at le a s t p o ten tia lly , the m o st e ffe c tiv e one. E m p h asis has to be p laced , how ever, on the lim ita tio n s im p lied by the te rm ' p oten tia l' . W hile b o n e -m arro w tran sp lan ta tio n can e a s ily and rep ro d u cib ly produce a change o f su rv iv a l fro m 0% to 100% in exp erim en ta l a n im a ls , the sam e p ro ced u re is s t i l l an e x trem ely u n certa in and dangerous undertaking in human p a tien ts , a t le a s t a s re g a rd s a llo g en ic b o n e-m arro w g ra ftin g . The conditions w hich prom ote a take of a llo g en ic bone m arrow have by now been w ell defined even fo r m an [1], but i t has b eco m e equally c le a r that the im m u n olog ical g ra ft -v e r s u s -h o s t re a c tio n which follow s such a take and w hich r e s u lts in seco n d ary d ise a se is alw ays m uch m o re se v e re in p rim a te s than in ro d e n ts .

As the problem o f seco n d ary d ise a se s t i l l re m a in s the m o st se r io u s b a r r ie r to the g e n era l ap p licab ility o f a llo g en ic b o n e -m a rro w tran sp lan tation in m an, our group has co n cen tra ted its e ffo rts on the study of th is a sp e ct of b o n e -m a rro w tran sp lan ta tio n . The seco n d ary d ise a se u su ally ob served in irra d ia te d rod ents a fte r tran sp lan ta tio n of a llo g en ic b o n e -m a rro w c e lls can b e ch a ra c te r iz e d a s fo llow s. A s u c c e s s fu l take o f the bone m arrow lead s to fu ll re c o v e ry o f the b o n e -m a rro w synd rom e, but is follow ed about 3 -4 w eeks a fte r g raftin g by the ap p earan ce of a new com p lex of d isease sym p tom s, which a re g e n era lly ca lled seco n d ary d is e a s e . A fte r s e v e ra l y e a r s of in ten se in v estig a tio n s and sp ecu la tio n s th is seco n d ary d ise a se was proved to be cau sed by an im m u n olog ical re a c tio n of the lym phoid com ponents of the g ra ft a g a in st the h o st [2 , 3 ]. The m o st v io len t and fa ta l attack is on e p ith elia l c e l l s , lead ing to c h a r a c te r is t ic le s io n s in the sk in , the e p ith elia l s tru c tu re of the in te stin a l t r a c t and the l iv e r (T ab le I).

A nother c h a r a c te r is t ic finding in c a s e s of prolonged seco n d ary d isease is s e v e r e atrophy o f a l l lym p hatic t is s u e s , which has been a s c r ib e d to

3 5

3 6 VAN BEKKUM

T A B L E I. SECO N D ARY D ISEA SE A F T E R FO R EIG N BO N E-M A RRO W TRA N SPLA N TA TIO N

Lesions ' Symptoms

C ell death in ep ithelia of:

seco n d ary exhaustion of the lym phoid c e lls in the co u rse of th e ir in ten se a n ti-h o s t re a c tio n [4 , 5 ]. T h is situ ation then lead s to a w eakening in defence ag a in st m ic ro -o rg a n is m s so that the e p ith e lia l le s io n s b eco m e seco n d arily in fected and in flam m ation m ay b eco m e the predom inating h is to lo g ic a l le s io n .

The f i r s t s u c c e s s fu l a llo g e n ic b o n e -m a rro w g ra fts in p atien ts re v e a le d that the g ra ft -v e r s u s -h o s t re a c tio n is unusually se v e re and rap id on its o n se t in hum ans [6], th ereb y p resen tin g an e n tire ly d iffe ren t p attern than in m ice and r a ts . It soon b ecam e apparent that the m onkey is v e ry s im ila r to m an in its su sce p tib ility to the im m u n olog ical co m p lica tio n s o f a llo g en ic b o n e -m a rro w tran sp lan ta tio n [7 ] . F o r th is re a so n the m onkey is m o st su itab le fo r p re c lin ic a l stu d ies and we sh a ll th e re fo re r e fe r to the human (and m onkey) type of seco n d ary d ise a se as the p rim ate type o r the acu te • fo rm o f seco n d ary d ise a se w hile the rod ent type w ill be r e fe r r e d to as the la te fo rm of seco n d ary d is e a s e . T h is e a r ly and se v e re seco n d ary d is e a se of the p rim ate type can be induced in m ice by g ra ftin g sp leen o r lym ph node c e lls along with the bone m a rro w . It was th e re fo re assu m ed that p rim ate bone m arrow d iffe rs in its com p osition fro m that o f the rodent by a h igh er content of im m u nolog ically developed c e l ls .

To fa c ili ta te in v estig a tio n s o f m e th o d s ‘to co n tro l the acu te fo rm of seco n d ary d ise a se , we developed a m odel sy stem in m ice [8 ] . T h is m odel c o n s is ts of le th a lly ir ra d ia te d (С ВА Х C 5 7 B L )F 1 hybrid m ic e , w hich r e ce iv e 2X 1 0 6 CBA sp leen c e lls i .v . The num ber of im m u no-com p etent c e lls in th is g ra ft is such that se v e re acu te seco n d ary d ise a se is in v ariab ly produced, but the sam e sp leen c e ll population contains ju s t enough h aem o p o ie tic stem c e lls to r e s to r e the m ic e , when and if the im m u nolog ically a c tiv e c e lls a r e s e le c tiv e ly e lim in a ted . A m e re red u ction of v iab le c e lls (in a n o n -s e le c tiv e way) in the g ra ft w ill n ev er red u ce m o rta lity o f the re c ip ie n ts b eca u se the num ber of h aem op oietic s tem c e lls in the g ra ft is a lim itin g fa c to r .

PREVENTION AND CONTROL OF SECONDARY DISEASE 3 7

We have u sed the m ouse m odel ex ten siv e ly in the p ast few y e a rs to s c r e e n a v a r ie ty o f agen ts fo r the prevention o r tre a tm e n t o f acu te seco n d ary d is e a s e . W hen p ro m isin g r e s u lts w ere obtained the p roced u re was subsequently in v estig ated in-m onkeys, and so fa r our im p re ssio n is that the m ethods w hich w ere highly e ffe c tiv e in m ice w ere u su ally a lso e ffe c tiv e in -m onkeys, w hile the le s s e ffe c tiv e m ethods fo r m ice proved to b e of l i t t le value in the m onkey.

2. P R E V E N T IV E M EA SU RES

In the follow ing pages only th o se ap p roaches to the prevention and tre a tm e n t of acu te seco n d ary d ise a se w ill be d iscu sse d w hich seem to have p o ss ib ilit ie s o f eventual c l in ic a l application and those which a re of th e o r e tic a l in te re s t .

The m ethods d ire c te d a t the donor of the c e lls b e fo re grafting a re l is te d a s p reven tive m e a s u re s in T ab le II, and w ill be b r ie fly d iscu ssed .

T A B L E II. PR EV E N TIO N O F A C U TE SECONDARY D ISEA SE

Methods Rodents Primates Investigators

1. Selection of Monkeys:com patible donors - . in progress Balner et al.

2 .

(H antigens)

Selection of Foetal liver Foetal liver not van Putten et a l . , 1968 [11 ]c e ll type Bone marrow feasible in man

3. Treatment of donor: Not effective - van Bekkum, unpublishedX - irra d ., chem o- Slightly effectiv e - van Bekkum, unpublishedth erap ,, ALS Effective Not effective van Bekkum et a l . , 1967 [1 2 ]

4 . Treatm ent of graft: tem p. (-70°, 4°, 37°), Partially effectiv e Not effective van Putten et a l. , 1964 [1 3 ]ALS

. Partially effectiv e Low selectivity van Bekkum et a l . , 1967 [12 ]

5 , Separation of cells Effective In progress D icke et a l . , 1968 [1 4 ]

M ethod 1: S e le c tio n of com p atib le donors is b eing attem pted with the m ethod of leu co cy te antigen typing s im ila r to that used in organ tr a n s plantation . To avoid seco n d ary d ise a se , how ever, a l l the im portant an tigens of the re c ip ie n t, should be p re sen t in the donor. To prom ote a take o f th e .b o n e -m arro w g ra ft , the sam e p rin c ip le s apply as fo r organ tran sp lan ta tio n , nam ely that a l l o f the im p ortan t an tigens o f the donor should be p re sen t in the re c ip ie n t. The value of leu co cy te antigen group m atch ing along th e se p rin c ip le s has been re ce n tly d em on strated in dogs re ce iv in g a llog en ic b o n e -m a rro w g ra fts [9 ].

3 8 VAN BEKKUM

With our m ethod of b o n e -m a rro w g raftin g in m onkeys - without donor se le c tio n — a take of the b o n e -m a rro w g ra ft can be obtained in 95% of the c a s e s , but the seco n d ary d ise a se is acu te and n e a rly alw ays fa ta l. Our f i r s t o b je c tiv e is th e re fo re to d e c re a s e the se v e r ity of the second ary d ise a se by app rop riate m atch ing of the b o n e-m arro w d onors.

So fa r exp loratory exp erim en ts have not been p erfo rm ed . We can only re p o r t that re tro sp e c tiv e typing of our two lon gest surviv in g ch im e ra s (160 and 400 days) has thus fa r re v e a le d the identity of a t le a s t two of the an tigen ic groups in the rh e su s m onkey. Although the exp ecta tio n s a re high reg ard in g the advantages of th is approach , it w ill be som e tim e b e fo re our p re sen t m ethods of typing a re p e rfe cte d [10].

M ethod 2: F o e ta l l iv e rs of m ice contain a low er proportion of c e llscapable of e lic it in g la te seco n d ary d is e a s e . The re s u lts o f tran sp lan ta tio n of fo eta l l iv e r c e lls in m onkeys have b een disappointing in that the la rg e num ber of c e lls req u ired fo r e ffe ctiv e repopulation of the re c ip ie n t n e c e s s ita te s pooling s e v e r a l fo eta l l iv e r s [11 ].

S in ce it cannot be expected that a num ber of fre s h fo e ta l human liv e r s w ill be av ailab le at the sam e tim e , the u se of fro zen l iv e r c e lls se e m s unavoidable. If one tak es-in to accou nt that pooled fo eta l l iv e r c e lls a r e about one q u a rte r as e ffectiv e as adult b o n e -m a rro w c e lls in re s to r in g a le th a lly irra d ia te d individual, and allow ing fo r a lo s s of two th ird s of the v iab le c e lls from the fre e z in g p ro ced u re, the equivalent of 4X 108 a llo g en ic b o n e -m arro w c e lls p er kg body weight of the re c ip ie n t (a dose re q u ired fo r full repopulation) is about 5 X 109 fo eta l l iv e r c e l l s . T h is is ap p roxim ately the num ber of c e lls p re sen t in the l iv e r of a human foetus 2 0 -2 6 w eeks of age, so that the pooled fro zen c e lls of 50 fo etu ses would be re q u ired to r e s to r e a 50-kg p atien t. It is c le a r ly im p o ss ib le to tra n sfu se th is amount of m a te r ia l sa fe ly , so that for a ll p ra c t ic a l purposes fo e ta l l iv e r c e lls cannot be em ployed in c lin ic a l tran sp lan ta tio n s.

M ethod 3 : T h e se ap p roaches a re aim ed a t se le c tiv e e lim in atio n in vivo of th o se c e lls that a re re sp o n sib le fo r the acu te g r a f t -v e r s u s -h o s t re a c tio n . T h e re a re m any re a so n s to su sp ect that th ese c e lls a r e id e n tica l with the s o -c a l le d antigen resp o n siv e lym phatic c e l ls , probably the sam e a s the sm a ll lym p hocytes. O bviously, if such an e lim in ation could be accom p lish ed by p re tre a tm e n t of the donor th is would only be ap p licab le to the c l in ic a l situ ation i f i t could be shown to c a r ry no undue r is k s fo r the donor.

L ym phocytes have long been con sid ered excep tion ally ra d io sen sitiv e c e lls and it was th e re fo re of th e o re tic a l in te re s t to p e rfo rm a te s t X - irra d ia tio n of the donor. We have re ce n tly com e to r e a liz e that the ra d io sen sitiv ity of lym phocytes is not su b stan tia lly g re a te r than that of h aem o p o ietic stem c e lls [15] and it was th e re fo re to be expected that irra d ia tio n would not be an e ffe ctiv e p ro ced u re (T ab le III).

C erta in ch em oth erap eu tic ag en ts, e .g . cyclophospham ide, a re slightly e ffe c tiv e when given in high d oses to the donors (T ab le IV ). T h is p ro ced u re was not tr ie d in m onkeys b e ca u se it se e m s doubtful w hether such tre a tm e n t of human donors w ill e v e r be a cce p ta b le .

R ecen tly we in v estig ated the e ffe c t of h etero logou s A LS (an ti-lym p h o- cyte seru m ) and its p u rified g am m a-g lobu lin p rep ara tio n s on the prevention and tre a tm e n t of acu te seco n d ary d ise a se in m ice and m on keys. O ur re s u lts [12 , 16] a re su m m arized in T ab le V .


T A B L E III. E F F E C T O F IRRAD IATIO N O F TH E S P L E E N -C E L L DONOR M IC E ON A C U TE SECONDARY D ISEA SE IN TH E IR R A D IA TED R E C IP IE N T S (a>

Dose No. of °jo 30-day survival of recipients(rad) spleen ce lls (24 h) (2 h)

0 2 X 10 6 20°¡o 050 2 X 106 0 0

100 2 X 106 0 10200 2 X 106 ‘0 0200 4 X106 0 0300 2 X 10® 0 0300 4 X 106 0 0

- 0

^ Recipients were (CBAx C57BL)F hybrids irradiated with 750 rad.Donors were CBA m ice,, irradiated 24 h or 2 h before sacrifice , 10 m ice per group.

T A B L E IV . E F F E C T O F T R E A T M E N T O F THE S P L E E N -C E L L DONOR M IC E WITH CYCLO PH O SPH A M ID E ON A C U TE SECO N D ARY D ISEA SE IN THE IR R A D IA TED R E C IP IE N T S (a)

Dose Interval (h) % 30-day survival of Number of(mg/kg) to sacrifice recipients m ice

50 24 0 10100 24 0 19150 24 30 10200 24 85 13

(a)Recipients were (CBA + Donors were CBA m ice,

C57BL)F1 hybrids irradiated with 750 rad. , 2 x 106 spleen ce lls per recip ient.

T A B L E V. E F F E C T O F A LS ON A C U TE .SECONDARY D ISEA SE

ALS — ) Donor Cells in vitro Recipient

Mouse Very good Slight Slight

Monkey Ineffective Slight Effective

4 0 VAN BEKKUM.

. T re a tm e n t of. the s p le e n -c e ll donor m ice proved to be unusually e ffe c tiv e , w hich has been a s c r ib e d not only to the im m u n o -su p p ressiv e activ ity of ALS but a lso to the cap acity of c e r ta in p re p a ra tio n s to s tim u la te h aem o p o iesis in the sp leen , as evidenced by an in c re a se d content o f co lo n y -fo rm in g un its in the s p le e n -c e ll population. U nexpectedly, such b e n e fic ia l e ffe c t was to ta lly ab sen t when m onkey donors w ere tre a te d with A LS b e fo re th e ir bone m arro w was used fo r g raftin g onto ir ra d ia te d re c ip ie n ts and the re a so n fo r th is d iffe ren ce betw een m ice and m onkeys has so fa r not been d isco v e red .

M ethod 4: A s e le c tiv e e lim in atio n o f c e lls re sp o n sib le fo r the g r a f t -v e r s u s -h o st re a c tio n fro m the c e ll population to b e g rafted h as b een attem pted by in -v itro tre a tm e n t. Such a p ro ced u re , i f e ffe c tiv e , would obviously o ffer a nu m ber of advantages o v er the tre a tm e n t of the donor o r the re c ip ie n t.

F re e z in g the c e lls a t low te m p e ra tu re s , s to ra g e fo r s e v e r a l days at 4°C and incubation at 37°C fo r one o r two h ou rs have b een found to be e ffe c tiv e to vary ing d eg ree s in the c a s e o f m ouse bone m arro w o r sp leen [ 8 ,1 7 ,1 8 ] . H ow ever, none of th ese m ethods proved to be of any value when applied to m onkey bone m arrow [13 ].

Incubation of m ouse sp leen o r m onkey bone m arrow with the ap p rop riate A LS did r e s u lt in a c e r ta in d egree of s e le c tiv e in activ atio n of the lym phoid c e l l s , but the s e le c tiv ity fa c to r was not v e ry la rg e , so that a rep ro d u cib le d e c r e a s e o f acu te seco n d ary d ise a se with con com itan t p re se rv a tio n of r e s to r a t iv e ca p a c itie s o f the g ra ft could not be obtained [12 ].

M ethod 5 : P h y s ic a l sep ara tio n and re m o v a l of the dangerous c e lls fro m the g ra ft b e fo re tran sp lan tatio n se e m s to be an id ea l m ethod of avoiding acu te seco n d ary d is e a s e . P ro m is in g r e s u lts have b een re ce n tly obtained by D icke [14] who em ployed cen trifu gatio n on a discontinuous bovine album in g rad ien t.. In the c a s e .of m ouse s p le e n -c e ll su sp en sio n s it has b een p o ssib le to c o l le c t up to 50% o f the to ta l num ber o f h aem op oietic stem c e lls in a 5 - to 1 0 -fo ld co n cen tra tio n in one of the fr a c t io n s . T h is sam e fra c tio n contains le s s than 0.3% of the g r a f t -v e r s u s -h o s t activ ity found in the o r ig in á l sp le e n c e l l su sp en sio n . The tran sp lan ta tio n of th is fra c tio n in our m ouse m odel re s u lte d in 100% su rv iv a l without any sym ptom s of acu te o r la te seco n d ary d is e a s e in the su rv iv o rs . P re lim in a ry e x p erim en ts using the sam e p roced u re with m onkey bone m arro w re su lte d in a 6 -fo ld co n cen tra tio n o f h aem op oietic s te m -c e l l ac tiv ity , but adequate re m o v a l o f im m u no-com p etent c e lls fro m that fra c tio n has probably not y e t been ach iev ed , s in ce a su b -acu te type of seco n d ary d ise a se developed in the r e c ip ie n ts . P e rfe c t io n of th is sep ara tio n techniqu e with m onkey b o n e -m a rro w p re p ara tio n s is being attem pted at p re s e n t. .

3 . T H E R A P E U T IC M EA SU RES . . . .

In T ab le V T th e-ap p ro ach es to the co n tro l of acute seco n d ary d ise a se by tre a tm e n t o f the re c ip ie n t a re lis te d .

M ethod 1: A s in the c a s e o f donor irra d ia tio n , s im ila r ra d io se n s itiv ity of the h aem op oietic and lym phoid c e lls seem ed to preclu d e p o sitiv e re s u lts with X - r a y s . N o n eth eless, th is approach w as in v estig ated b e ca u se a

T A B L E V I. T R E A T M E N T O F A C U TE SECONDARY D ISEA SE


Methods Rodents Primates Investigators

1 . Irradiation Not effective Unknown van Bekkum (unpublished)

2 . Chemotherapy Effective Effective Uphoff [1 9 ]Santos et a l. [2 0 ] M uller-Bêrat et a l. [2 1 ]

3 . ALS Slightly effective Effective van Bekkum et a l. [1 2 ]

d iffe ren ce in the p ro life ra tio n ra te of the two c e ll types could have p ro vided som e p re fe re n tia l in activ a tio n . S in ce X -ir ra d ia tio n of the re c ip ie n ts a t v ario u s in te rv a ls a f te r tran sp lan ta tio n was found to be com p letely in e ffe c tiv e with a v a r ie ty o f d oses in ou r m ouse m odel of acu te second ary d ise a se , th is p ro ced u re has not been in vestig ated in m onkeys.

M ethod 2: T h re e y e a r s ago, M u lle r -B é r a t , van Putten and van Bekkum [21] began to s c re e n a num ber of ch e m ica l agents in a m ouse m odel of second ary d ise a se follow ing the r e s u lts obtained by Uphoff [19] with m eth o trexate in m ice and by Santos [20] with cyclophospham ide in r a t s . M ore re ce n tly , a com p arab le te s t fo r su p p ression o f g r a ft -v e r s u s -h o s t d ise a se in newborn m ic e was p erfo rm ed by L em m el and Nouza [22] who re p o rted re s u lts p a ra lle l with o u rs .

In our f i r s t s e r ie s of e x p e rim e n ts , both cyclophospham ide and am eth o p terin w ere found to be highly e ffe c tiv e d rugs, but the tim e of ad m in istra tio n a fte r the s p le e n -c e ll g raftin g proved to be c r i t i c a l . Our m o re re c e n t re s u lts with a v a r ie ty of chem oth erap eu tic agen ts a re p re sen ted in T ab le VII [23 ].

When the two m o st e ffe c tiv e d rugs, cyclophospham ide and am ethopterin , w e re su b seq u e n tly te s te d in m o n k e y s , we found th em to b e e q u a lly e f fe c t iv e in the e a r ly phase of the d ise a se and, m o re o v e r, the op tim al d oses w ere p re c is e ly com p arab le when extrap ola tio n w as p erfo rm ed on the b a s is of body s u r fa c e . T h ese findings u n d erline the re lia b ility of ou r m ouse m odel o f acu te seco n d ary d is e a s e .

When the tre a tm e n t is s ta r te d im m ed iate ly a fte r b o n e -m arro w tr a n s plantation , i t s ig n ifica n tly delays the on set o f seco n d ary d ise a se without se r io u s ly inhib iting the p ro life ra tio n of the h aem op oietic c e l l s . The seco n d ary synd rom e, which ap p ears la te r , cannot b e e ffe c tiv e ly con tro lled by th ese drugs b eca u se they do not a c t in a su ffic ien tly s p e c if ic m anner a t that tim e: with low er d oses the m onkeys die fro m seco n d ary d isease and with h igher d oses fro m b o n e -m arro w a p la s ia . N on eth eless, the s u r v iv a l tim e of m onkeys re ce iv in g a tran sp lan t of a llo g en ic bone m arro w can b e co n sid erab ly prolonged by th is tre a tm e n t (T ab le V III).

M ethod 3 : S im ila r ly hopeful r e s u lts w ere re c e n tly obtained with A LS in m onkeys (T ab le V III), but h e re the continued su p p ression of seco n d ary d ise a se by high d osages of A L S lead s u n ifo rm ly to fa ta l v iru s in fectio n s,

4 2 VAN BEKKUM

T A B L E V II. R E S U L T S O F T E S T FO R SU P P R ESSIO N O F A C U TE SECONDARY D ISEA SE IN M ICE

Substance Dose (mg/kg per day)No. of

schedulesActivity

Cyclophosphamide 1 2 .5 - 50 40 ++

Mustine HCl 0 .2 5 - 1 16 -

Alkylating jMelphalan 0 .5 - 2 .5 8 -

agentsT .E .M . 0 .5 - 2 .2 5 18 -

Treminon 0 .1 - 0 .2 5 12 -

Chlorambucil 10 - 25 10 -

MetaphaseVincristin 0 .1 - 0 .5 9

to xic drugs

Amethopterin 2 .5 - 10 35 ++

M ethyl-gag 5 - 50 9 -

6-M P 50 - 250 15 _

Antim etabolites -Imuran 25 - 500 36 -

Thioguanine 25 50 7 -

Hydroxyurea 100 - 1000 23 +

■

Actinomycin D 0 .0 5 - 1 24 -

Antibiotics * Streptovltacine 0 .5 - 1 5 -

Actidione 30 - 60 7 -

Natulan 50 - 500 28 + .

Salicy late 100 - 1000 9 -

Other * Ercoquin 25 - 100 9 -

Thalidomide 1250 10 -

Cortisone 100 - 400 12 _


T A B L E V III. SU R V IV A L TIM E O F RH ESU S M ONKEYS FO LLO W IN G A LLO G EN IC BO N E-M A RRO W TRA N SPLA N TA TIO N ^)

E ffe c t o f p o st-tra n sp la n ta tio n tre a tm e n t

Agent

Survival tim e (d)

< 10 10-20 2 0 -30 3 0 -40 > 40

None 8 14 4(controls)

( b )Cyclo early 1 3 3

Cyclo earlyand i - 4 5Cyclo later J

Cyclo early 1and f 2 3 1 3Amethopcerin la te rl

Anti-lym phocyte serum 1 2

Cyclo early jand г 4 7 2 2ALS later J

^ 4 X 10® homologous bone-marrow cells per kg recipient body weight were in jected intravenously,24 hours after 800 rad whole-body irradiation.

^ Cyclo early: cyclophosphamide treatment on days 2 and 4 after irradiation only.

and a dosage schedu le w hich is capable o f co n tro llin g seco n d ary d ise a se and a t the sam e tim e of p erm ittin g su ffic ie n t re c o v e ry of the lym phatic t is s u e s to w ithstand in fe c tio n s has not b een d isco v ered so fa r [12 ].

T h e re is a cu rio u s d iscrep an cy betw een the r e s u lts obtained in m onkeys and m ic e , when the re c ip ie n ts a re tre a te d with A L S .

In m ice [16] su p p ressio n of seco n d ary d ise a se is v a r ia b le and the e ffe c t is m o re outspoken in d econtam inated m ice (G ram n egativ e in te stin a l } o rg a n ism s e lim in ated by a n tib io tic tre a tm e n t) . It is co n ceiv ab le that som e ■ e ffe c ts of the s e r a , e .g . haem agglutinating ac tiv ity , a r e p a rt ic u la r ly h a rm ful to m ice in the f i r s t few w eeks a fte r tran sp lan tatio n when b o n e -m a rro w ‘| repopulation is not y e t co m p le te . In the la te r s ta g e s , the tre a tm e n t with ' A LS m ay w ell in c r e a s e su sce p tib ility to in fectio n . B oth th e se e ffe c ts of the A LS can be exp ected to co u n teract the b e n e fic ia l in flu ence o f a d im in ished g r a f t -v e r s u s -h o s t re a c tio n .

4 . CONCLUSION

In m ice we have now a v a ilab le a t le a s t five o r s ix v e ry e ffe c tiv e m ethods of prevention o r tre a tm e n t o f acu te seco n d ary d is e a s e . T h ese agents a re not only capable of p reventing acu te seco n d ary d is e a s e , but

4 4 VAN BEKKUM

in m any c a s e s a lso p rev en t developm ent of the delayed type of second ary d ise a se in the surviv ing m ic e , so that we can tru ly s ta te that com plete con tro l of the hazard s of a llo g e n ic b o n e-m arro w tran sp lan tation has been ach iev ed .

So fa r , s im ila r ly com plete su cce ss , has not been obtained with any of th e se m ethods in the m onkey. The in c re a s e of su rv iv a l tim e is s ig n ifican t, so m e tim e s even im p re s s iv e , but usu ally the s ig n s and sym ptom s of seco n d ary d ise a se re ap p ear a fte r a c e r ta in tim e and continuing tre a tm e n t with any of the agents w ill not co n tro l the d ise a se s a t is fa c to r ily .

F u r th e r im provem ent of a t le a s t two of th ese m odifying p ro ced u res is to be expected in the n e a r fu tu re, nam ely donor se le c tio n and sep aratio n of dangerous c e ll types fro m the g ra ft . The r e s u lts of a com bined ap p lica tio n o f th ese im proved m ethods, supplem ented if n e c e s s a ry by p o sttran sp lan tation tre a tm e n t o f the re c ip ie n ts with chem oth erap eu tic agents and A L S , cannot be p red icted , but on th e o re tic a l grounds a co n sid erab le im p rovem en t o f the ch an ces of su rv iv a l o f p rim ate rad ia tio n ch im e ra s m ay be exp ected .

The b a s is of such exp ecta tio n s l ie s in our ex p erien ce with the fa c to rs that govern the developm ent of seco n d ary d ise a se in ro d e n ts . If, un exp ected ly , the te ch n ica l ad vances outlined above do not re s u lt in s a t i s fa c to ry co n tro l of seco n d ary d isease in p r im a te s , it w ill b eco m e n e c e s s a ry to in v estig a te m uch m o re c lo se ly the d iffe re n ce s betw een rod ents and p rim a te s as re g a rd s the m ech an ism s of lym phoid re a c tiv ity and p ro life ra tio n follow ing b o n e-m arro w tran sp lan tatio n .

R E F E R E N C E S '

[1 ] BEKKUM, D .W ., VAN, "Hostile grafts", Advance in Transplantation (Proc. 1st Int. Congr. Transplantation Soc. , Paris, 1967) (DAUSSET, J. HAMBURGER, J . MATHE, G ., Eds ), Munksgaard, Copenhagen (1968) 565.

[2 ] BEKKUM, D .W ., VAN, VOS, O . , WEYZEN, W .W .H ., The pathogenesis of the secondary disease after foreign bone marrow transplantation, J. natn. Cancer Inst. 23 (1959) 75.

[3 ] DORIA, G ., Homograft reaction in mouse radiation chim eras. II. Direct evidence for g raft-an tihost activity , J. Immun. 89 (1962) 459.

[4 ] CONGDON, C. C . , URSO, I. S . , Homologous bone marrow in the treatment of radiation injury in m ice , Am. J. Path. 33 (1957) 749.

[5 ] BEKKUM, D .W ., VAN, VRIES, M. J . , DE, Radiation Chimaeras, Logos Press/Academic Press, London/New York (19671).

[6 ] Ma t h e , G ., bern a rd I, j . , v r ie s , m . i . d e , Sc h w a r z e n b e r g , l . , l a r r ie u , m . j . , l a l a n n e ,' c . m . ,DUTREIX, A ., AMÏEL, J. L . , SURMONT, J . , Nouveaux essais dé greffe de m oelle osseuse homologue après irradiation totale chez des enfants atteints de leu cém ie aiguë en rémission. Le problème du syndrome secondaire chez l'hom m e, Revue Hémat. 15 (1960) 115.

[7 ] VRIES, M. J.D E , CROUCH, B. G. , PUTTEN, L .M . ,VAN, BEKKUM, D .W .,. VAN, Pathologie changes in irradiated monkeys treated with bone márrow, J. natn.Cancer Inst. 27 (1961) 67.

[8 ] BEKKUM, D .W ., VAN, The selective elim ination of imm unologically com petent ce lls from bone marrow and lymphatic c e ll mixtures I. Effect of storage at 4°C, Transplantation 2 (1964) 393.

[9 ] EPSTEIN, R, B . , STORB, R ., RAGDE, H ., THOMAS, E .D . , Cytotoxic typing antisera for marrow grafting in litterm ate dogs, Transplantation 6 (1968) 45 .

[1 0 ] BALNER, H ., DERSJANT, H ., LEEUWEN, A . , VAN, ROOD, J. J . , VAN, "Identification of twomajor leukocyte antigens of rhesus monkeys and'their relation to histocom patibility” , H istocompatibility Testing 1967 (CURTON1, E .S . , MATTIUZ, P. L ., T O SI,-R. M ., Eds, ) Munksgaard, Copenhagen (1967)'267 . •

PREVENTION AND CONTROL OF SECONDARY DISEASE 45

[1 1 ] PUTTEN, L. M ,, VAN, BEKKUM, D .W ., VAN, VRIES, M. J . , DE, "Transplantation of foetal haem opoietic ce lls in irradiated rhesus monkeys", Radiation and the Control of Immune Response (Proc. Panel, Paris, 1967) IAEA, Vienna (1968) 41 .

[1 2 ] BEKKUM, D .W ., VAN, LEDNEY, G .D ., BALNER, H ., PUTTEN, L. M ., VAN, VRIES, M. J . , DE, "Suppression of secondary disease following foreign bone marrow grafting with antilym phocyte serum", Antilymphocyte Serum, Ciba Foundation Study Group N o.29, Churchill, London (1967) 97..

[1 3 ] PUTTEN, L '.M ., VAN, "Secondary disease in different species and its m od ification ", La greffe des cellules hém atopoiétiques allogéniques, Colloques Internationaux du Centre National de la Recherche Scientifique n °,1 4 7 (1964) 323,

[1 4 ] DICKE, K .A ., HOOFT, J. I. M ., VAN, BEKKUM, D .W ., VAN, The selectiv e elim ination of immunologically com petent ce lls from bone marrow and lym phatic c e ll mixtures: II. Mouse spleen ce ll fractionation on a discontinuous albumin gradient, Transplantation 6 (1968) (in Press).

[1 5 ] BEKKUM, D. W.,VAN, "Im m unological and hem atological aspects of recovery: im plications for therapeutic use of bone marrow grafts", Brookhaven Symposia in Biology 20 : Recovery and Repair Mechanisms in Radiobiology (Rep. Symp. New York, 1967) Brookhaven N ational Laboratories, Upton,N. Y . (1968) 790.

[1 6 ] LEDNEY, G .D . , BEKKUM, D. W ., VAN, "Suppression of acute secondary disease in the mouse with antilym phocyte serum ", Advance in Transplantation (Proc. 1st Int. Congr. Transplantation Society,Paris, 1967) (DAUSSET, J . , HAMBURGER, J . , MATHE, G ., Eds) Munksgaard, Copenhagen (1968) 441.

[1 7 ] SCHWARZENBERG, L ., A MIEL, J . L . , TENENBAUM, R ., MATHE, G . , Effets de la conservation à -7 0 eC sur les pouvoirs myelorestaurateur et inducteur de syndromes secondaires de la m oelle osseuse allogénique, Revue franç. Etudes c lin . bio l. 8 (1963) 783.

[1 8 ] AMIEL, J. L . , MATHE, G . , A comparison of the sensitivity to storage at 37°C in Tyrode solution ofim m unologically com petent ce lls and of myeloid ce lls, Nature (Lond. ) 200 (1963) 1224.

[1 9 ] UPHOFF, D .E ., Alteration of homograft reaction by A-methopterin in lethally irradiated m ice treated with homologous marrow, Proc. Soc. exp. Biol. Med. 99 (1958) 651.

[2 0 ] SANTOS, G. W ., OWENS, A. H ., J r . , Production of graft-versus-host disease in the rat and its treatm ent with cytotoxic agents, Nature (Lond.) 210 (1966) 139.

[2 1 ] MULLER-BERAT, C .N ., PUTTEN, L .M ., VAN, BEKKUM, D .W ., VAN, Cytostatic drugs in the treatm ent of secondary disease following homologous bone marrow transplantation: extrapolation from the mouse to the prim ate, Ann. N .Y . Acad. S e i. 129 (1966) 340.

[2 2 ] LEMMEL, E. M ., NOUZA, K . , Runt disease as a model of immuno-suppressive therapy, Folia Biol. XII(1966 )4 , 253.

[2 3 ] BEKKUM, D .W ., VAN, PLATENBURG, M .G .C . , to be published.

EFFECTS AND COMPLICATIONS OF BONE-MARROW TRANSPLANTATION IN MAN*

G. MATHE, L. SCHWARZENBERG, J .L . A MIEL,M. SCHNEIDER, A. CATTAN, J.R . SCHLUMBERGER Institut de cancérologie et immunogénétique,Hôpital Paul Brousse, Villejuif,France

Abstract

EFFECTS AND COMPLICATIONS OF BONE-MARROW TRANSPLANTATION IN MAN*11 Allogenic bone- marrow grafting in 24 human leukaem ic subjects is described. The graft failed in 7 cases and took in 17 cases.In the latter group, a ll 17 cases were com plicated by the secondary syndrome which was-fatal in 13 cases and controlled in 4 cases. The immunogenetic and immunological factors determining the establishment and evolution o f haem atological radiochimeras in man are discussed.

T he choice of donor is fundamental. Three tests are e ffectiv e in donoi selection, the indirect histocom patibility test, the leu cocyte antigen test and the reaction o f donor and recipient leucocytes in the dermis of an irradiated hamster. When marrow from several donors is transfused, the recipient spontaneously selects the genetically nearest. It seems likely there is more chance o f finding a suitable donor among genetically related subjects than among those who are unrelated. The frequency of graft take seems slightly lower in recipients who have previously received blood transfusions.

T otal bone-marrow graft is associated with specific tolerance towards donor tissues. This is paralleled by the production in the chim era of immunoglobulinsproduced by the graft. The secondary syndrome seems, as in animals, to be related essentially to the graft-versus-host reaction. It is convenient to distinguish among its various manifestations, on the one hand, those lesions which are readily controlled such as hepatitis or erythrodermia associated with infiltration and proliferation o f im m unologically competent ce lls from the graft and, on the other hand, immune insufficiency with regard to micro-organism s, especially viruses and Candida albicans. This latter group, the mechanism of which is com plex, still eludes attempts at preventive and curative control.

The use of multiple donors and the administration o f cortisone during marrow transfusion and A-methopterin and/or cyclophosphamide in the days following transfusions; seem to have reduced the severity o f the secondary syndrome, which, however, still cannot b e satisfactorily controlled.

T he graft reaction against the leukaem ic cells is utilized as an anti-leu kaem ic treatm ent. In the four patients who escaped acute secondary syndrome, survival has been notably longer, in one case being 20 months when death from zoster encephalitis supervened. No c lin ica l signs or histological evidence of leukaem ia were present at autopsy.

* This work was supported by a grant from the Commissariat à l'énerg ie atom ique, Fontenay-aux-Roses, Contract No, 8133/R, and by a grant from the DRME, Contract No. 6 5 .3 4 .3 6 2 .0 0 ,4 8 0 .7 5 .0 1 ,

** Since a com plete manuscript was not available, this paper is published by abstract only.

ARE HAEMOPOIETIC STEM CELLS PRECURSOR CELLS IN SECONDARY DISEASE?

J .L . CHERTKOV

C entral Institute o f H aem atology and Blood Transfusion,Moscow, USSR

Abstract

ARE HAEMOPOIETIC STEM CELLS PRECURSOR CELLS IN SECONDARY DISEASE Î The paper gives data on acute secondary disease developing in supra-lethally irradiated dogs and monkeys after transplantation of allogenic bone marrow. On the basis of the experim ental data obtained, the author discusses the question whether haem opoietic stem ce lls play a role as first links in the histogenesis of the lymphoid elem ents responsible for acute secondary disease.

At p re sen t, seco n d ary d ise a se is widely believed to be cau sed by im m u nologically com petent c e lls in bone m arrow which are independent of stem c e l ls . T h is assum ption is the b a s is of num erous attem p ts to m ake se le c tiv e in activ ation of im m unocom petent (ra th e r than an tig e n -sen sitiv e ) c e lls when sto rin g stem c e l ls . T h e re is to date no d ire c t exp erim en ta l ev idence fo r the e x is te n ce of an independent lin e of a n tig e n -sen sitiv e c e lls in the bone m arrow that induce seco n d ary d ise a se . On the co n tra ry , d ire c t d eterm in ation s show that the content of im m u nologically com petent c e lls and c e lls with im m une m em ory in the bone m arrow is ra th e r low.

In form ation at th is la b o ra to ry obtained fro m dogs and m onkeys with acute second ary d ise a se does not support the assum ption that at le a s t two types of p re c u rs o r c e ll e x is t an bone m arro w : one to provide haem op oietic re co v e ry and the o th er to induce the in itia l step of h is to g e n es is of im m unolo g ica lly activ e c e lls that act again st re c ip ie n t c e lls th ereb y producing second ary d is e a s e . In irra d ia te d dogs with b o n e-m arro w a llo g ra fts , huge am ounts of h yp erb aso p h ilic c e lls and lym phocytes w ere ob serv ed 7 - 8 days a fte r tran sp lan ta tion . The lymphoid c e lls a re s p e c ific a lly im m une to re c ip ie n ts , as re v e a le d with the im m une ' lym phocyte tr a n s fe r t e s t 1 . In the p re sen ce of antigen overload ing , th e se c e lls undergo ' a l le rg ic death' as d em onstrated by e le c tro n m icro sco p y . How ever, no fe a tu re s of n orm al h aem op oiesis of the g ra ft w ere ob serv ed e ith er in bone m arrow o r in blood. . If the seco n d ary d ise a se and n o rm al h aem o p o iesis had had d ifferen t ce llu la r b a s e s , h aem o p o iesis re s to ra t io n would have been expected sid e by side with fo rm ation of im m u nologically activ e c e l l s . T h is does not o ccu r in re a lity .

S im ila r fe a tu re s have been ob serv ed in m onkeys. In th is c a s e , when an a llog en ic tran sp lan tatio n is p erfo rm ed the lym phoid c e lls appear to be s im ila r to c e lls ob serv ed in dogs in m o rp h olog ica l and s u b -m icro s co p ic fe a tu re s . It m ight be thought that in ten siv e p ro life ra tio n of im m u nologically com petent c e lls would o ccu r to prevent the n o rm al h aem o p o iesis of the g ra ft . How ever, te s ts by in je c tin g bone m arrow fro m s e v e r a l donors sim ultaneously d em onstrated that in ten siv e im m une re a c tio n against the re c ip ie n t did not prevent p ro life ra tio n of the bone m arrow an tig en ica lly c lo s e r to the re c ip ie n t.

4 9

50 CHERTKOV

Consequently the m o re obvious assum p tion would be that seco n d ary d ise a se and haem op oietic re co v e ry a re influenced by the sam e p re c u rs o r c e l ls , 's te m c e lls '. P re lim in a ry thym ectom y in re c ip ie n t dogs should prevent fo rm ation of lym phoid c e lls fro m the g raft and su p p ress n orm al h aem o p o iesis .

T h is re p o rt p re se n ts co n firm a to ry exp erim en ta l data on a seco n d ary d ise a se in a rad ia tio n ch im e ra . Stem c e lls respond to antigens but th is s e n s itiv ity is under s t r ic t co n tro l, p resu m ably fro m the lymphoid tis su e of the body. When an tig e n -sen sitiv e c e lls a re p re se n t in the body, d ire c t d if fe re n tia tio n of s tem c e lls to im m unocytes under antigen influence is stro n g ly r e s tr ic te d . That is why the im m une re sp o n se of the bone m arrow in the n orm al o rg an ism does not take p lace , even a fte r hyperim m unization .

On tran sp lan ta tio n into im m u nologically non -com p etent o rg an ism s (for exam p le stro n g ly irra d ia te d ), the d irectio n of h is to g e n es is of h aem op oietic tis s u e in ev ery c a s e should be dependent on the sen sitiv ity of s tem c e lls to antigens and on the le v e l of co n tro l fro m the lymphoid tis su e of the re c ip ie n t. In le th a lly irra d ia te d m ic e , th e re is p re fe re n tia l d ifferen tia tio n of s tem c e lls in the d irectio n of h aem o p o iesis . As haem op oietic re s to ra t io n o c c u rs , the fo rm atio n of lymphoid p re c u rs o rs fro m stem c e lls tak es p la ce . G radual d ifferen tia tio n of the la t te r into im m u nocytes re ta rd s the ap p earance of seco n d ary d is e a s e . In su p ra le th a lly irra d ia te d dogs, th e re is p re fe re n tia l d iffe ren tia tio n of stem c e lls into an tig e n -sen sitiv e c e lls . Acute seco n d ary d is e a s e ta k e s p lace w h ereas the h aem o p o iesis of the g ra ft is prevented b eca u se the stem c e lls d iffe ren tia te into im m u nocy tes. The e x is te n ce of som e lym phoid tis su e of the re c ip ie n t (for exam ple on a low er dose of irra d ia tio n ) p reven ts the lym phoid d ifferen tia tio n of the g raft and the la tte r a tta in s haem op oietic com p eten ce . The sam e re s u lts m ight be o b serv ed when tran sp lan ta tio n of bone m arrow is m ade 5 - 6 days a fte r irra d ia tio n . It ap p ears that in itia l r e s to ra t io n of the r e c ip ie n t 's im m u nogenesis p reven ts lym phoid d ifferen tia tio n of the g raft. In su p ra le th a lly irra d ia te d m onkeys, sim u ltaneou s d ifferen tia tio n of stem c e lls into e ith e r the lym phoid or the h a e m o p o ie tic d ir e c t io n is s o m e tim e s o b s e r v e d .

Although th is hypothesis a g re e s w ell with exp erim en ta l data it cannot be supported exp erim en ta lly o - ’y by the u se of heterogenou s c e ll populations such as bone m arro w . F u r th e r developm ents in m ethods of cloning and fra c tio n a tio n of bone m arrow should m ake it p o ssib le to te s t th is hyp othesis.

LYMPHOID TISSUE GRAFTS IN MAN

H .E .M . KAYRoyal Marsden H ospital,Institute of C an cer Research,London, United Kingdom

Abstract

LYMPHOID TISSUE GRAFTS IN MAN. Grafts of lymphoid tissue or o f lymphoid stem cells may be appropriate in the treatm ent of some congenital immune deficiency disorders. The reasons for preferring tissues of foetal origin are discussed and the evidence for foetal im munocompetence is briefly summarized. Methods of storing foetal liver ce lls and ce lls or fragments of thymus are mentioned, and the organization of the Foetal Tissue Bank of the Royal Marsden Hospital is described. C lin ical data from transplantation of lymphoid ce lls in various immune deficiency disorders are briefly presented.

1. INTRODUCTION

The im m u nological h azard s a r is in g fro m tran sp lan ta tio n of bone m arrow a re so g re a t, both in ex p e rim e n ta l an im als and in m an, that it m ay seem su rp ris in g even to co n sid er the tran sp lan ta tio n of c e lls — belonging to the lymphoid sy stem — whose p rim a ry fu nction is to engender im m une re a c tio n s .

N e v e rth e le ss , th e re a re c l in ic a l cond itions, with o r without e x p e r im en tal co u n te rp a rts , w here such a tran sp lan ta tio n is ce r ta in ly a lo g ica l p ro ced u re and m ay indeed prove an e ffe c tiv e fo rm of tre a tm e n t. T h is is the situation in the im m une d efic ien cy synd rom es (ID S), pred om inantly , of co u rs e , in the con gen ita l fo rm s but ju s t p o ssib ly in the long run in the a c quired v a r ie t ie s a lso . The th eo ry is s im p le : an im m une d efect p e rm its the tran sp lan ta tio n of fo re ig n c e l l s ; i f th e se a re im m u nologically com petent c e lls they w ill provide the re c ip ie n t with a new fu nctional im m une sy stem .The m ain hazard , of c o u rs e , is that of a g ra ft -a g a in s t-h o s t re a c tio n , but th is m ay, p erh ap s, be avoided in one o r m o re of s e v e r a l w ays: by t is s u e - typing fo r co m p atib ility , by induced to le ra n c e in the donor, by p a rtia l d rug- induced im m u nosu p p ression o r by the u se of p re -co m p e te n t im m une stem c e lls such as a re p resu m ed to be p re sen t in e a r ly fo e ta l c e l ls .

T h is la s t lin e of re aso n in g has led us in the p ast ten y e a rs to do two th in g s. F i r s t , to e s ta b lis h an org an ization fo r co lle c tio n , s to ra g e and d is trib u tion of human fo e ta l c e lls and, second ly , to try to d isco v e r the age of fo e ta l l ife at which im m une re a c tio n s can f i r s t be in itia ted .

The evidence on th is su b je c t is s t i l l v ery incom p lete and la rg e ly in d ire c t, but i t is c le a r that the foetu s of fiv e m onths and m o re is capable of v aried and vigorous im m une re a c tio n s [ 1 -4 ] . F u r th e rm o re , it is highly probable that, as e a r ly as th re e m onths and p o ssib ly e a r l ie r , som e im m une com p etence is p re se n t. It is tem pting to equate the g e n esis of im m une cap acity with the developm ent of a lym phoid thym us and with the em e rg en ce of s m a ll lym phocy te s in the blood s tre a m . The re le v a n t data have been published e lse w h e re .

The thym us of tw elve w eeks g estatio n o r le s s is sm a ll but can be d is sected out and im planted . The w hereabouts of o th er lym phoid stem c e lls is l e s s ce r ta in , but fro m the w ork of Tyan and h is co lleag u es [ 5, 6] and of

51

5 2 KAY

T a y lo r [7 ] they are probably lo cated in the l iv e r and perhaps a lso e lse w h e re . The data of Tyan and Cole [ 5] in d icate that e a r ly tran sp lan ta tio n e ffe ctiv e ly p reven ts g ra ft -a g a in s t-h o s t re a c tio n s but p e rm its developm ent into im m unocom petent c e lls that a re to le ra n t of th e ir p rim a ry host.

2. TR A N SP L A N TA BL E LYM PH OID T ISSU E S .

What a lymphoid tissu e .b a n k needs a re thym uses and su sp en sio n s of l iv e r c e lls fro m young healthy fo e tu se s . T h e re is som e evidence that thym ic c e lls may be p a rtia lly e ffe c tiv e in re s to r in g im m unity to thym ectom ized an im als, but m o st w o rk ers ag ree on the su p e rio rity of in ta c t thym us glands o r .at le a s t of frag m en ts which p re s e rv e the thym ic s tru c tu re . A fter im p lantation the tis su e undergoes c e n tra l n e c r o s is , but provided it does not exceed 2 - 3 mm in d iam eter — 1 mm is . probably id eal — th e re is re g e n e r a tion fro m the in ta c t p erip h ery and the e ss e n tia l thym ic s tru c tu re i s re s to re d .

T h is can ce rta in ly a lso o ccu r with the sm a ll human fo e ta l th y m u ses. P erh a p s the ca se quoted by C o ttier [ 8] is the b e s t evidence of th is . T h is sam e c a s e a lso shows that the fo e ta l thym us can undergo developm ent fro m a p rim itiv e sem i-d iffe re n tia te d s tru c tu re to fu ll d iffe ren tia tio n , including the appearance of H a s s a l l 's c o rp u s c le s .

- 3. P R ESE R V A T IO N O F LYM PH OID .TISSU ES

H itherto in human tran sp lan ts we have alw ays im planted fr e s h fo e ta l . thym us. W here tra n sp o rt involves som e d e la y ,. the .tissu e has been kept in ic e -c o ld t is s i je cu ltu re m edium fo r up to 24 hours and probably a .s o m e - what longer delay would not b e .d is a s tro u s . In m ice it was shown by P la y fa ir and D avies [ 9] that the thym us could be fro z en in an -in tact s ta te and that enough c e lls rem ain ed v iab le a fte r thaw ing and reim p lan ta tio n to re g e n e ra te a functioning thym us. S ince the e a r ly human fo e ta l thym us, divided if n e c e s s a ry into two o r th ree p a r ts , is o f,a s im ila r s iz e , we could no doubt em ploy the sam e p ro ced u re. The e s s e n tia ls a re the im m e rsio n of the thym us in- 10% dim ethyl sulphoxide (DMSO) at 0°C fo r 10 - 20 m inutes follow ed by the usu al con tro lled ra te fre e z in g p ro g ram . A fter ,rapid thawing the thym uses w ere washed in two ch an g es.o f cu ltu re m edium to .a llow dilution and diffusion of the DMSO b efo re re im p lan tatio n .

Our method of p rep arin g l iv e r c e lls in su sp en sion s .for fre e z in g and fo r in je c tio n is e x trem ely s im p le . The young fo e ta l l iv e r has a v ery tenuous stro m a fro m which the c e lls can e a s ily be te a se d . . A spiration , in and out of a sy rin g e b re a k s the tis su e up into sin g le c e lls and s m a ll clum ps, and th is suspension can be in jected in s m a ll q u an tities . F o r fre e z in g , a liquots of the susp ensions a re equ ilib ra ted at n e ar 0°C with 10% DMSO. in s m a ll p la stic am poules. The c e lls can be in je c te d in traven o u sly and the r is k of m ic r o em bo li is sm a ll; how ever, p y re x ia l re a c tio n s have o ccu rre d in one o r two c a s e s a fte r in travenous in je c tio n and nowadays we p re fe r the in tra p e rito n e a l ro u te , although fro m an im m u nological aspect, th is is only a second b e s t .

T h e re i s no d ire c t proof of the v iab ility of th e se fo e ta l c e lls a fte r t r a n s p lantation , but th e re is s tro n g su ggestiv e evidence in a few c a s e s . It has . been d ire c tly shown, how ever, by Z u ckerm an , Kay and H ockley [ 1,0] that fp eta l l iv e r c e lls do su rv iv e th is m ethod of freez in g , and thawing and w ill grow th e re a fte r in tis su e cu ltu re . . . . .

LYMPHOID TISSUE GRAFTS 53

4 . ORGANIZATION O F A F O E T A L . TISSUE: BANK

The F o e ta l T iss u e Bank at the R oyal M arsd en H ospital is fo rtu n ately situ ated som ew h ere n e a r the ce n tre of London and is thus able to draw upon a ca tch m en t a re a of a lm o st 10 000 000 people. Our co n tacts a re with ov er 70 h o sp ita ls in and around London w h ere o p e ra tio n s.fo r te rm in a tio n of p re g nancy a re p erfo rm ed . T h is is an uncom m on op eration in any sin g le h osp ita l, but by draw ing on such a la rg e a re a we a re able to en su re a re g u la r supply of fo e ta l t is s u e s . In p ra c t ic e , the dem ands of tran sp lan ta tio n fo rm only a m in or p art of the whole w ork and the m a jo r u s e r s of fo e ta l t is s u e s a re th o se who em ploy tis su e cu ltu re , e sp e c ia lly in routine and r e s e a r c h v iro lo g y .

At p re sen t we co lle c t.a n d d is s e c t about 6 0 .fo e tu ses p e r m onth. All th e se com e fro m the op eration of abdom inal h y stero to m y and excep t w here a v iru s d ise a se such as ru b e lla has. been the ind ication fo r te rm in a tio n the t is s u e s can be co n sid ered to be healthy and thus su itab le fo r both viro logy and fo r-tra n sp la n ta tio n . When c e lls a re s to red o r 'tra n sp la n te d d irec tly , blood is taken fo r r e d - c e l l grouping and i f the foetu s is too s m a ll fo r the sex to be identified with c e rta in ty , the gonads’ a re taken fo r sec tio n and skin is cu ltu red fo r ch ro m o so m al sex in g .

Scrupulous atten tion i s , of co u rs e , paid to b a c te r ia l s te r i l i ty in a ll the o p eration s of d isse c tio n and in the p re p ara tio n of c e lls fo r cu ltu re , fre e z in g o r tran sp lan ta tio n . F o r th is purpose a s p e c ia l su ite of ro o m s has been b u ilt and th ese a re ven tila ted by a ir f i lte re d to 0. 5 pm . So fa r , con tam in ation of cu ltu re has been exceed in g ly r a r e and th e re is no evidence of in fectio n in any of the tw enty o r m o re tra n sp la n t c a s e s .

5. R E S U L T S AND CONCLUSIONS

The r e s u lts of lym phoid tis s u e tra n sp la n ts up to 1966 have a lread y been su m m arized [ 11] and som e of the subsequent c a s e s have b een o r w ill be r e ported in g re a te r d eta il [ 12] . T h e re is no doubt that, in the re la t iv e ly com m on type of 1 S w iss ' IDS, som e tran sp lan ted c e lls have survived and have probably e x e rte d som e im m u n olog ical function [ 13, 14], including b en ef ic ia l e ffe c ts in ov ercom in g b a c te r ia l o r fungal in fe c tio n s . H ow ever, in many c a s e s th e re has been no d em o n strab le evidence of a tak e and th e re have been no la s tin g im p ro vem en ts in any c a s e .

B y co n tra s t, a sin g le ca se of thym ic a p la s ia (Di G eorge syndrom e [ 15]) has survived fo r one y e a r s in ce the im plantation of a 13-w eek thym us [ 16] and th e re was su b stan tia l im provem en t in im m u n olog ical a ctiv ity a fte r g ra fting. T h e re is no d ir e c t proof that th is g ra ft actu ally ' took ' and it i s , of c o u rse , v ery p o ss ib le that the g ra ft has now been r e je c te d but that the en hancem ent of the im m une sy stem p e r s is t s .

O bviously, m uch re m a in s to be lea rn ed about lym phoid tis su e t r a n s p lantation and the fu ture m ay w ell depend upon d ev ising new m ethods of t is s u e typing. If th is can be achieved we m ay eventually be ab le to g raft su cce ss fu lly in c a s e s of both con gen ita l and acq u ired d é fic ie n ce s of the im m une sy stem .

A C K N O W L E D G E M E N T S

The F o e ta l T iss u e Bank of the R oyal M arsd en H ospital is supported by a grant fro m the M ed ica l R e s e a rc h C ouncil of G reat B r ita in .

5 4 KAY

R E F E R E N C E S

[1 ] EICHENWALD, H .F ., SHINEFELD, H .R ., J . Pediat. 63 (1963) 870.[2 ] KAY, H .E .M ., PLAYFAIR, J .H .L . , WOLFENDALE, Margaret R . , HOPPER, P .K . , Proc. Int.

Symp. Bone Marrow Therapy in Primates, Rijswijk, Netherlands (1962) 169.[3 ] SILVERSTEIN, A .M ., LUKES, R .J . , Lab. Invest. П (1962) 918.[ 4 ] VAN FURTH, R. C ., The Formation of Immunoglobulins by Human Tissue in Vitro, Leiden, Netherlands

(1964).[5 ] TYAN, M .L ., COLE, L .J . , Transplantation 2 (1964) 241.[6 ] TYAN, M. L ., COLE, L . J . , NOWELL, P .C ., Transplantation 4 (1966) 79 .[7 ] TAYLOR, R .B ., Br. J . exp. Path. 46 (1965) 376.[8] COTTIER, H., Proc. Third Developmental Immunology Workshop, Sanibel, Florida (in Press).[9 ] PLAYFAIR, J .H .L . , DAVIES, A .J . S . , Transplantation 2_ (1964) 271.

[1 0 ] ZUCKERMAN, A . J . , KAY, H .E .M ., HOCKLEY, A . B . , J . C lin . Path.;, 21 (1968) 109.[1 1 ] KAY, H .E .M ., Proc. Third Developmental Immunology Workshop, Sanibel, Florida (in Press).[1 2 ] HONG, R ., KAY, H .E .M ., COOPER, M. D ., MEUWISSEN, H ., ALLAN, Marjorie.. J .G . , GOOD, R .A .,

Lancet (1968) 503.[1 3 ] HARBOE, M ., PANDE, H ., BRANDTZAEG, P ., TVETER, K . J . , HJORT, P .E ., Scand. J . H aem at. 3

(1966). 351.[1 4 ] SOOTHILL, J . F . , personal com m unication (1968).[1 5 ] Di GEORGE, A .M ., LISCHNER, H .W ., DACOU, Catherine, AREY, J .B . , Proc. Third Developmental

Immunology Workshop, Sanibel, Florida (in Press).[1 6 ] CLEVELAND, W .E ., FOGEL, B . J . , KAY, H .E .M ., Proc. Am. Soc. c lin . Invest. (inPress).

SUPPRESSION OF IMMUNOGENESIS WHEN USING GANGLERON AND PREDNISOLONE

K . A. ANTONYANA rm enian Institute o f H aem atology and Blood Transfusion,Y erev an , USSR

Abstract

SUPPRESSION OF IMMUNOGENESIS WHEN USING GANGLERON AND PREDNISOLONE. The paper summarizes the results of research into the effect on haemopoiesis of transplantation of allogenic bone marrow into dogs suffering from depressed haemopoiesis caused by ionizing radiation and a ch em ical agent (myelosan).

Myelotherapy was carried out with the ganglion-blocking agent gangleron (synthesized by the Fine Organic Chemistry Institute of the Armenian Academy of Sciences) and prednisolone. -This method of therapy has prevented the development of secondary disease and contributed to a high survival rate.

1. INTRODUCTION

One of the m ost im p ortant q u estions a r is in g in the tran sp lan ta tio n of hom ogenic bone m arrow is the im m u nological co n flic t betw een the donor and the re c ip ie n t.

The s u c c e s s attending hom ologous b o n e-m arro w tran sp lan ta tio n depends on m aking the optim um ch oice of donor in re s p e c t of t is s u e com p atib ility .

A utogenic and iso g en ic tran sp lan ta tio n s of bone m arrow without co n flict a re alw ays su c c e s s fu l; with hom ogenic and h etero g en ic b o n e-m arro w tr a n s plantations the situ ation is o th erw ise , s in ce the antigen fa c to r of the donor c e lls and the d ifferin g g en etic m ake-u p of donor and re c ip ie n t cau se v iolent r e a c tio n s . T h ese re a c tio n s a re m utual: (a) donor v e rsu s re c ip ie n t, and (b) re c ip ie n t v e rsu s donor.

The m o re pronounced the tis su e in co m p atib ility , the m o re violent are the re a c tio n s and the m o re freq u en tly is the fin al outcom e fa ta l.

It should be noted that the age of the anim al a lso p lays a p art in the appearance of re a c tio n s . T h e se re a c tio n s a re hardly ev er ob served in new born an im als o r in the em bryonic s tag e , when the im m u nological m echanism has not yet developed. D uring m atu rity , once the m ech an ism is organized , hom ogenic and h e tero g en ic tra n sp la n ts cau se v io len t re a c tio n s in the re c ip ie n t.

D etailed study of blood com p atib ility in re g ard to d ifferen t types and groups is a m a tte r of g re a t im p o rtan ce . It should be noted that the outcom e of the tran sp lan ta tio n depends not only on identity of the e ry th ro cy te s but a lso on the antigen re a c tiv ity of the leu co cy te s and the nu cleated b o n e-m arro w c e lls .

Today tran sp lan tatio n im m unology is in rapid developm ent. S u ccessfu l solution of a ll p ro b lem s a r is in g in hom ogenic tran sp lan ta tio n w ill depend on solv ing the co n flic t betw een donor and re c ip ie n t.

To red uce the im m u nological re a c tiv ity of re c ip ie n t and donor we have proceed ed as fo llow s:

5 5

5 6 ANTONYAN

(1) The re c ip ie n t was exposed to a le th a l dose of rad iatio n .(2) The o rg an ism s of re c ip ie n t and donor w ere p re tre a te d with the ganglion- b locking su b stan ce gangleron and the c o r t ic o -s te r o id horm one p red n iso lo n e.

2. IRRAD IATIO N O F TH E R E C IP IE N T

T ran sp lan ta tio n of hom ogenic b o n e-m arro w into irra d ia te d an im als at f i r s t p ro ceed s su cc e s s fu lly , the anim al em erg in g fro m a se v e re p ath o lo g ical s ta te and the p e rip h e ra l blood being rap id ly renew ed. D uring the second, th ird and fourth m onths, seco n d ary d is e a s e s e ts in and the an im al d ies .

In our e x p e rim e n ts , 25% of the su rv iv in g an im als died within 3 - 5 m onths a fte r com p lete re c o v e ry of the b o n e-m arro w fu nctions.

The sym ptom s ob serv ed in the an im als that subsequently died w ere an abrupt r i s e o f te m p e ra tu re , lo s s of appetite, vom iting, d ia rrh o e a , e m a c ia tion , hepatom egaly , lym phopenia, p la te le t d efic ien cy , h aem o ly tic anaem ia, d e rm a to s is and h y p e rk e ra to s is .

In the lite ra tu re th is phenom enon has been term ed hom ologous, seco n d ary o r tran sp lan ta tio n d is e a s e , and is e x p resse d in the co n flic t betw een the g ra ft and the o rg an ism of the re c ip ie n t.

With a view to p reventing seco n d ary d ise a se we have used as a ganglion- b lock in g and d ese n sitiz in g agent the su b stan ce gangleron, syn th esized in the F in e O rganic C h em istry In stitu te of the A rm enian Academ y o f S c ie n ce s by L . L . M idzhoyan, M em ber of the A rm enian Academ y of S c ie n c e s .

The b e s t re s u lts w ere obtained in an im als when gangleron was used in con ju n ction with the c o r t ic o -s te r o id horm one p red n iso lo n e.

3. ' P R E T R E A T M E N T O F ANIMALS

D uring the two w eeks p reced in g tran sp lan ta tio n of bone m arro w , donor and re c ip ie n t w ere p re tre a te d with g an glero ir and p red n iso lo n e, given in te r n ally with the feed .

T h e se su b stan ces w ere shown by o b serv a tio n to have no sp e c ia l sid e e ffe c ts in the an im als n or w as the p e r ip h e ra l blood p ic tu re changed. The only changes which w ere noted w ere in the blood seru m : the a ssa y of n a tu ra l and im m une antibodies (in s a lt and co llo id a l m edia) fe ll to z e ro . The fe rro g ra m showed som e red u ction in the gam m a-globu lin fra c tio n , which points to the su p p ressio n of im m u nogenesis follow ing the application of gangleron and p red n iso lo n e.

D uring the p ast five y e a rs we have c a rr ie d out 343 hom ogenic b on e- m arro w tran sp lan ta tio n s into 148 dogs, 78 of w hich w ere p rev io u sly i r r a d i ated w hile a p la s ia was induced in the re m a in in g 70 by an o v erd o se o f m ile ra n .

The b o n e-m arro w tran sp lan ta tio n s w ere c a r r ie d out m o re than once on each an im al, using d ifferen t p ro ced u res , both in travenou s and in tra -o s s e o u s .

Of the 148 dogs, 133 re co v e re d fro m the acu tely p ath o lo g ica l s ta te , the p e r ip h e ra l blood p ictu re and b o n e -m arro w p ictu re re v e rtin g to n o rm al. In 25% of the su rviv in g dogs (36 in nu m ber), sym ptom s of seco n d ary d ise a se developed fro m 4 to 11 m onths a fte r com p lete re c o v e ry and they a ll died.

A second group (48 dogs), p re tre a te d with gangleron and p red n iso lo n e, a ll su rviv ed a fte r irra d ia tio n and an ov erd o se of m ile ra n , show ing no sym p -

SUPPRESSION OF IMMUNOGENESIS 57

to m s of seco n d ary d is e a s e . T h is group inclu d es dogs w hich have been under our c a r e fo r m o re than 10 y e a r s .

T h e re a re in d ica tion s in the l ite r a tu r e that seco n d ary d is e a s e a ffe c ts som e an im als m o re s e v e re ly and m o re rap id ly than o th e r s . T o in v e stig a te th is we c a r r ie d out p a r a lle l e x p e rim e n ts on 48 r a ts and 48 ra b b its (in each c a s e 24 co n tro l an im als and 24 p re tre a te d with gangleron and p red n iso lo n e).

A fter irra d ia tio n and hom ogeneic b o n e-m arro w tran sp lan ta tio n , the p e r centage of seco n d ary d ise a se in the co n tro l an im als was 60% o r m o re while the an im als p re tre a te d with gangleron and p red n iso lo n e showed sym ptom s of seco n d ary d ise a se in only 4 - 6% of the c a s e s .

4 . CONCLUSIONS

(1) A fter tra n sfu sio n o f hom ogenic bone m arro w , 25% o f the an im als develop sym ptom s of seco n d ary d is e a s e .(2) In an im als p re tre a te d with gangleron and p red n iso lo n e, b o n e-m arro w a p la s ia p ro ceed s slow ly and tra n sfu s io n of hom ogenic bone m arro w gives b e tte r re s u lts .(3) A nim als which have been given gangleron and p red n iso lo n e o v er the sp ace of a m onth show no sid e e ffe c ts , the im m u n olog ical p ro c e s s being inhibited in .such an im a ls .(4) A d m in istra ticp o f gangleron and p red niso lone to an im als b e fo re and a fte r tra n sfu s io n of hom ogenic bone m arro w p rev en ts the ap p earan ce of seco n d ary d ise a se and re d u ce s the fa ta lity ra te .

STEM -CELL INACTIVATION ON TRANSPLANTATION OF

. HAEMOPOIETIC CELL SUSPENSIONS FROM GENETICALLY DIFFERENT DONORS

R .V . PETROV Institute o f Biophysics,M inistry o f Public H ealth o f the USSR,Moscow, USSR

Abstract

STEM-CELL INACTIVATION ON TRANSPLANTATION OF HAEMOPOIETIC CELL SUSPENSIONS FROM GENETICALLY DIFFERENT DONORS. The transplantation of a mixture of haem opoietic or lymphoid cells from two gen etically different m ice into lethally irradiated F j recipients results in marked or total inactivation of the colony-form ing units of the graft. This phenomenon is observed following transplantation of mixtures of spleen ce lls or bone-marrow cells from anim als of different genotypes: CBA + C57BL, A + CBA, A + C57BL, C3H + C57BL, CBA + (CBA x C57BL) Fr Maximum inactivation is observed when lymph-node cells of one genotype are transplanted with spleen or bone-marrow cells of another genotype. Use of non-syngenic kidney cells or lymphoid ce lls inactivated by irradiation as one component of the mixture shows that inactivation of gen etically heterogeneous stem cells requires the participation of viable lymphoid ce lls . The inactivation phenomenon is also observed with Jerne's method. This shows that inactivation affects not only colony-forming ce lls but also the im m unologically com petent precursors of antibody-producing ce lls .

Although the problem of b o n e-m arro w tran sp lan ta tio n sim ultaneously from se v e r a l donors to an irra d ia te d re c ip ie n t has m o re than th e o re tic a l in te r e s t , the in te ra c tio n s betw een c e lls of d iffe ren t genotypes have not been studied adequately .

A num ber of attem p ts have been m ade to understand the in te ra ctio n s betw een im m u nologically activ e c e l l s . In -v itro tra n sfo rm a tio n of lym phoc y te s in resp o n se to o th er an tig en ica lly d ifferen t lym phocytes is evidently s im ila r to the re a c tio n s involving c e lls explanted from sp leen s [1, 2] . The re a c tio n s of im m une lym phocytes with th e ir ta rg e t c e lls have been studied m o st thoroughly. The w ork of H ells tro m et a l. [3] has led to d isco v ery of the phenomenon of a llo g en ic inhib ition , which is c h a ra c te r iz e d by the supp re ss io n of c e ll a c tiv ity sim ply a s a re s u lt of the p re se n ce o f a llogen ic a n tig en s. A ll th is w ork co n trib u tes to our understanding of the p ro c e s s e s involved in im m u n olog ical re a c tio n s betw een c e lls in -v iv o .

In our previou s com m u nications [4, 5] it has been shown that t r a n s plantation of m ouse s p le e n -c e ll suspension m ix tu re s from two d ifferen t genotypes of p aren ta l natu re into leth a lly irra d ia te d hybrid (F j) re c ip ie n ts re s u lts in s ig n ifican t o r com p lete in activ atio n of co lo n y -fo rm in g activ ity [6] in the stem c e lls of the g ra ft .

T h is p aper re p o rts the exp erim en ta l data c h a ra c te r iz in g the phenomenon w ith c e ll m ix tu re s of d ifferen t t is s u e s fro m m ice of d ifferen t genotypes. The p art played by the quantitative ra tio of c e lls in the m ix tu re s has been an aly sed . The p o ss ib ility o f re g is te r in g the in activ atio n of the p re c u rs o rs of antibody-prod u cing c e lls by J e r n e 's m ethod [7] has been d em onstrated .

5 9

6 0 PETROV

The b a s ic e x p e rim e n ta l se t-u p is given in F ig . 1. The e ffe c t of c e ll in te ra c tio n w as estim ated by com p arin g th re e m ain grou p s: Standard I - the num ber of co lo n ies produced in the, re c ip ie n ts ', sp leen s when 1 o r 2 X 10 6 c e l ls o f one genotype (P i) w ere ad m in istered i. v. ; Standard II - the nu m ber of co lo n ies produced in the r e c ip ie n ts '.s p le e n s when 1 o r 2 X l O 6 c e lls o f an oth er genotype (P¿). w ere ad m in istered i .v . ; E x p e rim e n ta l group - the num ber of co lo n ies produced in the re c ip ie n ts ' sp leen s by the m ixtu re of c e l ls (p er 106 in je c te d c e l l s ) .

LD10o

»------ [B E S — D standard I

(P.-Pi)F,LD 100

fo'PjF,

fo-PjF,

LOl00

standard II ,

* • ( Calculated numberof CFU (8-9day)

F IG .l . Diagram showing experim ental method used for demonstrating the inactivation o f non-syngenic stem ce lls . • •

E ig h t o r nine days follow ing c e l l tran sp lan tatio n , the re c ip ie n ts w ere s a c r if ic e d . The iso la te d sp leen s w ere p laced into B o u in 's o r a c e tic ac id - ethanol solu tion and the num ber o f co lo n ie s w as counted.

In a ll ex p e rim e n ts the in activ atio n index (I. I . ) w as ca lcu la ted acco rd in g to .th e fo rm u la

' L L - l00. X2LÜML . .

w h ere X is the num ber of co lo n ie s in the "e x p e r im e n ta l" group, and-y is -.the nu m ber of co lo n ies in Standard I + Standard II. . . . .

T a b le I su m m arizes the data on in a ctiv a tio n .o f,s tem c e l ls when m ix tu re s o f c e ll su sp en sion s of d ifferen t t is s u e s fro m CBA and C 5 7 B L m ice w ere tran sp lan ted into the le th a lly irra d ia te d (C BA x C5.7BL) F j re c ip ie n ts . As is seen from T a b le I, tran sp lantation , of C B A .sp leen o r lymphoid c e lls m ixed with C 5 7 B L sp leen c e lls r e s u lts in in activ atio n of 81 to 100% of colony- fo rm in g units (C F U ). T ran sp lan ta tio n of. СВЛ lymph node c e l l s , m ixed w ith C 5 7 B L b o n e-m arro w c e l ls , is a lso follow ed by a high in activ atio n index am ounting to 95%. T ra n sp la n ta tio n .o f a m ixtu re o f b o n e-m arro w or

; em bryon ic l iv e r c e lls from m ice of the two indicated -genotypes r e s u lts in in activ a tio n of 35 - 36% of the stem c e l l s . .. . . ,

In 'f a ls e ' e x p e rim e n ts , when d ifferen t c e lls of the sam e genotype w ere .m ixed and tran sp lan ted , the in activ atio n e ffe c t w as ab sen t. -,

It should be noted that th e re a re no c e lls capable of co lo n y -fo rm in g in lym ph nod es. The ob served num ber of co lo n ies a fte r tran sp lan ta tio n of lym ph-node c e lls so m e tim e s did n ot.exceed the num ber o f spontaneous

TABLE I. STEM CELL INACTIVATION ON TRANSFER OF A MIXTURE OF CELL SUSPENSIONS FROMDIFFERENT TISSUES TO LETHALLY IRRADIATED (CBA x C57BL) F1 RECIPIENTS . . .

Interacting ce lls Observed number o f CFU Expected number o f . CFU in

mixture

Inactivation index(%)Standard I Standard II Standard I Standard II Mixture

CBA spl. C57BL spl. 2 .2 ± 0 .2 7 .7 ± 0 . 7 1 ¿9 ± 0 .2 9 .9 ± 0 .7 81

CBA b .m . C57BL b .m . 1 6 .5 ± 1 .5 3 .4 ± 1 .2 1 2 .8 ± 1 .0 " 1 9 .9 ± 2 .1 36

. CBA e . l . C57BL e . l . 1 3 .1 ± l . !3 3 .7 ± 1 .1 5 .4 ± 0 . 5 8 .4 ± 1 .7 35

CBA l . n . C57BL b .m . 0 .0 3 .4 ± 0 .6 0 . 1 ± 0 . 1 3 .4 ± 0 .6 ' 95

C B A l.n . C57BL spl. 0 .0 .8 .7 ± 0 .5 0 .0 8 .7 ± 0 .5 100

• C57BL 1 . n. CBA spl. 0 .2 ± 0 .1 5 .7 ± 0 .7 5 .4 ± 0 .4 5 .9 ± 0 .7 9 (n/s)

spl. = spleen, b .m . = bone marrow, e . l . = embryonic liver, 1.,n . = lym ph node.

STEM-C

ELL IN

AC

TIVA

TION

TABLE II. STEM CELL INACTIVATION ON TRANSPLANTATION OF A MIXTURE OF CELL SUSPENSIONSOF DIFFERENT GENOTYPES

Interacting ce lls Observed number o f CFU Expected-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- number o f Inactivation index

Standard I Standard II Standard I Standard II Mixture ^mixture

A spl. C57BL spl. 5 . 3 1 1 . 2 3 .2 ± 1 . 2 3 .2 ± 0 . 6 8 .5 ± 2 . 5 63

A spl. CBA spl. 1 9 .8 ± 1 .1 1 0 .6 ± 1 .7 1 3 .2 ± 0 .7 3 0 .4 ± 2 . 0 56

C3H spl. C57BL spl. 0 .6 ± 0 .3 1 .4 ± 0 . 9 0 .1 ± 0 .1 2 .0 ± 1 . 0 94

CBA spl. (CBAxC57) spl. 2 .7 ± 0 .4 9 .6 ± 0 . 8 ' 2 .8 ± 0 . 4 1 2 .3 ± 0 . 9 ' 77

C57BL spl. (CBA xC57)F1spl. 4 .4 ± 0 .6 9 .6 ± 0 . 8 1 2 .8 ± 1 . 1 1 4 .1 ± 1 . 0 9 (n/s)

STEM-CELL INACTIVATION 6 3

co lo n ie s d etected in the sp leen s of the co n tro l irra d ia te d a n im a ls . Consequ ently , the lo s s of co lo n y -fo rm in g a c tiv ity when tran sp lan tin g a m ixtu re of g e n etica lly d iffe ren t c e ll su sp en sio n s su g g ests that CBA lym ph-node c e l ls in activ a te the C FU which a re contained in sp leen and bone m arrow of C 5 7 B L m ic e . W hen C 57B L lym ph-node c e l ls m ixed with CBA sp leen c e lls w ere tran sp lan ted into le th a lly irra d ia te d re c ip ie n ts , the s te m -c e l l in a c tiv a tio n w as ab sen t (T ab le I, lin e 6).

Thus th e re is ev ery re a so n to b e lie v e that CBA lym phoid c e lls ac t as 'k i l l e r s ' and C 5 7 B L stem c e lls a s 't a r g e t s ' . T h is assum p tion is supported by the a n a ly sis of the re s u lts obtained on tran sp lan ta tio n of a sp leen or b o n e-m arro w c e l l m ix tu re of the two above-m entioned genotypes (T ab le I, lin e s 1-2) . The ob serv ed num ber of co lo n ies in the m ix tu re alw ays co rresp o n d s to the num ber of C FU o ccu rrin g in the CBA s p le e n -c e ll susp ension .

A s is shown above, tran sp lan ta tio n of s p le e n -c e ll m ix tu re s of p aren ta l natu re into le th a lly irra d ia te d hybrid re c ip ie n ts is accom p anied , in v ario u s ex p e rim e n ts , by in activ a tio n of 65 to 81% of the stem c e l ls contained in th e se m ix tu re s . W hen CBA lym ph-node c e lls m ixed w ith C 5 7 B L sp leen c e l ls a re used, 100% in activ atio n is o b serv ed . S ince the phenom enon in q u estion could be s p e c if ic only fo r th is com bination of s tra in s , exp erim en ts w ere p erfo rm ed with c e l l su sp en sio n s from m ice o f o th er genotypes.

The in activ atio n phenom enon w as con firm ed fo r c e l l com binations of a num ber of genotypes. The r e s u lts of the ex p e rim e n ts a re su m m arized in T a b le II. In a l l the e x p e rim e n ts d e scrib e d , F j hybrid m ice w hich did not r e a c t to the antigens of both p aren ta l s tra in s w ere used a s re c ip ie n ts .

We can se e that the phenom enon under co n sid era tio n is ob served not only on tran sp lan ta tio n of c e ll m ix tu re s including the C 57B L com ponent but a lso when m ix tu re s of A + CBA , A + C 5 7 B L , C3H + C 5 7 B L , CBA + C 57B L , CBA + (CBA x C 57B L ) F;l c e lls a re tran sp lan ted into le th a lly irra d ia te d re c ip ie n ts of corresp o n d in g hybrid n a tu re . F u r th e rm o re , the s te m -c e ll in activ atio n phenom enon is a lso ob serv ed when m ice of one of the in te r actin g genotypes a re used a s re c ip ie n ts .

In one ex p erim en t with CBA + A sp leen c e l l m ix tu re , we used C B A T 6T 6 d onors. As re c ip ie n ts we used (A x С В А Т б Т б ^ m ic e . C h rom osom e a n a ly sis of the co lo n ies showed that a l l co lo n ies a re A genotype, so in th is system the CBA c e lls a r e ta rg e t c e l l s . In the above-m entioned sy s te m , CBA + C 5 7 B L , the ro le of ta rg e ts is played by the CBA c e l l s . L in e s 4 and 5 of T a b le II con firm th is assu m p tion .

To o b serv e the in activ ation of im m unocom petent p r e c u r s o r s in the tran sp lan ted m ixed c e l l su sp en sio n s, we used an analogous exp erim en ta l schedule (F ig . 2).

The d iffe ren ce was th a t ,p r io r to th e ir tran sp lan ta tio n into the i r r a d i ated re c ip ie n ts , sheep e ry th ro cy te s w ere added to the c e l ls . T h is antigen- stim u lated the p r e c u r s o r s of an tibo d y -form in g c e l ls , w hich could a lso be d eterm ined q u antita tively .

The c e ll m ix tu re s w ere in je c te d i . v . into le th a lly irra d ia te d re c ip ie n ts a t a dose of 25 X 106 nu cleated c e lls of each genotype. S ix o r seven days la te r , the re c ip ie n ts w ere s a c r if ic e d and the num ber of h aem oly sin - producing c e lls in th e ir sp leen s w as counted. The num ber of th ese c e lls in the sp leen of each re c ip ie n t w as counted tw ice and the m ean value was d eriv ed . The in activ a tio n index fo r the p re c u rs o rs of antibody-producing c e lls w as d eterm ined acco rd in g to the above-m entioned fo rm u la .

6 4 PETROV

The re s u lts of the ex p e rim e n ts a r e g iv en 'in T a b le III . T he tab le shows that in je c tio n of a standard am ount of CBA sp leen c e l ls m ixed with sheep e ry th ro cy te s lead s, s ix days la te r , to an accu m u lation of 2 .6 X 1 0 6 antibody- producing c e l ls in the re c ip ie n ts ' sp lee n s . B y day 7 th e ir num ber am ounts to 23 . 3 X 106. The sam e am ount of C 5 7 B L sp leen c e l l s , when tran sp lan ted , g iv es 2 .3 X 106 and 2 4 . 4 X 1 0 6 antibody-prod u cing c e lls by days 6 and 7, re s p e c t iv e ly . A m ixtu re o f c e l l su sp en sio n s of CBA and C 5 7 B L m ice under s im ila r cond itions g ives an accu m u lation of only 0. 5 X 1 0 6 antibody- producing c e l ls on the six th day and 10. 8 X 106 on the seven th . T he in ac tiv a tio n index ob serv ed o v er th is period is equal to 77-90% .

FIG. 2 . Experimental schedule for the demonstration of s tem -ce ll inactivation as determined by Jem e plaque count following in-vitro incubation o f spleen ce lls .

In a ll the exp erim en ts d escrib e d above, the ra tio o f in te ra c tin g c e l ls w as 1:1. In a s e r ie s of subsequent ex p e rim e n ts , the quantitative ra tio of c e l ls in the m ixtu re w as changed. A s is see n from T ab le IV , the num ber o f C 5 7 B L sp leen c e lls rem ain ed con stan t at 2 X 106. T he num ber of CBA lym ph-node c e l ls v aried from 0. 2 X 106 to 2 X 106. T he ra tio of in te r ac tin g c e l ls w as 1 :1, 1 :2 , 1 :5 and 1 :1 0 . W ith the c e ll ra tio in the 1 :1 - 1 :5 ran g e , in activ atio n of betw een 100 and 93% w as ob serv ed . W ith the ra tio of the two c e l l com ponents 1:10, th e re w as no in activ atio n of stem c e l l s .

The exp erim en ts d escrib ed have thus shown that the in activ a tio n e ffe c t is ob serv ed on tran sp lan ta tio n of c e l l m ix tu re s from d ifferen t t is s u e s : lym ph nodes and sp leen , lym ph nodes and bone m arrow , and m ix tu re s of sp leen , b o n e-m arro w and em bryon ic c e l l s .

It should be em phasized that not only colony-prod u cing c e l ls o f h aem op o ie tic tis su e but a lso im m unocom petent p re c u rs o rs of antibody-prod ucing c e l ls a re s u b je c t to in activ a tio n . C onsequently , d iffe ren t p ro life ra tin g c e l l s , p r e c u rs o rs of a given h aem op oietic o r lymphoid tis su e , a re su b je c t to in activ a tio n .

T ran sp lan ta tio n of sp leen c e lls m ixed with o th er n on -syn gen ic c e l ls , in cap able of p ro life ra tio n , p a rt ic u la r ly with kidney c e lls o r ir ra d ia tio n - in activ a ted lymphoid c e l ls , g iv es negative r e s u lts . The in activ atio n e ffe c t is a lso ab sen t when h aem op oietic c e l l s a r e tran sp lan ted m ixed w ith m ic r o b ia l antigens. T h is te s t if ie s a g a in st the assu m p tion that lo s s of co lon y - fo rm in g a b ility is the re s u lt of a change in the d ire c tio n of c e l l d if ie re n -

Count of antibody forming cells (ploques).

6-7 doys öfter inoculation.*

T A B L E III . IN A CTIVA TIO N O F P R E C U R SO R S O F A N T IB O D Y -FO R M IN G C E L L S ON TR A N SPLA N TA TIO N O F C E L L M IX T U R E S

Interacting cellsObserved number

o f antibody-form ing ce lls (XlO6 )Expected

number o f ’ antibody-

forming ce lls in mixture

In activ ation in d ex ;(%) ■'

Standard I Standard II Standard I Standard II Mixture

CBA spl. C57BL spl. 2 3 .3 i 3 .8 2 4 .4 ± 3 .8 1 0 .8 ± 1 .7 ' 4 7 .7 ± 5 .5 77

CBA spl. C57BL spl. 2 .6 ± 0 .7 2 .3 ± 0 .4 0 .5 ± 0 . 2 4 .9 ± 0 . 8 90

T A B L E IV . E F F E C T O F Q U A N TITA TIV E R A TIO O F C E L L S IN TH E M IX T U R E ON TH E S T E M C E L L IN A C TIVA TIO N PHENOMENON

Number o f interacting cells ( x 106)

Observed number o f CFUExpected

number of Inactivation index

(%) .Standard I(CBA 1 . n.)

Standard II(C57BL sp l.)

Standard I Standard II Mixture.CFU in mixture

2 2 _ 1 6 .4 ± 0 .9 0 .0 .1 6 .4 ± 0 .9 100 . ’

1 2 - 1 2 .5 ± 1 .0 0 .4 ± 0 .1 1 2 .5 ± 1 .0 97/

0 .4 2 - 1 0 .0 ± 1 .0 0 .7 ± 0 . 1 ' . .1 0 .0 ± 1 .0 93 ■

0 .2 2 - 1 1 .3 ± 1 .3 1 0 .9 ± 1 . 6 1 1 .3 ± 1 .3 3 (n/s)

STEM-C

ELL IN

AC

TIVA

TION

6 6 PETROV

tia tio n under the e ffe c t of fo re ig n an tigen ic s tim u li, o rig in atin g fro m the second com ponent of the m ixtu re being tran sp lan ted .

T he e ffe c t is probably connected with inhib ition of p ro life ra tio n of stem c e l l s of one genotype under the in fluence of lymphoid c e l ls of the second genotype. H ere the q uestion a r is e s w hether a m utual in activ ation of c e lls o ccu rs o r w hether c e lls of only one of the genotypes s e r v e as 'k i l l e r s ' . A fu rth e r problem is w hether in activ atio n of stem c e lls is the con seq u en ce of a g r a f t -v e r s u s -g r a f t re a c tio n o r w hether it tak es p lace ■ through a g r a ft -v e rs u s -h o s t re a c tio n .

R E F E R E N C E S

[1] BAIN, B. e t a l . , Blood 23 (1964) 108.[2] FESTENSTEIN, H ., Ann. N. Y . Acad. S e i. 129 (1966) 567.[3] HELLSTROM, K. E. e t a l . . Nature (Lond.) 208 (1965) 4 58 .[4] PETROV, R .V ., SESLAVINA, L .S . , D okl.Akad.N auk SSSR 176 (1967) 170.[5] PETROV, R .V . et a l . , Nature (Lond.) 217 (1968) 558.[6] TILL, J .E . , MacCULLOCH, E .A ., R adiat.Res. И (1961) 213 .[7] JERNE, N .K ., NORDIN, A .A ., Science 140 (1963) 4 05 .

EFFECT OF MASSIVE BLOOD TRANSFUSION ON THE THERAPEUTIC EFFICIENCY OF HOMOGENIC BONE MARROW IN ACUTE RADIATION ILLNESS

V . SERAPHIM OV-DIM ITROV, Z . DÉCHEVA, M . NEDYALKOVA Institute o f H aem atology and Blood Transfusion,S o fia , Bulgaria

Abstract

EFFECT OF MASSIVE BLOOD TRANSFUSION ON THE THERAPEUTIC EFFICIENCY OF HOMOGENIC BONE MARROW IN ACUTE RADIATION ILLNESS. Simultaneously with bone-marrow transplantation, the authors replaced the blood of the lethally irradiated recipient animals with blood from the bone-marrow donor. From experiments on dogs and rabbits it becam e clear that replacing 86% of the recipient's blood with blood from the bone-marrow donor considerably reduces the therapeutic e ffect of bone-marrow transplantation. The authors consider that the m ain cause of the anim als' early death in experiments combining bone-marrow transplantation and massive donor blood transfusions is a secondary syndrome resulting from the graft-versus-host reaction. This does not exclude the inverse possibility - that the development of a host- versus-graft reaction is due to the presence of a massive number of antigens of the donor blood in the blood o f the recipient.

T ran sp lan ta tio n of bone m arrow is the m o st e ffe c tiv e method fo r tre a tm e n t of acute rad ia tio n i lln e s s [ 1 , 2 ] . A ccord ing to our exp erim en ta l data and the data of a num ber of o th er w o rk e rs , in c e r ta in conditions blood exchange can a lso have a b e n e fic ia l th erap eu tic e ffe c t on le th a lly and sub- le th a lly irra d ia te d an im als [ 3 , 4 ] . We have acco rd in g ly in our exp erim en ts sim u ltan eou sly tran sp lan ted bone m arrow and re p laced the blood of le th a lly irra d ia te d an im als with blood taken fro m the b o n e-m arro w donor. We hoped that to ta l tran sp lan ta tio n of h aem op oietic tis su e and p e rip h e ra l blood would c r e a te b e tte r conditions fo r grow th of the g ra ft , provided th e re was re la t iv e ly good com p atib ility betw een the tissu e antigens of donor and re c ip ie n t [ 5 , 6 ] .

T h e re a re a num ber o f stu d ies rep o rted in the lite ra tu re which show that p r io r ad m in istra tio n of hom ologous blood im m u nizes the re c ip ie n t and sh arp ly red u ces the th erap eu tic e ffe c t o f b o n e-m arro w tran sp lan ta tio n [ 7 , 8 ] . It w as our hope that th is e ffe c t would not o ccu r in our exp erim en ts owing to the strong su p p ressio n of the im m une re a c tio n in the re c ip ie n t 24 hours a fte r le th a l irra d ia tio n when blood tra n sfu sio n w as p erfo rm ed as w ell as b o n e-m arro w tran sp lan ta tio n . We a lso reckoned that the seco n d ary su p p ression of rad iatio n -in d u ced im m unity would be enhanced owing to the m a ss iv e num ber o f tra n sfu se d blood an tig en s, w hich, it is known, m ay in c e r ta in conditions re s u lt in im m u nological to le ra n c e , even w here th e re is a hom ologous re la tio n sh ip betw een donor and re c ip ie n t [9 , 10] .

The data obtained in our exp erim en ts did not b e a r out our exp ecta tio n s. Thanks to w ork done on dogs [6] , it b ecam e c le a r that the re p lacem e n t of 86% of the blood of the re c ip ie n t with blood fro m the b o n e-m arro w donor co n sid erab ly red u ces the th erap eu tic e ffe c t of b o n e-m arro w tran sp lan ta tio n (T ab le I).

6 7

T A B L E I. SURVIVAL O F E X P E R IM E N T A L ANIMALS (%)

6 8 SERAPHIMOV-DIMITROV et al.

Dogs Rabbits

Group (treated) Number of animals

SurvivedSurvival"

№

' Number of animals

SurvivedSurvival

№

Control (untreated) 28 0 0 14 • • 0 0

Bone-marrow transplantation 15 9 60 18 9 50

Bone-marrow transplantation + massive blood transfusion

10 2 20 20 6 30

Bone-marrow transplantation+ blood transfusion(10°jo o f the blood volume)

11 3 « 3 0

The sam e e ffe ct was found in an ex p erim en t with ra b b its [11] . If 63% of the blood of le th a lly irra d ia te d an im als w as rep laced with donor blood im m ed iate ly b efo re tran sp lan ta tion of bone m arrow fro m the sam e donor, th e re w as a s im ila r drop in the th erap eu tic e ffe c t of tran sp lan tatio n (T ab le I).It can be seen fro m th e se data that a sin gle hom ologous tran sp lan tatio n of bone m arrow into le th a lly irra d ia te d dogs and rab b its re s u lts in the su rv iv al of m o re than h alf of the exp erim en ta l an im als (a ll co n tro l an im als dying), w hile the addition of m a ss iv e q u antities of donor blood notably red u ces the th erap eu tic e ffe c t. In o th er in v estig ation s it was found that a s im ila r e ffe c t is obtained if b o n e-m arro w tran sp lan ta tio n is accom panied by the t r a n s fusion of to ta l blood in an am ount 10% of the volum e of the blood of the ex - . p erim en ta l an im al o r by the tran sfu sio n of le u c o c y te -fr e e blood [12] .

In the exp erim en ts with ra b b its , we in vestig ated the su rv iv al of bone- m arrow g ra fts by d eterm in ing the p ercen tag e of g ran u lo cytes with sex ch ro m atin ( 'd r u m s t ic k s ') . T h is was p o ssib le b ecau se a ll the donors studied in th ese exp erim en ts w ere fe m a le s . The re s u lts obtained showed that the la r g e s t p ercen tag e of b on e-m arrow su rv iv a l (44%) was found in exp erim en ta l an im als which had been tre a te d so le ly by b o n e-m arro w tran sp lan tatio n . In- ra b b its which had b ee n .tre a te d with b o n e-m arro w and m assiv e qu an tities o f blood, the su rv iv a l ra te w as low er (30% ). The growth of the g ra ft (reckoned by the in c re a s e in the num ber of granu locytes with sex ch ro m atin in the blood) in an im als which had re ce iv e d only bone m arrow was w eak o r m edium (p e r cen tage o f gran u lo cy tes with sex ch ro m atin 2 -3% ), while it was co n sid erab ly m o re in an im als w hich had re ce iv e d bone m arrow and m assiv e qu an tities of blood (the p ercen tage of leu co cy te s with sex ch ro m atin was 5-6% — alm ost the sam e as in fem ale ra b b its ) (T ab le II).

At the sam e tim e , exp erim en ts showed that by the ninth day a fte r irra d ia tio n the bone m arrow in the co n tro l an im als had not re co v ere d its a b ility to in co rp o ra te 3H -thym idine. Y et the bone m arrow of the e x p erim en ta l an im als on w hich b on e-m arrow tran sp lan ta tio n o r t r a n s plantation plus m assiv e blood tran sfu sio n had been p erfo rm ed contained

TABLE II. SURVIVAL OF BONE-MARROW GRAFTS

Number of animals

SuccessfulDegree o f transplantation growth (according to the percent o f the sex chromatin)

Group (treated) transplantation

№High

proliferation(5-6% )

Meanproliferation

( 2 - 3 »

Lowproliferation

(1 -1 .5 % )

Graft did not take(below 1 °jo)

Bone-marrow transplantation 18 44 0 4 4 10

Bone-marrow transplantation + massive, blood transfusion

20 30 6 0 0 14

EFFECT OF

BLOOD TR

AN

SFUSIO

N


Ж

I

F IG .l . Incorporation of radioactive thymidine in bone marrow of irradiated rabbit, 9 days after bone- marrow transplantation.

F IG .2 . Incorporation of radioactive thymidine in bone marrow of irradiated rabbit, 16 days after bone- marrow transplantation.

c e ll com ponents cap ab le of in co rp o ratin g rad io activ e thym idine (F ig s . 1 and 2) [13] .

F a ce d with the a d v erse e ffe c t of sim ultaneous tran sfu sio n of la rg e or m a ss iv e qu an tities of donor blood on the th erap eu tic e ffe c t of b o n e-m arro w tran sp lan ta tio n into le th a lly irra d ia te d a n im a ls , we fe lt obliged to see k m eans of elu cid ating the natu re of th is phenom enon. It was n atu ra l to suppose that it has an im m u nological c h a ra c te r and is due to the g ra ft - v e rs u s -h o s t re a c tio n . We th e re fo re deem ed it im portant to study the im m u nological com petence o f the bone m arrow of n orm al an im als and an im als exposed to le th a l d oses of rad ia tio n , during the e a r ly sta g e s fo l lowing b o n e-m arro w tran sp lan ta tio n .

i

EFFECT OF BLOOD TRANSFUSION 7 1

In exp erim en ts with n o rm al m ic e , using the Je rn e m ethod, we found that a sin gle in je c tio n of sheep e ry th ro cy te s does not cau se the fo rm ation of haem olysin -p rod u cin g c e lls in the bone m arro w . Such c e lls o ccu r in ap p reciab le qu an tities only a fte r a second stim u lation with th is antigen (F ig . 3) [ 14 ] .

In o th er exp erim en ts we found that in the bone m arrow of rab b its hyperim m unized to Salm on ella typhim urium th e re appear antibody-producing c e lls which a re capable of ca rry in g o v er and continuing the antibody- producing p ro ce ss in le th a lly irra d ia te d rab b its [15] . T h is fa c t shows unam biguously that the bone m arrow of a p rev iou sly hyperim m unized donor contains im m u nologically com petent c e lls w hich a re cap ab le o f producing antibodies independently. In addition to th is , we found that in the e a rly s ta g e s follow ing tran sp lan ta tio n of bone m arro w , a le th a lly irra d ia te d o rgan ism gives a p rim a ry immune re sp o n se . T hu s, when iso g en ic bone m arrow is tran sp lan ted into le th a lly irra d ia te d m ice of B 10D 2 inbred s tra in , we found that betw een the 4th and 7th days follow ing tran sp lan ta tio n of the bone m arrow and stim u lation by sheep e ry th ro cy te s the sp leen contained a co n sid erab le num ber of h aem oly sin -p rod u cin g c e l l s ; the sam e was found with the tran sp lan tatio n of hom ologous bone m arrow (F ig s 4 and 5) [16] .

HPC

В из B1 H M .1 2 3 4 5 6 7 9 11 13 d a y s

FIG. 3. Mean and individual number of haemolysin-producing cells (HPC) per 107 bone-marrow cells following secondary immune response.

Po “ Pi < 0 - oí» ? ! - p3 < o .o o i , P3 - P 4 < 0 .0 1 .

Н Р С /spleen

spleen сellulority

4th day 7th day

FIG .4 . Changes in total number of spleen cells and number of haemolysin-producing cells in immunized, lethally irradiated white m ice following transplantation of homogenic bone-marrow and spleen cells.P--------spleen ce lls, P-------------- haemolysin-producing ce lls , • • individual assay o f haemolysin-producingcells.


H ow ever, the num ber of such c e lls in the sp leen is m uch g re a te r when th ere is an iso logous re la tio n sh ip betw een the donor and re c ip ie n t," probably owing to the ab sen ce of the 'hom ogenic tran sp lan ta tio n re a c tio n . :

T hu s, in studying-the im m unocom petent function of bone m arrow and the im m u nological com p etence o f the irra d ia te d o rg an ism a fte r b o n e-m arro w tran sp lan ta tio n , we obtained two groups of fa c ts .

4th day 7th day

FIG. 5 . Changes in total number of spleen cells and number o f haemolysin-producing spleen ce lls in imm unized,lethally irradiated V10 D2 and V10 LP m ice following transplantation o f isologous bone marrow. P---------spleence lls , P ---------haemolysin-producing spleen ce lls, • • individual assay of haemolysin-producing spleen ce lls.

On the one hand, the bone m arrow of a n o rm al o rg an ism did not respond to the p rim a ry antigen s tim u lu s; the im m u n olog ically com petent c e lls o c cu rre d in bone m arrow only when th e re w as a seco n d ary antigen s tim u lu s.F o r th is re a so n it is lo g ic a l to assu m é that bone m arrow can en su re t r a n s fe r im m unity only when th e re has been, p rior, im m u nization of the donor.

On the o th er hand, a fte r b on e-m arrow tran sp lan tatio n an irra d ia te d o rg an ism respond s to a p rim a ry antigen stim u lu s, i . e . it e x p e rie n ce s a ' p rim a ry im m une re sp o n se . B ut why should one and the sam e organ — b o n e-m arro w - fa il to r e a c t when it is in the donor o rg a n ism , yet give a p rim a ry im m une resp o n se when it e n te rs the sp leen of the re c ip ie n t?

T h e re a re th re e p o s s ib ili t ie s :

(1) The im m u nolog ically com petent c e lls of the g ra ft find in the sp leen fav ou rable conditions fo r m atu ration and d iffe ren tia tio n , 'which m akes them prone to respond to a p rim a ry antigen stim u lu s.

(2) On en terin g the irra d ia te d o rg a n ism , the im m u nologically com petent c e lls of the g ra ft a re com pelled v ica r io u sly to respond to the antigen s tim u lu s , owing to the in su ffic ien cy o f the rem ain in g lym p hoid -tissu e.

(3) The tran sp lan ta tio n ca u se s d isap p earan ce of a hypothetical re g u latin g e ffe c t of lym phoid tis su e (C hertkov) on the im m u nolog ically com petent p art of the bone m arrow .

We co n sid er the f i r s t p o ss ib ility m o st lik e ly .T h e se data c le a r ly show that a b o n e-m arro w g ra ft contains c e lls o r

.ce ll p re c u rs o rs which a re cap able of re a c tin g to a p rim a ry antigen s tim u lu s. ¡-Consequently, a re a c tio n of the .graft ag a in st the antigens o f the re c ip ie n t is p e rfe c tly p o ssib le in e a r ly s ta g e s (up to 10 to 12 days), which is when

EFFECT OF BLOOD TRANSFUSION 73

the an im als in ou r exp erim en t died. It m ay be supposed that th is re a c tio n is enhanced by the p re sen ce o f a la rg e num ber of im m u nologically com petent c e lls of the donor type introduced into the irra d ia te d org an ism by a m a ss iv e blood tra n sfu sio n im m ed iate ly p reced in g b o n e-m arro w tran sp lan ta tio n . We e stim a te that in our e x p erim en ta l conditions betw een 45 and 66 m illio n lym phoid c e l ls p er kg of body w eight a re introduced with the blood.

A ll th is g ives ground fo r b eliev in g that the m ain cau se of e a r ly death of the an im als in com bined b o n e-m arro w tran sp lan ta tio n and m a ss iv e donor blood tra n sfu sio n s is a seco n d ary synd rom e resu ltin g fro m the g r a f t -v e r s u s - h ost re a c tio n . T h is does not exclude the in v e rse p o ss ib ility — the d evelopm ent of a h o s t -v e r s u s -g r a f t re a c tio n owing to the p re sen ce of a m a ss iv e num ber of antigens of donor blood com ponents in the blood of the re c ip ie n t.

R E F E R E N C E S

[ 1 ] LORENZ, E. et a l . , J . natn. cancer Inst. 12 (1951) 1.[2 ] MATHE, G . et a l . , Rév. fr. é t . clin . biol. 4 3 (1959) 226.[3 ] SERAPHIMOV-DIMITROV, V . , Vopr. hem . krovopr. 8 (1961) 37 .[4 ] STOITCHKOV, I. , Vopr. hem . krovopr. 11 (1965) 191.[5 ] SERAPHIMOV-DIMITROV, V . , DETCHEVA^ Z . , Vopr. hem . krovopr. 8 (1961) 47 .[6 ] SERAPHIMOV-DIMITROV, V. et a l . , Vopr. hem . krovopr. 10- (1963) 55.[7 ] DA COSTA, R. et a l . , Vox sang. 9 4 (1964) 420.[8 ] PUTTEN, L .M . et a l . , Blood 30 6 (1967) 749.[9 ] HASEK, M. et a l . , Proc. Xth Congr. Int. Soc. Blood T ransí., Stockholm (1964) 192.

[1 0 ] PUZA, A . , GOMBOS, A ., Transpi. Bull. 51 (1958) 30.[1 1 ] DETCHEVA, Z . , Exp. m ed. morph. VI 3 (1967) 180.[1 2 ] DETCHEVA, Z . , Vopr. hem . krovopr. 12 (1967) 95.[1 3 ] DETCHEVA, Z . , Vopr. hem . krovopr. 12 (1967) 89.[1 4 ] NEDELKOVA, M . , Exp. m ed. morph, (in Press).[1 5 ] NEDELKOVA, M . , Roentg. rad. (Bu lg.) VI (1967) 14.[1 6 ] NEDELKOVA, M . , Thesis, Univ. Sofia (1968).

сCONSERVATION AND STORAGE

OF BONE-MARROW CELLS, WHITE CELLS AND THROMBOCYTES

CYTOTOXIC AND AGGLUTINATING PROPERTIES OF SERA FROM .MULTI-TRANSFUSED AND NORMAL PERSONS

M arth a M . EIBL

I I . M ed iz in isch e U n iversitätsk lin ik

and

H . EIBL

ö s te rre ic h is c h e s In stitu t für H a e m o d e riv a te G e s .m .b .H , ■

V ien n a, A ustria

Abstract

CYTOTOXIC AND AGGLUTINATING PROPERTIES OF SERA FROM MULTI-TRANSFUSED AND NORMAL PERSONS. The presence o f cytotoxic and agglutinating antibodies against bone-marrow ce lls and peripheral leucocytes enriched with lymphocytes was tested in serum from both healthy and multi-transfused persons. Agglutinating antibodies against bone-marrow ce lls were more frequently found in multi-transfused persons as compared with those against peripheral leucocytes. The importance o f these prelim inary findings for the transplantation o f haem opoietic ce lls is discussed.

1. INTRODUCTION

S u cc e ss fu l au to tran sp lan tation of h aem op oietic c e lls has been known fo r som e tim e [ 1 , 2 ] . T h e re a r e a lso s e v e r a l.r e p o r ts on s u c c e s s fu l i s o gen ic tran sp lan ta tio n of h aem op oietic c e lls in id e n tica l tw ins without the subsequent ap p earan ce of seco n d ary d is e a s e [ 3 - 8 ] .

If th e re is no p o ss ib ility of an iso g en ic tran sp lan ta tio n and c lo s e ly re la ted d onors a r e a v a ila b le , a fu n ction ally s u c c e s s fu l h aem op oietic g ra ft m ight be ach iev ed , but the d anger of seco n d ary d ise a se o ccu rrin g is p re sen t [ 9 - 1 1 ]

S u cc e ss fu l a llo g en ic h aem op oietic g raftin g has been re p o rted , in p a rticu la r c a s e s w h ere thé h o s t-v e r s u s -g r a f t re a c tio n o f the re c ip ie n t had been im peded o r su p p ressed by su itab le m e a s u re s . T he g r a f t -v e r s u s - host re a c tio n follow ing a llo g e n ic b o n e-m arro w tran sp lan ta tio n depends on the d iffe re n ce betw een the h isto co m p atib ility antigens of donor and re c ip ie n t [12 - 16]. !

S e n sitiz a tio n of the re c ip ie n t cau sed by p reviou s organ tra n sp la n ta tio n , m u lti-tra n s fu s io n [17] , p re g n an cies o r by c e r ta in b a c te r ia l a n tigens [18] m ust be con sid ered a s an ag grav ating fa c to r .

It is ad v isab le th a t, in addition to h isto co m p a tib ility te s tin g , the p re se n ce of an tibod ies in the re c ip ie n t seru m ag a in st donor c e lls should be d eterm in ed . A ntibodies in d ica te in su ffic ien t h isto co m p a tib ility m atching w hether they o ccu r b e fo re o r a f te r tran sp lan ta tio n . ■

7 7

7 8 EIBL and EIBL

2. 1. C o llectio n of seru m sam p les

Seru m sam p les w ere obtained fro m m u lti-tra n sfu se d p atien ts in h aem o d ia ly sis who had re ce iv e d betw een 15 and 500 blood tra n sfu s io n s .The blood w as obtained by venipuncture, kept fo r 4 hours a t room te m p e ra tu re and 16 to 20 h ou rs at +4°C a fte r being cen trifu ged . T he s e r a w ere in activ ated fo r 30 m in a t 56°C and fro z en in sm a ll u n its . They w ere fro zen and thawed only once p r io r to u se . H ealthy blood donors w ere used as co n tro ls .

2 . 2 . ' B o n e -m a rro w c e lls

S te rn a l puncture w as p erfo rm ed on ch ild ren and ju v en iles not su fferin g fro m m alignan t d is e a s e s . F iv e m il l i l i t r e s of sa lin e with 1000 un its H eparin w ere used in a 1 0 -m l hypoderm ic sy rin g e fo r a sp ira tio n of ap p roxim ate ly 3 m l bone m arro w . The c e lls thus obtained w ere shaken v ig o ro u sly in the sy rin g e and q u ick ly in jected through a hypoderm ic needle into a s te r i le g la ss co n ta in e r . T he b o n e-m arro w c e ll su sp ension obtained in th is way w as used w ithin 2 h ou rs.

To obtain a su sp en sio n only s lig h tly contam ined by e ry th ro c y te s , equ al volu m es of a 2% d extran so lu tion (m ol. wt 250 000) w e re added and sed im ented into a g la ss tube of 10 -m m d ia m ete r . A fte r sed im en tation fo r two hours at room te m p e ra tu re , the uncoloured c e ll la y e r was a sp ira te d .

F o r agglutination and fo r the cy to to x ic ity t e s t , a su sp en sio n of app ro x im ate ly 107 c e lls/ m l w ith le s s than 10% e ry th ro cy te s w as used . A gglutination w as read m ic ro s c o p ic a lly .

2 .3 . L y m p h o cy te-en rich ed leu co cy te p rep aratio n

B u ffy -co a t c e lls w ere sep ara ted by sed im en tation in d ex tran , fu rth e r pu rified by h aem o ly sis and w ashed by H BSS, containing 2% c a lf seru m .A su sp en sio n of 107 c e lls w as used .

2 .4 . A gglutination

A 0 . 1 - m l b o n e-m arro w c e ll su sp en sio n o r leu co cy te susp ension w as m ixed with 0.1 m l of the seru m being te s ted in a te s t tube of 5 -m m d ia m e te r and incubated fo r 30 m in at 37°C; agglutination w as read m ic r o s co p ica lly .

2. 5 . C yto tox icity

0.1 m l of the above-m entioned b o n e-m arro w c e ll o r leu co cy te s u s pension w as m ixed with 0.1 m l of the seru m being te s ted and 0 .05 m l gu in ea-p ig com plem ent and incubated fo r 60 m in. Then 0.1 m l of a fre s h ly p rep ared 2% trypan blue solution w as added and the m ixtu re was incubated fo r 20 m in at 37°C and cen trifu ged . T he sed im ent w as then ob served fo r stained c e lls .

2 . M E T H O D

CYTOTOXIC AND AGGLUTINATING PROPERTIES 7 9

T A B L E I. M IC R O SC O PIC A LLY O B SE R V E D AGGLUTINATION O F ALPH A-AGGLU TININ -CO N TAIN IN G SER A W ITH E R Y T H R O C Y T E -P O O R BO N E-M ARRO W C E L L S FR O M GRO UP A DONOR

Bone marrow ce lls from donor group Serum T itre 6 p

A x A z

1 : 16 0 0

1 : 4 ± 0

1 : 512 0 0

1 : 512 0 0

SK-5

SK-6

Test serum anti-A No. 360165

Test serum anti-A No. 370565

T A B L E II. COM PARISON O F AGGLUTINATION O F BO N E-M ARRO W C E L L S AND P E R IP H E R A L L E U C O C Y T E S B Y SE R A D ERIV ED FRO M NORMAL AND M U L T I-T R A N SF U SE D PERSO N S

Sera Number of seraBone marrow from normal persons with blood group

A 0

Peripheral leucocytes from normal persons with

blood group

A 0

Normal sera

(isoagglutinins present)

f

Beta 7 13/0a 12/0 12/0 18/0

Alpha, beta

Sera from m ulti

transfused persons

2 2/0 4/0 2/0 3/0

Alpha 1 1/0 2/0 I/O' 1/0

Beta 9 13/3 20/6 13/2 11/1

Alpha, beta 3 6/3 6/2 7/2 5/1

a Ratio of number of tests performed to number of positive results.

8 0 EIBL and EIBL

3. R E SU L T S : - h ■ . .

When e ry th ro cy te -p o o r b o n e-m arrq w .'ce lls ; fro m .blood-group A donors w ere te s ted fo r agglutination w ith two d ifferen t lo ts of a n ti-A b lood-grouping seru m and s e v e r a l a lp h a-agg lu tin in -con tain in g co n tro l s e r a , no agglutination w as ob serv ed (Table: I).

T h is finding se e m s to be of som e im p o rtan ce , s in ce s e r a of m u ltitra n sfu se d p atien ts often show a re la t iv e ly high isoagglu tin in t i t r e .

As shown in T a b le II, agglutination of pu rified b o n e-m arro w c e lls and p e rip h e ra l leu co cy te s is independent of the p re se n ce of iso agg lu tin in s in the p a tie n ts1 seru m .

O f the 13 s e r a fro m m u lti-tra n sfu se d p atien ts under in v estig atio n ,8 showed agglutinating antibod ies ag ain st b o n e-m arro w c e lls w h ereas only 5 of th e se had agglutinating antibodies ag a in st the p e rip h e ra l le u co cy tes fro m the sam e c e ll d onor. F o u r of the th ir te e n s e r a w ere p o sitiv e in the cy to to x ic ity te s t ag a in st b o n e-m arro w c e lls and 3 of the ГЗ ag a in st p e rip h e ra l leu co cy te s (T a b le s III and IV). One of the s e r a te s ted showed

T A B L E III. C Y TO TO X IC AND AGGLUTINATING P R O P E R T IE S O F P A T IE N T SE R A AGAINST BO N E-M A RRO W C E L L S AND P E R IP H E R A L L E U C O C Y T E S \ , .

No. PatientAgglutination Cytotoxicity

■ Bone-marrow ce lls Peripheral leucocytes Bone-marrow ce lls 'Peripheralleucocytes

1. BES. 3/1 ■ ' 2/0 ' 3/2 2/0

2 . BIR. 3/0 2/0 3/0 2/0

3. HEI. 5/1 5/1 5/0 5/0

4 . LUW. 3/0 2/0 . 3/2 2/1;

5. WEI. 3/1 2/0 3/0 ......... 2/0 ■

6. REI. 3/3 2/2 3/0 2/0

7. STR. 3/3 2/0 3/1 , • 2/0 ■

8. WIE. 5/0 5/0 5/0 5/0 . ' 1

9. ZAN. 3/0 2/0 3/0 2/0

10. VRB. 3/0 2/0 3/1 2/0

11. MAN. 3/2 2/0 3/0 2/0

12. WEI. 4/2 5/1 4/0 5/1

13. w o s . 5/3 5/2 5/0 5/1

CYTOTOXIC AND AGGLUTINATING PROPERTIES 81

T A B L E IV .. C Y TO TO X IC AND AGGLUTINATING P R O P E R T IE S O F P A T IE N T SE R A AGAINST BO N E-M A RRO W C E L L S AND P E R IP H E R A L L E U C O C Y T E S

■ Agglutination Cytotoxicity

Bone-marrow ' ce lls

Peripheralleucocytes

Bone-marrowcells

Peripheralleucocytes

Multi-transfused patients.46/16 38/7 43/6 38/3

Total tests to positive 3 5% 18 % ■ 14 % 8%tests.

Control persons.32/0 31/2 32/2 31/3

Total tests to positive< -3% 6 % 6 % 10%

. tests.

Multi-transfused patients.Total patients tested to 13/8 13/5 13/4 13/3number of patients with 61% 38 % 30 % 23 %positive test.

Control persons. Totalpersons tested to number 11/0 11/2 11/2 ’ ■ 11/2of persons with positive < 9 <% ' 18 % 18 % 18 %test.

agglutination of a ll the b o n e-m arro w c e ll and p e r ip h e ra l leu co cy te p r e p aration s te s te d . T h is w as the seru m of a patient who had had stap hyloco ccu s s e p tice m ia s e v e r a l t im e s during the p reviou s s ix m onths but was in good health when the seru m sam p le w as taken .

4 . DISCUSSION

F r e s h ly obtained b o n e-m arro w c e lls in iso to n ic h ep arin b u ffer , hom ogeneously suspended, g e n e ra lly contain la r g e r qu an tities of e ry th ro c y te s . Such su sp e n sio n s , obtained fro m individuals of blood group A, agglutinate m a c ro s c o p ic a lly on the addition of a lp h a -iso a g g lu tin in - containing s e ru m . M ic ro s c o p ic a lly , p a rtic ip a tio n of b o n e-m arro w c e lls in the ag g lu tin ates can be re co g n iz e d . T h is is co n sid ered as m ixed agglutination [19] .

If the b o n e-m arro w c e l l su sp en sio n is m ixed with d ex tran , the ery th ro cy te s can be sep ara ted by sed im en tatio n . The b o n e-m arro w c e lls purified in th is way show no m a c ro s c o p ic a lly v is ib le agglutination when alp h a- iso ag g lu tin in -co n ta in in g s e r a fro m n o rm al p e rso n s o r a n ti-A b lood- grouping seru m w as added. M ic ro s c o p ic a lly , an agglutination of the ery th ro cy tes is s t i l l p re sen t w ith e ry th ro b la s t p a rtic ip a tio n , w h ereas the m ain p art of the b o n e-m arro w c e l ls show s no agglutination . Thus a scre en in g of the agglutinating an tibod ies a g a in st b o n e-m arro w c e l l s , r e g a rd le s s of A BO -blood g ro u p s, is p o ss ib le .

8 2 EIBL and EIBL

T he re s u lts show that in m u lti-tra n sfu se d p atien ts agglutinating a n tibodies ag ain st b o n e-m arro w c e lls o ccu r m o re freq u ently than those ag a in st p e rip h e ra l le u co cy te s . T h is lead s to the con clu sion that bone- m arrow c e lls m ight be m o re sen sitiv e fo r d eterm in ation of agglutinating antibodies than p e rip h e ra l leu co cy te s . It se e m s fe a s ib le to te s t the bone- m arrow c e lls of the donor ag ain st the re c ip ie n t seru m when haem op oietic c e lls a re tran sp lan ted , even if they a re not obtained fro m bone m arrow . T he d ivergent agglutination of b on e-m arrow c e lls and p e rip h e ra l le u co cy te s of the sam e individual is not n e c e s s a r i ly b ased on the d iv e rsity of the antigen s tru c tu re of th e se c e l ls . It is p o ss ib le to p rep are hom ogenous b on e-m arrow ' c e ll su sp en sion s without the addition of p lasm a p ro te in s . L eu co cy te su sp en sio n s show spontaneous agglutination i f p rep ared in th is way.

Im m unization te s ts w ith leu co cy tes that have been w ashed s e v e r a l tim e s s t i l l show fo rm ation of an a n ti-fib rin o g en , which in d ica tes a strong binding of c e r ta in p lasm a p ro te in s to th e se c e lls [20] . It m ight be that in hom ogenous leu co cy te su sp en sion s the c e ll su r fa ce is co n sid erab ly m o re hyd rop hilic than the c e ll s u rfa ce of b on e-m arrow c e lls and fo r th is re a so n such antigen-antibody com p lexes show le s s tendency toward ag g reg atio n .

A ll the s e r a that agglutinate p e rip h e ra l leu co cy te s a lso agglutinate b o n e-m arro w c e lls of the sam e c e ll donor. T h is was not the c a s e in cy to to x ic ity te s tin g .

R E F E R E N C E S

[ 1 ] KURNICK, N .B ., Autologous and isologous bone marrow storage and infusion in the treatment of myelo-suppression, Transfusion 2 (1962) 178.

[2 ] MANNICK, J . A. , LOCHTE, H .L. , Jr . , THOMAS, E .D . , FERREBEE, J . W ., In vitro and in vivo assessment of viability of dog marrow after storage, Blood 15 (1960) 517.

[3 ] MILLS, S .D ., KYLE, R .A ., HALLENBECK, G .A ., PEASE, G .L ., CREE, I . C . , Bone marrow transplant in an identical twin, I . Am. med. Assoc. 188 (1964) 1037.

[4 ] PEGG, D .E ., A quantitative study of bone marrow grafting im plications for human bone marrow infusion, Br. J . Cancer 1£ (1962) 4 00 .

[ 5 ] PILLOW, R .P ., EPSTEIN, R .B ., BUCKNER, C .D ., GIBLETT, E .R ., THOMAS, E .D ., Treatment of bone marrow failure by isogenic marrow infusion, New Engl. J . Med. 275 (1966) 94.

[6 ] ROBINS, M .M ., NOYES, W .D ., Aplastic anemia treated with bone marrow transfusion from an identical twin, New Engl. J . Med. 265 (1961) 974.

[7 ] RUSSELL, E .S . , G enetic aspects of implantation o f b loo d -fo rm in g tissue. Fed. Proc. 19 (1960) 573.[8 ] THOMAS, E .D ., PHILIPS, J .H . , FINCH, C .A ., Recovery from marrow failure following isogenic

marrow infusion. J . Am. med. Assoc. 188 (1964) 1041.[9 ] BEILBY, J .O .W ., CADE, I . S . , JELLIFFE, A .M ., PARKIN, D .M ., STEWART, J .W ., Prolonged

survival of a bone marrow graft resulting in a blood-group chim era, Br. med. J . I (1960) 96.[1 0 ] MATHE, G ., AMIEL, J .L . , SCHWARZENBERG, L ., CATTAN, A ., SCHNEIDER, M ., DEVRIES, M .J . ,

TUBIANA, M ., LALANNE, C . , BINET, J .L . , PAPIERNIK, M ., SEMAN, G ., MATSUKURA, M .,MERY, A .M ., SCHWARZMANN, ü . , FLAISLER, A ., Successful allogenic bone marrow transplantation in man: Chimerism, induced specific tolerance and possible anti-leu kem ic effects, Blood 25 (1965) 179.

[1 1 ] STEWART, J .W ., Haemopoietic chim era, Br. med. J . I (1964) 304.[1 2 ] JACOBSON, L .O ., Evidence for humoral factor (or factors) concerned in recovery from radiation

injury, Cancer Res. 12 (1952) 315 .[1 3 ] LORENZ, E ., CONGDON. C .C . , UPHOFF, D ., Modification of acute irradiation injury in m ice and

guinea-pigs by bone marrow in jections, Radiology 58 (1952) 863.

CYTOTOXIC AND AGGLUTINATING PROPERTIES 8 3

[1 4 ] MATHE, G ., SCHWARZENBERG, L ., AMIEL, J . L . , SCHNEIDER, M ., CATTAN, A .,SCHLUMBERGER, J .R . , TUBIANA, M ., LALANNE, C . , Immunogenic and im m unological problems of allogenic haem opoietic radio-chim eras in man, Scand. J . H aem at. 4 (1967) 193.

[1 5 ] MULLER-BERAT, C .N ., VAN PUTTEN, L .M ., VAN BEKKUM, D .W ., Cytostatic drugs in thetreatm ent of secondary disease following homologous bone marrow transplantation: extrapolation from the mouseto the prim ate, Ann. N .Y . Acad. S e i. 129 (1966) 340.

[1 6 ] THOMAS, E .D . , PLAIN, G .L ., GRAHAM, T .C . , FERREBEE, J .W ., Long-term survival of lethally irradiated dogs given homografts of bone marrow, Blood 23 (1964) 4 88 .

[1 7 ] DA COSTA, H ., AMIEL, J .L . , MATHE, G ., Immunisation contre des greffes de ce llu les hém ato- poiétiques allogéniques par des transfusions de sang antérieures, Vox Sang. £ (1964) 420 .

[1 8 ] RAPAPORT, F . 'T . , CHASE, R .M . , J r . , The induction of homograft sensitivity with bacterial antigens, Vox Sang. 11 (1966) 345.

[1 9 ] COOMBS, R .R .A ., BEDFORD, D ., ROUILLARD, L .M ., A and В blood group antigens on human epiderm al ce lls demonstrated by mixed agglutination, Lancet i (1956) 461 .

[2 0 ] EIBL, M .M ., EIBL, H ., Unpublished observation.

SUB-MICROSCOPIC ORGANIZATION AND FUNCTIONAL PROPERTIES OF CELLS STORED IN A BANK FOR FROZEN LEUCOCYTES AND PLATELETS

F .R . VINOGRAD-FINKEL, E .I . TERENTIEVA, V .A . LEONTOVICH,S .B . SKOPINA, N. N. ABEZGAUZ, A .A . TO TSK A YA C entral Institute o f H aem atology and Blood Transfusion,Moscow, USSR

Abstract

SUB-MICROSCOPIC ORGANIZATION AND FUNCTIONAL PROPERTIES OF CELLS STORED IN A BANK FOR FROZEN LEUCOCYTES AND PLATELETS. Massive transfusions of leucocyte mass containing young . and stem cells in addition to adult ce lls are carried out successfully in the treatm ent of patients suffering from depressed haemopoiesis. To m eet the needs of clin ics a leucocyte mass bank-has been established and methods have been developed for the long-term storage of leucocyte mass by keeping it in the frozen state at -196°C . Various carrier solutions containing cryoprotective substances (dimethylsulphoxide, glycerine, polyvinyl pyrrolidone) have been proposed. These make it possible to conserve from 70 to 90°jo of liv e cells for as long as two years or more.

The v iability o f the frozen cells is proved by supravital staining and luminescent microscopy, and also by determining the extent to which their functional properties are preserved, the phagocytic activity of the gran ulocytes and the a b ility o f th e ly m p h ocy tes to produce young forms in tissue culture. It has also been observed that their g lycolytic activ ity is preserved (up to 60%). .All these data correlate with the data obtained by electron microscopy regarding the extent to which the sub-m icroscopic organization of frozen leucocytes is preserved.

The bank is also used to store a frozen platelet mass, dimethylsulphoxide being used as the cryoprotectiv e agent. The ultrastructure of the platelets and their functional properties (re tractile activity) suffer least impairment.

It has been d efin ite ly esta b lish e d that tra n sfu sio n s of leu co cy te and p la te le t m a s s , as w ell as m ed ullotherapy, can be used su c c e s s fu lly during a p la s ia s cau sed by rad ia tio n , drugs and o th er fa c to r s . It has a lso been e sta b lish e d that granu locytopenia and in fectio n , which o ccu r during acute leu k aem ia and rad ia tio n leu cop en ia , in m any c a s e s a re su c c e s s fu lly o v e r - ' com e by tra n sfu sio n s of the p e rip h e ra l blood leu co cy te s [ 1 -4 ] . T h e re is evidence that th is s u c c e s s is due to the p re se n ce of the stem c e lls in p erip h e r a l blood, capable of p ro life ra tin g and d ifferen tia tin g into m atu re lym phocy te s , g ran u lo cy tes and e ry th ro cy té s [5] .

M o reo v er, a fte r the tra n sfu sio n s of le u co cy te s , Sch w arzen berg et a l . [6 ] ob serv ed re m is s io n s in a num ber o f p atien ts affected by acu te le u c o s is . T h ese authors exp lain such a n ti- le u c o s is e ffe c ts by the im m une resp o n se of im m u nolog ically com petent c e lls (b la s t e le m e n ts )p re s e n t am ong the t r a n s fused leu co cy te s .

V ario u s in v e s tig a to rs [7 , 8], as w ell as som e s c ie n tis ts at our in stitu te [ 9 - 1 1 ] , have p roved 'the p re se n ce of young c e lls in the p e rip h e ra l blood of healthy p erso n s that a re cap ab le of fu rth e r repop ulating the le u co cy te s .The p ercen tag e of th e se c e lls in the p e rip h e ra l blood is low and, s in ce p o sitive r e s u lts have been m o re often attained during m a ss iv e tra n sfu s io n s ,

85

86 VINO GRAD-FINKEL et al.

it is con sid ered n e c e s s a ry to in je c t v ery la rg e am ounts of le u co cy te s , up to ten s and even hundreds of b illio n s of white c e lls , fo r therap y.

It is th e re fo re n e c e s s a ry to have av a ilab le a g reat num ber of donors of the sam e blood group and R h -fa c to r fo r a sin gle tran sfu sio n of le u co cy te s .

Iso la tio n of leu co cy te s and p la te le ts fro m whole blood re q u ire s a num ber of com plex p ro ced u res and is tim e-co n su m in g , and fo r the tim e ly p ro v ision of p atien ts with such tra n sfu s io n m edium s, sto rag e in sp e c ia l banks is th e r e fo re req u ired .

M ethods fo r prolonged sto rag e of leu co cy tes and p la te le ts in the s ta te of an ab io sis by m eans of deep fre e z in g [ 12, 13] w ere developed at our Institu te and we rep o rted a m ethod of leu cocyte fre e z in g at the X I C o n g ress of Blood T ran sfu sio n , Sydney, 1966 [14] .

Our exp erien ce with b lo o d -ce ll fre e z in g shows that each type of c e ll r e q u ire s an individual approach fo r the developm ent of a fre e z in g m ethod. D iffe ren ces in stru ctu re and functions put th e ir im p rin t on the c e ll resp o n se to in flu en ces of vario u s kinds, including deep fre e z in g . The s im p le r c e lls , fo r exam ple anucleate e ry th ro cy te s , a re re la tiv e ly cry o to le ra n t, w h ereas nucleated c e lls with a com p licated e n d o ce llu lar s tru ctu re re q u ire s p e c ia l fre e z in g conditions, which can only be d eterm ined by individual exp erim en ts . T h is is p a rticu la rly tru e of leu co cy tes and p la te le ts .

R ecen t notions of the m echanism of le s io n by deep coo ling of liv in g c e lls w ere the b a s is fo r m ethods we developed. C u rren t m ethods of p revention of c e ll d estru ctio n by m eans of c e ll p ro tectio n and, p a rticu la r ly , e ffe c tiv e c ry o - p h y lactic agents and su itab le cooling r a te s w ere a lso used .

How ever, the m ethods developed fo r the fre e z in g of leu co cy tes and p la te le ts a re distinguished by a num ber of p e c u lia r itie s a sso c ia te d with the p ro p e rtie s of th ese c e l ls . F o r leu co cy tes a la rg e ch o ice of cry o p h y lactic agents is p o ssib le , w h ereas fo r p la te le ts only dim ethylsulphoxide (DMSO) has been proven e ffic ie n t. O ther agents exam ined d eterio ra ted the c e llu la r s tru c tu re even b efo re fre e z in g .

An in cre a se d tendency of neutrophils to aggregate during sto ra g e and e sp e c ia lly at low te m p e ra tu re s made it n e c e s s a ry to add an activ e an ticoagu lant to the p ro tectiv e solution.

ED TA is even m o re n e c e s s a ry in the p ro tectiv e m edium during the fre e z in g of p latelets', s in ce th e se m ore re ad ily agglutinate than do le u co cy te s . B eca u se of the e la s tic ity of white c e lls and th e ir ab ility to co n tra c t and sw ell in an an isoton ic medium without form in g le s io n s , the question of the need fo r s p e c ia l trea tm en t fo r thaw ing leu co cy te s b e fo re tra n sfu sio n is decided upon is e a s ie r than in the c a s e of e ry th ro cy te s .

In the p ro c e s s of se a rch in g fo r the optim um thawing cond itions, nam ely, the se le c tio n of solutions activ e ly p ro tectin g the c e lls to g eth er with fre e z in g and thawing ra te s and ways of c e ll tre a tm e n t b e fo re tran sfu sio n , we used rapid m ethods of d eterm in ing the ce ll function e ffic ie n cy : su p rav ita l stain ing fo r leu co cy tes and the r e tr a c t i le activ ity e stim atio n fo r p la te le ts .

As the re s u lt of our stu d ies on leu co cy te s , th ree p ro tectiv e so lutions with d ifferen t c ry o p h ilactic agents a re p roposed : g ly cerin e and d im eth ylsulphoxide (DMSO) as en d o cellu lar agen ts, and p o lyv in y l-p irro lid o n e (P V P ), which has an e x tra c e llu la r e ffe ct. T h is solution is m ixed with leu co cy tes in equal volum es suspended in the p lasm a.

We used a program m ed cooling re g im e : 1 degC/min to -15° С and then 10degC /m in to - 1 9 6 ° C. T h is p ro g ram p e rm its a fa s te r cooling ra te , for exam ple 4 degC/min.

PROPERTIES OF STORED FROZEN CELLS 87

Thaw ing m u st be rap id . B e fo re the tran sfu sio n , with the aim of lo w e ring the high o sm o la r ity within the c e ll and w ithdraw ing the g re a te r p art of the p ro te c tiv e agent, thawed c e lls a re diluted by a ca rb o h y d ra te -sa lin e iso to n ic so lution . D uring the u se o f th is m ethod a su ffic ie n tly high p ercen tag e o f r e co v ered c e lls (70 - 90%) is attained even a fte r a prolonged s to ra g e of up to two y e a rs and m o re (F ig . 1).

£C3

оо

100

908070GO50ад3020100

GLYCEROL 15 V.

DMSO15'/.

П BEFORE LJ FREEZING

■ AFTER THAWING

Kg AFTER ADDITION “ OF WASHING SOLUTION

Я AFTER REPLACING “ IN PLASMA

PVP 20 V.

FIG. 1 . Survival of frozen and thawed leucocytes.

F o r the fre e z in g of p la te le ts only a 20% solution of DMSO in a 1% solution o f NaCl has proven to be u se fu l. T h is solution is m ixed with fre s h ly iso la ted p la te le ts and suspended in the p lasm a in equal v o lu m es. A fter the fre e z in g , the r e t r a c t i le activ ity d e c r e a s e s by 20-30% of the in itia l value and prolonged s to ra g e (up to two y e a rs ) ca u se s an in sig n ifican t change of that activ ity (by an additional 10%).

In the p ro c e s s of s e le c tin g optim um fre e z in g cond itions the in v e stig a to rs used d ifferen t m ethods of evaluation , w hich enabled the c e ll e ffic ie n cy in the m ain m a s s to be e sta b lish e d .

H ow ever, until re ce n tly no co m p ariso n of function with a d etailed study o f fro zen leu co cy te s and p la te le ts at the s u b -m ic ro s c o p ic le v e l has been conducted. The r e s u lts of our :study should th e re fo re m ake p o ssib le a b e tte r c h a ra c te r iz a tio n of the m orphologic and fu nctional e ffic ie n cy and in te g rity of the blood c e lls a fte r fre e z in g , and th ereb y help p e r fe c t m ethods fo r th e ir s to ra g e at low te m p e ra tu re s .

In th is re p o rt we sh a ll give only a v ery b r ie f d iscu ss io n of our r e s u lts .The question of the fre e z in g e ffe c t on the u ltra s tr u c tu re of leu co cy tes

is ra th e r an in tr ic a te one. One has to d eal sim u ltan eou sly with d ifferen t nu cleated c e l l ty p es, the su b -m ic ro s c o p ic organ ization of which is polym orphic and is su ffic ie n tly s p e c if ic fo r each c e ll ca te g o ry . T h is lead s to the follow ing b r ie f d iscu ssio n on the deep coo lin g e ffe c t on the su b -m ic ro s c o p ic s tru c tu re of white blood c e lls .

We w ere p r im a rily in te re s te d in the e ffe c t of the p ro te c tiv e agents on the fine c e ll s tru c tu re even b efo re fre e z in g .

We sh a ll f i r s t d iscu ss the e ffe c t of g ly cerin e and dim ethylsulphoxide. E ven at the beginning som e c e ll changes b eco m e evident. In g e n era l, the changes m ay be ch a ra c te r iz e d in the follow ing way. The sy stem of the c e ll m em brane s tru c tu re s is the m o st m odified one and th is is tru e of the cy to p la sm ic and n u cle a r m em b ran e and of in tra c e llu la r .m e m b ra n e s such as the

8 8 VINO GRAD-FINKEL et al.

end oplasm ic re ticu lu m . W ithin the c e lls many v acu oles appear and so m e tim e s th e se fu se with each o th er . Not in frequ en tly , e sp e c ia lly in lymphoid c e l l s , an in c re a s e in s iz e of the p e rin u c le a r zone tak es p la ce . A ll of the above in d ica tes the o sm o tic c h a r a c te r of the changes in th e se cond itions.

How ever, to g eth er with the com m on fe a tu re s re la t iv e to the e ffe c ts of the cry o p h y lactic agents on c e l ls , c e r ta in s p e c if ic changes a lso o c c u r . The DMSO e ffe c t is evidenced m o stly by sw ellin g of the end oplasm ic re ticu lu m and e sp e c ia lly of the c e ll s u r fa c e s , and c la sm a c y to s is and p in o cy to sis a re ob serv ed freq u en tly . The m itoch on d ria a lso sw ell to som e extent and the p e r in u c le a r sp ace in c r e a s e s .

The g ly cerin e e ffe c t on the end oplasm ic re ticu lu m is som ew hat le s s pronounced. A fe a tu re c h a r a c te r is t ic of th at agent is the ap p earan ce on the c e ll s tru c tu re s of som ething lik e a v e il. It is e sp e c ia lly evident in the n u clear s tru c tu r e s , w hich give the appearance of being smudged and lo s e th e ir c le a r - cut co n to u rs.

An ex fo lia tio n of m em b ran es of s p e c ia l gran u les and p a rtia l ly s is of cy to p lasm atic s tru c tu re s can be d em onstrated in eosin o p h ils .

The d iffe ren ce s in the e le c tro n m ic ro s c o p ic im age follow ing the u se of DMSO and g ly cerin e on leu co cy te s su ggest a som ew hat d ifferen t m ech an ism of e ffe c t by th e se two ag en ts. H ow ever, it is an accep ted view that the m ech an ism s of the p ro te ctiv e e ffe c t of g ly cerin e and DMSO on c e lls during fre e z in g a re id e n tica l. Both agents e a s ily p en etra te into the c e lls and both bind w ater, th ereb y p resu m ably preventing it fro m fre e z in g .

L a te ly , how ever, som e data have been accu m ulating thât in d ica te c e rta in d iffe re n ce s in the e ffe c ts of th e se ag en ts. It is thought that DMSO m o re e ffe c tiv e ly binds the c e ll w ater and p rev en ts its re c ry s ta lliz a t io n as w ell as fo rm atio n of the la rg e ic e c r y s ta ls . It i s a lso w ell-know n that DMSO p e n e tra te s through the c e ll m em b ran es m o re e a s ily than g ly cerin e .

Our findings allow us to assu m e that th e se agents p e n e tra te the c e ll by d ifferen t ways o r a re d istrib u ted in leu co cy te s in d ifferen t m an n ers ,DMSO ap p ears p r im a r ily to b lo ck the s tru c tu re s of end oplasm ic re ticu lu m , w hile g ly cerin e is d istrib u ted d iffu sely in the c e ll cy top lasm and nu cleus, h en ce a som ew hat d ifferen t p ro tectiv e e ffe c t r e s u lts .

F u r th e r , it was im p ortan t to d eterm in e the e ffe c t of the fre e z in g p ro c e s s on the in te g rity of the white c e ll s tru c tu r e . It has been shown that a fte r the fre e z in g of leu co cy te s with en d o ce llu lar p ro tectin g agents the su b - m ic ro s c o p ic s tru c tu re of m o st c e lls u n d ergoes sm a ll changes as com pared with the p r e -fre e z in g im ag e. T h is is c le a r ly d em onstrated by e le c tro n o g ra m s.

A leu co cy te a fte r fre e z in g with g ly cerin e has the sam e sm ooth nucleus s tru c tu re as b e fo re fre e z in g , while the end oplasm ic re ticu lu m p attern is not m arked .

In F ig . 2 the c e lls a fte r fre e z in g with DMSÓ a re shown. H ere a lso , as b e fo re fre e z in g , one can s e e an activ e c e ll m em b ran e and c la s m a c y to s is . F ig u re 3 shows a lym phocyte fro z en with DMSO w hich show s som e changes of the sa m e c h a ra c te r , but th e se a re le s s m arked .

The s ta te of the u ltra s tr u c tu re s of leu co cy te s fro z en with P V P , an e x tr a c e llu la r p ro tectiv e agent, has a lso been studied.

,-Here it should be f i r s t of a ll em phasized that tre a tm e n t (b efo re fre e z in g ) by P V P changes the s u b -m ic ro s c o p ic .c e ll im age le s s than do g ly cerin e and DM SO. Only a sligh t in c r e a s e of c e ll d en sity is o b serv ed ; som e P V P clo d s, c irc u m sc r ib e d by the m em b ran e and p o ssib ly phagocytized , a re seen .

PROPERTIES OF STORED FROZEN CELLS 89

F IG .2 . Electron microscopic characterization of granulocytes after DMSO treatm ent and freezing, (a) Before freezing; activation of citoplasm ic membrane and clasm acytosis. (b) After freezing; active citoplasm ic membranè phagosomes and insignificant changes in the sub-m icroscopic structure as compared with the pre- freezing im age.

A fter fre e z in g th e re is a v ery s lig h t in c re a s e in the p e rin u c le a r zone and an in sig n ifican t vacu oliza tion of the cy top lasm .

A fter fre e z in g , leu co cy te s a re b e tte r p re se rv e d when p re tre a te d with so lutions of DMSO and P V P than with g ly cerin e . H ere it should be m entioned that, although m o st leu co cy te s re m a in in tact a fte r thaw ing, som e c e lls (m ostly g ran u lo cy tes) do show ly s is of the cytop lasm and, m o re r a r e ly ,

90 VINOGRAD-FINKEL et al.

( a ) à -

FIG. 3 . Sub-microscopic structure of lymphocytes frozen with DMSO. (a) Lymphocytes after DMSO treatm ent (unfrozen); mitochondria are swollen; enlarged perinuclear zone, (b) Lymphocytes with DMSO after freezing. Insignificant changes in sub-m icroscopic structure as compared with the pre-freezing im age.

n u cle ar s tru c tu r e s . The m orphologic c e ll in te g rity is im p aired , but the p ercen tag e of such c e lls is not la rg e .

It should be noted that with a ll m ethods of c e ll p ro tectio n both m ito chondria and Golgi zones a re w ell co n serv ed . H ow ever, g ran u les, e sp e c ia lly th o se of eosin o p h ils , have a tendency to sw ell. The m axim um changes a re ob serv ed in cy to p lasm ic m em b ran es.

PROPERTIES OF STORED FROZEN CELLS 9 1

( c )

(a ) (b)

<S ' W ittfc j ä(d ) (e ) ( f ) .■

F IG .4 . Lymphocytes after freezing. Different stages of transformation in culture with PGA. Blast ce lls ((a), (b) and (d)) and mitosis ((c), (e) and (f)) are seen.

As to the m orp hologic in te g rity and function of d ifferen t c e ll types a fte r fre e z in g , it should be em phasized that of a ll c e ll types lym phocytes a re the b est p re se rv e d , the m onocytes the next b e s t p re se rv e d and the g ran u lo cy tes the le a s t p re se rv e d .

B e t te r re ten tio n of the m orp hologic in te g rity of lym phocytes a fte r f r e e z ing, as com pared with g ran u lo cy tes, was shown by m eans of lu m in escen t m icro sco p y with a crid ih e orange as a flu o ro ch ro m e. T h is m ethod p e rm its a sim u ltaneou s d eterm in ation of the num ber of n o n -v iab le c e lls and id e n tifica tion as to th e ir type. B y th is m ethod it was e sta b lish e d that of the to ta l of leu co cy te s d estroy ed , the g ran u lo cytes co m p rised the la r g e s t p art w h ereas lym phocytes co m p rised at m o st 10%.

Our findings on the in te g rity of the su b -m ic ro s c o p ic leu co cy te o rg a n iz a tio n a re co n sis te n t with the r e s u lts that we obtained w hile studying the ce lls ' fu nctional activ ity .

F i r s t of a ll, we d eterm ined the p re se rv a tio n of the g ly co ly tic activ ity of the leu co cy te m a ss as a w hole. It has been proved that, a fte r tre a tm e n t of c e lls by g ly cerin e o r DMSO, th e ir g lu co se -sp litt in g a b ility shows a m arked d e c r e a s e . The fre e z in g ca u se s a fu rth e r low erin g of the g ly co ly tic activ ity . N e v e rth e le ss , in the thawed c e l l s , that ab ility is co n serv ed at the 60 - 70% le v e l as com pared with the p rim itiv e , p re -fre e z in g value.

Sim ultaneou sly , individual e stim a tio n s w ere m ade of the in te g rity of fu nctional p ro p e rtie s in thawed g ran u lo cytes (phagocyte activ ity ) and ly m phocytes (the ab ility to produce young fo rm s in tis s u e cu ltu re ).

9 2 VINOGRAD-FINKEL et al.

FIG. 5 . Sub-m icroscopic structure of thrombocytes after DMSO treatment and freezing. No changes o f ultrastructure either before (a) or after (b) freezing. Only the appearance of numerous vacuoles can be demonstrated after freezing.

T h e se in v estig ation s a lso showed th at g ran u lo cy tes re ta in the p o o rest in te g rity , b ecau se a fte r fre e z in g only 50% of the c e lls , which had displayed phagocytic activ ity b e fo re fre e z in g re ta in ed that function in v itro .

At the sam e tim e , n e a rly a ll lym phocytes re ta in th e ir ab ility to t r a n s fo rm into b la s t c e lls (se e F ig . 4) in the p re se n ce of phytohaem agglutinin.

A fter 48 hours in cu ltu re ,la rg e c e lls of the b la s t type pred om inate .T h e se have ty p ica lly a la rg e nucleus and nu cleolus and lig h t-b lu e cy to p lasm . O ccasio n a lly c e lls in m y to sis can be see n . T h e se c h a r a c te r is t ic s i llu s tra te the v iab ility of lym phocytes p re serv e d in a fro z en s ta te .

D uring our in v estig atio n s on the u ltra s tru c tu re of p la te le ts , we e s ta b lish ed that the addition of g ly cerin e changes the s u b -m ic ro s c o p ic o rg a n iz a -

PROPERTIES OF STORED FROZEN CELLS 9 3

tion of p la te le ts even b efo re fre e z in g . Even an 8% co n cen tra tio n of g ly cerin e d estro y s p la te le ts b e fo re the e ffe c t of the deep fre e z in g o c c u rs . H yalom ers a re e sp e c ia lly v u ln erab le . Ruptured m e m b ra n e s , ab sen ce of g ran u les and m ytochond ria , and ap p earan ce of au tosom es m ay be ob serv ed in th e s e . The r e t r a c t i le activ ity is low ered by 40% . A fter fre e z in g , a ll s u b -m ic ro s c o p ic changes b eco m e even m o re m arked . The r e t r a c t i le a ctiv ity is lo w ered by up to 60% of the in itia l value.

DMSO does not im p air the p la te le t u ltra s tru c tu re e ith e r b e fo re or a fte r fre e z in g (F ig . 5). Only s lig h t changes can be d em on strated , w hich a re e x p re sse d in the ap p earan ce of num erous v a cu o les . The re tr a c t io n of the coagulum d e c r e a s e s slig h tly , but not by m o re than 20-30% .

In th is way e le c tro n m ic ro s c o p ic r e s u lts and o th ers have con firm ed that the b e s t p la te le t p ro tectin g agent is DMSO.

Our stu d ies in d ica te that p re se rv a tio n of the s u b -m ic ro s c o p ic o rg a n iz a tion and fu nctional a ctiv ity of leu co cy te s and p la te le ts s to red in a deep fro zen sta te is (in p rin cip le ) fe a s ib le .

At our in stitu te a bank of le u co cy te s and p la te le ts fo r tra n sfu sio n s has been e sta b lish e d . H ere the c e lls a re kept at u ltra -lo w te m p e ra tu re s (up to -196° C) fo r u se in the c lin ic [ 15, 16]. L o w -te m p e ra tu re equipm ent has been sp e c ia lly designed and co n stru cte d to provide fo r the fre e z in g and p ro longed sto rag e of the c e lls in liquid n itrogen .

In the bank we p re s e r v e :(1) L eu co cy te s iso la te d fro m the blood of healthy d onors.(2) The co n cen tra ted leu co cy te m a s s , taken fro m many donors of one

group by the m ethod developed at our in stitu te by N. N. C h ern eshova.(3) L eu co cy tes fro m p atien ts a ffected by ch ro n ic m y e lo le u co s is a re

c o lle c te d by p la sm a cy to p h o re s is . T h e se a re used only during tra n sfu s io n s fo r p atien ts affected by d iso rd e rs of the sam e group during the p eriod of h aem op oietic a p la s ia , which develops as a re s u lt of ch e m o - and rad ia tio n therap y.

Since the foundation of the bank about 2 thousand b illio n white c e lls of the p e rip h e ra l blood (2 X 10 12) have been s to red . M ost of th e se have a lread y been tra n sfu se d fo r c l in ic a l p u rp o ses. T ra n sfu s io n s of the thawed leu co cy te s , which have been sto red fo r a period of one month to two y e a rs , a re to le ra te d w ell by the p atien ts , and b rin g about the d isap p earan ce of fe v e r and an in c re a s e in the num ber of le u co cy te s .

R E F E R E N C E S

[ 1 ] SHAPIRO, Z .A ., C lin . Med. (Moscow) 4 (1950) 91.[2 ] BAGDASAROV, A .A ., VINOGRAD-FINKEL, F .R .. RODINA, R .I . , ILYHIN,, A .V .» Proc. Vlllth

Congr. europ. Soc. H aemat. (1961), Karger, Basel, New York (1962) 521.[3 ] FREIREICH, E . J . , MORSE, E .E :, J . clin . Invest. 41 (1962) 1359.[4 ] LEVIN, R .H ., WHANG, J., CARBONE, P .P . , FRÈIREICH, E . J . , Blood 26_ (1965) 587.[5 ] GOODDMAN, J .W ., HODGSON, C .S .,. Blood. 19 (1962) 702.[6 ] SCHWARZENBERG, L ., MATHE, G ., AMIEL, J . L . , CATTAN, A ., Am . J . Med. 43 2 (1967) 206.[7 ] EFRANI, P . , ROZENSZAJN.L., Blood 16 1 (1960) 1012.[8 ] RUBINI, I .R . , BOND, V .P . , KELLER, S ., J . Lab. c lin . M ed. 58 5 (1961) 751 .[9 ] KOSINETS, G . I . , ALPEROVICH, V .V . , CHERNESHOVA, N .N ., Questions of Blood Transfusion and

C lin ical M edicine3, Kirov (1965) 127.[1 0 ] KOSINETS, G .I . ."FAINSHTE1N, F . I . , ALPEROVICH, V .V . , CHERNESHOVA, N .N ., Xlth Congr.

in t. Soc. H aem at., Sydney, 1966, Abstract 108.

9 4 VIN OGRAD -FIN KEL et al.

[1 1 ] CHERNESHOVA, M .N ., ALPEROVICH, V .V ., Current Problems of Haematology and Blood Transfusion, Compiled Works 39 (1966) 373.

[1 2 ] ABEZGAUZ, N .N ., LEONTOVICH, V .A ., Probl. H aemat. Blood Transfus. 9 (1966) 24 .[1 3 ] SCOPINA, S .B . , KOROLUCK, K .I . , Probl. H aem at. Blood Transfus. 9 (1966) 30.[1 4 ] VINOGRAD-FINKEL, F .R ., KISELYOV, A ., LEONTOVICH, V .A ., ABEZGAUZ, N .N ., Proc. Xlth

Congr. int. Soc. Blood Transfus., Sydney, 1966; Bibl. haem at. 29 , part 3, Karger, Basel, New York (1968) 719.

[1 5 ] VINOGRAD-FINKEL, F .R ., LEONTOVICH, V .A ., ABEZGAUZ, N .N ., KRUTICOV, V .A ., 43rd Conf. Inst. Haemat. and Blood Transfus., Moscow, Abstr. 1 (1967) 54.

[1 6 ] CHERNESHOVA, N. N ., TOORBINA, N .S . , Proc. 43rd Conf. Inst. H aem at. and Blood Transfus,, Moscow, Abstr. II (1967) 378.

VIABILITY TESTS FOR FRESH AND s t o r e d h a e m o p o i e t i c c e l l s -

т . м. FLIEDNERAbteilung für klinische Physiologie,Zentrum für Klinische Grundlagenforschung,Universität Ulm,Ulm, Federal Republic of Germany

Abstract

VIABILITY TESTS FOR FRESH AND STORED HAEMOPOIETIC CELLS. This paper reviews current methods of measurement of the viability of fresh and stored haem opoietic ce lls . The life expectancy of granulocytes and monocytes after transfusion can be studied by in-vitro labelling with 3H-DFP and subsequent autoradiography. The evaluation of data in about 30 patients with various haem opoietic conditions indicates a wide variation of the disappearance half-tim e of granulocytes. 3H -cytidine labels essentially a ll lymphocytes in vitro, predominantly in their RNA. Transfusion of 3H -cytid ine-labelled lymphocytes enables one to measure the lower lim it of their life-expectan cy as well as their rate of RNA m etabolism . If bone-marrow cells are labelled in vitro with 3H-thymidine and subsequently transfused, their capability to circu late , to reach the haemopoietic tissue of the host, to proliferate and to mature can be demonstrated. However, the repopulating capacity of frozen and thawed marrow is independent of the ability of 3H -TD R-labelled marrow cells to circulate, proliferate and mature. It is assumed that bone-marrow cells capable of repopulating depleted haem opoietic tissue are resting under steady-state conditions and can be labelled by means of 3H-TDR only using special conditions. Thus the only viability tests for fresh and stored bone-marrow cells at present appear to be bioassay methods at the anim al experim ental lev e l. The results indicate the need for the development of reliable viabilitv tests for stem cells applicable in both experim ental and clin ica l conditions.

1. INTRODUCTION

The tran sfu sio n of b o n e-m arro w and blood c e lls has been found e ffe ctiv e in the tre a tm e n t of rad iatio n -in d u ced b o n e-m arro w fa ilu re . B on e- m arrow c e lls w ill r e s to r e the b lo o d -c e ll fo rm in g cap acity of rad iatio n d epleted m arrow if given in su ffic ie n t q u antities and i f th e re is a su ffic ien t d eg ree of t is su e com p atib ility [ 1 -4 ] . In exp erim en ts in dogs and m ice it has been found that the b lo o d -ce ll form in g cap acity of the m arrow can also be re s to re d i f blood leu co cy te s a re used , ind icating the p re se n ce of h aem op o ietic stem c e lls among the nu cleated c e lls of norm al blood in 'th e se sp e c ie s [5] . At p re sen t th e re is a lso co n sid erab le in te r e s t in the c lin ic a l use of granulocyte tra n sfu sio n , s in ce it has been shown that se v e re b a c te r ia l in fectio n s o ccu rrin g as a consequence of granu locytopenia induced by rad iatio n or other m eans can be su cce s s fu lly tre a te d i f su ffic ie n t numb e rs of g ranu locytes a re tra n sfu se d [ 6 - 8 ] . A nim al exp erim en ts have in d icated that the in cre a se d re s is ta n c e of the body re su ltin g fro m such a t r a n s fusion o ccu rs even if the num ber of c ircu la tin g c e lls cannot be kept at a high lev e l [ 9]. The use of p la te le t tra n sfu sio n has b eco m e a stand ard p ro cedu re in many h o sp ita ls as a m eans of tre a tin g throm bopénie bleeding.

* Research supported by the Association Contract "Haem atology" between the G esellschaft für Strahlenforschung mbH and the European Atomic Community (EURATOM).

95

96 FLIEDNER

It has been shown ex p erim en ta lly that b leed ing as a consequence of r a d ia tion-ind u ced throm bocytopenia can be 'tu rn e d o ff' a lm ost instantaneou sly i f a m e a su ra b le p la te le t le v e l in the blood can be achieved by p la te le t t r a n s fu sion . L yophilized p la te le ts , how ever, a re of no value [ 1 0 -1 2 ].

T h e se advances in the use of co rp u scu la r blood e le m e n ts in rep lacem en t and su bstitu tion th erap y of rad iatio n -in d u ced h aem op oietic fa ilu re w ill be of even g re a te r in te re s t if the developm ent of su itab le h isto co m p atib ility te s ts con tin u es, which c a lls fo r w id espread in tern a tio n a l co -o p e ra tio n . F u r th e rm o re , p ro g re ss in su itab le p re se rv a tio n and s to ra g e p ro ced u res fo r b o n e-m arro w stem c e l ls , blood leu co cy te s — re ta in in g e ith e r s te m -c e l l p o te n tia lit ie s , a n ti-b a c te r ia l and perhaps a n ti-v ira l p o ten tia ls , o r im m unolo g ic a l com p eten ce — and blood p la te le ts and, la s t but not le a s t , red c e lls w ill soon n e c e s s ita te the esta b lish m e n t of a chain of b lood- and b o ite-m arro w c e ll banks that a re in terco n n ected to exchange c e lls of a su itab le type throughout the w orld.

In th is context, the p roblem of su itab le te s ts fo r the v iab ility of c e lls subm itted to v ario u s p re se rv a tio n and s to ra g e p ro ced u res has to be con s id e re d . M ost of the in -v itro te s ts em ployed so fa r (dye e xclu sio n te s ts , te s tin g of D N A -syn th esiz in g cap ab ility , t is s u e cu ltu re te s ts , e tc . ) a re in adequate, s in ce th e re does not seem to be a d ire c t p ro p ortio n ality betw een the num ber of c e lls ' a liv e ' by th e se c r i t e r ia and, fo r in sta n ce , th e ir r e populating ab ility [ 13-16] .

It is thus the purpose of th is paper to in d icate how the l ife exp ectan cy of g ran u lo cy tes, m onocytes and lym phocytes can be m easu red by rad io activ e la b e llin g m ethods in the human being as a m eans to te s t the fe a s ib ility of p re se rv a tio n and sto rag e p ro ced u res . The m ethods em ployed w ill be s im ila r to th o se used in the d eterm in ation of r e d -c e l l and p la te le t su rv iv a l [ 17] by m ean s of c e ll la b e llin g with ch ro m iu m -5 1 . F u r th e rm o re , som e re a so n s fo r the d ifficu ltie s in c o r re la tin g in -v itro v iab ility te s ts of h aem op oietic s tem c e lls to th e ir d em onstrated in -v iv o p otential w ill be d iscu ssed to g e th e r with p ro m isin g te s ts su itab le fo r c l in ic a l u se .

2. L IF E E X P E C T A N C Y O F BLO O D L E U C O C Y T E S AS A SIGN O F V IA B IL IT Y

F o r s e v e r a l y e a rs the l ife exp ectan cy of red c e lls and of p la te le ts has b een co rre la te d to the d isap p earan ce h a lf-t im e of ch ro m iu m -5 1 -la b e lle d red c e lls or p la te le ts infused into a su itab le re c ip ie n t. T h e se te s ts have been su cce s s fu lly em ployed to in v estig ate p re se rv a tio n and sto rag e m ethods [ 18, 19].

F o r blood le u co cy te s , the f i r s t re lia b le v iab ility te s t of th is kind was p ro posed by Athens et a l. [ 20], . who used D F 32P -la b e llin g of blood leu co cy te s with th e ir subsequent tra n sfu sio n . The D F P (d iisopropylfluorophosphate) fo rm s an ir r e v e r s ib le bond with leu co cy te en zym es, y ield ing a d iiso p ro p y l- p hosp hate-enzym e m o lecu le . If 32P is used as a rad io activ e m a rk e r , the c e ll-ra d io a c tiv ity can be m easu red m o st re lia b ly by liq u id -sc in tilla tio n counting d ev ice s . It has been found in norm al human v o lu n teers that D F 32P l a b elled gran u lo cytes d isap p ear from the blood s tre a m a fte r in -v itro lab e llin g and subsequent iso tra n sfu sio n with a h a lf-tim e of about 7. 5 h ou rs. How ever, th is m eans of m easu rin g leu co cy te ra d io a ctiv ity in a liq u id -sc in tilla tio n cou n ter b eco m es u n reliab le i f the granu locyte le v e l is low, such as in leu co - pen ic con d ition s, o r d ifficu lt to in te rp re t if im m atu re c e lls a re p resen t

V IA B IL IT Y T E ST S 97

(s in ce n eu trop h ilic band fo rm s , m etam y elo cy tes and m y elo cy tes a re lab e lled as w ell). In a' s e r ie s of stu d ies in p atien ts with v ario u s h aem ato lo g ica l co n d ition s, tr it ia te d D F P (^ - D F P ) w as em ployed as a leu co cy te la b e l and the rad io activ e c e lls w ere d etected and evaluated in au torad iog ram s of leu co cy te co n cen tra te s m e a r s . - ;

When 500 m l of A C D -blood w ere incubated with 150 /jg ^ - D F P fo r 60 m inutes at room te m p e ra tu re , it was found that not only a ll g ran u lo cytes but a lso a ll m onocytes w ere la b é lled . Thus it appeared that th is method could be used to m e a su re the life -e x p e c ta n c y of both g ran u lo cy tes and m onocytes under v ario u s cond itions. The value of the m ethod was m o st c le a r ly d em onstrated in one patient with a c y c lic neu trop enia , in whom granu locyte and m onocyte su rv iv a l was m easu red during the phase of a d ep ressed and a n o rm al gran u lo cy te le v e l. D eta ils of th is study a re rep o rted e ls e w h e re .[ 21]. The blood leu co cy te changes are -d em o n stra ted in F ig . 1. It is c le a r th a t-th e re was a se v e re granu locytopenia about ev ery 25 - 27 days with a re tu rn to m o re n o rm al valu es in betw een. In c o n tra s t, the m onocyte le v e ls w ere h igher during the granulocytopenic phase than in the n o rm al o r n e a r n orm al granulocyte p h ases . The granu locytopenic p h ases w ere a sso c ia te d with fe v e r , but th e re was no evidence of b a c te r ia l in fectio n .

F IG .l . Total leu cocyte, granulocyte and monocyte count related to the body temperature in a.patient with cy c lic neutropenia.

D uring the granu locytopenic phase (re la p se ) and during the phase of m o re n orm al granu locyte num ber (re m is s io n ), 500 m l of blood w ere rem oved and incubated with 150 /ug 3H -D F P (0. 8 цCi/ml). The c e lls w ere then re in fu sed and leu co cy te co n cen tra te s m e a rs p rep ared at re g u la r in te rv a ls . In F ig . 2 the d isap p earan ce of 3H -D F P -la b e lle d g ran u lo cy tes is shown fo r the phases of re la p se and re m is s io n . D uring th e 's e v e r e granu locytopenic phase, a ' very sh o rt T ^ 2 of only a l it t le m o re than 2 hours was found,, while the Tj/2 of about 5 hours fo r the re m iss io n phase is c lo s e to n o rm al. In -F ig . 3, the d isap p earan ce h a lf-t im e of ^ I -D F P -la b e lle d m onocytes in the p h ases of granu locyte re la p se and r e m is s io n is d em on strated and in d ica tes a m arked

9 8 FLIEDNER

C V C U C N E U T R O P E N IA { P o t . H .K . )

FIG ,2 . Fate of 3H -D FP-labelled granulocytes after isotransfusion in the patient with cy c lic neutropenia through the phase of granulocyte remission and relapse.

C Y C L IC N E U TR O P E N IA (P a t. H .K )

D is a p p e a ra n c e o f ^ H -O F P la b e le d

HOURS AFTER R E IN F U S IO N

F IG .3 . Fate of 3H -D FP-labelled monocytes in the patient with cy c lic neutropenia, determined at the tim e of granulocyte remission and relapse.

difference between the period for which the granulocyte count is about normal (7 hours) and the period for which it is very low (14 hours). In Tables I and II the disappearance half-times of 3H-DFP-labelled granulocytes and monocytes are indicated under various conditions. The significance of these variations is discussed in detail elsewhere [22]. In the context of this paper it appears sufficient to indicate that the 3H-DFP. labelling of granulocytes and monocytes and their subsequent transfusion into human beings and autoradiogram evaluation of leucocyte concentrates is a suitable method to measure the life expectancy of these cells and thus provides a suitable means to test various preservation and storage procedures under clinical conditions. It remains to be seen how the life expectancy of these cells after transfusion can be correlated to their functional potentiality, such as increasing the resistance of the organism to bacterial invasion.

In the 3H-DFP labelling studies of blood leucocytes, no label was found in the blood lymphocytes. However, the fate of these cells after transfusion

VIABILITY TESTS 99

TABLE I. DISAPPEARANCE HALF-TIMES OF 3H-DFP-LABELLED GRANULOCYTES IN SEVEN INDIVIDUALS WITH DIFFERENT HAEMATOLOGICAL CONDITIONS

Patient

Granulocyte Disappearance (3HDFP)

Diagnosis (Haem. )

T i2(h)

Ig -I Neutrop. (Relapse) 2 .3

■ Ig .II Neutrop. (Rem iss.) 5

Eg. Neutropenia 5

Gii. Neutrop. (Splenom .) 8 .5

Wa. PCV 14. 5

Stö. PCV 2 3 .5

Ha. CML 56

TABLE II. DISAPPEARANCE HALF-TIMES OF 3H-DFP--LABELLEDMONOCYTES IN NINE INDIVIDUALS WITH DIFFERENTHAEMATOLOGICAL CONDITIONS

Monocyte Disappearance (3HDFP)

Patient Diagnosis T l(H aem .) (h)

Bo. normal 10

Kii. "norm al" (Li-C a) 1 4 .5

Gr. "norm al" (Lu-Ca) 9 .5

Ig-I Neutrop. (Rem iss.) 7

Ig .II Neutrop. (Relapse) 14

Ku. Neutrop. (Splenom. ) 5

Gii. Neutrop. (Splenom .) 1 0 .5

Stö. PCV 3 .5

Wa. PCV 10

can be studied if tritiated cytidine (3H-cyt. ) is employed as a cell label. It has been shown that the in-vitro incubation of blood of normal human beings as well as of patients with chronic lymphatic leukaemia with tritiated cytidine results in close to 100% labelling of all lymphocytes [ 23,24]. Details of these studies are given elsewhere [23]. Cytidine is incorporated into both DNA and RNA [23]. However, since the fraction of blood leucocytes capable of DNA synthesis at any one time is very small indeed (it has.been shown that there are normally about 5 mononuclear cells or lymphocytes per mm3 blood that incorporate 3H-thymidine and hence synthesize DNA), the incorporation of 3H-cytidine into lymphocytes is mainly the result of the continuous RNA synthesis of the lymphocytes, as can be shown by extraction

1 0 0 FLIEDNER

methods (with perchloric acid or RN-ase). Thus, if 3H-cytidine-labelled lymphocytes are transfused and their fate followed, the RNA-turnover characteristics of these cells are utilized. In Fig. 4, the fate of 3H-cytidine-labelled lymphocytes is demonstrated after isotransfusion into three patients with chronic lymphatic leukaemia. In all instances, 500 ml of blood were removed and incubated in vitro at room temperature with 500 /nCi/3H-cytidine for 45 minutes. This resulted in a labelling index of practically 100% of the lymphocytes. It has been shown that the cells do not loose their RNA-synthesizing capability while remaining at room temperature for 45 minutes or for several hours [23]. After isotransfusion, leucocyte concentrate sm ears are made at regular intervals and the fraction of labelled cells as well as their labelling intensity is determined with autoradiograms. An autoradiographic study of labelled lymphocytes is essential if the vitality of these cells is to be studied: the 3H-cytidine label in lymphocytes is not stable because of the RNA turnover of the cells. Thus, as has been described elsewhere [ 23, 24], the label disappears from circulating lymphocytes in a multi-component fashion, the half-times of three major components being of the order of minutes, hours and days. The disappearance of labelled lymphocytes is thus only in part a reflection of their life expectancy. It is mainly a reflection of their RNA turnover and gives only the lower limit of the lymphocyte life expectancy. However, for the viability testing of preservation and storage procedures, both aspects — the RNA turnover and the lower limit öf life expectancy in the circulating blood — may be of considerable interest but can be determined adequately only in autoradiographic studies. It will be of particular interest to investigate the relationship between lymphocyte RNA turnover and the immunological competence of these cells and the influence of preservation and storage on them. Furthermore, since it must be assumed that haematological stem cells are among the cells classified as lymphocyctes or at least as mononuclear cells in the blood stream, future research has to clarify whether the survival characteristics of ^-cytidine-labelled lymphocytes or of a particular fraction of mononuclear blood cells after auto- transfusion can be correlated in any way with to the repopulating ability of a blood leucocyte suspension.

DAYS A F T E R IS O T R A N S F U S IO N

( IN -V IT R O 3H -C Y T ID IN E LA B E LE D LYMPHOCYTES)

• P a t E . Ffct. к . P a l. a

FIG .4 . Fate of 3H -cytid ine-labelled lymphocytes in three patients with chronic lym phocytic leukaem ia after in-vitro labelling and subsequent isotransfusion.

VIABILITY TESTS 1 0 1

There is no doubt that the only reliable method at present of ascertaining the influence of preservation and storage procedures on the repopulating ability of a bone-marrow cell suspension is bioassay in the spleen-colony- induction method in the mouse [ 13]. As has been demonstrated by. several authors, there is no good correlation between the number of viable cells as' determined by the dye-exclusion test, the ^-thymidine incorporation into DNA or the in-vitro culture system and the repopulating ability of a bone- marrow cell suspension [13-16]. •

The difficulties involved in testing the repopulating ability of a bone- marrow cell suspension in human beings after storage at low temperatures were.demonstrated clearly a few years ago [25]., Patients with advanced neoplasia but no marked disturbance of haemopoiesis were given high doses of nitrogen mustard (0. 8 mg/kg body weight) that were felt to cause such a profound depression of the cell-forming ability of the bone marrow that only the administration of bone-marrow cells could help repopulate-the bone- marrow spaces. Between 300 and 500 ml of bone marrow were therefore collected, labelled with 3H-thymidine in vitro, mixed with glycerol and stored at a low temperature (-79° C). The administration of nitrogen mustard to the patients resulted in very severe leucocyte and platelet depression which reached dangerously low levels around day 10. It is known from whole-body irradiation that dramatic leucopenia and thrombopenia within 10 days of radiation exposure is indicative of such severe marrow damage that spontaneous marrow recovery cannot be expected for several weeks, if at all. Thus, since nitrogen mustard had been assumed to be ' radiomimetic' , it was felt that the stored isogenic marrow should be reinfused at the time of the most severe blood-cell depression. Some investigators have already reinfused such stored isogenic marrow shortly after nitrogen mustard treatment to allow a number of days for the marrow to become repopulated from the stem cells assumed to be among the stored cells [ 26-28]-. In the first four cases we studied [25], the stored marrow was thawed rapidly and reinfused. Since it was labelled with-tritiated thymidine, frequent blood samples were taken to look for the reinfused labelled cells. The careful autoradiographic study did not reveal good evidence for the circulation of the 3H-thymidine-labelled bone-marrow cells that had been kept for about 2 weeks in a deep-frozen state. However, a marked blood-cell recovery (leucocytes-and platelets) became evident a few days after bone-marrow transfusion as has been observed also by other investigators [26-28]. However, since we did not find labellëd cells after reinfusion of the labelled frozen and thawed marrow cell suspension and since blood-cell recovery was much faster and earlier than could be accounted for on the basis of cytokinetic data for human blood-cell formation and maturation, we had doubts about the causative relationship between marrow cell transfusion and blood-cell regeneration. Thus, in the next series of patients, the same dose of nitrogen mustard (0. 8 mg/kg body weight) and even higher doses (1 .2 mg/.kg body weight) were given but the isotransfusion of stored bone marrow withheld. Despite the fact that the marrow transfusion was withheld, the same rapid blood-cell regeneration was observed as in the cases with marrow transfusion. The conclusion was therefore reached that the isotransfusion of stored marrow was of little, if any, significance for marrow repopulation .after high doses of nitrogen

.mustard [25]. In subsequent animal studies it was shown that bone marrow,

3. V IA B IL IT Y T E S T S O F BO N E-M A R R O W C E L L S IN HUMAN BEIN GS

1 0 2 FLIEDNER

rendered aplastic with high doses of nitrogen mustard, is still capable of repopulating depleted marrow if transfused into a lethally irràdiated recipient [ 29].

The conclusion from these studies in large animals and human beings on the testing of bone-marrow cell viability after preservation and storage is that there is as yet no good model for it and that the spontaneous repopulation ability of the human marrow has to be carefully considered before an observed effect after marrow transfusion is correlated to it.

The present methods for testing the viability of bone-marrow cells by in-vivo or in-vitro labelling with tritiated thymidine and subsequent tran sfusion of fresh or frozen and thawed cells into lethally irradiated recipients have also been proven unsatisfactory.

In studies in dogs, isogenic bone-marrow transfusions were given after whole-body irradiation [30-32]. The effect on the leucocyte and platelet count is shown in Fig. 5(a) and (b). There is a very rapid fall of leucocytes and platelets, indicating an almost complete block of any new formation of granulocytes, lymphocytes or platelets compatible with a radiation dose of

FIG. 5 . . Leucocyte and platelet count changes in two dogs given 1200 R 60Co whole-body irradiation.(a) Pattern of b lo od -cell recovery after transfusion of fresh bone-marrow cells (isogenic).(b) Pattern o f b lo od -cell recovery after transfusion o f frozen and thawed (DMSO) bone-marrow cells (isogenic).

VIABILITY TESTS 10 3

1200 R, from which a spontaneous marrow recovery has never been observed. In one animal (836), marrow was removed shortly before radiation and la belled in vitro with 3H-thymidine. This marrow was kept at 4° С during radiation and then reinfused immediately thereafter. In a second animal (891), the marrow was removed several days before irradiation, labelled in vitro with tritiated thymidine, and stored in dimethylsulphoxide at -80° C. After whole-body irradiation, the frozen marrow was thawed rapidly to 37° С and isotransfused. In both animals (836 and 891), frequent blood samples were taken to follow the fate of the reinfused 3H-thymidine-labelled bone-marrow cells. Marrow samples were also taken at frequent intervals to investigate the pattern of marrow regeneration. In both animals, a nearly complete recovery of the number of leucocytes and platelets occurred. In animal 836, which received fresh 3H-thymidine-labelled bone-marrow cells, there was no difficulty in detecting many immature 3H-TDR-labelled marrow cells in the circulating blood shortly after marrow reinfusion (Fig. 6). Subsequent blood-cell studies indicated that these labelled cells must have undergone at least one division and then maturation: labelled mature granulocytes were found 1 - 2 days after bone-marrow reinfusion (Fig. 7). However, a very careful evaluation of the bone-marrow autoradiograms did not reveal any evidence that the obvious regeneration was associated with or resulted from 3H-TDR-labelled m arrow -cells. In dog 891, which received 3H-TDR- labelled frozen and thawed marrow cells, it was obvious that the reinfused thawed cells did circulate in the blood. Many were detected during the first few hours after reinfusion. However, all labelled and unlabelled immature bone-marrow cells disappeared from the blood within one day (Fig. 6). In contrast to dog 836, there was no evidence that reinfused labelled bone- marrow cells did divide or mature into segmented neutrophils (Fig. 7).Neither was there any evidence in this animal that the marrow regeneration was caused by labelled precursor, although it could have been argued that

104 ;

оо «__ . DOG 836 IFRESH M l

о— о DOG 891 (THAWED M.)m

о3

0 4 8 12 16 20 24

HOURS AFTER MARROW TRANSFUSION

FIG. 6 . Disappearance o f imm ature m yelocytic precursors from the blood after transfusion o f fresh or thawed marrow cells (isogenic).

1 0 4 FLIEDNER

their number would have been.too small to make the discovery of evidence for labelled ' stem cells1 úndergoing division, and thus dilution of the label, very likely.

7 .

100

80

0 A 8 . 12 16 20 24 28 32

HOURS AFTER MARROW TRANSFUSION .

0¡«—« LAB. E 5 AS V. OF LAS. E 0;x—* LAB. M 6 - M 8 AS % OF LAB. M

F IG .7 . - Appearance of labelled granulocytes and normoblasts after transfusion of in-vitro 3,H rTD R-labelled, fresh bone-marrow cells and after transfusion o f 3H -TD R-labelled , frozen and thawed bone-marrow ce lls .

. . . From these studies it was concluded that the process of freezing and thawing does not interfere with the repopulating ability of a marrow suspension but with the ability of 1 cells in cycle1 to undergo further division and maturation. It is .felt that this is why all 3H-thymidine .labelling methods have so far been unsatisfactory in testing the viability of stored bone-marrow cells.- The studies also.support the hypothesis that the repopulating capacity of a marrow cell suspension is associated with cells that are rarely, if ever, in a reproductive cycle under steady-state conditions but are 1 resting1 . Thus the only way of testing the viability of stem cells in vitro would be the development of a method to trigger resting stem cells'into proliferation and differentiation. This might be achieved if human marrow cells could be tested in a heterospecific bioassay system and if a suitable animal were found to be tolerant to human cells.

4. CONCLUDING REMARKS

A major task to ensure successful preservation and storage of haemopoietic cellular elements is the development of suitable tests for cell viability. From all studies performed so far it does not- appear to be sufficient to prove that a cell is ' alive' . Methods are essential to test the cells in their particular functional task. For granulocytes it may be sufficient to test their phagocytic and/or -lytic potentiality and their life expectancy after transfusion. The latter can be tested by labelling the cells with 3H-DFP, and the former by a standardized phagocytic test [33]. The same is true for monocytes that can also be labelled in vitro with 3H-DFP and their fate studied after transfusion. ' It may be possible to utilize this granulocyte and monocyte transfusion technique also as a test for the-histocompatibility of donor and recipient for bone-marrow transfusion or organ transplantation:

VIABILITY TESTS 1 0 5

one would expect that incompatible cells would have a markedly reduced life expectancy after transfusion. The life expectancy of lymphocytes and their RNA synthesizing capacity can be tested with the 3H-cytidine labelling technique and may be .a function of their own true viability. It remains to be determined whether the lymphocyte transfusion technique can also be .employed as an in-vivo histocompatibility test that is easier and faster than skin transplantation. Furthermore, it should also be determined whether lymphocyte transfusion can be employed therapeutically in the treatment of certain diseases of immunological incompetence and whether lymphocyte life characteristics (RNA turnover, life expectancy or disáppearance rate after transfusion) can be correlated to the marrow repopulating ability of blood'leucocytes. For testing bone-marrow cell viability or stem rcell potential, the bioassay method in small rodents appears to be the only reliable approach. An important task will be to utilize the same principle for human marrow if a suitable heterospecific system can be found.

R E F E R E N C E S

[1 ] URSO, P ., CONGDON, C .C . , Blood 12 (1957) 251 .[2 ] THOMAS, E .D ., COLLINS, J .A . , HERMAN, E .C . , J r . , FERREBEE, J .W . , Blood 19 (1962) 217 .[3 ] LOUTIT, J . F . , MICKLEM, H .S ., Tissue Grafting and Radiation, AIBS-Monograph, Academ ic Press.[4 ] VAN PUTTEN, L. M ., BALNER, H ., MÜLLER-BERAT, C .N ., DEVRIES, M .J . , VAN BEKKUM, D .W .,

"Progress in the treatm ent and prevention o f secondary disease after homologous bone marrow transplantation in monkeys. Effect of chemotherapy and of donor selection by histocom patibility testing, X I. Congr. int. Soc. Blood Transfus,, Sydney, Plenary Session (1966) 283 .

[5 ] CAVINS, J .A . , SCHEER, S . C . , THOMAS, E .D ., FERREBEE, J .W ., Blood 23 (1964) 38 .[6 ] CRONKITE, E .P ., BRECHER, G ., Ann. N. Y . Acad. Se i. 59 (1955) 815.[7 ] BRECHER, G ., WILBUR, K .M ., CRONKITE, E .P . , Proc. Soc. exp. B io l. Med. 84 (1953) 54.[8 ] FREIREICH, E . J . , LEVIN, R .H ., WHANG, J . , CARBONE, P .P ., BRONSON, .W ., MORSE, E .E .,

Ann. N. Y . Acad. S e i. 113 (1964) 1081.[9 ] CRONKITE, E .P ., BRECHER, G ., WILBUR, K .M ., M ilit. Surg. 111 (1954) 359.

[1 0 ] CRONKITE, E .P ., BRECHER, G ., Acta radiol. (Suppl. 116) (1954) 376.[1 1 ] FLIEDNER, T .M . , SORENSEN, D. K . , BOND, V .P . , CRONKITE, E .P ., JACKSON, D .P . , ADAMIK, E .,

Proc. Soc. exp. B io l. Med. 99 (1958) 731.[1 2 ] JACKSON, D .P . , SORENSEN, D .K ., CRONKITE, E .P ., BOND, V .P . , FLIEDNER, T .M . , J . c lin .

Invest. 38 (1959) 1689.[1 3 ] LEWIS, J . P . , TROBAUGH, F .E ., J r . , Ann. N. Y . Acad. Se i. 114 (1964) 677.[1 4 ] PYLE, H .M ., BOYER, H ., Ann. N. Y . Acad. S e i. 114 (1964) 686.[1 5 ] VAN PUTTEN, L .M ., Ann. N. Y . Acad. Se i. 114 (1964) 686.[1 6 ] LEWIS, J . P . , FARNES, M .P ., ALBALA, M ., TROBAUGH, F .E . , J r . , Ann. N. Y . Acad. S e i. 114

(1964) 701.[1 7 ] ODELL, T . T . , J r . , in Blood Platelets, Little Brown and C o., Boston, Mass. (1961).[1 8 ] COHEN, P ., GARDNER, F . , in Blood P latelets, Little Brown and C o., Boston, Mass. (1961).[1 9 ] COHEN, P ., GARDNER, F. H ., J . c lin . Invest. 41 (1962) 10.[2 0 ] ATHENS, J .W . , MAURER, A .M ., ASHENBRUCKER, H ., CARTWRIGHT, G .E ., WINTROBE, M .M .,

Blood 14 (1959) 303.[2 1 ] MEURET, K ., FLIEDNER, T .M . , in preparation,1968.[2 2 ] MEURET, K ., FLIEDNER, T .M . , SCHÜTZ, W ., JANSEN, J . , in preparation, 1968.[2 3 ] BREMER, K ., Inaugural Dissertation, University of Ulm , 1967.[2 4 ] FLIEDNER, T .M . , T he Lymphocyte in Immunology and Haemopoiesis, Edward Arnold (Pu bl.) L td .,

London (1967).[2 5 ] MEYER, L ., FLIEDNER, T .M . , CRONKITE, E .P ., Ann. N. Y . Acad. S e i. 114 (1964) 499.[2 6 ] KURNICK, N .B ., FEDER, B .H ., MONTANO, A ., GERDES, J . C . , NAKAMURA, R ., Ann. intern. Med.

51 (1959) 1204.

1 0 6 FLIEDNER

[2 7 ] McFARLAND, w . , GRANVILLE, N .в . , DAMESHEK, W ., Blood 1 4 (1 9 5 9 ) 503.[2 8 ] BLACK, M .M ., SPEER, F .D . , STONE, M. L . , Ann. intern. Med. 51 (1959) 517.[2 9 ] FLIEDNER, T .M . , THOMAS, E .D ., FACHE, I . , THOMAS, D ., CRONKITE, E .P ., "Pattern of re

generation o f nitrogen mustard treated marrow after transfusion into lethally irradiated homologous recipients", in La Greffe des C ellules Hématopoiétiques Allogêniques, Editions du Centre National de la Recherche Scientifique, 15 Quai A natole-France, Paris (1965).

[3 0 ] FLIEDNER, T . M ., THOMAS, E .D ., MEYER, L .M ., CRONKITE, E .P ., Ann. N .T . Acad. S e i. 114 (1964) 510.

[3 1 ] MANNICK, J .A . , LOCHTE, H .L ., ASHLEY, C .A ., THOMAS, E .D ., FERREBEE, J .W ., Blood 15 (1960) 255.

[3 2 ] CAVINS, J .A . , KASAKURA, S . , THOMAS, E .D ., FERREBEE, J .W . , Blood 20 (1962) 730.[3 3 ] ROWE, A .W ., COHEN, E ., XIXth Scien tific Meeting of the Protein Foundation.

PRESERVATION OF BONE-MARROW CELLS, LEUCOCYTES AND PLATELETS AT LOW TEMPERATURES A Review

M .J . A SH W O O D -SM ITH

M e d ic a l R ese a rch C o u n cil,

R a d io b io lo g ica l R esearch U n it,

H arw ell, Berks, U n ited K ingdom

Abstract

PRESERVATION OF BONE-MARROW CELLS, LEUCOCYTES AND PLATELETS AT LOW TEMPERATURES:A REVIEW. The basic principles of cryobiology are discussed and applications of these principles by numerous workers in attempts to preserve marrow ce lls , leucocytes and platelets at low temperatures are reviewed.It is concluded that:

(1) Lymphocytes from animals and man can be stored for long periods of tim e at low temperatures when cooled slowly in 10 -1 b°Jo DMSO or glycerol and thawed rapidly. Recovery figures are high and function is in tact and unaltered. DMSO is probably a better preservative than glycerol, and storage life at -196°C is probably indefinite for a ll practical purposes. Other leucocytes can be stored with sim ilar techniques but recoveries after freezing and thawing are probably lower than with lymphocytes.

(2) Bone-marrow ce lls o f several anim als including mouse, rabbit and dog can be preserved at -196°C , probably indefinitely, and sim ilar procedures to those used for lymphocytes give the best results. The com prehensive studies of Lewis and Trobaugh indicate that under carefully controlled conditions 9&7o of the stem cells in mouse marrow are viable after freezing and thawing. Opinions are divided over the efficacy o f the two preservatives, DMSO and glycerol, but in view of the w ell documented accounts of the lack of toxicity of glycerol it would seem advisable for the momenf to use this agent with a ll human marrow samples. The usefulness of PVP as a preservative for marrow is still to be resolved. Human marrow after freezing and thawing probably behaves in a similar manner to mouse marrow both in vitro and in vivo. However, it would be wise to consider that there might be differences which could cause wrong assessments of freezing procedures.

(3) Platelet preservation, clin ica lly perhaps the most useful procedure discussed in this review, is still to a large extent in the experim ental stage. Much work has been done but even the best methods available permit relatively low recoveries o f v iable p latelets. Preservation of human platelets in 12% glycerol associated with slow freezing and fast thawing and followed by very careful deglycerolization procedures yields a product with 23% of the original activ ity . This is clin ica lly acceptab le perhaps, but biologically it leaves much to be desired. Studies with rat platelets and combinations of various protective agents such as dextrose and DMSO and dextrose and dimethyl acetam ide are encouraging, but to xicity problems may prove difficult in human studies.

(4) The evidence from many different bacteria l and anim al c e ll studies indicates that -79°C is not low enough for long-term preservation. Temperatures below -1 3 0 °C should be used whenever possible and precautions taken to exclude radiation from natural and cosm ic sources when preservation in terms of years is considered.

1. INTRODUCTION

The clinical merits of transplanting bone marrow, leucocytes or platelets will not be discussed here. The major part of this review is devoted to a consideration of the efforts made to preserve haemopoietic cells and platelets at low temperatures. However, a broad description of the basic principles of low temperature biology or, as it is now called, cryobiology, would first be appropriate.

107

1 0 8 ASH W OO D -SM ITH

During the last few years the applications of cryogenics to biology and medicine have been considerable and varied. In many instances these applications have relied on empiricism and it is only now that a more scientific approach to cryobiology is evident.

Biologists and physicians who may wish to store cells for long periods must use low temperatures. Storage at temperatures just above 0°C is not practicable for long periods, even though most enzymatic reactions are considerably reduced. The life of a cell depends on the harmonious interplay of many different enzyme systems and not all of them respond in quite the same quantitative manner to a small change in temperature. Also, the ionic environment of a cell is dependent on the enzymatic pumping of ions in and out of the various sub-cellular components, so that when the temperature falls these processes cease, although normal diffusion processes are but little affected by the same relatively small temperature change. Thus there is a finite limit to the length of time that cells can be stored above 0°C. Fo r cells to be stored indefinitely they must be kept in the solid state at.temperatures well below zero.-

Water is by far the largest single component in a cell. Much of this water is not only acting as a solute for the innumerable cellular constituents and as a medium in which many chemical reactions take place but is also playing a very important part in the chemistry of the cell. The ability of water to form hydrogen bonds with other water molecules and with proteins is of great importance in biology. The subject of hydrogen bonds and the part played by wa'ter is a relatively new one in biology, but already many scientists are convinced that an understanding of hydrogen bonding is the key to many diverse biological phenomena.

To enable biologists to keep cells at temperatures low enough to preclude'chemical reactions, and thus the slow disorganization of the cell, consequences associated with the freezing of water must be minimized, as these and others dependent on them are the main reasons for the death of animal cells subjected to freezing and thawing. Water in the laboratory rarely freezes at 0°C, but usually supercools by at least five or six degrees. The presence of small amounts of salts and other solutes depresses the freezing point of most biological materials by about 0. 5°C.A suspension of red blood cells in a dilute salt solution, 'if cooled slowly, will supercool to about -6 °C and then freeze. The temperature will then rise, with the liberation of the latent heat of freezing, to the freezing point of the suspending fluid which will be - 0 . 5°C. Once this heat has been dissipated, the frozen sample will .continue to cool but not at the same rate as before, since the thermal conductivity of the frozen sample will differ from that of the unfrozen sample. There is little evidence that the release of heat causes any biological damage. The change in viscosity of water when frozen is again unlikely, per se, to be responsible for biological damage. ’ The actual formation of ice crystals has been suggested by several cryo- biologists to be a likely cause of cell death. The faster the rate of thawing the smaller the ice crystals formed. A very rapidly frozen sample of blood, cooled at a velocity of several hundredths of a degree per second, will have few if any ice crystals formed in it, and when thawed equally fast will still possess viable red blood cells. -However, with the possible exception of certain plant cells, most biologists do not regard the part played by ice-crystal formation inside or outside a cell as being a direct cause of cellular death during freezing and thawing.

PRESERVATION OF CELLS AT LOW TEMPERATURES 1 0 9

The most favoured idea, which also has the most experimental evidence to support.it, is that cellular injury associated with freezing and thawing concerns the destruction of lipoprotein membranes by high concentrations of salts which build up inside and outside cells after the initiation of ice formation and before the eutectic temperature of the salt is reached and the salt itself separates as a solid. Many biological fluids contain sodium chloride, the eutectic temperature of which is -2 1 .5 ° C . Much experimental evidence indicates that the critical temperature for mammalian cells is the region between - 2 еС and -3 0 °C . Cells progressing through this temperature range are subjected to high concentrations of various salts which can cause changes in protein and lipoprotein structures. Just as water nearly always supercools when it is cooled below its freezing point, so many inorganic solutes pass below their own eutectic temperature before complete phase separation occurs. In practice this means that the

F IG .l . Loss of viability of Escherichia co li B/r WP2 suspended in M/15 phosphate buffer (pH 7) during storage at low tem perature. (Courtesy of Dr. Bryn A. Bridges, unpublished observations.)

critical temperature range for most mammalian cells suspended in biological fluids often extends to temperatures lower than would be indicated by a knowledge of the eutectic temperatures of the various components in the system. It goes without saying that storage of biological materials in this temperature range, although perhaps offering longer life in terms, of . lower temperature coefficients for chemical reactions, is not to be re commended as it-can be equivalent to storage in brine solutions. Thus we have two concepts to concern us. We must in some way modify, limit or abolish the damage to cells associated with exposure during cooling to temperatures within the range of - 2 to - 30 or -4 0 ° С and, secondly, we must store our safely piloted cells at the lowest practical temperature.As an example of the effect of storage temperature on viability, the progressive decrease in the number of bacteria kept at various low temperatures is illustrated in Fig. 1. There are other reasons for this latter statement, which will not be dealt with here, but which relate to the migratory recrystallization of ice at sub-zero temperatures above -130°C .

Other changes which occur during the freezing of cells and tissues are changes in the solubility of gases and alterations in the concentrations of

1 1 0 ASH W OOD-SM ITH

various solutes as a result of their differing solubilities and eutectic temperatures. These changes can have very profound effects on a biological system, and two examples should be sufficient to illustrate this point. The hydrogen ion concentration of nearly all biological systems, whether they be individual cells, tissues, organs or fluids containing cells, is kept fairly constant by a number of different and, in some cases, very complicated buffer systems at around neutrality, pH 7 .4 . Deviations from this value by more than one pH unit usually result in cell death, and smaller changes than this can cause considerable alterations in the performance of an organ such as the heart or the kidney. Cells or organs which are to be frozen are usually surrounded or bathed in blood or artificial fluids which have been designed by biologists to contain molecules similar in type and kind to those normally required by cells and present in blood. The pH of these fluids is dependent on the buffering capacity of bicarbonate ions and various phosphate salts. As the cells and their bathing fluid are cooled, the solubility of the carbon dioxide increases until water freezes when the gas abruptly comes out of solution. The bicarbonate ion has a eutectic temperature of -1 .5 ° C and the two major components of the phosphate buffer system have differing eutectic temperatures. Thus the pH of the water remaining available as a solvent, before the final separation of the solute with the lowest eutectic temperature, varies in a complex and not always easily predictable manner.

The second example concerns the question of chemical reaction rates as a function of temperature. Most biochemical reactions double in rate for a 10 degC rise in temperature. This is a simple statement which is not necessarily true, as the final synthesis of a biochemically important compound may be the result of several linked reactions, each one of which may have different temperature characteristics. A fall in temperature during the early stages of cell cooling and freezing will result in a much lower rate of chemical reaction as far as the temperature coefficient is concerned. However, once more the importance of eutectic temperature has to be considered, since before this temperature is reached the number of molecules available for reaction in a given volume will be much higher and thus the mass action will be higher. In some cases it is possible that the reaction rate for the reaction being studied, i .e . the experimentally observed rate, may in fact be higher, even though the temperature is lower. An example of this can be borrowed from chemistry, although it is of considerable interest in biochemistry and radiation biology. This concerns the photochemical dimerization of thymine with ultra-violet light. This reaction takes place only very slowly at normal room temperatures when solutions of thymine are exposed to u .v . light at 2537Â. However, frozen solutions of thymine and other pyrimidine bases are very efficiently dimerized. It would appear that temperature per se in this instance is not responsible, but the necessary concomitant of the lower temperature, namely a solute (thymine) concentration during the separation of water molecule as ice. Thymine molecules are brought sufficiently and correctly close to one another for efficient dimerization to occur. This example is one of several recent ones from chemistry which would indicate that at low temperatures in the solid state not all is as quiet as the grave! There are other instances of chemical reaction being either more efficient at low temperatures or taking placé in a different manner. Hydrolysis of penicillin solutions occurs more readily in frozen solutions than in non-frozen

PRESERVATION OF CELLS A T LOW TEMPERATURES 1 1 1

solutions. There is also a suggestion that in some reactions ice may act as a catalyst. These thoughts should concern the biologists who think in term s of life eternal in suspended animation, or anabiosis, as it may not be possible to exclude the possibility of all chemical reactions at very low temperatures. Cosmic radiation and radiations from the small amounts of naturally occurring isotopes present in most materials must be considered a hazard to biological materials stored for long periods at low temperatures. Radiation damage will accumulate at a slow rate and will become manifest when the samples are thawed. Most cells have very complicated repair systems which operate on radiation-induced damage, but these systems are based on enzymes which would not function at low temperatures and which would probably be incapable of dealing effectively with all the accumulated damage when the cells are thawed.

Before 1949, attempts had been made to freeze blood and certain other cells very quickly in the hope that a rapid passage through the critical temperature zone would result in little or no damage. Blood, if frozen and thawed without special regard to freezing and thawing rates, is haemolysed. Luyet [1] and his colleagues in America were able to show that if thin films of blood on slides were immersed in liquid nitrogen at -196° С and then thawed rapidly a large percentage of the cells was undamaged. Attempts to use this technique with other cells have not been successful. Various modifications of this basic rapid-freeze and rapid- thaw method for red-cell preservation have been investigated. Blood can be injected through a fine nozzle directly into a rotating flask containing liquid nitrogen. When this is done the blood freezes very rapidly and forms small spherical beads of frozen blood which can be kept for many years at -196°C . When required these beads may be dropped into warm saline where they thaw rapidly and the blood returns to its normal, or almost normal, pre-frozen condition. This very simple and inexpensive method is used in many blood banks as a method for maintaining small quantities of many different bloods that can be used for blood typing. The frozen blood serves as a bank of reference antigens. This method cannot be adapted for freezing large quantities of blood for transfusion purposes. Sterility problems would be considerable if this particular method were to be upgraded for pint quantities of blood. Various very ingenious methods have been adopted to handle, under sterile conditions, large volumes of blood suitable for transfusion purposes with very rapid freezing methods and with the blood in specially designed containers. The need for frozen blood for the transfusion service is normally not great. However, wastage of transfusion blood is fairly high, as 14 to 21 days represents the maximum length of time which blood, suitably treated, can be kept at 4° С at one of the regional transfusion centres. After this period it cannot be used clinically. Stockpiling rare bloods and blood to cover holiday periods, a time when traffic accidents, especially in the United States of America, are often very high, is now a feature in several of the large American hospitals. The blood bank at the Massachusetts General Hospital in Boston, under the control of Dr. Charles Huggins, was one of the first to pioneer the use of frozen blood in clinical practice using a most ingenious freezing method, developed by Dr. Huggins [2]. The blood bank at the Chelsea Navy Hospital in Boston was perhaps the first large centre to use frozen blood which had been treated with glycerol [3]. Clinical use of such blood at this hospital did much to establish its safety and efficacy for trans

1 1 2 ASH W OOD-SMITH

fusion. The New York.Blood Centre has stocks of frozen blood and the ■ United States Services have stockpiled frozen blood for military use. Blood frozen according to Huggins' method or according to one of the various' methods'of rapid freezing with closed containers and developed by the Linde Corporation of America has been used in the treatment of military and civilian casualities in Viet-Nam.

Erythrocytes are exceptional in that ultra-rapid freezing and thawing procedures can be used to preserve them at low temperatures. Nearly all other mammalian cells do not survive this treatment. In 1949, Polge,Smith and Parkes [4] made the discovery that fowl spermatozoa, normally killed when frozen and thawed, could be shown to be motile after such procedures provided about 10 or 15% glycerol was added to the suspending medium. This observation was quickly followed by the demonstration that such sperm were fertile; and the application of the glycerol mèthod, as it became known, resulted in a revolution in the cattle-breeding industry: Numerous investigators in the United Kingdom; the United States of America and Australia were able to develop methods for*long-term preservation of bull spermatozoa at low temperatures and the subsequent use of this frozen semen for artificial insemination. This frozen semen could be shipped around the world, after long-term storage at -7 9 ° С or -196°C . This latter temperature, which involves liquid nitrogen, .has been shown to give better long-term preservation than .-79° С for nearly all types of cells that have so far been successfully frozen. ,

The freezing and thawing rates for the successful resurrection of sperm required careful control. The so-called two-stage slow freezing method followed by a rapid thaw, ¡which was first used by Dr. Audrey-Smith and ' her colleagues, was and still is the basis for most of the methods. Apparatus designed around these methods is produced commercially by several firm s. This is not the place to enter into detail, but briefly the method used for most animal sperm and many other cells consists- of adding either glycerol or dimethyl sulphoxide (DMSO) to the appropriate suspending medium to a final concentration of-about 10 or 15%. Before the freezing process is started, a certain time is allowed to elapse to allow penetration of the protective compound into the cell. The sample, sealed in a glass . ampoule-, is then cooled slowly at the rate of about 1 or 2 degC/min until ' a temperature reading in the sample indicates about -1 5 °C . The sample is then cooled a t.5 degC/min for the rest of the cooling-regimei Once a temperature of - 50 or - 60°C has been reached, the sample can be plunged directly into-liquid nitrogen. When the time has come for the sample to be thawed it is plunged into a water bath at 40° C, and when thawed the contents are used either directly for artificial insemination or the glycerol or DMSO are diluted before use. This is a very much simplified account of the procedures,; and the cells of different animals-may require different methods. Many types of sperm have been preserved at'low temperatures by these techniques. Human, fowl, bull, horse, -goat-and certain fish sperm are fertile and have been used for artificial insemination after freezing and thawing. However, the use of frozen animal semen outside the cattle-breeding industry has-at times met with prejudice. Mouse sperm and rabbit sperm have been much more difficult to :freeze. This is a great pity, as much biological research is conducted with these animals and central gene banks would be most helpful. The establishmentof banks of -human sperm for eugenic use has been mentioned-.as a possibility from


time to time and the long-term preservation of the spermatozoa of great men is quite practicable. Whether it is socially desirable is another matter!

The discovery of the glycerol method of sperm preservation was followed by attempts to apply the principle to. a wide variety of other animal cells and tissues. Much of this pioneering work was done at the National Institute for Medical Research, London, by Dr. Smith, the scientist associated with the original discovery of the protective effect of glycerol, and her colleagues. A series of papers published in the early 1950's clearly demonstrated that blood (red cells), cells grown in tissue culture, skin and many hormone-producing tissues such as the ovary, pituitary, testis and embryonic tissues could also be frozen, stored at and thawed from -7 9 ° С with suitable modifications of the glycerol technique [5].

How does glycerol prevent freezing damage? The simplest explanation suggests that glycerol acts as a protective agent by virtue of-its colligative properties. In the presence of glycerol, the amount of salts concentrating during the freezing process is less at any given temperature than in the absence of glycerol. Thus the denaturing effect of a high salt concentration is modified. This simple explanation, first suggested by Lovelock [6] in. 1953, has much to recommend it. Thus it follows that any low molecular weight, non-toxic chemical which can readily pass into and out of cells should possess protective properties. Many compounds have been found that have limited protective properties, but only glycerol and DMSO are in general use. Other explanations to account for the action of glycerol and similar compounds concern the known facts that these compounds alter the form of the ice crystals which form during freezing processes and that they also interfere with nucléation of intracellular water by extracellular ice.

This short introduction to some of the basic ideas of cryobiology will, it is hoped, help toward understanding the peculiar problems of leucocyte, platelet and bone-marrow cell preservation which are now to be considered.

2. THE LOW-TEMPERATURE PRESERVATION OF LEUCOCYTES

Studies on leucocyte preservation have in some instances failed to differentiate between lymphocytes, granulocytes and circulating blood stem cells. The assay used by authors to assess viability should be sufficient, perhaps, to indicate the nature of the cell type in question.

Studies on bone-marrow preservation had indicated that the lymphoid tissue of irradiated animals injected with frozen bone marrow recovered, and this and other evidence suggested that lymphocytes present in bone marrow had survived freezing and thawing in the presence of suitable protective agents. Several reports concerning the preservation of animal and human lymphocytes have provided ample evidence of the effectiveness of properly controlled freezing procedures. Atkins [7] preserved human leucocytes suspended in Parker '199.' with 15% glycerol at - 8 0 ° С using a slow freezing and rapid thawing procedure. After thawing, 35% glucose was added to balance the osmotic pressure inside the cells during the subsequent dilution process. Culture of the preserved cells indicated normal karyotypes after storage at - 8 0 ° С for two months. Atkins' results demonstrated better preservation with slow freezing than with fást freezing. The number of cells recovered was less after two months than after 24 hours


of storage at -80°C ; a loss of nearly 50% was recorded over this period. Ashwood-Smith [8] demonstrated that mouse lymph node lymphocytes suspended in Parker 4 99’ tissue culture fluid and with added serum were viable after slow freezing and rapid thawing in the presence of 15% DMSO. Under optimal conditions, frozen and thawed lymphocytes were capable in vitro of jjrotein synthesis rates approaching nearly 50% of the rates of unfrozen lymphocytes (Fig .2). The thawed cells showed normal motility when cultured for short periods on specially prepared agar slides. When these lymph node lymphocytes ('A' strain) were injected into sub-lethally X-irradiated LAFi mice, splenomegaly developed (Fig .3). Cells which had been frozen and thawed without added protective agent caused no splenomegaly. Cells frozen with glycerol had some biological activity, but the extent of the splenomegaly seen after the injection of cells frozen with DMSO was nearly as great as that seen with the same dose of fresh cells. Lymphocytes treated with 10 or 15% DMSO and kept at - 165°С for three months after slow freezing were tested and it was concluded on the basis of the splenomegaly in injected animals that at least 50% of the cells responsible for causing a graft-versus-host reaction were viable after low- temperature storage (Fig. 4).

PER CENT CONCENTRATION OF D IM E TH Y L SULFOXIDE ( v /v )

F IG .2 . Protein synthesis of fresh and frozen mouse lymphocytes (incorporation of DL - valine - 4 - 14С into proteins during in-vitro incubation;' 1 hour at 37°C ).

Symes [9 -1 1 ] and his colleagues in Bristol, United Kingdom, have developed a simple and reliable method for the preservation of human spleen cells. The use of spleen cells for the immunological treatment of tumours, for stem -cell replacement in patients treated with immunosuppressive drugs and for the production of anti-lymphocyte serum were factors which initially caused the Bristol group to investigate preservation techniques for small and medium lymphocytes. Carefully prepared suspensions of spleen cells were suspended in Parker '199' tissue-culture solution to which had been added 5% DMSO and 15% AB serum and cooled at a rate of 1 degC per minute to -40°C when the aluminium ampoule was removed and placed in liquid nitrogen. When required for use the ampoules were thawed by immersion in saline at 37° С and the cells after centrifuging gently resuspended in fresh tissue-culture fluid containing 25% AB, R+ serum. Symes


feels that this high concentration of serum stops the cells from being damaged during the removal of the DMSO. This suspension of cells is then ready for injection. Tests in vitro on frozen and thawed cells indicated a high recovery. Nearly 80% of the small lymphocytes in the spleen-cell suspension responded to stimulation with PHA and after freezing and thawing and removal of DMSO about 7 0% of the lymphocytes could be transformed.

n o ' * 1 FRESHC E L LS FROZEN A N D TH A W E D C E L LS CELLS

C E L L S FROZEN AND THAW ED C E L LS CELLS

F IG .3 . Spleen and lymph-node weights in sub-lethaUy X-irradiated LAF2 (fem ale) m ice in jected with fresh and frozen lymphocytes ( ’ A ’ m ice , fem ale); 5 . 4 x lO 6 ce lls per mouse.

The Bristol group estimated that about 30% of the nucleated cells were lost during the freeze-thaw cycle, but the survivors were normal in every aspect of their physiology that was tested. Microdensitometry of frozen and thawed cells indicated no change in DNA content and upon subsequent culturing no changes were seen in chromosome number. Frozen and thawed cells showed no lag in respect of DNA synthesis when the uptake of tritiated thymidine was measured. Freezing and thawing spleen cells without DMSO

1 1 6 ASH WOOD "S M IT H

resulted in nearly 100% of the cells being destroyed. Different concentrations of DMSO and differing freezing and thawing rates were less good than the regime of using 5% DMSO with slow freezing and rapid thawing. Symes et al. are now using this method successfully to preserve human peripheral blood lymphocytes. The lymphocytes are first separated from

3 0 0

N 0 FRESH PRESERVED CELLS C ELLS CELLS 7.6 x 1 0 s

N 0 FRESH PRESERVED CELLS CELLS CELLS 7 6 * 1 0 *

F IG .4 . Spleen and lymph-node weights in sub-lethally X-irradiated LAFt (fem ale) m ice in jected with fresh lymphocytes and lymphocytes ( ’A'-strain fem ale) which had been stored for 3 months at ,-196°C in the presence of dimethyl sulphoxide.

red cells, platelets and granulocytes by filtration through a long glass column containing cotton wool. Pegg [12] has described a method for obtaining, separating and preserving Human peripheral blood leucocytes. The suspending medium was Parker 4 99' tissue-culture fluid with 10% autologous serum and DMSO at a final concentration of 10%. Ampoules containing cells were cooled to -8 0 ° С at .a rate of about 1 degC/min and then transferred to a liquid nitrogen refrigerator. Cell counts after the cells


had been at - 196° С for 24 hours and then rapidly thawed indicated a mean loss of about 10%. Storage over a three-month period increased this loss by about 5%. The staining reactions of the various cell types were normal after low-temperature storage, although there was some reduction in the staining reaction for leucocyte alkaline phosphotase. Comparison of samples before and after three.months' storage, when the samples were stained with PAS, peroxidase-and Sudan Black B, showed no detectable change. Similar studies for non-specific esterases and lactic dehydrogenase made before and after eight weeks' storage showed no obvious change.Other tests on frozen cells, such as supravital staining with acridine orange and motility studies, indicated normal response; Lymphocytes also responded to treatment with PHA and the mitotic index was unchanged by the freeze-thaw procedures. Cultures of lymphocytes with abnormal karyotype were the same after freezing as before. Pegg was satisfied that in addition to normal mixed leucocytes and lymphocytes he was able to preserve, without change, cells from patients with the following ..conditions: chronic myeloid and lymphatic leukaemia, monocytic leukaemia, polycythaemia rubra vera, myelofibrosis, Kleinfelter's syndrome and Downs' syndrome. Davies, Coulson and Smith [13] have obtained transformation of peripheral blood lymphocytes after slow freezing in the presence of 10% DMSO added directly to the cell suspension without prior dilution. Playfair and Davies [14] have demonstrated that mouse neonatal thymus can be successfully preserved in 10% DMSO by slow freezing and rapid thawing.Storage was for two weeks at - 7 9 ° С and the viability of the frozen thymus tissue was assessed by grafting procedures.

A recent study by Bouroncle [15] included information on the toxicity of DMSO to various leucocytes at various temperatures above zero. According to Bouroncle it is best to add DMSO immediately before commencing the freezing procedures. This advice is contrary to that of several other observers, who claim that a necessary prerequisite to s u c c e s s f u l freezing is a short but definite time of incubation with DMSO to allow it to penetrate. Bouroncle concludes that with slow freezing and fast thawing most normal and leukaemic leucocytes are best preserved when in the presence of 12.5% DMSO. Viability in these studies was assessed by cell culture methods and the uptake of tritiated thymidine as an indication of DNA synthesis. The more mature cells, such as granulocytes and monocytes, were less well preserved than the more primitive blast and reticulum cells.

Human granulocytes have been successfully preserved at low temperatures by Cavins, Djerassi, Roy and Klein [16]. Human buffy coat from normal donors was used in this study of freezing in plasma containing varying amounts of DMSO and dextrose. Samples were cooled at the rate of 1 degC/min within the critical range 0° to -30°C and then at a rate of about 20 degC/min down to -195°C . The period of storage varied from one to several days and then the cell samples were thawed by immersion in a water bath at 37° C.. After removal of the DMSO to below 2% by slow dilution, the ability of the granulocytes to phagocytose polystyrene particles was determined and compared with unfrozen control cells.Cavins et al. concluded from these studies that preservation of granulocytes in 10% or 15% DMSO resulted in cells with appreciable phagocytic ability. However, it is not absolutely clear from their results how well the phago-

1 1 8 ASH WOOD “SM ITH

cvtic ability was preserved after freezing when compared with the same sample before freezing.

Rowe, Kaczmarek, and Cohen [17] studied the preservation of granulocytes with the idea of using such preparations for the treatment of septicaemia in leukaemic patients. Rabbit and human leucocytes were frozen in 10% to 15% DMSO at a controlled rate of 1 degC/min and stored at -196°C . Polystyrene latex particles were used to test phagocytic activity. Frozen rabbit granulocytes thawed rapidly at 37°C possessed respiration rates of 50 -60% of the control values for unfrozen cells; phagocytosis was also evident. When leucocytes from patients with chronic lymphocytic leukaemia were frozen according to the same regime, respiration rates of thawed cells approached 90% of the control values. These frozen and thawed cells were agglutinated by the sera of patients demonstrated to contain leucoagglutins.

An extensive study on the preservation of leukaemic white cells and especially chronic myelogenous leukaemic cells has recently been undertaken by Shohet and Mohler [18]. These authors, who also critically re viewed the literature on the subject of leucocyte preservation, came to the conclusion that DMSO at a concentration of about 8% was considerably better than glycerol as a preservative. Macromolecules such as PVP were also investigated but were considered inferior as cryoprotective agents. Tritiated thymidine incorporation into cellular DNA was used to judge the efficacy of the preservation regime, and, in accord with most other work on the subject, slow freezing and rapid thawing of the cell samples was advocated. Overall recovery figures of between 50 and 60% were obtained by Shohert and Mohler and they consider that cell loss in the overall preservation process occurs primarily in handling steps after freezing and thawing and is "apparently due to latent damage induced in the freezing process, which renders the cell more susceptible to subsequent mechanical traum a". Albright, Makinodan and Mazur [19] made a careful study of the low-temperature preservation of antibody-producing (haemagglutinating antibodies) spleen cells from preimmunized mice. The method of choice consisted of suspending spleen cells in Tyrodes solution with added DMSO (10%). Th'e temperature was decreased slowly to -50°C and followed by storage at -196°C . After rapid thawing the antibody-producing ability of the cells was unimpaired. Glycerol was far less effective as a protective agent (Fig. 5). Prolonged storage of cells in DMSO at - 196°C resulted in no loss of capacity to produce antibodies (Table I).

Cavins, Scheer, Thomas and Ferrebee [20] have reported on the preservation of autologous dog leucocytes preserved at -80°C in 10% DMSO and stored for various lengths of time at -8 0 °C . The ability of these peripheral blood leucocytes to prevent radiation death in mongrel dogs was assessed and at the same time the return of the peripheral white-cell and platelet counts to normal levels was measured. It is not possible to obtain much quantitative information on the comparative effects of fresh and frozen leucocytes from this publication, but Cavins et al. state that the frozen leucocyte infusions (2Х10Ю cells) took nearly three weeks to produce a return to normal blood leucocyte and platelet values compared with the same dose of bone-marrow cells. Perry, Malinin, Kerby and Dolan [21] have investigated the protection afforded to lethally X-irradiated guinea pigs treated with fresh and frozen allogenic peripheral blood leucocytes. These authors suspended separated peripheral blood leucocytes in saline


containing 6% bovine, fibrinogen and 15% DMSO. The cells were cooled at 1 degC/ min to - 30°С and then at a rate of 10 degC to - 100°C or -150°C ; the cells were then stored at -150°C until used. Guinea pigs were irradiated with 550 rad (LD9g, 30 days). Frozen cell samples were thawed rapidly by immersing the ampoules in a water bath at 40° С and injected into the irradiated animals without removal of the DMSO. Results of these experiments demonstrated that 68% of animals injected with 1X108 fresh allogenic leucocytes survived 30 days. Frozen leucocytes (1X109) protected 83% of the small number of guinea pigs irradiated.

FIG. 5 . Antibody production of spleen ce lls frozen by decreasing temperature at 1 degC/min to -5 0 2С fo l- lowed by storage for 1 hour at -1 9 6 °C . Cells suspended in varying concentrations of either glycerol or dimethyl suiphoxide. Results based on 3 ex p er im en ts utilizing 10 or 20 recipient m ice per point. 95% confidence lim its shown. (From Albright, J . F . , Makinodan, T . , Mazur, P ., Proc. Soc. exp . B iol. Med, 114 (1963)489.)

TABLE I. ANTIBODY PRODUCTION OF SPLEEN CELLS STORED FOR PROLONGED PERIODS IN 10% DMSO AT - 196°C

T im e of StorageMean titre

Clog)No. of samples

0 1 1 .3 15

1 h 1 1 .2 10

1 d 1 1 .2 10

4 d 1 0 .9 11

8 d 1 1 .4 11

135 d 1 1 .6 15

(Table from Albright, J . F . , Makinodan, T . , Mazur, P ., Proc. Soc. exp. M ed. 114 (1963) 489,


Goodman [22] has established that frozen mouse leucocytes stored at - 196° Cj after freezing in 15% glycerol (cooling rate 1 degC/ min to - 25°С followed by liquid nitrogen; rapid thawing was achieved by immersion of ampoules in water at 37°C) are viable. Preservation of immunological competence was demonstrated by injecting frozen or fresh cells into mice treated with allogenic marrow cells. Mice thus treated died from rejection of the allogenic graft. If the leucocytes were given 2000 rad of X -rays no rejections took place. The frozen leucocytes also possessed erythropoietic function as. judged by 59Fe uptake in irradiated mice treated with frozen leucocytes.

3. PLATELET PRESERVATION

Morrison [23] has summarized the reasons for blood platelet preservation as follows:

"(1) Satisfactory treatment for haemorrhage due to most types of thrombocytopenia requires transfusion of viable platelets in adequate number;

(2) The useful shelf life of platelets at 4°C is very short;(3) The routine blood bank has great difficulty in responding to the

intermittent, usually unforeseen, and urgent call for platelets;(4) There are enormous quantities of platelets being wasted in routine

ly banked blood;(5) Treatment of disease with myelotoxic agents is severely hampered

by the development of thrombocytopenia unless supportive platelet therapy is available; ’

(6) The concept of specific blood component therapy is gaining general acceptance.

(7) The succèss with cryopreservation of red cells has been an encouraging experience."

Problems of platelet preservation have received much less attention than those of blood and bone-marrow preservation. Many of the earlier attempts to preserve animal and human platelets at low temperatures were unsuccessful. Baldini, Costea and Dameshek [24] froze human platelets slowly in the presence of 9% glycerol. When thawed, 80% of the platelets were destroyed and the authors suggested that glycerol per se had some toxic effect even without freezing. Cat platelets were studied by Cohen and Gardner [25]. Platelets suspended in glycerol and plasma and frozen slowly lost 50% of their original activity. A series of additives including glycerol, DMSO, dextrose, sucrose, gelatin, PVP and albumin were investigated as protective agents for rat platelets by Djerassi, Färber and Roy [26]. A combination of 5% dextrose and 5% DMSO in plasma resulted in preservation of platelet integrity. Platelets stored at -195°C for periods ranging from one to four weeks when infused into rats circulated with an efficiency as high as 8 9%. Platelet counts in rats 24 hours after infusion were higher than the pretreatment levels. Abnormally prolonged bleeding times of the animals were reduced to normal values following the transfusion of platelet preparations. lossifides, Geisler, Eichman and


Tocantins [27] showed that 15% DMSO in the presence of plasma preserved the clot retraction action of frozen and thawed human platelets; cooling rates in these experiments were as high as 10 degC/min.

PLATELET COUNT

XIOJ

1000-

9 0 0

8 0 0

7 0 0 -

6 0 0 -

5 0 0 -

4 0 0 -

3 0 0 -

200-11

10'

rO'---------------------------1-----------------------1------------------------------ 1-------------------- 1--------------------------------1----------------------1 I [— i

I 2 3 4 5 6 24

TIME (HOURS) AFTER INFUSION

F IG .6 . In-vivo circulation of platelets frozen to -1 9 5 eC in 5% dextrose and b°]o DMSO. Duration of storage D, F, G - 1 week; A - 2 weeks, B, C , E - 3 weeks; H - 4 weeks. (From Djerassi, I . , Roy, A . , Blood 22 (1963) 7 0 3 .) i _

A comprehensive study of the best conditions for the successful low- temperature preservations of rat platelets has been made by Djerassi and Roy [28]. These authors used rats made thrombocytopenic by whole-body exposure to 700 rad of X -rays for in-vivo tests of platelet viability. Initial experiments were made to determine platelet morphology after freezing and thawing in the presence of additives and circulation studies in vivo were made only on preparations showing 90-100% morphologically preserved platelets. Fast cooling rates of 300 degC/min and 84 degC/min were used4 and the best results were obtained (70 -80% survival) with 5% DMSO and 5% dextrose in plasma. Results of experiments in which platelets had been preserved at - 195°C for various periods can be seen in Fig. 6. Studies of the bleeding time before and after infusion of preserved platelets suggested that some of the haemostatic functions of platelets were also pre

MEAN OF FRESH PLATELET EXPT'S

STAND DEV. OF FRESH PLATELET EXPT'S

5 % OH.S. ♦ 3% DEXTROSE

1 2 2 ASH WOOD-SMITH

served and when the circulating platelet levels increased to more than 300 000/mm3 , bleeding times returned to normal. Perhaps even better results might have been obtained by Djerassi and Roy if they had used much slower freezing rates. Weinherth and Huggins [29] have studied the procedures used for the collection of human platelets which, they insist, are a necessary prerequisite for freezing procedures designed for long-term preservation of platelets at low temperatures. Differences in collection technique can, according to Weinherth and Huggins, result in variable degrees of aggregation and contamination of platelets with leucocytes. Aggregation would reduce the effectiveness of platelets in vivo for transfusion.The presence of lymphocytes in an infusion can sensitize the recipient to leucocyte and platelet antigens. Several different methods oí platelet concentration were studied and the absolute yield of platelets, degree of aggregation, and the effectiveness of erythrocyte and leucocyte separation were used to judge efficacy. The most efficient method for platelet concentrations when these criteria were used for assessment was one based on the collection of 500 ml of blood in 60 ml of ACD-A with 15 ml of ACD-A added to the platelet-rich plasma. Upon subsequent centrifugation, a platelet re covery figure of 1011 platelets per unit of blood was achieved with a leucocyte separation efficiency of 99. 9%. The platelets were normal in morphology and were not aggregated. Djerassi, Roy, Färber, Cavins and Klein [30] have recently studied a variety of protective agents with rat platelets because of the relatively disappointing results obtained when human platelets preserved with DMSO and glycerol were used clinically. Infusion of frozen platelets gave only 30 -50% of the increase in circulating blood platelets seen after transfusion of the same number of fresh platelets. These authors cooled rat platelets at 1 degC/min after the addition of various concentrations of DMSO, dimethyl acetamide, glycerol or propylene glycol, alone or combined with dextrose. The samples were stored at - 196°C for 5 to 14 days and when thawed (the rate of thawing is not mentioned) platelets w e r e in f u s e d into rats m a d e t h r o m b o c y t o p e n i c with X-irradiation. The presence of 5% dimethyl acetamide plus 5% dextrose proved to be better than 5% DMSO plus 5% dextrose in its ability to protect rat platelets from the effects associated with cooling to, storage at and thawing from -196°C . Propylene glycol and glycerol were not considered by Djerassi et al. to be very promising cryophylactic agents for platelets.

Cohen and Gardner [31] have made a detailed study of the preservation of human platelet concentrates by controlled slow freezing in glycerol media. Blood platelets were separated from whole blood by centrifugation but the ACD solutions employed during these procedures had been specially selected [32]. Platelet-rich plasma was labelled with 51Cr of high specific activity. The effect of varying concentrations of glycerol on platelets was studied before other experiments were undertaken. These experiments demonstrated that glycerol did not penetrate all human platelets with equal facility. Perhaps there are different populations of circulating platelets, and the authors suggest that at least three morphological types predominate with respect to glycerol penetration. When 51 Cr-labelled platelets were exposed to glycerol concentrations between 2 and 4%, no damage was done. With higher concentrations of glycerol more damage to platelets was evident. If, however, partial deglycerolization and osmotic buffering were carried out before transfusion and after exposure of platelets in vitro to glycerol (10-12% ), then 50% yields of platelets were obtained and these had normal


life-spans when transfused. The yields of viable platelets diminished progressively when samples were exposed to 14-24% glycerol, despite deglycerolization and osmotic buffering. The most effective range of glycerol concentration to achieve preservation of erythrocytes is perhaps outside the range tolerated by the platelet. When the maximum concentration of glycerol (12%) that permitted normal radioactivity yields after deglycerolization was used and the platelets frozen and thawed, radioactivity yields of 23% were obtained after deglycerolization and buffering and before transfusion. The rate of cooling was 1 degC/min to -3 0 ° С and then 5 degC/min to -8 0 °C . Thawing rates were not very rapid and were achieved by plunging plastic bags containing the platelets into a cold water bath. Eight thrombocytopenic patients were given transfusions of unlabelled platelets frozen and thawed in the presence of 12% glycerol. Four of these patients were given platelets which had been thawed on the same day as the freezing procedure had been performed and the other four patients received platelets which had been stored for one month at - 80° . Results of these experiments by Cohen and Gardner are summarized in Table II. The platelet counts of all the patients were raised to levels significantly above base-line values for periods of one to three days.

Cohen and Gardner consider that their system of human platelet preservation is the best yet devised and they emphasize that their normal yields of platelets after freezing and thawing with glycerol are always higher than the results from labelling with 51Cr would indicate. They suspect that elution of the label may be influenced by several factors not directly connected with platelet viability. It is felt by Cohen and Gardner that the lack of toxicity of intravenously administered glycerol and its long-term clinical usage [33] are advantages which other compounds such as DMSO, lactose, sucrose, PVP and polyglycol, all of which have been used for cell preservation, do not possess.

4. BONE-MARROW PRESERVATION

The subject of the low-temperature preservation of bone-marrow cells has been reviewed by Ashwood-Smith [34] and recently an excellent account of the theory and practice of marrow transplantation and preservation has been published by Pegg [35]. This book gives a detailed account of the animal and human research in this subject and also concerns itself with clinical problems of the collection, low-temperature storage and transfusion of bone-marrow cells. Pegg states that "if the potentialities of other sources (other than allogenic marrow) of haemopoietic tissue (surgical operations, autopsy and foetus) are to be realized, then storage is essential".

Preservation of cells at temperatures between IoС and 15°С is only effective over a relatively limited time span. Even after 5 days at 2 -5 °C , syngenic mouse marrow still has some ability to protect irradiated mice [36], and after storage for only 48 hours no difference could be demonstrated between fresh and stored marrow. When the storage temperature was as high as 22 - 25°C, the protective effect of marrow to lethally X-irradiated mice fell after 24 hours and was no longer detectable after 48 hours.

TABLE II. E FFE C T OF TRANSFUSION OF UNSTORED AND STORED FROZEN PLATELETS(a)

PatientW eight of recipient

(kg)

Plateletstransfused

(units)

Storage interv al (week)

T otalp latelet

count(X 109)

Base-linelevel

Level 1 h after

transfusion

1 h p latelet

concentration

Unstored:

1 3 8 .6 l<b> 352 1500 119500 118 000

2 6 3 .6 l(b) - 367 106 000 184 500 78 500

3 4 9 .8 3 - 146 73 000 90 000 17 000

4 7 7 .3 !<b) - 312 35 000 130 000 95 000

Stored:

1 5 7 .8 4 4 199 63 000 130 000 67 000

2 5 4 .5 4 11 186 77 000 99 500 22 500

3

4 5 5 .4 6 9 370 82 500 110 000 27 500

5 4 9 .4 6 8 288 52 000 7 8000 26 000

T able from Cohen, P . , and Gardner, F .H ., New England Journal of M edicine 274 (1966) 1400 . (b) polycythem ia vera.

124 A

SHW

OO

D-SM

ITH


Changes in the tissue-culture fluid can, however, increase the period of useful storage at 25°C [37, 38] to four days. Preservation of human stem cells at above zero temperatures has not been adequately studied, as in-vitro tests alone, Pegg suggests, should not have too much reliance placed upon them. A paper in which Hampel and Levan [39] reported that prolonged cold treatment of human cell lines in tissue culture resulted in an increase in the incidence of chromosome breaks and "break constrictions" adds a note of caution to ideas for storage at temperatures above freezing point. Preservation of marrow cells in the frozen state is the only adequate way to achieve conservation of stem -cell activity over very long periods.

There will be no attempt here to cover the now fairly considerable literature on marrow preservation but rather to consider several points which perhaps are of general interest. A central question in the mind of a cryobiologist is: "Is this cell dead or alive, or is it nearly dead but perhaps under suitable conditions restorable to full function?" Thus the question of cell viability is of great concern and for the assessment of the effectiveness of the preservation methods used on human marrow it is of the essence. Any one test of viability in vitro is suspect and some tests such as the eosin exclusion technique are very suspect. However, if with animal marrow cells four or five differing tests of viability, in vitro, give similar results and if when tested in a suitable in-vivo system this preserved marrow is functional, then it is perhaps reasonable to assume that human marrow cells behaving in a similar manner after freezing may function upon transfusion into patients.

Various tests for marrow cell function, in vitro, have been derived and all of them have certain advantages and disadvantages. Pegg [35] has suggested that the following criteria for in-vitro viability tests of human marrow be adhered to:

"(1) Since the effective stem cells cannot at the present time be distinguished with certainty, the test should either not discriminate between cell types at all, or, if it does, it should select a group of cells which definitely includes the effective cells.

(2) The test should be one of general cell physiology rather than one depending on a single system: tests of single enzymes, for instance, might easily be affected by irrelevant factors and are, therefore, suspect.

(3) The test should be sensitive to cell injury caused by freezing and thawing, and in particular it should register quantitative differences between different preservation techniques similar to those established in vivo in experimental animals. "

The tests in vitro used to assess bone marrow after freezing and thawing or after treatment with preservative agents such as glycerol or DMSO are listed below.

(a) Routine morphology

Staining with the usual haematological stains is unsatisfactory as both glycerol and DMSO treatment make fixation difficult and colour rendering is not normal.


(b) Fluorescence microscopy

Treatment of live cells with fluorochromes such as acridine orange and examination of these cells with blue-violet light gives about as much information as that which can be obtained by careful phase-contrast examination, although the information is delivered in a very beautiful and pleasing manner [40]. Intact and viable cells after treatment with1 : 20 000 acridine orange or euchrysine show green nuclear fluorescence, while cytoplasmic particles fluoresce red. Cells without cytoplasm are obvious as no red fluorescence is visible and cells in the process of dying, when cytoplasmic liquefaction is apparent, are easily identified by the rapid movement of red granules.

E F F E C T O F D IM E T H Y L S U L P H O X ID E AN D G L Y C E R O L O N T H E P R O TE IN S Y N T H E S IS O F M O U S E B O N E M ARROW C E L L S In vitro

IO O

UJ

P E R C E N T C O N C E N T R A T IO N C V / ^ ]

F IG .7 . Protein synthesis measured in mouse bone-marrow ce lls in vitro by analysis of incorporation-o f 14С into protein fraction from 2\iCi of sodium aceta te - 1 - 14C for 1 hour in Parker ’ 1 9 9 ’ tissue-culture medium at 37°C . Inhibitory e ffe ct of DMSO and glycerol expressed as per cent of the control value. (From Ashwood- Sm ith, M . J . , Ann. N .Y . Acad. S e i.141 (1967) 4 5 .)


(c) Phase-contrast examination

Short-term culture of bone-marrow cells on slides coated with tissue- culture fluid solidified with agar [41] and followed by phase-contrast examination, perhaps combined with motility studies, can be helpful in assessing the state of marrow samples. Motility has to be considered carefully as the most active cells are the more mature members of the granulocytic series. The most important cells in the bone marrow, the blast cells, show little motility although pseudopodial movements are usually quite apparent. Long-term tissue-culture methods for assessing viability are at the present time useless. However, techniques based on the semi-solid agar method developed for studying colony-forming cells of the granulocytic series might prove most helpful.

(d) Dye exclusion tests

Nearly all workers are agreed that tests based on the exclusion by intact viable cells of dyes such as eosin and nigrosin are probably very unreliable. Although simple, this test can give very misleading results as dead cells in the presence of protein often fail to take up stain.

(e) Biochemical tests

Various aspects of the metabolism of frozen and non-frozen cells have been studied by several groups. Synthesis of lipids, proteins and DNA has been followed by means of appropriate radioactive tra ce rs . Respiration rates of frozen and thawed cells have also been studied. One essential point that must be realized with all experiments is that, in many instances, the protective agents themselves have appreciable effects on enzyme systems when used in the concentrations necessary to protect cells from freezing damage. These inhibitory effects are largely reversible, however, at least with DMSO and glycerol. The effect of DMSO and glycerol on protein synthesis in mouse bone-marrow cells is shown in Fig. 7.Studies on mouse marrow cells cooled slowly (1 degC/min) in the presence of various amounts of DMSO to -7 9° С and then thawed rapidly are illustrated in Fig. 8. The DMSO was removed after thawing when the cells were incubated with 14C glycine. The amount of activity in the protein fractions was then estimated after 1 hour at 37° С. The effect on lipid synthesis is illustrated in Fig. 9. Experiments of this sort can give an indication of the effectiveness of the freezing procedures and correlate fairly well with other measures of viability both in vitro and in vivo [42].

Several groups have studied DNA synthesis of bone-marrow cells before and after freezing. Uptake of tritiated thymidine with subsequent autoradiography of cells or uptake of tritiated thymidine followed by actual analysis of the DNA fraction have been the two methods used. The essence of this test is based on the premise that stem cells are dividing cells. However, two reservations must be made. F irst, stem cells are not necessarily always dividing. Secondly, cells after injurious treatments, even though destined to survive, may not undergo DNA replication for a considerable time after thawing. It is known, for example, that after X-irradiation damaged cells do not undergo DNA replication and division


MOUSE BONE MARROWE F F E C T OF FR EEZ ING ТО-79® С ON INCORPORATION OF 14C IN T O PRO TE IN : IN VITRO INCUBATION PARKER199 ' ■ A C E T A T E ~ l4C FOR I HOUR AT 37° С

□ NORMAL CELLS

■ FROZEN CELLS

PER CENT DIMETHYL SULPHOXIDE

FIG. 8 . E ffect o f freezing mouse bone-marrow cells in the presence of different concentrations o f DMSO on protein synthesizing ab ility . (Incorporation of 14C from aceta te into protein fraction; 1 hour, 37 °C in Parker '1 9 9 ' tissue culture fluid.)

MOUSE BONE MARROWEFFECT OF FREEZING Т О ? Л ON INCORPORATION O F ,4C lN T O LIPID : tnVitrO INCUBATION

PARKER'i 99*+ ACETATE-I-,4C FOR S HOURS A T У7°С

П NORMAL CELLS

FROZEN CELLS

5 10 15 20

PERCENT DIMETHYL SULPHOXIDE ■

F IG .9 . Effect of freezing mouse bone-marrow cells in the presence of different concentrations of DMSO on lipid synthesizing ab ility . (Incorporation of 14C from acetate into lipid fraction; 1 hour, 37°C in Parker '1 9 9 ’ tissue culture fliiid .)


until after cellular damage has been repaired. However, with these re servations in mind, studies on the DNA synthesis of frozen marrow cells have yielded much useful information. Workers at Cooperstown in New York State have used this test extensively for assessment in vitro of frozen marrow [4 3 -4 5 ]. In general, these tests have indicated very low DNA synthesis rates with marrow frozen without preservatives. The addition of glycerol or DMSO gives higher synthesis rates after freezing and thawing and results agree reasonably well with those from in-vivo studies.

EFFECT OF FRESH A N D PRESERVED

BONE MARROW O N S U R V IV A L OF

L E T H A L L Y X — I R R A D IA T E D MICE

FR E S H

M A R R O W

NO

MARROW FROZEN M ARRO W KEPT AT - 7 9 " C D M O N T h D

MEDIUMO N LY

+ '5 7 . ,+ '5% O 'G L Y C E R O L SU LPH O X IO E

FIG. 10 . Survival (30 day) of lethally X-irradiated CBA m ice in jected with fresh or preserved (1 month at -79°C ) syngenic marrow ce lls ( 5 x l 0 6) . (From Ashwood-Smith, M .J . , Proc. 8th Congr. European Soc. for Haematology, Vienna 68 (1 9 6 1 ).)

1 3 0 ASHWOOD-SMITH

(f ) Tests for marrow function in vivo

For several years after the original publication of Barnes and Loutit [46] in 1955 on the survival of mouse spleen cells treated with glycerol and cooled slowly to -70°C , the most favoured and perhaps most important test for stem -cell activity was to test for the survival of lethally X -irrad i- ated animals treated with spleen, marrow or foetal-liver cells. Several workers noted that the freezing of mouse marrow cells without the protection of additives destroyed nearly all the haematological properties [42, 43]. A few treated animals often survived beyond 30 days after X -ir -

DAVS AFTER X -R A Y S

FIG. 11 . Effect of syngenic fresh marrow and marrow preserved at -79°C with dimethyl sulphoxide on the recovery of blood leucocytes in lethally X-irradiated m ice . (From Ashwood-Smith, M . J . , Nature (Lond.) 190 (1961) 1 2 0 4 .)


radiation, which suggested that a small proportion of marrow stem cells survived slow freezing and thawing in the presence of serum without added DMSO or glycerol [42]. However, the addition of glycerol or DMSO [42] to the medium gave results which made it difficult to tell apart fresh and frozen marrow preparations (Fig. 10). More exact comparisons could be made by adjusting the dose of fresh and frozen cells and thus making the test more sensitive [43, 47]. Measurement of the rate at which haematological recovery takes place in irradiated m i c e treated with fresh and frozen marrow cells (Figs 11 and 12) has been used by Ashwood-Smith [42] and when this was done, and comparisons made with the results of tests in vitro, good agreement was obtained between the various regimes. Davies, Playfair and Cross [48] found that freezing mouse marrow in 10% DMSO at 1 degC/min to -79°C followed by rapid thawing diminished the therapeutic potential by 90%, the capacity to restore peripheral blood components by 50% (little difference between the various components was observed) and the potential for giving rise to spleen colonies by 50%.

FIG. 12. Recovery of blood leucocytes in lethally X-irradiated m ice treated with syngenic marrow preserved for one month at - 7 9 ’ C in either 15<Уо glycerol or 15% dimethyl sulphoxide. (From Ashwood-Smith, M . J . , Proc. 8th Congr. European Soc. for Haematology, Vienna 68 (1 9 6 1 ).)


FIG. 13 . Protective e ffe ct of fresh and thawed syngenic marrow ce lls in lethally X-irradiated CAF1 m ice . Mean survival and 95°/o confidence lim its from studies in '400 anim als. (From Lewis, J . P . , Trobaugh, F .E ., Ann. N .Y . Acad. Sei. 114 (1964) 6 7 7 .)

16

-^e x p e cte d c o l o n i e s

LO G 10 No. c e l l s i n j e c t e d 10 ,0 0 0

M ean o b s e r v e d c o l o n i e s , f r e s h c e l l e M ean o b s e r v e d c o l o n i e s , th aw ed c e l l s

R e g r e s s i o n e q u a t io n , f r e s h c e l l s -9 = .2. 3 0 8 9 + 3. 3 747 x

R e g r e s s i o n e q u a t io n , thaw ed c e l l s -

Y 1. 8b S 5 + 3. 3 747 x

- 4 -10.000 20 I 000 3 0 , 0 0 0 4 0 , 0 0 0 5 0 , 0 0 0 6 0 , 0 0 0 7 0 , 0 0 0 8 0 , 0 0 0

N u m b e r of C e l l s I n j e c t e d

F IG .14. Regression lines for spleen’colonies of fresh and thawed syngenic marrow, (From Lewis, J . P . , Trobaugh, F .E . , Ann. N .Y . A cad .-Sei. 114 (1 9 6 4 ); 677. )


There is no. doubt, however, that, the most definitive work on bone- marrow preservation techniques using in-rvivo tests has. been done by Lewis and Trobaugh [49] with a modification of the Till'and McCulloch [50] spleen-colony assay system to investigate the number of surviving cells able to form colonies after preservation. When slow freezing and thawing in glyc.erol was used with mouse bone-marrow cells, 74% of the colony-. forming potential remained. The optimal concentration of glycerol was found to be 15%. Injection of differing numbers (greater than 50 000) of cells into lethally X-irradiated mice resulted in the same number of survivors as injection of fresh marrow cells. This is a good example of the lack of sensitivity of the test system, u'hich uses 30-day survival of X-irradiated animals. The response of lethally X-irradiated CAP} mice injected with different numbers of fresh and frozen/thawed cells is illustrated in Fig. 13, which is taken from the 1964 paper by Lewis and Trobaugh; note that the effectiveness of thawed marrow is virtually identical with fresh marrow. Regression lines relating to the number of colonies formed after injection of different numbers of fresh and frozen cells were constructed by Lewis and Trobaugh and are shown in Fig. 14. The relative potency of the thawed cells was 73. 9% with 95% confidence limits of 63. 7% and 85.6%. This method of analysis was used by these authors [49] to demonstrate that DMSO was inferior to glycerol for preservation of mouse marrow at low temperature, a result which does not accord with the observations of Ashwood-Smith [42]. Differences could be due to the different techniques of assay used or the osmotic'buffering usedduring the treatment of thawed cells. A recent paper by Lewis, Passovoy and Trobaugh [51] has reviewed their previous work on marrow preservation with the spleen- colony method. Lewis et al. concluded that marrow cells, like bull spermatozoa, are not sensitive to "thermal shock" above 0°C and that they are not sensitive to the temperature at which freezing is initiated. When, however, cooling rates are appreciably faster or slower than 2 degC/min, the repopulating potential of the marrow is adversely affected. Lewis et al. have outlined a technique with mouse marrow cells suspended in Hank's solution containing 12% glycerol and ,4% calf serum. Marrow samples were cooled at a rate of 2 degC/min to -100°C , freezing being initiated at -1 2 °C . Thawing was done rapidly by immersion of ampoules in water at 40°C. Deglycerolization was achieved by a careful regime which included the use of 35% glucose and 6% dextran. This procedure yielded marrow cells of a relative potency of 94.54% with 95% confidence limits of 81.8% and 109.2%; regression lines are shown in Fig. 15. Lewis et al. conclude that "If human haematopoietic cells respond similarly, and there is no' evidence either that they will or will not, the means for preserving human-marrow for indefinite periods of .time are at hand". What is'the proof that .stored human' marrow is viable? Pegg [35] has summarized the evidence as follows:

"(1) It has been shown by the examination of preserved bone marrow in short-term tissue cultures that living motile cells are present.

(2) It has been shown that stored autologous, bone marrow will accelerate the haematological recovery of patients treated with cancer chemotherapeutic agents.; the m o s t c o n v in c in g of these were the reports ' concerning mannitol mustard [52] and very large doses of mustine - :hydrochloride [53]. . . r , . . .

1 3 4 ASHWOOD-SMITH

(3) It has been shown that irradiated human recipients of stored allogenic bone marrow have sustained temporary marrow grafts with the production of donor-type erythrocytes [54, 55]. "

It would be reasonable to assume that human marrow behaves in a manner not dissimilar to other animal marrow cells, although Van Putten's work is interesting in this respect.

= * Fresh morrow ™ 1 Froien ond thowed morrow

16 -

95% C.L.(R.R) = 0.818 and 1.092

Fresh Thowed TotalRep. 3 3Dose lev. 3 3An./dose 7 7lanimol 63 63 126

. . i ______i___10.000 2 0 .0 0 0 4 0 .0 0 0

Number of Cells Injected

FIG. 15 . Bioassay of the repopulating potential of syngenic marrow which has been frozen and thawed. (From Lewis, J .P . , Passovoy,'M . , Trobaugh, F .E ., Cryobiology 3 (1966) 4 7 .)

Van Putten [56] has investigated the best conditions for freezing and thawing mouse marrow and foetal-liver cells and also for monkey cells.He made a careful study of the recovery of lethally X-irradiated mice and of monkeys. Cooling rates were 1 degC/min between + 20°С and -4 0 °C . Storage was in liquid nitrogen at -196°C (1 week to 3 months). Thawing was rapid and was achieved by immersion of ampoules in water at 38°С .Van Putten found that PVP alone gave good protection to mouse cells in agreement with Persidsky and Richards [57, 58]. This finding is at variance with that of Bender, Tran and Smith [59] who found PVP to be ineffective. Combinations of PVP (10%) with glycerol (10%) and DMSO (10%) were very efficient. Foetal mouse liver cells were less well preserved. Autologous monkey cells were poorly preserved except when glycerol (10%) and PVP (10%) were used in combination. Approximately 50% of the cells


survived under optimal conditions. The main point of interest in Van Putten's studies is the strong indication that species differences may be important and preclude, in Van Putten's opinion, dependable recommendations for storage of human bone marrow.

No attempt has been made here to cover all of the references on bone- marrow preservation at low temperatures or to consider the interesting results obtained when PVP [57, 58] or various inorganic salts [60, 61] are used as protective agents. Reports on the longevity of stored marrow cells are few. Malinin [62] has found that marrow cells stored at -7 0 ° С show progressive morphological changes as storage time increases, but at -150°C only minimal to moderate changes were observed. Smith and Tran [63] have examined the protective efficiency of m o u s e m a r r o w c e l l s preserved with glycerol and injected into lethally X-irradiated mice after storage at various sub-zero temperatures for various periods of time. After 1 hour at -30°C , survival figures for mice were 83%; after 4 weeks, 57% and after 25 weeks, 0%. With a storage temperature of - 7 0 ° G no difference could be detected between 1 hour and 22 weeks and in both instances 80% survival values were obtained. Although cells preserved for 57 months at -196°C were partly clumped on thawing, 88% of mice injected with these cells survived.

Are there any changes in the biological characteristics of frozen marrow cells? Schwartzenberg and colleagues [64] have demonstrated a marked reduction in secondary disease of lethally X-irradiated mice injected with allogenic cells preserved at -70°C with DMSO. However, Iossifides et al. [65] have failed to show differences between injections of frozen syngenic and allogenic marrow cells and the occurrence of secondary disease.

5.' CONCLUSIONS

(1) Lymphocytes from animals and man can be stored for long periods of time at low temperatures when cooled slowly in 10-15% DMSO or glycerol and thawed rapidly. Récovery figures are high and function is intact and unaltered. DMSO is probably a better preservative than glycerol and storage life at - 196°С is probably indefinite for all practical purposes. Other leucocytes can be stored with similar techniques but recoveries after freezing and thawing are probably lower than with lymphocytes.

(2) Bone-marrow cells of several animals including mouse, rabbit and dog can be preserved at - 196°C, probably indefinitely, and procedures sim ilar to those used for lymphocytes give the best results. The comprehensive studies of Lewis and Trobaugh indicate that under carefully controlled conditions 95% of the stem cells in mouse marrow are viable after freezing and thawing. Opinions are divided over the efficacy of the two preservatives, DMSO and glycerol, but in view of the well documented a c counts of the lack of toxicity of glycerol it would seem advisable for the moment to use this agent with all human marrow samples. The usefulnessof PVP as a preservative for marrow is still to be resolved. Human marrow after freezing and thawing probably behaves in a similar manner to mouse marrow both in vitro and in vivo. However, it would be wise to consider that there might be differences which could cause wrong assessments of freezing procedures.

1 3 6 ASHWOOD-SMITH

(3) Platelet preservation, clinically perhaps the most useful procedure discussed in this review, is still to a large extent in the experimental stage. Much work has been done but even the best methods available allow for relatively low recoveries of viable platelets. Preservation of human plateletsin 12% glycerol associated with slow freezing and fast thawing and followed by very .careful- deglycerolization procedures yields a product with 23% of the original activity. This is clinically acceptable, perhaps, but biologically it leaves much to be desired. Studies with rat platelets and combinations of various .protective agents such as dextrose and DMSO and dextrose and dimethyl acetamide are encouraging but toxicity problems may prove difficult in human studies.

(4) The evidence from many different bacterial and animal cell studies indicates that - 79°G is not low enough for long-term preservation. Temperatures below -130°C should be used whenever possible and precautions be taken to exclude radiation from natural and cosmic sources when preservation in term s of years is considered.

R E F E R E N C E S

[1] LUYET, B . J . , Biodynamica 7 (1949) 217.[2] HUGGINS, C .E . , Ann. Surg. 160 (1964) 643. . . .[2a] HUGGINS, C .E . , Proc. IXth Congr. in t. Soc. Blood Transfus., Stockholm, 1964, Karger, Basel-

New York (1965) 662. ! .[31 HAYNES, L . L . , TULLIS, J .L . , PYLE, H 'ÍM ., SPROUL, M . T . , WALLACH, S . , TURVILLE, W .C .,’ • ’ J . Am , med. Assoc. '173 (1960) 1557.[4] POLGE, C . , SMITH, A .U ., PARKES, A. S ., Nature (Lond. ) 164 (1949) 666.[5] SMITH, A .U ., Biological Effects of Freezing and Supercooling, Edward Arnold, London (1961).[6a] LOVELOCK, J .E . , Biochim . biophys. Acta 11 (1953) 28 .[6b] LOVELOCK, Ï .E . , Biochim . biophys. Acta 10 (1953) 414 .[7] ATKINS, L . , Nature (Lond.) 195 (1962) 610T[ 8 ] . .ASHWOOD-SMITH, M .J . , Blood 23 (1964) 494 .[9] SYMES, M .O .', RIDDELL, A .G ., HILL, R¿ D . , Lancet 1 (1968) 1052.

[10] SYMES, M .O ., MEEK, E .S . , RIDDELL, A .G ., in Lymphocytes in Immunology and Haemopoiesis(Yoffey, Ed.) Edward Arnold, London, (Г967) 279.

[11] MEEK, E .S . , SYMES, M .O ., RIDDELL, A .G ., Immunology 12 (1967) 339.[12] PEGG, P . J . , Br. J . Haemat. U (1965) 586.[13] DAVIES, J .D . , COULSON, A ^S:, SMITH, A .P ., Cryobiology 2 (1966) 263.[14] PLAYFAIR, J .H .L . , DAVIES, A . J . S. ,. Transplantation 2 (1964) 271.[15] BOURONCLE, B. A . , Cryobiology 3 (1967) 445.[16] CAVINS, J .A . , DJERASSI, L . , ROY, A .J . , KLEIN, E . ,. Cryobiology 2 (1965) 129.[17] ROWE, A .W .', KACZMAREK, C .S . , COHEN, E ., Fedn Proc. 22 (1963) 170.[18] SHOHET, S .B . , MOHLER, W .C ., Cryobiology 4 (1967) 47 .[19] ALBRIGHT, ' J . F . , MAKINODAN, T . ,■ MAZUR, P '., Proc. Soc. exp. Med. 114 (1963) 489 .[20] CAVINS, J .A . , SC H E E R ,-S .C ., THOMAS, E .D ., FERREBEE, J .W . , Blood 22 '(1963 ) 505.[21] PERRY, V .P . , MALININ, T . I . , KERBY, C :C . , DOLAN, M : F . , Cryobiology 1 (1965) 233 .[22 ] GOODMAN, J . W ., Blood 21 (1963) 7 77 , .[2 3 ]. MORRISON, F . S . , Cryobiology 3 (1967) 377. . . .[24] BALDINI, M ., COSTEA, N .. DAMESHEK, N .. Blood 16 (1960) 1669.[25] COHEN, P ., GARDNER, F . H . , J . Lab. c lin . Invest. 41 (1962) 10.[26] DJERASSI, I . , FÄRBER, S . , ROY, A ., Blood 20 (1962) 762.[27] IOSSIFIDES, I .A . , GEISLER, P ., EICHMAN, ”m .F . , TOCANTINS, L .M ., Blood’ 20 (1962) 762 .[28] DJERASSI, I . , ROY, A ., Blood 22 (1963) 703. _[29] WEINHERTH, J . , HUGGINS,- O .E . , Cryobiology 2 '(1 9 6 6 ) 310.[30] DJERASSI, I . , ROY, A ., FÄRBER, S . , CAVINS, J . , KLEIN, E . , Cryobiology 2 (1966) 299.


[31] COHEN, P . , GARDNER, F .H ., New Engl. ] . M ed. 274 (1966) 1400.[32] COHEN, P . , COOLEY, M .H ., GARDNER, F .H ., New Engl. J . M ed. 2T3 (1965) 845.[33] HAYNES, L .L ., J . Am. m ed. Ass. 173 (1960) 657.[34] ASHWOOD-SMITH, M . J . , Fedn Proc. 24 2, Part 3, Suppl. 15 (1965) S -299 .[35] PEGG, D .E ., Bone Marrow Transplantation, Lloyd-Luke, London (1966).[36] URSO, I . S . , CONGDON, C .C . , ] . appl. Physiol. 10 (1957) 314,[37] BILLEN, D ., Nature (Lond.) 179 (1957) 574.[38] BILLEN, D ., J . natn. Cancer Inst. 23 (1959) 1389.[39] HAMPEL, K .E . , LEVAN, A ., Hereditas 51 (1964) 315.[40] ASHWOOD-SMITH, M . J . , YOUNG, M .R ., J . R. m icrosc. Soc. 80 (1962) 191.[41] PULVERTAFT, R . J .V . , HUMBLE, J .G . , Rev. Hém at. 11 (1956) 349.[42] ASHWOOD-SMITH, M .J . , Nature (Lond.) 190 (1961) 1204.[43] FERREBEE, J .W ., BILLEN, D ., URSO, I . M ., LU, W .C ., THOMAS, E .D ., CONGDON, C .C . ,

Blood 12 (1957) 1096.[44] LOCHTE, H .L ., FERREBEE, J .W ., THOMAS, E .D ., J . Lab. c lin . M ed. 53 (1959) 117.[45] MANNICK, J .A . , LOCHTE, H .L ., THOMAS, E .D ., FERREBEE, I . W ., Blood 15 (1960) 517.[46] BARNES, D .W .H ., LOUTIT, J .F . , ] . natn. C ancer. Inst. 15 (1955) 901.[47] IOSSIFIDES, I . , Fundamental and C lin ical Aspects of Radiation Protection and Recovery, ORNL N o.4

(1961) 27.[48] DAVIES, A . J .S . , PLAYFAIR, J .H .L . , CROSS, A .M ., Transplantation 2 (1964) 241.[49] LEWIS, I . P . , TROBAUGH, F .E . , Ann. N .Y , Acad. Sei. И 4 (1964) 77 .[50] TILL, J .E . , McCULLOCH, E .A ., Radiat. Res. 14 (1961) 213.[51] LEWIS, J .P . , PASSOVOY, M ., TROBAUGH, F .E . , Cryobiology 3 (1966) 47 .[52] PEGG, D .E ., HUMBLE, J .G . , NEWTON, K .A ., Br. J . Cancer 16 (1962) 417 .[53] CLIFFORD, P ., CLIFT, R .A ., DUFF, J .K . , Lancet 1 (1961) 687.[54] CONSTANDONLAKIS, M ., Ph.D . Thesis, Univ. London (1959).[55] THOMAS, E .D . , LOCHTE, H .L ., LU, W .C ., FERREBEE, J .W . , New Engl. J . M ed. 257 (1957) 491 .[56] PUTTEN VAN, L .M ., Europ. J . Cancer 1 (1965) 15.[57] PERSIDSKY, M .D ., RICHARDS, U ., Blood 23 (1964) 337.[58] PERSIDSKY, M .D ., RICHARDS, U ., LEEF, J . , Cryobiology 2 (1965) 74 .[59] BENDER, M .A ., PHAN THE TRAN,SMITH, L .H ., J . appl. Physiol. 15 (1960) 520.[60] PHAN THE TRAN, BENDER, M .A ., Proc. Soc. exp. M ed. Biol. 104 (1960) 388.[61] PHAN THE TRAN, BENDER, M .A ., Exp. ce ll Res. 20 (1960) 651.[62] MALININ, T . I . , Cryobiology 2 (966) 345.[63] SMITH, L .H ., PHAN THE TRAN, N aturettond.) 205 (1965) 4970, 503.[64] SCHWARTZENBERG, L . , AMEIL, L . J . , JENEBAUM, R ., MATHE, G . , Revue fr. Etud. clin . biol. 8

(1963) 783,[65] IOSSIFIDES, I .A . , BRAND, M ., TOCANTINS, L .M ., in Fundamental and C lin ical Aspects of Radiation

Recovery and Protection, ORNL N o.6 (1963) 11.

POSTHUMOUS BONE MARROW ANDITS SIGNIFICANCE FOR TRANSPLANTATION

N. G. KARTASHEVSKY, Т.К. MAMYSHEVAResearch Institute for Haematology and Blood Transfusion,Leningrad, USSR

Abstract

POSTHUMOUS BONE MARROW AND ITS SIGNIFICANCE FOR TRANSPLANTATION. Bone marrow obtained by aspiration from the chest and iliac crest of adults who had died suddenly, or by forcing it out o f their vertebrae, has been studied at the Leningrad Research Institute for Haematology and Blood Transfusion since 1959, with morphological, functional and biochem ical methods.

It has been found that the vital activ ity of the bone-marrow cells depends on the tim e which has elapsed since death, the optimum period being the first six hours after death. As can be seen from a number of indices (phagocytic activity , capacity for granulopoiesis o f v ita l dyes, luminescent microscopy and energy metabolism ) posthumous bone marrow removed during this period differs little from donor bone marrow.

The number of bone-marrow cells taken from corpses is several times greater than the number that can be taken from live donors. Experimental and c lin ic a l results show that the transplantation o f posthumous bone marrow has a stimulating e ffe ct on haemopoiesis.

On the basis-of the research that has been carried out, bone marrow obtained within six hours o f death can be regarded as valuable, bio logically a c t iv e tissue suitable for transplantation.

Research results of recent years indicate that bone-marrow transplantations have an extremely favourable effect on the restoration of the haemopoietic function in a series of diseases of the blood system, especially in radiation illness.

Use of bone marrow in the clinic necessitated obtaining and preserving it in considerable quantities to create a permanent store. That this was possible was mainly due to the homologous nature of bone marrow.

As is well known, the amounts of bone marrow that can be obtained from donors are relatively very small. In view of this, great interest and importance attaches to studying the possibilities of using bone marrow obtained from corpses.

In the Soviet Union the investigation of bone marrow taken posthumously from adults was first undertaken in the Leningrad Institute for Haematology and Blood Transfusion in 1959. This work was carried out concurrently in several laboratories and in the haematological clinic by specialists of various types (T. K. Mamysheva, L .I . Spizharskaya, M. N. Blinov). The technique of obtaining bone marrow from corpses was worked out, as well as methods of preserving it. The viability of the cells was determined by various methods. Morphological, functional and biochemical tests were used as well as an original tissue-culture technique. In addition, posthumous bone marrow was transplanted into patients with blood diseases and into animals that had been given experimentally a lethal dose of radiation.

In this paper we shall briefly treat the data which allowed us to evaluate bone-marrow cells obtained posthumously and to draw the co rre sponding practical conclusion.

1 3 9

1 4 0 KARTASHEV SKY and MAMYSHEVA

The bone marrow was obtained from corpses of human beings (303), from 16 to 88 years of age, at various times after sudden death (1 .5 to 48 hours).

The bone marrow was obtained by aspiration under vacuum from the chest and iliac crest and by forcing it out of the vertebrae. (Both these methods were developed in the Leningrad Institute for Haematology and Blood Transfusion by N. G. Kartashevsky and Т .К . Mamysheva).

Fo r determination of the vital activity of cells of the posthumous bone marrow the following methods were used:

(a) Morphological methods;(b) Luminescent microscopy.

In addition, the functional state of the cells was investigated by:

(c) Determination of their phagocytic activity:(d) Establishment of their mobility with the aid of phase-contrast

microscopy; and(e) Determination of their granulopoietic ability.

Some biochemical indices of energy metabolism of the cells of the posthumous bone marrow were investigated, such as respiration, glycolysis, synthesis of glycogen, content of ATP and some enzymes participating in the synthesis of glycogen.

The vital activity of posthumous bone-marrow cells was established by the phenomenon of experimental splenomegaly (after transplantation of posthumous bone-marrow cells into the chorionallantois of 9- to 10-day chicken embryos).

The results of the research on posthumous bone-marrow cells showed that the number of bone-marrow cells depends on:

(1) Where they were obtained:

from the chest, on the average - ,n ' m

after 60 years, on the average

The morphological and functional state of nucleated cells of the posthumous bone marrow is determined mainly by the period of time elapsing . from the moment of death. Even when the corpse is stored ,at a temperature of +4°C, the autolytic processes in myelokaryocytes noticeably increase with the lengthening of the posthumous period. Three hours after death about 40% deformed cells are already determined; after 6-10 hours, 60.8%; after 10-15 hours, 69.9%; after 15-20 hours, 70. 34%; after 20 hours, 85. 11%.

In investigating some functions of nucleated elements of posthumous bone marrow (TableI), the presence of amoeboid mobility of the cells was

from the iliac crest from the vertebrae

(2) The age of the subject:

up to 30 years, on the average

POSTHUMOUS BONE MARROW 1 4 1

TABLE I. COMPARISON OF FUNCTIONAL CHARACTERISTICS OF POSTHUMOUS AND DONOR BONE MARROW

Method of investigation Posthumous bone marrow Donor bone marrow

Phagocytic activity 6 9 .4 - 7 2 .4 % 6 4 .1 0 - 77 .20%

Phagocytic index 9 .2 - 1 1 .1 1 0 .7 6 - 12 .8 8

Capacity for granulopoiesis of vital dyes

7 5 .0 8 - 8 5 .9 % 8 4 .9 - 96 .5% ■ ■

Content o f viable ce lls according to luminescent microscopy data

8 1 .2 2 - 90 .84% 9 0 .4 6 - 94 .70%

discovered, and their activity in phagocytic reaction was noted. Cells were capable of devouring and digesting micro-organisms and this ability differed little from that of the cells in the donor bone marrow. The phagocytic activity of myelocaryocytes of posthumous bone marrow is from 69.4 to 72.4% and the phagocytic index is from 9.2 to 11. 1, while donor bone marrow gives 64.10 - 77.20% and 10.76 to 12.88, respectively.

The same is true in regard to the capacity for granulopoiesis of vital dyes. In posthumous bone marrow this capacity is found in 75. 08 - 85. 9% of cells, and in donor bone marrow in 84. 9 - 96. 5% of cells.

With luminescent microscopy of the bone marrow, it was established that the content of viable cells in posthumous bone marrow is 81.22 - 90.84%, while in donor bone marrow it is 90.46 - 94. 7%. The character and structure of the haemopoietic elements of posthumous and donor bone marrow are identical.

A good index of the functional state of cells is their energy metabolism. In the investigation of some indices of the chemism and metabolism of posthumous bone-marrow cells, it has been established that myelokaryocytes prepared in the first four hours after death are characterized by moderate respiration (Q*gl = 4 .8 ), high aerobic glycolysis (QŸi = 16.8) and intensive

2 N 2anaerobic glycolysis (Qr i = 33. 1); ATP content per 109cells is

2

711 ± 43 ß g and glycogen content is 3287 ± 257 ß g . Intensive glycogen metabolism in myelokaryocytes of posthumous bone-marrow indicates that the bone marrow retains the enzyme systems participating in the resynthesis of glycogen. All this shows that in the cells of posthumous bone marrow active metabolic processes are taking place, and therefore the cells have not lost their vital activity.

In studying the viability of posthumous bone-marrow cells by implanting them in the chorionallantois of chicken embryos, the Splenomegalie reaction was clearly demonstrated. The essence of this phenomenon is that the vital cells of the bone marrow, transplanted into the chorionallantois of 9- to 10-day chicken embryos, proliferate and stimulate the growth of spleen.

The foregoing information testifies to the fact that bone marrow obtained from corpses up to six hours after death is biologically active, and

1 4 2 KARTASHEVSKY and MAMYSHEVA

we may therefore assume that after transplantation it will have the same stimulating effect on the haemopoietic organs as donor bone marrow. The correctness of this supposition has been confirmed by the results obtained in the transplantation of posthumous bone marrow in the clinic (by K.M. Abdulkadyrov) and in experiments (b y l.R . Petrov, I.V . Ilyinskaya, T.N . Astakhova and T. K. Mamysheva).

In the haematological clinic at this institute 30 transplantations into 19 patients with various diseases of the blood system have been carried out. The therapeutic effect of these transplantations was noted in patients with hypoplastic anaemia. Posthumous bone marrow, both fresh and preserved (by freezing), was also transplanted into rabbits exposed to lethal doses of radiation. The results showed that posthumous bone marrow, whether fresh or preserved, has a stimulating effect on the haemopoiesis of irradiated animals, increasing their ability to survive, raising the natural resistance of the organism and preventing the development of subsequent anaemia.

By means of this paper we have sought to focus attention on the fact that bone marrow obtained from corpses up to six hours after death is a valuable biologically-active tissue which may be utilized for transplantation into patients. One very important factor in this connection is that, with proper organization of the work, from one corpse considerably more bone-marrow c e l l s m a y b e obtained (50-80 X 109) than from a donor (16-18 X 109).

BONE-MARROW STORAGE AND TRANSPLANTATION"

0 . COSTÄCHEL, I. CORNECI, T . ANDRIAN, I. KITZULESCU,N. POPESCU, D. PASCU, E. BUZI, N. VOICULETZ Oncological Institute,Bucharest, Romania

Abstract

BONE-MARROW STORAGE AND TRANSPLANTATION. The authors present some results from their experiments on bone-marrow storage and transplantation. The m ain problems with preservation o f stored bone marrow are the duration, temperature, adjuvant substances and the significance of v iability tests during the conservation processes.

The results showed th at:

Storage o f bone marrow at + 4 eC produces a progressive decrease in its restoring capacity versus storage tim e.While bone marrow stored for 24 h is able to restore 100% o f dogs lethally irradiated with 600 rad, after 10 days of storage only 20% o f the animals can be restored.No correlation exists between the actual survival of dogs and that calcu lated by dye exclusion tests, which indicate a rather high (70%) v iability , even after 10 days bone-marrow storage at + 4 °C .DNA degradation (depolym erization) measurements o f the bone marrow may be used as a supplementary test for checking the v iability or restoration potency of bone-marrow cells after storage.In the freezing process, the optimum contact tim e between glycerol and the bone-marrow cells is 15 m in.

Results of experiments regarding certain bone-marrow transplantation problems showed th at:

The best tim e to administer bone marrow is between 24 and 48 h after irradiation.No survivors were obtained with dogs lethally irradiated with 600 rad by administering autogenic or allogenic DNA extracted from bone marrow, spleen or liver.H istocompatibility related to sex may play an important role in the bone-marrow graft. The lowest survival o f C57BL m ice was obtained when the donors were m ales and the recipients fem ales.In radioprotection with foetal haem ocytopoietic tissues, the donor's age represents one of the main factors. The best results were obtained in experiments on rats, with 19 - to 20-d ay foetal liver (period of com plete and maximum haem ocytopoietic activ ity ).

•The tissues mentioned below may be connected with the appearance of certain typical signs o f secondary

syndrome.

Transfusion of blood or allogenic splenic ce lls did not influence noxiously the take of autogenic bone- marrow grafts in lethally irradiated dogs.After repeated irradiations and recovery of dogs (1 -3 tim es) following treatm ent with autogenic bone- marrow, late chromosomal lesions were observed.

1. INTRODUCTION

This paper is a brief summary of the work carried out at the Oncological Institute and is intended to cover the main topics discussed at this Panel and to present some conclusions.

Supported in part by the International Atomic Energy Agency (Vienna) Contract 226/RB

1 4 3

1 4 4 COSTÄCHEL et a l.

The storage and transplantation of bone-marrow cells pose complex problems that have not yet been solved. Thèse problems mainly concern duration, temperature and substances added to improve survival as well as the significance of viability tests on the stored bone-marrow cells and their haemopoietic capacity.

2. DURATION AND TEMPERATURE OF STORAGE SUBSTANCES ADDEDAND SIGNIFICANCE OF VIABILITY TESTS

Bone marrow stored for four days at 2-5° С or for two days at room temperature affords good protection to lethally irradiated mice [1,2] .

Autogenic dog bone marrow stored for 24 h at +4°C saves 100% of animals lethally teleirradiated; after four days of storage, even much higher doses of cells save only a few. In parallel with the decreased re populating capacity, the DNA synthesizing capacity of these cells also decreases after 24 h to 30-40% and after 96 h to approximately 10% [3] .

The same rapid decrease in the DNA synthesizing capacity has been observed in both rabbit and human bone marrow stored at room temperature or at 37°C [4-6 ].

Suspensions whose DNA synthesizing capacity decreases in vitro may, however, proliferate in vivo and save irradiated animals [7] . .

There is a serious discrepancy between the results obtained with viability and in-vitro tests and those obtained in vivo, the latter being the results that really express the viability of medullary cells after they have been stored and processed [8-11] .

Below is a summary of our results on duration of storage temperature, significance of viability tests and DNA degradation tests.

The restorative capacity of autogenic bone marrow, stored at + 4 °С for 1-10 days, was tested on 90 mongrel dogs that had been lethally irradiated with a whole-body dose of 600 rad.

DAYS

FIG. 1. Survival o f dogs by day 30 after whole-body teleirradiation (600 rad) and therapy with autogenic bone marrow stored for 1-10 days at +4°C. Black columns represent actual survival of dogs; the hatched columns show the expected calcu lated survival based on the eosine test and the white columns that based on the trypan blue test. Figures show the minimum and maximum infused ce ll doses per kg body weight (figures in brackets).

STORAGE AND TRANSPLANTATION 1 4 5

From Fig. 1, depicting animal survival (black column), it can obviously be concluded that autogenic bone marrow harvested before the animals were irradiated, stored for 24 h at a temperature of +4°C and then infused into the irradiated dogs restored 100% of the animals; this proves the efficiency of a minimal dose of 1.5 X 109 medullary cells per animal or 13.8 X 107 cells per kg body weight.

However, the restorative capacity of the bone marrow progressively decreased from ten out of ten (10/10) animals after 24-h storage to 2/10 animals after 10 days' storage.

In the same graph a lack of correlation can be observed between the actual survival and that calculated by two dye-exclusion tests (trypan blue and eosine), which indicated a high cell viability, even after 10 days' storage.

This would mean that the bone marrow, subjected to relatively longterm storage at +4°C, contained a great number of cells that maintained one vital quality, integrity of cell membrane selectivity, while losing that of division and proliferation. Tennant [12] called this the "twilight zone".

FIG. 2 . DNA dosage according to Schmidt-Tannhauser and radioactivity of DNA and soluble acid fractions.

О ---------О DNA mg/gД -----Д DNA fraction radioactivity• • • • ■• soluble acid fraction radioactivity.

Changes in the viability of cells stored at +4°C were also studied by the labelled DNA degradation test (3H-TDR administered to mice before sacrifice).

Results of the DNA degradation tests show that at 24 h the cellular content in highly polymerized DNA starts to decrease and the acid-soluble fraction to increase (Fig. 2).

Chromatographic study (Fig. 3) shows a change in the distribution of fractions characterized by various molecular weights. In our chromatographic system, fraction 6 represents DNA with a molecular weight of over2 X 106, fraction 5 having a weight of 1-2 X 106 and fraction 4 below 106. There isa decrease of radioactivity in fraction 6 and a subsequent increase in fractions of lower molecular weight between 48 and 72 h.

This process reflects the depolymerization of DNA by cellular lysis and the occurrence of fine changes during storage.


DNA depolymerization therefore constitutes one of the mechanisms that lead to the loss of the capacity to proliferate and to the death of cells.

The study of DNA degradation (depolymerization) of bone-marrow cell suspensions may be useful as a supplementary viability test and as an estimate of the restorative potential of stored bone-marrow cells. However, the risk presented by the in-vivo labelling and long-term storage of labelled cells must be considered.

F IG .3 . Cromatographic pattem of DNA extracted from bone marrow cells stored at various time intervals.r unabsorbed fraction

As = total soluble acid fraction J fraction 1 I fraction 2

3 - eluated fraction in 0 .5 M NaCl solution4 = eluated fraction in 1 M_ NaCl solution5 = eluated fraction in 0 .2 iv£NH3 + 2 M NaCl

, . Г а ) eluated in 1 M NH3 + 2 M NaCl6 = fraction J — —

I b) eluated in 1 M_Na OH

FPt = total polymerized fraction.

3. OPTIMUM INTERVAL AFTER IRRADIATION FOR THEADMINISTRATION OF BONE-MARROW CELLS

Few data on the optimum administration of bone marrow after irradiation are available in the literature of the last few years.

Useful work in this respect is that of the Van Bekkum group [13], during which the authors experimented on two strains of mice, CBA and C57BL, and established the optimum interval for administration as 24 h.

In connection with this problem, Costachel et al. [14] conducted a series of studies on the A strain (isologue). The results are g iv en in Table I.

As is clear from Table I, it may be concluded that, under experimental conditions where the cell dose represents a constant and the time factor a variable, the maximum efficiency of myelotransfusion is a function of the time after irradiation. Myelotransfusion between 24 and 48 h after irradiation with 800 or 900 rad gives the best survival of mice (strain A). In addition, with the same dose of irradiation the efficiency of myelotransfusion decreases when it is performed before 24 h or after 48 h.

STORAGE AND TRANSPLANTATION 147

TABLE I. POST-IRRADIATION MYELOTRANSFUSION OF MICE AND MEAN SURVIVAL TIME (LD100/30)

Mice(Nos)

Dose (whole-body

irradiation in rad)

C ell dose(X 106 )

Tim e o f myelotransfusion (hours after irradiation)

Increase of mean survival tim e per

group against control

(%)

20 (control) 900 - ■ - 0

20 900 10 within 1 h after irradiation 2 5 .5

20 900 10 24 7 1 .0

20 900 10 48 7 2 .9

20 900 10 72 2 7 .1

20 900 10 96 8 .0

20 (control) 800 - - 0

20 800 10 within 1 h after irradiation 3 8 .5

20 800 10 24 8 2 .8

20- 800 10 48 8 0 .6

20 800 10 72 3 1 .8

20 800 10 96 11 .0

4. METHOD FOR PRESERVATION OF BONE-MARROW CELLS WITHCRYOPHYLACTIC AGENTS

The ability of cryophylactic agents to protect bone-marrow cells during long-term freezing at -80°C is attributed to the property they possess of impeding the formation of intracellular ice crystals [15] and/or increasing the electrolyte concentration during freezing [16, 17].

The efficiency of the freezing techniques depends on the manner in which the factors influencing the different phases of the freezing process are controlled. These factors are: the freezing and defreezing rate; the preserving temperature; the presence of low molecular weight cryophylactic agents in the cell [18] .

Of particular importance with regard to the last-mentioned factor is the contact time between the cell and the agent, during which the latter penetrates the cell and dehydration takes place (the so-called ’equilibrium period1 ).

Andrian, Corneci and Buzi [19] studied the action of glycerol on dog marrow cells as well as on two types of tumour cell subjected to various durations of contact. In the former, the capacity of the cells to restore animals lethally irradiated was studied and in the latter the ability to form ascites (see Table II).

1 4 8 COSTÀCHEL et a l.

TABLE II. SURVIVAL OF DOGS IRRADIATED WITH 600 RAD AND INOCULATED WITH AUTOGENIC BONE MARROW AFTER VARIOUS PERIODS OF CONTACT WITH GLYCEROL BEFORE INOCULATION

Lot No.(and number o f animals)

Marrow ce ll dose/kg

body weight (X 108)

Duration of contact with glycerol

Incubationtemperature

(deg°C)

Survival by day 30 (N o.)

1 (10) 2 .0 - 3 .5 _ 10/10(a)

2 ( 5 ) 2 .5 - 3 .5 15 min 4 5/5

3 ( 5 ) 2 .5 - 3 .5 15 min 25 5/5

4 (5 ) 2 .6 - 3 .5 1 h 4 3/5

5 (5 ) 2 .6 - 3 .5 1 h 25 2/5

6 (5) 2 .5 - 3 .8 6 h 4 0/5

7 (5) 2 .5 - 3 .8 6 h 25 0/5

Bone marrow from 5 anim als was preserved at + 4°C up to the moment of inoculation, and marrow from 5 other animals was kept at room temperature for 6 h before administration.

Results show that (a) the optimum equilibrating time is 15 min,(b) survival decreases significantly after 1 h of incubation (when incubation was extended to 6 h no animals survived), and (c) in dog bone marrow glycerol has a noxious effect on nucleated cells from the marrow if the contact between it and the cells exceeds 15 min.

The results are in accordance with Kurnick's observations on his in-vitro studies [20, 21] . With the dye exclusion test he established that the decrease of marrow cell viability is proportional to the length of glycerol contact time before freezing.

5. DIFFERENCES IN SENSITIVITY

Differences in sensitivity between mice and monkeys were demonstrated by Van Putten [22] . Standard slow freezing in 30% glycerol-tyrode solution, rapid thawing and the Sloviter procedure give the mice about 60% p reservation as the first protective effect. Fo r m o n k e y s the preservation percentage seems, in preliminary studies, to be below 10, and it was impossible to protect any animal with autogenic bone marrow after frozen storage.

Another experimental model followed the optimum equilibrating time of glycerol with two types of ascites tumour cell: OIA and T8(Table III).

From this experimental model it was determined that:

(a) neoplastic cells are much more resistant to the action of glycerol than the bone-marrow cells;

(b) ascites cells suspended in glycerol medium behaved identically to those suspended in the medium without cryophylactic agents up to 72 h after incubation;


TABLE III. EFFEC T OF GLYCEROL ON THE GRAFTING CAPACITY OF TUMOUR CELLS AFTER VARIOUS PERIODS OF CONTACT BEFORE INOCULATION

TumourInoculatedm aterial

Lethality in m ice after various contact periods between tumour ce lls and glycerol

type(ce ll suspensions)

15 min 1 h 6 h 24 h 48 h 72 h 120 h

OIA

Without glycerol 1G/16 15/15 14/15 16/16 16/16 15/15 -

With '1 r/’/yglycerol 14/15 16Д 6 15/15 15/16 14/14 14/14 -

AscitesWithout glycerol 13/13 15/15 16/16 16Л 6 14/14 15/15 12/16

T8With 15% glycerol 15/15 16/16 14/14 13 Д 4 15/15 16Д 6 0/16

(c) only when incubation was extended to 120 h (T8 tumour) was no ascites obtained in animals which had received incubated suspensions with glycerol, while the controls died due to ascites, but after a longer period of survival.

G. ROLE OF HAEMOTRANSFUSION IN BONE-MARROW PROTECTION

Various authors [23-32] showed that secondary disease may be aggravated as a result of allogenic marrow contamination with immunocompetent cells from the peripheral blood.

In recent works, Vos [33] showed that the higher the number of immunocompetent cells associated with foreign bone marrow, the shorter is the survival time of the animals. Willard and Smith [34] showed that there is a certain gradation in the immunological competence of various types of cell.

Van Putten [22] irradiated and then treated monkeys with mixtures of blood and allogenic bone-marrow cells or splenic or lymph node cells together with marrow and was not able to reproduce the results obtained in rodents.

Corneci et al. (unpublished paper) investigated the effect of total blood transfusion and suspensions of splenic cells from allogenic donors on the restorative capacity of autogenic bone marrow in lethally irradiated dogs.

The plotting of the experiment is given in Table IV.Results demonstrate that: (a) no significant differences exist between

the survival of dogs transfused only with autogenic bone marrow and that of dogs which, besides this therapy, also received total blood transfusion or splenic cells; (b) all animals irradiated with 600 rad and treated with marrow or marrow and transfusions survived (in our experience the dose is 100% lethal); (c) the negative effect of immunocompetent cells on the take of a medullar graft, in our experimental model, might be due to


TABLE IV. SURVIVAL OF DOGS LETHALLY IRRADIATED AND TREATED BY AUTOGENIC BONE MARROW TOGETHER WITH TOTAL FRESH BLOOD TRANSFUSIONS OR SUSPENSIONS OF SPLENIC CELLS FROM ALLOGENIC DONORS

Lot No. (and number

of animals)

Radiationdoses(rad)

No. of marrow cells

inoculated/kg body weight

(X IO 8)

Amount of blood

administered

No. of splenic cells

administeredAg body weight

(X 10s )

Survival

day 30 to day 120

1 (5) 600 2 - 3 0 0 5

2 (5 ) 600 2 - 3 3 X 400 ml ■ 0 5

3 (10) 600 1 .5 - 3 0 3 .5 - 6 .9 9

4 ( 6 ) 1000 3 - 4 0 0 5

5 (5) 1000 2 . 8 - 4 3 X 400 m l 0 4

6 (5) 1000 3 .2 - 4 .5 0 4 - 7 .5 5

N um ber o f an im als

FIG. 4 . Results in splenectomized and untreated dogs and those treated with autologous DNA or autologous bone marrow after whole-body irradiation (600 rad).

competition between the two eell populations. In this case the host performs an autoseparation of inoculated cells, recognizes and prefers those more closely related genetically (hence an autogenetic preference) which take and proliferate while the foreign cells are rejected.

This phenomenon is similar to that described by Mathé et al. [35], who used marrow from multiple human donors, the receiver spontaneously selecting the genetically most appropriate cells.


Some attempts to prevent radiation disease by using xeno- and/or allogenic DNA have been made with more or less favourable results [36-48] .In other experiments, and especially in the more recent ones,. all the results have been negative [49-51],

Our results have also been negative [52]. Throughout we used DNA extracted from autogenic bone marrow and spleen in large doses (from one spleen and all the bone marrow of one dog, i .e. 12.7 - 4 6 . 7 mg/kg body weight in a single or repeated dose). Figures 4 and 5 show the survival and blood recovery rates.

The same negative results (Fig. 6) were obtained with allogenic DNA extracted from whole spleen, bone marrow and liver (50-70 mg/kg body weight).

7. P R O T E C T IO N A F F O R D E D B Y BO N E-M A RRO W (S P L E E N , L IV E R ) DNA

FIG. 5 . Peripheral white blood c e ll counts on four groups of splenectom ized dogs that received 600 rad of whole-body irradiation only or followed by infusion of autologous DNA or autologous bone marrow.

8. RELATIONSHIP BETWEEN HISTOCOMPATIBILITY AND SEX IN THE GRAFT OF HAEMOCYTOPOIETIC TISSUE.

Research conducted by Eichwald and Silmser [53] proved that even with a syngenic strain perfect compatibility of the graft does not exist. Such skin grafts from male donors to female recipients from an inbred strain of C57 BL or A/Yax mice are often rejected,' while grafts from male to male, female to female and female to male almost always take.

1 5 2 COSTÄCHEL et al.

The phenomenon was explained by the fact that on the odd segment of chromosome 4 a gene of histocompatibility might exist which is absent in the female and which releases the immunological reaction of the host against the graft.

Experiments by Lengerova [54] proved that the Eichwald-Silmser phenomenon is blocked immunologically when irradiated syngenic animals are used.

Days after irradiationFIG. 6 . Results in survival of dogs irradiated with 600 rad and treated with allogenic DNA against nontreated control.

0 Treated lotВ Irradiated control lo t .

TABLE V. SURVIVAL AFTER MYELOTRANSFUSIONS IN DIFFERENT SEX-DONOR-RECIPIENT COMBINATIONS (MICE C57 BL AND H)

Lot No.No. o f animals

Donor RecipientX-ray doses

(rad)

No. of in jected elements

(X 106)

Survival by day 60

Percentage

С 54 BL Strain

a 15 _ 900 - 0/15 0

2 15 M M ■ 900 7 10/15 66.6

3 15 F F 900 7 9/15 60

4 15 M F 900 7 4/15 26

5 15 ' F M 900 7 8Д 5 53

H Strain

1 15 - 900 - 0/15 0

2 15 M M 900 7 7/15 4 6 .6

3 . ■ ; 15 ■ F F. 900 7 6/15 40

4 15 M ■ F 900 7 8/15 53

5 15 F M 900 7 7/15 4 6 .6


Costächel et al. [52] attempted to determine whether the Eichwald- Silmser phenomenon occurs in the OIB H mice strain as compared with the C57 BL strain (Table V).

From Table V it can be seen that the best survival in the treated groups of C57BL strain was obtained in combinations where donors and recipients were of the same sex, or when the donors were females and the recipients males. When the donors were males and the recipients females the percentage survival was lower than in the previous groups.

Among the groups of H strain there is no significant difference in the behaviour in all donor-recipient combinations.

Our experiments confirm those of Lengerova on the C57BL line and, moreover, show that the Eichwald-Silmser phenomenon does not take place in all strains of mice.

The exception that we recorded with the strain does not seem to have the capacity of diminishing the importance of sex identity in conditions of transfusion.

9. PROTECTIVE EFFEC T OF HAEMOCYTOPOIETIC EMBRYONICTISSUE

A series of treatments has been attempted against the secondary syndrome, e.g. replacement of adult bone marrow with embryonic haemocytopoietic tissues [55-59].

To observe the influence of the age of embryonic tissue, Costächel et al. used allogenic foetal haemocytopoietic tissues (liver, spleen) in the treatment of lethally irradiated rats and dogs (950 rad) and observed an increase in the development of secondary disease (Tables VI and VII).

TABLE VI. INFLUENCE OF DONOR AGE (RATS) ON PROTECTION WITH HAEMOCYTOPOIETIC EMBRYONIC TISSUE

N o. o f animalsX -ray Survival by day 30

1 Treatm entdose (rad) (N o.) (°Jo)

40 950 Saline solution 0/40 0

15 950 Whole embryo o f 10-12 days 2/15 13(2 X 108 cells)

15 950 Embryo liver o f 12-13 days 3/15 20

15 950 Embryo liver of 18-20 days 7/15 47(2 X 10s cells)

15 950 Embryo liver of 19-20 days 8/15 53(2 X 10s cells)

15 950 Embryo liver + spleen o f 7/15 4719-20 days (2 X 108 cells)


TABLE VII. INFLUENCE OF DONOR AGE (DOGS) ON PROTECTION WITH HAEMOCYTOPOIETIC EMBRYONIC TISSUE

No. of animalsX-ray

dose (rad)Treatment

Survival byday 30

Remarks

10 450 Antibiotics 0/10

10 750 Antibiotics 0A0

10 1350 Antibiotics

H aemocytopoietic tissue

0Д0

3 450 (liver) 2 .5 X 1010 foetal cells (7 weeks) + antibiotics

Haemocytopoietic tissue

1/3

3 750 (liv er)2 .5 X 1010 foetal cells (7 weeks) + antibiotics '

Haemocytopoietic tissue

l/ö

Dead after 46 days33 1350 (liver) 2.5 x 1010 foetal cells

(7 weeks) + antibiotics

Haem ocytopoietic tissue

2/3 and 53 days with secondary syndrome

3 450(liver) 2 .5 X 1010 newborn nucleated cells (24 h) + antibiotics

H aemocytopoietic tissue

0/3

3 750(liver) 2 .5 X 1010 newborn nucleated cells (24 h) + antibiotics

Haem ocytopoietic tissue

0/3

3 1350(liver) 2 .5 x 10:0 newborn nucleated cells (24 h) + antibiotics

0/3

Results show that:

(1) Allogenic foetal haemocytopoietic tissue (liver, spleen, whole embryo) administered to rats favourably modifies the development of radiation disease (survival up to 66%).

(2) The age of the embryo from which the haemocytopoietic tissueis taken influences the degree of protection. In rats as well as in mice the best protection is given by 19- to 20-day foetal liver haemocytopoietic tissue, which is at its period of maximum haemocytopoietic activity when all blood-element precursors are produced.

Results on dogs showed that administration of foetal haemocytopoietic tissues may produce secondary immunological reactions. This is the case


FIG. 7 . Dog 2841. Metaphases from femur bone marrow 9 months after irradiation with a single 600-rad dose and treatment with autogenic bone marrow. Part o f giant polyploid metaphase possibly resulted from endore duplication. Note apparent'stickness*.

Ж # * vЖ

Ж

FIG. 8 . Dog 2936 . Irradiated twice and transplanted with autogenic bone marrow (1 year after irradiation). AC = attraction at centrom ere.BR = break in a chromatid of a large concentric chromosome.R = rearrangement.

1 5 6 COSTACHEL et a l.

with the two animals which, after surviving the 30-day period, died on days 46 and 53, respectively, with lesions typical of the secondary syndrome.

Urso et al. [55J claim that late immunological reactions (weaker than those produced by adult allogenic tissues) may be encountered in mice after administration of foetal allogenic tissues, and Barnes et al. [59] found this to be true even after administration of foetal isogenic tissues. They discovered that 30% of CBA mice irradiated and then treated with CBA foetal liver (syngenic) die of secondary disease.

F IG .9 . Dog 2936. Metaphases 16 months after the last irradiation of a 3 X 600 rad total dose and treatment with bone marrow.В = chromatin bridge between two chromosomes.D = deletion on the X chromosome.R = rearrangement between three chromosomes.AC - centromere attraction.

1 0. LATE CHROMOSOMAL LESIONS IN DOGS IRRADIATED ONCE AND/OR REPEATEDLY AND SUCCESSIVELY TREATED WITH BONE MARROW AND/OR AUTOGENIC SPLENIC TISSUES (FIGS 7-9 AND TABLES VIII-X)

Barnes et al. [60] observed cells with chromosomal abnormalities in irradiated mice after they had rejected allogenic medullary grafts and had been repopulated by their own haemocytopoietic cells; Nowell et al. [61] found 10-80% chromosomal abnormalities in the bone marrow, spleen, thymus and lymph nodes of mice 8-10 months after sub-lethal irradiation.


Yu et al. [62] made a similar observation in hamsters. Goodman and Bender [63] showed an increase in the percentage of chromosomal aberrations in lethally irradiated mice that were restored by allogenic irradiated bone marrow. ,

TABLE VIII. CHROMOSOMAL ABERRATIONS IN IRRADIATED DOGS TREATED WITH BONE MARROW

Dog No. Dose (rad) TreatmentTim e after irradiation

Metaphasescounted

Aberrantmetaphases

m

848 600Autogenic bone marrow

8 months 50 86


10 months 60 80


14 months 50 54


17 months 50 52


19 months 50 30


1 year 90 80^a )


6 days 50 92


14 months 50 46

1738 600Autogenicbone marrow

18 months 50 48

1411 1000Bone marrow + transfusion

17 months 50 78

891 1000Bone marrow + transfusion

9 months 50 44

891 1000Bone'marrow + transfusion

9 months 50 48

814 1000 Bone marrow 9 months 50 54

70 1200 Bone marrow 1 month 50 92

70 1200 Bone marrow 5 months 50 80

^ These data have already been presented elsewhere [52] and were also used in the present tables to obtain a com plete record of a ll rim e intervals.

15 8 COSTÄCHEL et a l.

TABLE IX. CHROMOSOMAL ABERRATIONS IN DOGS AFTER TWO AND THREE IRRADIATIONS WITH 600 RAD AND BONE-MARROW TREATMENT

DogNo.

Total irradiation dose (rad)

No. of irradiations

Treatm entTim e after

last irradiation

Metaphasescounted

Aberrantmetaphases

(%)

2626 600 2Bonemarrow

8 months 50 62


9 months 50 90

600Bonemarrow

1 year 96 8 3 .5 <a)

600 Bonemarrow

15 months 50 84


1 year 50 86

600Bonemarrow

1 year 35 S2


9 months- 50 80

600Bonemarrow

1 year 50 80

600Bonemarrow 16 months 50 74


2 months 50 94

600 Bonemarrow

1 year 94 90(a)

(a ) These data have already been presented elsewhere [52] and were also used in the present tables to obtain a com plete record of all tim e intervals.

Costachel et al. [64] claimed the existence of chromosomal lesions in lethally irradiated dogs that had been repeatedly restored (at one-year intervals) by autogenic bone marrow.

In our work, mongrel dogs were teleirradiated one to three times, by single doses of X -rays (600-1000 rad). These were restored after each irradiation by transfusion of autogenic bone marrow or splenic cells.

At variable intervals (up to 19 months) after each restoration the karyotypes of medullary cells in these dogs were compared with those of non-irradiated control dogs.

In those dogs restored by two types of autogenic cell, modifications were found consisting of a high number of aneuploid cells and chromosomal


TABLE X. CHROMOSOMAL ABERRATIONS IN DOGS AFTER ONE IRRADIATION WITH 600 RAD AND SPLENIC CELL TREATMENT

Dog No. Dose (rad) TreatmentT im e after irradiation

Metaphasescounted

Aberrantmetaphases

(%)

74 600Spleniccells

7 months 50 88

74 600Spleniccells

1 year 50 40

79 600.Spleniccells

7 months 90 80(a)

79 600Spleniccells

1 year 50 34

829 600 17 months 50 38

2003 600Bone marrow + splenic cells

1 day 32 94


1 month 50 90

2003 ■ 600Bone marrow + splenic ce lls

10 months 50 70


14 months 50 40

T h ese data have already been presen ted elsewhere [52] and were also used in the present tables to obtain a com plete record o f a ll tim e intervals.

aberrations persisted for a long time. These took the form of breaks, ring chromosomes, deletions, attraction of centromere, stickiness, re joinings, chromatin bridges, minute or extra long chromosomes, etc.

Lesion frequency for the same time interval was higher in animals repeatedly irradiated and restored.

The progressive accumulation of altered cells (to be found at some time interval after each of the repeated irradiations and restorations) is difficult to explain.

In the case of dogs restored many times the marrow and splenic cells contain both irradiated and non-irradiated cells. The origin of chromosomal lesions in the marrow cells can be explained in two ways:

(1) Repopulation of marrow is by the dogs' own cells and not by the inoculated cells [65], whereas the non-aberrating cells come mainly from the inoculated cells. The high frequency of chromosomal aberrations after two or three irradiations and successive myelotransfusions support this hypothesis. Certain cells harvested after the restorations following the


first and second irradiations and administered after a new irradiation of the dogs already contain a high percentage of aberrations.

The paradoxical situation in which the frequency of aberrations is less after 1000 rad during the same time interval than in dogs receiving only 600 rad is attributed to the fact that after irradiation with 6 00 rad a higher number of cells with biocompatible lesions survive. These contribute to repopulation to a greater extent together with the infected cells, whereas after 1000 rad repopulation is accomplished almost totally by the injected cells.

(2) Cells introduced into the irradiated organism may undergo chromosomal aberrations by an indirect radiobiological effect [66] . This second possibility is supported by the observation by Barschi et al. [6 7] that one cell type nucleus occurred in two different stem -cell lines and by Fadei's observation [68] that nuclear transfer between cells takes place in irradiated tumours in vitro.

R E F E R E N C E S

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12 (1957) 1096.[8] LEWIS. J .P . , TROBAUGH, F .E ., Jr ., A nn.N. Y . Acad. Sei. 114 1 (1964) 677.[9] LEWIS, J . P . , FARNES, M .P ., ALBALA, M ., TROBAUGH, F .E ., Jr ., A nn.N. Y . Acad. S e i. 114 1 (1964)

701 .[10] KURNICK, N .B .. Ann. N .Y . Acad. Sei. 114 1 (1964) 713 .[11] VAN PUTTEN, L .M ., A nn.N. Y . Acad. Sei. 144 1 (1964) 695.[12] TENNANT, J .R . , Transplantation 2 6 (1964) 685.[13] VOS, O ., CROUCH, B .G ., VAN BEKKUM, D .W ., Int. J . Radiat. Biol. 3 (1961) 337.[14] COSTACHEL, O ., CORNECI, I . , ANDRIAN, T . e t a l . , Revta sanit. m ilit., Special Issue (1965) 357

(Vllth M edico-M ilitary Conference, Bucarest, 4 -9 O ct. 1965).[15] MAZUR, P ., A n n .N .Y .A ca d .Sei. 85 (1960) 610.[16] LOVELOCK, J .E . , Ann. N .Y . Acad. Sei. 85 (1960) 610.[17] FERRANT, J . , Nature (Lond.) 205 (1965) 1284.[18] HUGGINS, C . , Fedn Proc. 24, part III (1965) 2.[19] ANDRIAN, T . , CORNECI, I . , BUZI, E ., Paper presented at the 6th Annual Meeting o f the Society

for Radiation Biology, Interlaken, Switzerland, 5 -8 June 1968.[20] KURNICK, N .B ., in Diagnosis and Treatm ent of Acute Radiation Injury. WHO, Geneva (1961) 309.[21] KURNICK, N .B ., Transfusion 2 (1962) 3 .[22] VAN PUTTEN, L .M ., Proc. Int. Symp. on Bone Marrow Therapy and Chem ical Protection in irradiated

Primates (1962) 137.[23] COLE, L . J . , GARVER, R .M ., OKUNEWICK, J . , TransplantnBull. 6 2 (1959) 429.[24] COLE, J .L . , GARVER, R .M ., Nature (Lond.) 184 (1959) 1815.[25] COLE, L . J . , GARVER, R .M ., A m .J.P h ysio l. 200 (1961) 1.[26] CONGDON, C .C . , DUDA, Dorothy В ., ArchsPath. 71 (1961) 311.[27] GOODMAN, J .W ., CONGDON, C .C . , Radiat.Res. 12 (1960) 439.[28] GOODMAN, J .W ., CONGDON, C .C ., ArchsPath. 72 (1961) 18.[29] PORTER, K .A .. CHAPIUS, G ., FREEMAN. M .K ., Ann. N. Y . Acad. Sei. 99 3 (1962) 416 .[30] SMITH, L .H ., CONGDON. C .C . . Lab.Invest. 10 (1961) 3 .[31] VOS, O ., DE VRIES, M .J ., COLLENTEUR, J .C . , VAN BEKKUM, D .S . ,J .n a tn Cancer In st.23 (1959) 53.


[32] VOS, O ., WEYZEN, W. W. , Transplantn Bull. 30 4 (1962) 501/111 - 507/ Ш .[33] VOS, O ., J.n a tn Cancer Inst. ^6 3 (1966) 431 .[34] WILLARD, H .G ., SMITH, L .H .. Transplantation 4 1 (1966) 56.[35] MATHÉ, G ., SCHWARTZENBERG, L ., AMIEL, J .L . , SCHNEIDER. M ., C A T TA N .-A .,

SCHLUMBERGER, J .R . , TUBIANA. M ., LALLANE, C ., Scan d .J.H aem at. 4 (1967) 193.[36] KANAZIR, D .T ., CECUK, O. Z . . KRAJINCANIC, B .N ., HUDNIK, T .A . . Bu ll.In st.n u cl. Sei.

"Boris Kidrich" (Belgrade)^ (1959) 133.[37] KANAZIR, D .T . , BECAREVIC, A ., PANJEVAC, B ., S1MIC, M ., RISTIC. G ., Bu ll.In st.n u cl. Sei.

"Boris Kidrich" (Belgrade) 9 (1959) 145.[38] PANJEVAC, B ., RISTIC, G ., B u ll.In s t.n u cl.Sei. "Boris Kidrich” (Belgrade) 8 (1958) 159.[39] PANJEVAC, B ., RISTIC, G ., KANAZIR, D ., 2nd int. Conf. peaceful Uses atom . Energy

(Proc.Conf.G eneva, 1958) 23, UN, New York (1958) 64.[40] PANTIC, V . , STOS1C, N .7"k ANAZIR, D ., BECAREVIC, A ., JOVICKI, G ., Nature (Lond.) 193

(1962) 83.[41] PANTIC, V . , STOSIC, N .. KANAZIR, D ., BECAREVIC, A ., JOVICKI, G ., Nature (Lond.) 193

(1962) 993.[42] SIMIC, M .M ., PETKOVIC, M .Z . , MANCtó, D .D ., B u ll.In st.N u cl.Sei. "Boris Kidrich" (Belgrade)

9 (1959) 155.[43] KANAZIR, D ., PANJEVAC, B ., CECUK. O .,'R IS T IC , G ., KRAJINCANIC. B ., Radiat.Res. 9

(1958) 137.[44] SAVCOVIC. N. V . , Nature (Lond.) 203 (1964) 1297.[45] SAVCOVlC, N .V ., HAJDUKOVIC, S . I . , I n t . J .Radiat .B io l. 9 (1965) 361.[46] PETROVIC, D . ,B r . , J . Radiol. 39 (1966) 640.[47] WILCZOK, T . , MENDECKI, J . , I n t . J .R adiât.B iol. 9 (1965) 201.[48] POPESCU, G. e t a l . , Revta san itari m il i t . , Special number (1965) 409.[49] KARPFEL, Z ., PALECEK, E ., SLOTOVA, J . , 2nd Int.Congr. Radiat.Res.. Abstracts o f Papers (1962)

148.[50] SMITH. L .H .. CONGDON, C .C . , in Radiation Protection and Recovery, (HOLLAENDER, A, E d .) ,

Pergamon Press, Oxford, London, New York, Paris (1960) 242.[51] SCHMIDT, F . , HUBER, R .. COUTELLE, R ., Acta b io l.m ed .germ . 3 (1959) 624.[52] COSTÄCHEL, О .. CORNECI. I . , ANDRIAN. T . , KITZULESCU, I . . PASCU. D .. Expl H ematol.

12 (1967) 9 .[53] EICHWALD, E . J . . SILMSER, C .R ., Transplantn Bull. 2 (1955) 148.[54] LENGEROVA, A .. CHUTNA, Folia b io l. 5 (1959) 24.[55] URSO, I . S . , P roc .Soc.ex p l B io l.M ed. 100 2 (1959) 395.[56] DUPLAN, J . F . , C .R . S o c .B io l. 155,4 (1961) 712.[57] WOLF. N .S . . C .R .S o c .b io l. 155 10 (1961) 1895.[58] UPHOFF, D .E ., J.n a tn Cancer Inst. 20 (1958) 625.[59] BARNES, D. W .H .. LOUTIT, F . J . . MICKLEM. S .H ., A n n .N .Y . Acad. Sei. 99 3 (1962) 374.[60] BARNES, D .H . W .. FORD. C .E .. LOUTIT, J . F . , Sang 30 (1959) 762.[61] NOWELL, P .C . , HUNGERFORD. A .D ., COLE, J . L . , A n n .N .Y .A cad .S ei. 114 1 (1964) 252 .[62] YU, K .C . , SINCLAIR, K. W ., Science 145 (1964) 508.[63] GOODMAN, J .W ., BENDER, M .A ., Transplantation 2 3 (1964) 134.[64] COSTÄCHEL, O .. CORNECI, I . , ANDRIAN. T . . MAT ACHE. A ., BUZI, E .. POPP. I . , Expl Hem atol.

15 (1968) 126.[65] BARNES, D. W .H ., FORD, C .E . , GRAY, S .M ., LOUTIT. J . F . . Prog.nucl.Energy. Ser.V I 2_(1959) 1.[66] COSTÄCHEL, О ., Hlth Phys. (inPress).[67] BARSCHI, J . , BELEHRADEK. Y . . Expl C e ll Res. 29 (1963) 1 11 .[68] FADEI, L ., Archs A n at.m icrosc. 25 4 (1963) 615.

PRESERVATION OF BONE MARROW FOR CLINICAL USE

A .G . FEDOTENKOVBone-M arrow Preservation and Culture Laboratory,C entral Institute of H aem atology and Blood Transfusion,M oscow, USSR

Abstract

PRESERVATION OF BONE MARROW FOR CLINICAL USE. The author describes the results of many years' research into the problems of obtaining and preserving bone marrow in the quantities required for c lin ica l use. Particular attention is paid to the preservation and long-term storage o f bone marrow at ultra- low temperatures ( -1 9 6 aC ), its separation from the protective medium and methods of determining whether the biological functions of thawed bone marrow have been impaired.

Many experimental and clinical data now available confirm the effectiveness of bone-marrow transplantation in the treatment of radiation injury, hypoplastic anaemia, and other diseases accompanied by depression of haemopoiesis. Special importance therefore attaches to the problem of obtaining the necessary amounts of bone marrow and to the development of satisfactory methods for its long-term preservation.

■ Although the literature contains a number of reports on work devoted to the preservation of bone marrow, many difficulties associated with obtaining and preserving bone marrow in the amounts required for clinical use are still unsolved.

This is especially the case as regards methods of obtaining bone marrow from cadavers, the preservation of bone marrow at positive and negative temperatures, processes for freezing and thawing bone marrow, storage periods, the organization of bone-marrow banks, etc. The studies carried out by the scientists of the Bone-Marrow Preservation Laboratory have for the last few years been devoted to precisely these vitally important problems.

The results obtained indicate that donors at present constitute the main source of bone marrow for homotransplantation, while adult cadavers provide an additional source. For autotransplantation, bone marrow is obtained from patients who have to be given chemo- or radiotherapy in h ig h doses.

Since, at present, sufficient bone-marrow donors are not available, special attention is being paid to the development of methods of obtaining bone marrow from cadavers.

On the basis of the studies carried out it has been shown that, for clinical purposes, bone marrow can be obtained from suddenly deceased persons within five hours after death has occurred. These findings are in almost complete agreement with those of Thomas, Ferrebee and the workers at the Leningrad Institute of Blood Transfusion.

The morphological preservability and functional activity of the bone- marrow cells have been established with supravital staining, phase-contrast microscopy, tissue-culture methods and autoradiography employing 3H- thymidine.

1 6 3

1 6 4 FEDOTENKOV

The quality of the bone marrow depends to a large extent on the way in which it is obtained. We have developed a simple closed method of obtaining bone marrow from the iliac bones and the sternum by perfusion with a preserving solution. In brief, the method, which has no traumatic effect on the cells, is as follows. The iliac bones or sternum are punctured with two special needles, one of which is connected to the preserving solution and the other to an empty flask, which in turn is connected to a vacuum pump. A single closed system with a negative pressure of 150-200 mmHg draws the bone marrow, washed out of the bones by the preserving solution, into the empty flask.

With this method it is possible to obtain from one cadaver up to 500 ml of bone-marrow suspension, containing 3 - 9000 million biologically intact nuclear elements (up to 2000 million from the sternum and up to 7000 million from the iliac bones).

With this method practically no destruction of cells (in particular of erythrocytes) is observed, so that haemolysis in the bone-marrow suspension obtained is usually no more than 14 mg %. The viability of the cells (according to the eosin test) was 85 - 95% during the first 2 - 3 hours following death and 65 - 70% after 4 - 5 hours.

It should be noted that the initial bone marrow obtained from the cadaver is morphologically somewhat different from that obtained from donors. The cadaver bone marrow contains substantially fewer mature elements of the granulocytic series, which can be explained by the poor stability of these cellular elements and their partial destruction before the bone marrow is taken.

It is obvious that the restricted preservation periods of bone marrow at positive temperatures limit its therapeutic use in clinical practice. For the past nine years we have therefore directed our attention in a more promising direction, namely the preservation of bone marrow at a very low temperature (-196° C) in the amounts necessary for clinical use.

During the first stage of our work we gave special consideration to the selection of a suitable protective substance. Like a number of other workers we decided on glycerol, since it is non-toxic and is reliable for use in connection with cell freezing. Chemically pure glycerol is produced industrially in large quantities in many countries, and this is an important factor in the adoption of the freezing method for medical purposes.

_c G lucose : 0.4-с

о .2Sucrose 5 .4EDTA : 0 .1

11 D o u b le -d is tille d waterup to 50 mi

10% g e la tin e , ca lc iu m -fre e ; 35m l Sod ium c itra te : 0 .6 D o u b le -d is tille d w ater up to 50m l

FIG. 1. Composition of solution TSOLIPK N o.3 .

Since the No. 1 and No. 3 sugar-glucose-gelatin solutions developed by us have a favourable effect on the cells, we used the approved solution Tsolipk No. 3 (Fig. 1) for storing the bone marrow prior to freezing and also for dilution of glycerol. The constituents of this solution, especially the mono

BONE-MARROW PRESERVATION 1 6 5

saccharides and disaccharides, give good protection against the damaging effects of low temperatures (N. I.Kalabukhov, 1933, 1946; Bender et al., 1960), and in addition the disaccharides, which do not penetrate into the cells, help to save the latter from destruction during thawing by maintaining the osmotic pressure outside the cell. Gelatin, being a colloidal substance, enhances cell-preservation and in addition — this should be stressed — it promotes the precipitation and separation of a large number of erythrocytes from the bone- marrow cells before they are frozen; this is very important in reducing haemolysis in the bone-marrow suspension after thawing.

This procedure enabled us to transplant into a patient, in one operation, up to 400 ml of thawed and prepared bone marrow from 2 - 5 donors, without the appearance of any post-transfusion haemoglobinuria.

It was confirmed that a 15% glycerol concentration is best for preparation of the bone marrow for freezing, with the marrow being exposed to the glycerol for 30 minutes. These findings were substantiated not only by supravital staining of the cells of the thawed bone marrow, but also by electron- microscope studies carried out in collaboration with E. I. Terenteva. With this period of exposure the structure of the undifferentiated and immature cells hardly changes, the greatest modification being found in the mature granulocytes.

With longer exposure times the harmful effect on the haemopoietic cells increases. After 2 - 5 hours some of the cells are hardly changed, but the vast majority of the cytoplasmic and nuclear cell structures (endoplasmic network, mitochondria, Golgi apparatus) undergo pronounced alterations: they are either completely lysed, or lose their normal morphological structure. Such cells are consistently found to contain many lipid inclusions.

These studies showed that in all cases the.mature granulocytes are most vulnerable, followed by the immature cells of the granulocytic series. The erythropoietic elements and the lymphocytes, are the most stable. The most sensitive sub-microscopic structures are the cytoplasmic membrane structures (endoplasmic network, mitochondria membranes), followed by special granulation. The nuclear structures' are more stable, but not the nuclear membrane.

The electron-microscope studies thus also indicate the need to shorten the time during which the bone-marrow cells are exposed to giycerol before freezing. The data obtained show once more that exposure to glycerol is a very important stage in preparing the bone marrow for freezing.

The rate at which the bone marrow is frozen is highly significant for cell preservation. So far, however, no uniform opinion can be derived from the literature regarding the optimal freezing conditions. This is particularly so as regards large volumes of bone marrow (120 - 150 ml). After developing the equipment for regular freezing and making containers for freezing large volumes of bone marrow, we studied different freezing conditions in. which the freezing chamber was cooled down from ambient temperature to -196° С at rates of 1 degC/min (Regime 1), and 10degC/min (Regime 5),. or in which cooling was effected in two stages, 1 degC/min down to -25°C or -13°C, and then 10degC/min down to -196°C (Regimes 3 and 4).

The best results were obtained with Regimes 4 and 5 (Fig. 2), when the crystalization time was hot longer than two minutes. In these experiments 80 - 85% of the cells were preserved, i .e . there was a decrease of only10 - 15% compared with the initial value.

1 6 6 FEDOTENKOV

Г С

F IG .2 . Freezing of bone marrow at a rate o f 1 degC/min down to -13°C , and then at 10 degC/min to -196°C (Regime 4 ). Initial bone-marrow temperature, 23°C; in itia l room temperature, 15°C.

FIG. 3 . Aluminium containers for freezing 120 ml of bone marrow.


With Regimes 3 and 1, where the second period was 7 - 1 1 minutes, the results were somewhat poorer: after thawing, 70 - 72% of the bone-marrow cells survived, i. e. there was a decrease of 23 - 25% compared with the initial figure.

A comparative analysis of the results of studies on cell preservation and freezing rate at all stages down to -196° С with Regimes 1, 3, 4 and 5 indicates that cooling down to -11° С at a rate of 0.3 -4 degC/min (Regime 4) and 1 - 6 degC/min (Regime 5) does not have an adverse effect.on bone-marrow cell preservation. In all these experiments the percentage cell preservation was higher than with the other freezing conditions which we studied.

Freezing in a third stage at a rate of 2 - 26 degC/min caused no reduction in bone-marrow cell preservation. These findings are supported by studies employing Regimes 4 and 5, in which certain parts of the bone marrow were cooled at 2 - 26 degC/min; the percentage cell preservation was then high (8 0 - 85).

In subsequent work on the freezing of bone marrow for clinical purposes we used Regime 4, i .e . the freezing chamber was cooled at 1 degC/min from 15 to -13°C and thereafter at 10degC/min down to -196°C.

The successful preservation of bone marrow at a temperature of -196° С depends largely on the container used for freezing and on the storage and transport conditions. In trials with different types of aluminium container which we developed (flat or corrugated surface), the best results were obtained with a container having corrugated offset walls (volume 170 - 200 ml) and a cross-section of not more than 1 - 1 . 5 cm (Fig. 3). With a container of this type the number of cells preserved was 4 - 10% lower than the initial quantity, whereas with a container of cross-section 2 cm the reduction was11 - 23%.

The preservation of bone marrow at low temperatures is likewise strongly affected by the conditions of thawing and of washing out the protective substance. Ferrebee et al. showed this clearly in 1957, when they were the first to propose the use of a glucose-saline solution to wash the glycerol from the bone marrow.

We studied three solutions for washing the bone marrow: glucose- saline solutions Nos 1 and 2, and glucose-saccharose solution No. 3, which we suggeste'd in 1960.

The introduction of saccharose into solution No. 3 instead of the saline components is, in our opinion, justifiable since the literature contains data (N.B. Chernyak, 1957) showing that isotonic solutions of sodium chloride suppress leucocyte respiration.

Saccharose, of course, has a pronounced stabilizing effect on the protein systems of blood under preservation (P. S. Vasilev, 1946), which makes the cell structure resistant to destruction.

The method of washing the glycerol off the bone-marrow cells is as follows: 120 ml of thawed bone marrow are transferred f r o m the aluminium container to a sterile 500 ml PVC bag, to which we add \volume (compared with the volume of bone-marrow) of a 48. 6% solution of glucose, diluted with an 8% saccharose or Hank1 s solution. After two minutes, l| volumes of the second part of solution No. 2 or No. 3 are added twice to the bone-marrow. suspension. This results in dilution of the bone marrow by a factor of 4. 5, and gives a final glycerol concentration of 3. 3% and a glucose concentration of 5.4%. The bag with the diluted bone-marrow suspension is centrifuged

for 15 minutes at 1200 rev/m in. The supernatant liquid along with the free haemoglobin and erythrocyte stroma is then transferred by pressure into another vessel.

The bag now contains 100 - 125 ml of bone-marrow suspension, which is suitable for transplantation after filtering.

For washing the glycerol off'the bone marrow the best results were obtained with the glucose-saccharose solution. The percentage of cell preservation is then reduced by 6%, compared with 23% with glucose-saline solution'.'

The advantage of the glucose-saccharose solution is particularly obvious when the ultrastructure of the cells is studied with an electron microscope. After washing the bone, marrow with a glucose-saccharose solution.the cells do not undergo any further alterations; on the contrary, the ultrastructural characteristics of many of the cell elements hardly differ from the normal. When washing with a glucose-saline solution, the nucleus and nuclear membrane of some cells undergo lysis, and sometimes pyknosis of the nuclei and lysis of the cytoplasmic structures are noted.

Since, after thawing, the bone marrow must be'prepared for transfusion (glycerol removal), which is not possible in all therapeutic establishments, we..studied the possibility of transporting thawed bone marrow already prepared for transplanting.

It was found that the cells of such bone marrow survive for 24 hours at .a temperature of 4 - 5°C. Transferring the thawed bone marrow into a thermos bottle with ice (+4°C) for a period of 8 hours has no marked effect on cell preservation. .

To evaluate the biological properties of frozen bone marrow, the latter was subjected to a thorough study.

Before dealing with this, however, I should like to point out that in studying bone marrow-preserved by freezing'(with glycerol as a protective agent); it is not possible to apply all the methods used for studying the biological properties of fresh bone marrow-or bone marrow preserved in solutions. This is due to the fact that the normal methods of studying cell elements in bone-marrow smears are not suitable for the study of bone marrow frozén with glycerol. Once the bone marrow has been thawed and the protective substances washed off, the cells may, owing to their fragility, be easily traumatized when preparing sm eárs. This may lead to an exaggerated estimate of the number of cells' destroyed, an estimate which is refuted by various tests,' in-particular those involving eosin, and phase-contrast and electron microscopy, which reveal the high proportion of morphologically preserved cells.' Apart from this, cells treated with glycerol take up the stain in-excess amounts,' so that-it is impossible to differentiate the cellular elements. The methods of tissue culture and autoradiography and also of myelogram analysis are therefore not suitable for determining the biological quality, of frozen bone marrow. . Our experience shows that a rapid and simple working test in studying.-the morphological preservation of frozen bone marrow consists of applying supravital staining with, a 1%. eosin solution and

■determining the nucleated elements. Phase-contrast and electron microscopy may be used .to study cellular structure. . The viability of frozen bone marrow may be determined under clinical conditions by transplanting bone marrow from genetically different donors and marking the donor leucocytes with 'drum sticks'.

1 6 8 FHDOTENKOV

i


The most objective and convincing evidence regarding the.viability of frozen bone marrow is illustrated by the accumulated data from in-vivo experiments:

(1) the survival of animals after irradiation with superlethal doses and transplantation of preserved autologous bone marrow;

(2) the preservation of parent (stem) cells in the bone marrow; these can be revealed by the cloning of haemopoietic tissue in the spleen of lethally irradiated mice.

Let us consider now some of the data obtained in our studies on the viability of frozen bone marrow. By using supravital staining with a 1% eosin solution and simultaneous determination of the total number of myelokaryocytes it was shown that donor bone marrow stored for 1 - 4 years at a temperature of -196° contains up to 85% of preserved cells.

In an electron-microscope study, the sub-microscopic changes in the cellular elements of the bone marrow were either completely absent or only slight. Regardless of the périod of storage, intact cells of th erythrocytic and granulocytic series were found in cells in which varying degrees of destructive change had taken place. Among the well-preserved cellular elements we encountered all types of cell (from the undifferentiated cells and immature forms of the granulopoietic and erythropoietic series to the rod-nucleated and segment-nucleated granulocytes and normoblasts).

The changes observed in the cells, as we said above, are related to the effect of the glycerol before freezing.

Data were obtained in transplanting frozen bone marrow into dogs irradiated with superlethal doses (1000 - 1200 R) are of interest. This work was carried out in conjunction with J . L. Chertkov and M. N. Novikova. Autologous bone marrow, having been frozen and stored for periods between 15 days and 2 years, was transplanted into dogs in doses of 2. 6 - 6000 million cells and preserved the life of the animals which had been given doubly lethal doses of X -rays, just as effectively as fresh bone marrow. In animals given frozen bone marrow, however, the restoration of bone-marrow haemopoiesis and of the peripheral blood picture occurs somewhat later (Fig. 4).

It is difficult to determine the reason for this difference. It may be that the thawed parent haemopoietic cells recover their proliferative activity only by degrees.

The viability of frozen bone marrow stored for long periods was confirmed in an experiment using the method of cloning of haemopoietic tissue in the spleen of lethally irradiated inbred mice. With this method it was shown that in bone marrow frozen with 15% glycerol and kept for six months (the period of observation) at an ultra-low temperature (-196°C) half the stem cells existing in fresh bone marrow are preserved. These cells also bring about a repopulation of bone-marrow elements in the recipient organism. In a histological study elements of the granulopoietic and erythropoietic series and the megakaryocytic and compound series were found. The microscopic structure of the colonies, and the quantitative relation between the different types of colony obtained through the introduction of frozen bone marrow, were the same as when using fresh bone marrow (40% erythropoietic, 20% granulocytic, 20% megakaryocytic, 20% compound). Only the number of colonies varied, as a function of preservation.

1 7 0 FEDOTENKOV

The biological quality of frozen bone marrow has also been clinically substantiated in the treatment of patients suffering from Hodgkin's disease, malignant neoplasms (with hypoplastic haemopoiesis after chemo- or radiotherapy), and hypoplastic anaemia.

More than 150 transplants have been made with bone-marrow suspension which had been stored at -196° С for between 14 days and 3. 5 years. The doses ranged from 100 to 400 ml, containing 2 - 12 000 million bone-marrow cells, obtained from 1 - 5 donors. Reactions were obtained in 12. 3% of the cases. Most of these were slight, consisting only of a temperature increase of about 1 degC. Sometimes, however, the reactions were characterized by chill, temperature increase up to 39°C, and severe headache.

500- KHb ------- ^ 100’^

¿00 r\ 90 J / 80 - г300

/ \1 \ 60-/

f 60-/

200 /7Л io ¿0-

100- / / ' Л 20 20-

0 / / ( g ) n ( h ) „ ( i )0 12 2£ 36 ¿0 60 72 0 12 % 36 48 60 7'l Ó 12 21 3’6 ¿8 60 Í2

FIG. 4 . Haemopoiesis during period o f activ e recovery in supralethally irradiated dogs given transplantations of autologous bone marrow. Mean values ( in per cent of in itia l value) for the absolute number of bone-marrow and blood ce lls per mm3.

------- Freshly prepared bone marrow------ Preserved bone marrow

Along axis of abscissas— tim e after irradiation (days); along axis of ordinates — number of ce lls (°fc). (a ) M ye- lokaryocytes, (b) immature granulocytic ce lls , (c) erythroblast cells, (d) leucocytes, (e ) lymphocytes, (f)(f) thrombocytes, (g) reticulocytes, (h) erythrocytes, ( i) mean values of haemoglobin content (in per cent of in itia l value).


The th erap eu tic e ffe c t o b served in p atien ts has a lso co n firm ed the b io lo g ica l quality of bone m arrow p re se rv e d at -1 9 6 ° C. F , E . F a in sh te in has alread y rep o rted on th is .

The w ork c a r r ie d out has a ll serv ed as a b a s is fo r e s ta b lish in g a fro zen b o n e-m arro w bank at the C en tra l In stitu te of H aem atology and B lood T r a n s fu sion , w hich w ill o ffe r additional fa c i l i t ie s fo r applying m yelo therap y in m ed ica l p ra c t ic e .

B I B L I O G R A P H Y

BENDER, M .A ., TRAN, P. T , , SMITH, L .H ., Preservation of viable bone-marrow cells by freezing, J . appl. Physiol. 15 (1960) 520.

CHERNYAK, N .B ., Izucenie processov dyhanija i g likoliza lejkocitov (A study of respiration and glycolysis in leucocytes), Voprosy m edicinskoj him ii (Topics in m edical chemistry) III 3 (1957) 218.

FEDOTENKOV, A, G ., Konservirovanie kostnogo mozga dlja kliniëeskih ce le j (T h e preservation of bonemarrow for clin ica l purposes), Doctorate thesis, Moscow (1967).

FEDOTENKOV, A. G ., DANILOVA, L. A ., DISHKANT, I. P ., PAFOMOV, G. A ., KULAGIN, I .N ., Novyj sposobzagotovki trupnogo kostnogo mozga, prednaznafcennogo dlja transplantacii (A new method of obtaining cadaver bone marrow for transplantation), Problemy gem atologii i perelivanija krovi 2 (1963) 28.

FEDOTENKOV, A. G ., DANILOVA, L. A ., MEFEDOVA, N. A. , DISHKANT, I. P . , Konservirovanie kostnogo mozga v plastmassovoj tare (The preservation of bone marrow in plastic m aterial), Problemy gem atologii i perelivanija krovi 1 (1964) 35.

FEDOTENKOV, A. G ., MEFEDOVA, N .A ., DISHKANT, I. P ., Voprosy zagotovki i konservirovanija kostnogo mozga (Obtaining and preserving bone marrow), Abstracts o f papers at 39th Plenary M eeting o f Academic Council of Tsolipk, Moscow (1960) 68 .

FEDOTENKOV, A. G . , MEFEDOVA, N .A ., DISHKANT, I. P ., Voprosy zagotovki i konservirovanija kostnogo mozga (Obtaining and preserving bone marrow), Problemy gem atologii i perelivanija krovi 2 (1961) 46 .

FEDOTENKOV, A. G ., SHISHKINA, I .D . , DANILOVA, L. A ., ZMIEVSKAYA, K .M ., VORONTSOVA, E. I . ,GERASIMOVA, N .A ., Konservirovanie kostnogo mozga pri nizkih temperaturah dlja ego klinifceskogoprimenenija (The low-temperature preservation o f bone marrow for clin ica l use), Problemy gem atologii i perelivanija krovi 2 (1966) 45 .

FEDOTENKOV, A. G ., SHISHKINA, I .D . , LEVITSKAYA, L .A ., Z-amorazivanie kostnogo mozga dlja konservirovanija ego pri nizkih temperaturah (The freezing of bone marrow for low-temperature preservation), Problemy gem atologii i perelivanija krovi 5 (1963) 16.

FERREBEE, J .W ., ATKINSON, L ., LOCHTE, H .L ., McFARLAND, R .В ., RICHARDSON JONES, A .,DAMMIN, G . J . , THOMAS, E .D ., The collection , storage and preparation of viable cadaver marrow for intravenous use, Blood Ы (1959) 140.

KALABUKHOV, N .I . , Spjafcka zivotnyh (Anim al sleep) M. -L . (1946).

NOVIKOVA, M .N ., FEDOTENKOV, A .G ., CHERTKOV, I . L ., Transplantacija svezego i konservirovannogo autologicnogo kostnogo mozga sverhsmertel’ no obluÜennym sobakam (Transplantation of fresh and preserved autologous bone marrow to superlethally irradiated dogs), Problemy gem atologii i perelivanija krovi 13 2

(1968) 16.

PEGG, D .E ., Bone marrow transplantation, London (1966).

SPIZHARSKAYA, L .M ., MAMYSHEVA, Т . К . , К vozmofcnosti ispol'zovanija trupnogo kostnogo mozga v kliniceskoj praktike (T h e possibility of using cadaver bone marrow in c lin ica l practice), Problemy gem atologii i perelivanija krovi 2 (1961) 42 .

TIEL, J . E . , McCULLOCH, E. A ., A direct measurement of the radiation sensitivity o f normal mouse bone-marrow c e lls . Radiât. Res. 14 2 (1961) 213.

PRESERVATION OF BONE MARROW BY DEEP FREEZING WITH POLYVINYL PYRROLIDONE (PVP)

S . S . LAVRIK

R esearch In stitu te o f H a e m a to lo g y and Blood Transfusion,

K ie v , USSR

Abstract

PRESERVATION OF BONE MARROW BY DEEP FREEZING WITH POLYVINYL PYRROLIDONE ( PVP).The author presents original data on the preservation of human bone marrow at -196°C with polyvinyi pyrrolidone (PVP) of low m olecular weight. For bone-marrow preservation the author has used a combined solution of PVP of low m olecular weight - 17%, glucose - 10%, homologous blood serum ~ 10% heparin 2500 int. units, levom ycetin 0.01-5 g and up to 100 ml of double-distilled water. The ratio of bone marrow to protective medium is 1 :1 . The final PVP concentration is between 8 .5 and 9%.

Using morphological, biochem ical and historadiographic investigations the author has shown that the viability of the ce lls is not appreciably lowered during long-term storage. Stress is laid on the necessity of keeping constant low-temperature conditions during prolonged storage of bone marrow.Keeping the low temperature constant, the author succeeded in storing bone-marrow ce lls in liquid nitrogen for a period of four years. This is the longest period for which human bone marrow lias yet been stored.Bone marrow which had been stored for lengthy periods was used for autotransplantation into patients suffering from haem opoietic hypoplasia following use of cytostatic substances and a pronounced therapeutic e ffect was observed.

The problem of b on e-m arrow tran sp lan ta tio n is in sep arab ly linked with w orking out ways of p re serv in g bone m arrow and sto rin g it l'or long p e r io d s .

If bone m arrow can be p re se rv e d , it w ill of co u rse find w ider ap p lica tion , thanks to the p o ss ib ility of e stab lish in g r e s e r v e s and, m o reo v er, prep aring in advance and sto rin g fo r subsequent au totran sp lan tation , bone m arrow fro m people who w ork in the rad ia tio n indu stry or with apparatus fo r rad iatio n th erap y and p atien ts req u irin g therapy with cy to sta tic subs ta n ce s o r rad ia tio n therap y .

The overw helm ing m a jo r ity of r e s e a r c h w o rk ers cu rre n tly re ly p r im a rily on variou s co n cen tra tio n s of g ly ce ro l as a p ro tectiv é medium in the p re se rv a tio n of bone m arrow by fre e z in g .

E x istin g data in the l ite ra tu re and our own data show that a 15% solution of g ly ce ro l in com bination with hom ologous blood seru m and tis su e cu ltu re fluid p o s s e s s e s ra th e r high p ro tectiv e p ro p e rtie s . Howe v e r , the use of g ly ce ro l has c e r ta in se r io u s d isad van tages, m ainly due to the n e c e s s ity of w ashing off the g ly ce ro l a fte r thawing the c e ll susp ension .

We have acco rd in g ly been studying in th is lab o ra to ry s in ce 1962 the p o ss ib ility of using polyvinyl pyrro lid one (P V P ) so lutions as a p ro tectiv e m edium fo r b o n e-m arro w p re se rv a tio n . T o d eterm in e the e ffica cy of P V P fo r th is purpose we have c a r r ie d out s e v e r a l s e r ie s of te s ts .

In p a r t ic u la r we have studied the p ro tectiv e p ro p e rtie s of an aqueous solution of P V P by i t s e l f and in com bination with hom ologous blood • se ru m , H an k 's sa lin e so lu tion , g lu co se , e tc . The freez in g was su p er-

1 7 3

1 7 4 LAVRIK

T A B L E I. V IA B IL IT Y O F DONOR BO N E-M A RRO W C E L L S A F T E R STO RA G E A T T° -196°C (%)

Donors 12 months 24 months 48 months

1. I 89 89 89

2. G 90 90 87

3. P 91 90 88

4. S 90 90 89

5. E 90 90 86

6. P 90 92 87

quick, quick and slow (program m ed ). The d eg ree of cooling achieved w as T° -7 8 .5 °C (dry ice ) and T° -196°C (liquid n itrogen ).

We have studied the in flu ence of P V P so lutions on b o n e-m arro w c e lls at te m p e ra tu re s above fre e z in g , the p ro te ctiv e p ro p e rtie s of P V P when so lu tions a re fro zen to T° -78°C and T° -196°C and the e ffe c t of d iffe ren t ra te s of fre e z in g .

T he bone m arrow to be p reserv ed was taken fro m ra b b its by a s p ir a tion and cu re tta g e . In the c a s e of human su b je c ts the bone m arrow was taken fro m the sternum and i l ia c bone by rep eated a sp ira tio n s .

T h e se in v estig atio n s have shown that a p ro te ctiv e m edium containing P V P does not dam age the c e lls at te m p e ra tu re s above fre e z in g . Unlike a g ly ce ro l m edium , the b on e-m arrow c e lls m ay be kept in P V P solution fo r m o re than 24 h o u rs , and a fte r the p ro ced u re a ll the c e ll com ponents a re a liv e (T ab le I). The bone m arrow w as kept a t T° +2°C fo r a period of 24 h o u rs .

When b on e-m arrow c e lls tre a te d with an aqueous solution of P V P (fin al co n cen tra tio n 8 .5%) a re d ee p -fro z e n , the av erag e num ber of v iable c e lls surviv in g is 57%. Addition of hom ologous blood seru m to the P V P aqueous so lu tion in c r e a s e s the c e ll yield to 72% a fte r fre e z in g .

Addition of Hank’ s sa lin e solution to the P V P solution plus hom ologous blood seru m gives a low er yield of v iab le c e lls (67% ). Conseq u ently , instead of Hank1 s sa lin e so lu tion we have added to the p ro te c tiv e m edium a g lu cose so lu tion , w hich re su lte d in the yield of v iable b o n e-m arro w c e lls in cre a s in g to 95%.

As a re su lt of our t e s t s , the follow ing com p osition is suggested fo r a p ro te c tiv e m edium :

P V P , m o le cu la r w eight 12.6 : Hom ologous blood seru m : G lu co se :L ev om ycetin :H eparin :D o u b le-d istilled w ater:

17 g 10 g 10 - 12 g0 .015 g2500 in tern atio n al units up to 100 m l

DEEP FREEZING WITH PVP 1 7 5

Follow ing th e se p o sitiv e r e s u lts with rab b it b on e-m arrow c e l l s , in our next s e r ie s of te s ts we studied the p o ss ib ility of p re serv in g human bone m arrow and sto r in g it fo r lengthy p erio d s.

O ur e x p erim en ts showed that with program m ed fre e z in g (down to T° -1 5 to -18°C at a ra te of 1 degC /m in, and then down to T° -78°C at a ra te of 10 degC/m in) 94±3 .4% v iab le c e lls su rv iv e a fte r s to ra g e in dry ic e fo r 30 d a y s .

When fro zen bone m arrow is kept at T° -78°C fo r 12 m onths, a s many as 85±5.9% b o n e-m arro w c e lls re ta in th e ir v ia b ility .

The e ffe c tiv e n e ss of the suggested p ro te ctiv e m edium fo r human bone m arrow fro zen in liquid n itrogen (T° -196°C ) was studied s im u ltaneou sly . The fre e z in g w as c a r r ie d out at a ra te of 1 degC/m in down to T° -1 5 to -1 8 °C , at 10 degC/m in fro m T° -15°C to T° -60°C and th e r e a fte r at 20 degC/m in down to T° -196°C .

It was found that if the su sp en sio n is sto red in n itrog en fo r th ree m onths the yield of v iab le c e lls is 92±2.6% , a fte r one y e a r 90±1.9% , a fte r two y e a rs 90±2.4% and a fte r four y e a rs 89%.

T A B L E II. M YELO G RA M O F DONOR G

Conditions of bone-marrow ce ll storage C ell elem ents °

Initial data 24 hours

1. Haemohisti oblasts 0 .8 0 .2

2 . Haemocytoblasts 0 .8 0 .6

3 . Premyelocytes 0 .8 1 .0

4 . Myelocytes 8 .6 8 .2

5. M etam yelocytes 14 .0 1 4 .0

6. Neutrophiles stab-like ' 2 0 .0 1 8 .0

7. Neutrophiles segmented 2 5 .2 2 6 .0

8. Eosinophils 0 .8 0 .6

9. Basophils 0 .2 0 .2

10. Monocytes 1 .6 1 .2

11. Lymphocytes 6 .0 7 .2

12. Reticulocytes 0 .6 0 .2

13. Plasmacytes 1 .0 0 .4

14. Pre-erythroblasts 0 .8 0 .4

15. Erythroblasts 4 .2 3 .0

16. Normoblasts 1 4 .2 1 9 .0

17. Megakaryocytes 0 .4 0 .0

18. Indeterm inate ce lls 0 .0 0 .0

1 7 6 LAVRIK

T h e se data su ggest that:

(a) The yield of v iab le c e lls a fte r s to ra g e in liquid n itrogen fo r four y e a rs is h ig h er than a fte r s to ra g e in d ry ic e fo r one y e a r ;

(b) The yield of v iab le c e lls is not reduced ap p reciab ly a fte r being fro zen down to T° -196°C and stored fo r a long tim e in liquid n itrogen .T h is su g g ests that the s to ra g e tim e s rep o rted by us a re not lim itin g v a lu es . It is p o ssib le that if the lo w -tem p era tu re conditions a re kept s t r ic t ly constan t it w ill be p o ssib le to s to re bone m arrow ' in a b io lo g ica lly un im paired s ta te fo r s e v e r a l y e a rs , p o ssib ly even d ecad es. T a b le s II,III and IV give m yelo gram s of the th ree d ifferen t d onors.

In addition to the conventional m o rp h olog ical m ethods of in vestig atin g the v ia b ility of p re serv e d c e lls (eosin te s t , lu m in escen t and p h a s e -c o n tra s t m icro sco p y , m yelog ram count, e tc .) we have a lso studied the fu nctional a c tiv ity of the c e l ls , in p a r tic u la r the d istrib u tio n and re s id e n ce tim e of p re se rv e d b o n e-m arro w c e lls in the o rgan ism of healthy and irra d ia te d ra b b its . T he values obtained w ere s im ila r to th o se obtained w ith lab elled c e lls fro m fre s h donor bone m arrow .

To d eterm in e the c l in ic a l ap p licatio n s of b o n e-m arro w c e lls p r e s erv e d by the u se of P V P a fte r long p eriod s of s to rag e we c a rr ie d out au to tran sp lan tation s on 16 p atien ts su fferin g fro m acu te d ep ressio n of

T A B L E III. M YELO GRA M O F DONOR P A F T E R TH E P R E SE R V E D BO N E-M A RRO W C E L L S HAD B E E N K E P T F O R A Y E A R

C ell elements %

1. Haemohistioblasts 0 .0

2 . Haemocytoblasts 0 .5

3. Premyelocytes 0 .0

4 . Myelocytes 2 .5

5. M etam yelocytes 1 1 .0

6. Neutrophiles stab-like 1 3 .0

7. Neutrophiles segmented 3 6 .5

8 . Eosinophils 0 .5

9. Basophils 0 .0

10. Lymphocytes 1 7 .5

11. Monocytes 0 .5

12. Plasmacytes 0 .0

13. Reticulocytes 0 .0

14. Pre-erythroblasts 0 .0

15. Erythroblasts 1 .0

16. Normoblasts 1 7 .0

DEEP FREEZING WITH PVP 1 7 7

T A B L E IV . M YELO G RA M O F DONOR N B E F O R E TH E BO N E-M A RRO W C E L L S HAD B E E N FR O Z E N AND A F T E R T H E Y HAD B E E N STO R ED F O R A Y E A R

C ell elem ents Before treatmentAfter treatment before freezing

After storagefor 365 days

1 . Haemohistioblasts 0 .3 0 .0 0.0

2 . Haemocytoblasts 0 .5 0 .2 5 1 .0

3 . Premyelocytes 1 .0 0 .7 5 0 .5

4 . Myelocytes 9 .0 10 .25 1 4 .0

5 . M etam yelocytes 6 .4 1 0 .5 11.0

6 . Neutrophiles stab-like 9 .5 18 .2 5 1 4 .0

7 . Neutrophiles segmented 3 3 .3 2 3 .5 2 9 .0

8 . Eosinophils 1 .7 2 .0 1 .0

9 . Basophils 0 .0 0 .2 5 0 .0

10. Lymphocytes 1 7 .8 9 .75 5 .5

11 . Monocytes 1 .6 0 .2 5 0 .0

12. PlasmacyteS 0 .5 1 .0 1 .5

13. Reticulocytes 0 .2 0 .5 0 .0

14. Pre-erythroblasts 2 .1 0 .7 5 0 .0

15. Erythroblasts 7 .4 9 .7 5 7 .0

16. Normoblasts 8 .7 12 .25 1 5 .0

h aem o p o iesis follow ing chem otherap y fo r m alignant tu m o u rs. In 12 out of the 16 p atien ts the leu co cy te count reach ed the in itia l (p re-ch em o th erap y ) le v e l 2 0 -3 0 days a fte r tran sp lan ta tio n of autologous p re se rv e d bone m arro w . T h e re w as a lso an in c r e a s e in the num ber of p la te le ts .

T h e se c l in ic a l o b serv a tio n s show that bone m arrow p re se rv e d by u se of P V P m ay be given to p atien ts without rem oving the P V P fro m the b o n e-m arro w c e ll su sp en sio n a fte r thaw ing.

Thus our e x p e rim e n ts , tak en to g e th e r , su gg est that iso la te d Ь д е - m arrow c e lls tre a te d w ith a p ro te c tiv e m edium containing P V P can , by • deep fre e z in g , be kept fo r s e v e r a l y e a rs in a s ta te su itab le fo r tra n sp la n ta tio n .

D

SCIENTIFIC AND ORGANIZATIONAL PROBLEMS OF BONE-MARROW CELL BANKS

SCIENTIFIC AND ORGANIZATIONAL PROBLEMS CONNECTED WITH THE ESTABLISHMENT OF BONE-MARROW AND BLOOD-COMPONENT BANKS

A .£ . KISELEVCentral Institute of Haematology and Blood Transfusion,Moscow, USSR

Abstract .

SCIENTIFIC AND ORGANIZATIONAL PROBLEMS CONNECTED WITH THE ESTABLISHMENT OF BONE- MARROW AND BLOOD-COMPONENT BANKS. The author describes the results of studies carried out with a view to devising rational means for the long-term preservation of bone-marrow and blood cells at ultra- low temperatures. Attention is especially directed to freezing conditions and cryoprotective agents.- The. paper stresses the significance o f the apparatus that has been constructed for programmed freezing operations and analyses the data regarding bone-marrow donors.

The present lev el of knowledge in regard to the preservation of bone-marrow and blood components makes it possible to organize banks for their long-term storage, thereby permitting more extensive c lin ica l use of bone-marrow transplantation and haemotherapy.

E xten siv e in form ation has been obtained on the e ffe c tiv e n e ss o f bone m arro w , blood and blood com ponents sto red at v ery low te m p e ra tu re s .B y v irtu e of th is fa c t a lon e , the question of estab lish in g banks fo r the ' lo n g -te rm sto ra g e of such su b stan ces should be co n sid ered .

Nobody now doubts the e ffe c tiv e n e ss of autologous bone m arrow 'w hen h aem o p o iesis is d ep ressed in p e rso n s with m alignant grow ths resu ltin g from ' rad ia tio n tre a tm e n t arid chem otherap y. '

It m ust a lso be b orne in mind th at, with the w id espread use of n u clear energy in v ario u s b ran ch es of s c ie n ce and technology , one cannot co m p lete ly exclude irra d ia tio n a cc id e n ts among p erso n s whose w ork involves rad ia tio n s o u rc e s . N eith er can one exclude the p o ss ib ility o f e x c e s s iv e exp osu re to rad iatio n as sp ace r e s e a r c h tak es m an beyond o rb ita l to in te r p lan etary f lig h ts . . It is obvious that in such c a s e s au tom y elo tran sp lan ta- tions a re p o ss ib le only i f one has banks fo r the lo n g -te rm sto ra g e of bone m arro w . T h e re is c l in ic a l ev idence that the tran sp lan ta tio n of a llog en ic bone m arrow is to a c e r ta in extent e ffe c tiv e in the tre a tm e n t o f su b -acu te hypoplastic an aem ias cau sed by v ario u s fa c to rs (including rad iation) having ■ a d ep ress iv e in fluence on haem opoiesis-. The o rig in of th is type of d isease is s t i l l u n certa in .

F o rtu n a te ly , th e re have so fa r been only e x tre m e ly r a r e , iso la te d c a s e s of acute rad iatio n d ise a se in m an. N e v e rth e le ss , a llo m y elo tran sp lan ta tio n shows co n sid erab le p ro m ise as a m ethod of tre a tin g acute rad ia tio n d is e a s e s . T h is has been con firm ed by m any exp erim en ta l in v estig atio n s c a r r ie d out on la rg e an im a ls . O bviously, the e x is ten ce of banks fo r th e 'lo n g -te rm s to ra g e o f bone m arrow w ill help to en su re that tran sp lan ta tio n s a re p e r form ed in tim e , p a rticu la r ly when it is n e c e s s a ry to use bone m arrow fro m s e v e r a l d onors. ' 1

The accu m ulated ch e m ica l ev idence in d ica tes that blood and blood com ponents — e ry th ro c y te s , leu co cy te s and th rom b ocytes — can be sto red

1 8 1

1 8 2 KISELEV

at low te m p e ra tu re s o v er long p eriod s without p re ju d ice to th e ir b io lo g ica l p ro p e rtie s .

B y e stab lish in g banks fo r the lo n g -te rm sto ra g e of haem otherap eu tic su b stan ces it w ill be p o ssib le to m eet the g re a tly in cre a se d m ed ica l demand a r is in g out of the need fo r m a jo r b lo o d 'tra n s fu s io n s . T h is b eco m es e s p e c ia lly im portant when it is a question of r a r e blood groups such as rh e su s - n eg ativ e , and is p a rtic u la r ly so in the c a s e of the A B(IV ) group.

F o r exam p le, 5 -7 l i t r e s o f blood (o r even m o re) a re u su ally req u ired in h e a rt op eration s involving e x tra c o rp o re a l c ircu la tio n o r a change of blood. A rh e su s-n e g a tiv e patient can obtain th is quantity of blood fro m 10 -1 5 rh e su s-n e g a tiv e d onors.

In addition, we should not ov erlook the fa c t th at, when blood p lasm a is p rep ared on a la rg e s c a le and vario u s th erap eu tic p rep aratio n s a re d eriv ed fro m it , th ere n a tu ra lly rem ain s ig n ifica n t q u antities of e ry th ro cy te s fo r which th e re is freq u en tly no c l in ic a l u se . B y sto rin g th ese e ry th ro cy te s in the fro z e n s ta te one can p re s e rv e th e ir th erap eu tic p ro p e rtie s fo r long p e rio d s. F u r th e r evidence has been obtained reg ard in g the p o s s ib ilit ie s of using le u c o c y te s . F o r exam p le, in c a s e s of d ep ressed h aem o p o iesis m a ss iv e doses o f leu co cy te s fro m s e v e r a l donors a re so m e tim e s ad m in iste re d ; th is tre a tm e n t is based on the fa c t that a s m a ll num ber of s tem c e l l s , capable of repop ulation , c irc u la te among the leu co cy te s in the p e rip h e ra l blood of healthy p e rso n s . I f the p roblem of p re se rv in g the b io lo g ica l p ro p e rtie s of e ry th ro cy te s fo r long period s is solved , it w ill be p o ssib le to p rep are in advance the la rg e q u an tities n e c e s s a ry fo r c l in ic a l ap p lica tio n s.

It fo llow s that the estab lish m en t o f banks fo r the lo n g -te rm sto ra g e o f bone m arro w , blood and blood com ponents is a p roblem of p rim a ry im p o rtan ce .

O bviously, now that m any of the p rin cip a l a sp e c ts o f the p roblem of lo n g -te rm sto ra g e have been so lved , the conditions e x is t fo r se ttin g up such banks.

It is g e n e ra lly recogn ized that the m ost p ro m isin g m ethod of p r e s e r v a tion is s to ra g e at e x tre m e ly low te m p e ra tu re s (-1 9 6 °C ), w hich co m p lete ly in h ib its m etab o lism , in the c e lls and consequ ently en ables them to be kept . in an a n a b io tic .s ta te .

H ow ever, we now know th a t, although it su p p resse s c e ll m e ta b o lism , deep fre e z in g ca u se s v e ry su b stan tia l dehydration and e x c e s s iv e co n cen tra tio n of s a lts and o th er d isso lved su b stan ces insid e and outside the c e ll due to the fo rm ation of ice c r y s ta ls .

The .m ost im portant event in the study ,of c e ll p re se rv a tio n by fre e z in g has undoubtedly been the d isco v e ry of c ry o p h ila c tic com pounds w hich p r e vent c e ll dam age fro m lo w -tem p e ra tu re e ffe c ts .

Of the cry o p h ila c tic com pounds tha.t have been developed, dim ethyl sulphoxide, g ly ce ro l and polyvinylpyrrolidone have found the w id est a p p lic a tion . In v estig atio n s at our In stitu te have d em onstrated the advantage, fro m the point of view of m aintain ing the o sm o tic equ ilib riu m of the c e l l , of using in com bination v ario u s p ro tectiv e agents capable of actin g in sid e and outside the c e ll .

W ithout going into the s p e c ia l fe a tu res o f the individual c ry o p h ila c tic com pounds, it is worth noting that such com pounds a re c h a ra c te r iz e d by a com m on p ro tectiv e m ech an ism based on the a b ility of a p a rtic u la r su bstance to p en etra te into the c e ll and to form sta b le bonds with w a te r , th ereb y p r e venting the fo rm ation of re g u la r ice c r y s ta ls .

MARROW AND BLOOD-COMPONENT BANKS 1 8 3

E x p e rie n ce has shown th at, although no s ig n ifica n t d iffe ren ce is o b serv e d betw een the p ro p e rtie s o f c e l ls p re serv e d with the help o f d ifferen t p ro te ctiv e a g en ts , th e re a re grounds fo r p re fe rr in g dim ethyl sulphoxide and p olyvinylpyrrolidone to g ly c e ro l.

L eav in g asid e the d eta ils of the v ario u s fre e z in g s y s te m s , we would sim p ly m ention that cooling to -196°C ap p ears to give the b e s t re s u lts ; with th is m ethod the c e lls p ass ráp id ly through the c r i t i c a l te m p e ra tu re zone and only s m a ll ice c r y s ta ls a r e fo rm ed . T h is has been v e r ifie d by X - r a y stu d ies of the s tru c tu re of e ry th ro cy te s a f te r rapid fre e z in g in liquid n itrogen .

A point w orth noting is th at, ' with the u ltra -lo w fre e z in g o f la rg e am ounts of bone m arrow and blood it is n o t .n e c e s s a ry to u se p ro tectiv e agents in high con cen tratio n s'. T h is is p a rt ic u la r ly im p ortant when one is using g ly c e ro l, w hich has to be w ashed off a f te r d efreez in g by m eans of e x tre m e ly involved p ro ced u res .

In v estig atio n s conducted a t our In stitu te o v er a num ber o f y e a rs have cu lm inated in the developm ent of ra tio n a l m ethods fo r thawing bóne m arrow and blood and fo r w ashing off the p ro tectiv e com pounds (F ig . 1). In p a r t ic u la r , we have e sta b lish e d that g lu c o s e -s u c r o s e so lu tions á re b e tte r than g lu c o s e -s a lt so lu tions fo r w ashing g ly c e ro l o ff bone m arrow in that a g re a te r proportion (80-85% ) o f the m y elo k ary o cy tes re m ain in ta ct.

V ario u s co n ta in e rs have been developed fo r the fre e z in g , lo n g -te rm sto ra g e and tra n sp o rta tio n of bone m a rro w , blood and blood com ponents. A lum inium co n ta in e rs with co rru g ate d s u r fa c e s and a c e r ta in c r o s s - sec tio n have proved b e s t . The. idea of using T eflo n sa c k s fo r such p u rp o ses is a v e ry a ttr a c tiv e one.

An e s s e n tia l re q u irem en t of lo n g -te rm sto ra g e banks is equipm ent fo r the p rogram m ed fre e z in g of bone m arro w , blood and blood com ponents (F ig . 2). Equipm ent fo r p rog ram m ed fre e z in g is m anu factu red by L inde AG; in the Soviet Union, s im ila r equipm ent is produced in M oscow , K harkov and T b il is i . In addition, bunkers have been co n stru cted fo r the s to ra g e of la rg e am ounts of such th erap eu tic m a te r ia ls in liquid n itrog en .

C om p rehensive in v estig a tio n s 'h av e con firm ed that a high proportion (85-96% ) of b o n e-m arro w and blood c e l ls s to red in liquid n itrog en fo r 1 -4 y e a rs re ta in ed th e ir b io lo g ica l p ro p e r tie s .

T h is can be shown by analysing the data obtained in stu d ies involving sta in in g w ith a 1% solution of eo sin w ith p a ra lle l counting of the to ta l num ber o f n u cleate c e l ls and the r e s u lts o f e le c tro n -m ic ro s c o p e c e ll s tu d ies .

It is w orth noting’ that m o re th an .70% of 51C r -la b e lle d e ry th ro c y te s , d efrozen a fte r lo n g -te rm s to ra g e , take and c ir c u la te fo r the n o rm al period in the blood of the re c ip ie n t (T|. > 30 days)/

W hile not w ishing to b e litt le the value of such data, we would em p hasize that the pronounced th erap eu tic e ffe c t o b serv ed when m y elo k ary o cy tes and blood c e lls a re usèd a fte r long p eriod s of s to ra g e (up to 4 y e a rs ) is probably the m o st s ig n ifican t evidence fo r the fa c t that they have re ta in ed th e ir b io lo g ic a l p ro p e r tie s . ........ '

In d iscu ss io n s o f ¡the o rg an iza tio n a l and s c ie n tif ic a sp e c ts o f estab lish in g banks fo r the lo n g -te rm sto ra g e of bone m a rro w , sp e c ia l atten tion should be paid to the donor q u estio n . At p re se n t, m y elo k ary o cy tes fo r a llo m y elo - tran sp lan ta tio n p u rp oses a r e e a s ily obtained fro m d onors.

PART .1

GLUCOSE : Ö.ASUCROSE ■ 4.3EÖTA N02 : 0.1DOUBLY-D fô T iU ED WATER TO

PART II

10V. GELATIN SOLUTION NEUTRAL SODIUM CITRATE LEVOMYCEÍIN DOUBLY- DISTILLED MAKE UP 50 m l

STERNUM

SEPARATION OF GLYCERIN FROM BONE MARROW BY WASHING

80NE MARROW TRANSFUSION

FIG. 1. Schem atic diagram o f the sequence o f stages in freezing, storage and thawing o f bone marrow.

184 K

ISELEV

MARROW AND BLOOD-COMPONENT BANKS 185

LIQUID NITROGEN UNDER LOW PRESSURE

ELECTRIC CABLE

TEMPERATURERECORDINGDEVICE

SENSING DEVICE TO - GAUGE TEMPERATURE DIFFERENCE

THERMOSTAT TO CONTROL CHAMBER TEMPERATURE

HEATER

FAN TO CIRCULATE GAS INSIOE CHAMBER

TRIP (R E LIE F) VALVE OR VENT

FIG. 2 . Layout o f BF-4 freezing system (from +25°C to -1 5 0 °C ).

In the Sov iet Union, bone m arrow is obtained fro m donors on the sam e b a s is a s blood, the b a s ic p rin cip le being "n o h arm to the health o f the donor — m axim um b en efit to the p atien t" .

B o n e -m a rro w donors a re s e le c te d by the s ta f f o f blood supply s e r v ic e s , by m e m b ers of the Red C ro s s So cie ty and by d o cto rs a t the m ed ica l e s ta b lis h m ents w here the p atien ts req u irin g a llo m y elo tran sp lan ta tio n a re h o sp ita lized .

B o n e -m a rro w donors a r e g e n e ra lly s e le c te d fro m am ong re g u la r blood d on o rs; how ever, bone m arrow is so m e tim e s taken fro m p erso n s who have n ev er given blood, including frien d s and re la tio n s of the p atien t.

Only healthy p erso n s 2 0 -4 0 y e a rs of age a r e accep ted a s b o n e-m arro w d on o rs. They a r e exam ined m e d ica lly by the' d o cto rs o f the blood supply s e r v ic e b e fo re each b o n e-m arro w re m o v a l. The h ae m ato lo g ists at whose re q u e st the bone m arrow is to be rem oved a r e a lso p re sen t at such e x a m inations .

The d o cto rs m u st s a tis fy th e m se lv e s that the condition o f the donor is such that bone m arrow can be rem oved w ithout p re ju d ice to h is h ealth .E a s ily e x c ita b le , em otion ally u n stable p erso n s a r e not accep ted as d onors. T he m ed ica l exam in ation inclu d es o b lig a to ry haem oglobin s tu d ies , e ry th ro cy te , le u co cy te , th rom b ocyte and re tic u lo c y te cou n ts, a d iffe ren tia l le u c o cy te count and evaluation of the e ry th ro cy te sed im en tation ra te .

It m ust a lso be esta b lish e d that the donor has n ev er had sy p h ilis , m a la r ia o r h e p a titis .

Up to 200 m l o f b o n e-m arro w su sp en sio n , containing (2 -4 ) X 108 m y e- lo k a ry o cy te s , m ay be taken fro m a donor at one tim e , by m ean s o f 7 -1 0 punctures o f the s tern u m and the i l ia . The donor m ay give blood four m onths la te r provided he p a s s e s a com p lete c l in ic a l and h aem ato lo g ica l exam in ation . A fu rth e r rem o v a l of bone m arrow is p erm itted only a fte r a y e a r i f the donor h as continued to give blood and a fte r s ix m onths i f he has not.

O bserv ation s involving the study of h aem o g ram s and m y elo g ram s and of vario u s h aem op oietic fa c to rs have shown that the re m o v a l o f bone m arrow in p e rm is s ib le am ounts and a t ap p rop ria te in te rv a ls does not produce any harm fu l e ffe c t on the o rg an ism of the donor.

1 8 6 KISELEV

Further research will be directed towards establishing the maximum permissible amount of bone marrow which may be removed and the minimum intervals of time between removals.

.With the establishment of bone-marrow banks, a special group of donors has come into.being — people whose bone marrow is stored for possible use in automyelotransplantation. They include persons with malignant tumours who are about to undergo radiation treatment and chemotherapy. Obviously, the health requirements for normal bone-marrow donation purposes do not apply to this group.

We have been able to discuss in only very general terms the principal questions connected with the establishment of banks for the long-term storage of bone marrow, blood and blood components. We feel we have presented convincing evidence that, in the present state of knowledge regarding the effectiveness of these substances, the use of freezing to preserve the biological properties of cells and the absence of danger to the donor, suitable conditions now exist for the establishment of such banks, which will undoubtedly contribute to the more widespread use of myelotransplantation and haemotherapy.

BASIC CONSIDERATIONS FOR THE ESTABLISHMENT OF A BONE-MARROW BANK

R. KLENUniversity Hospital, Tissue Bank,Hradec Králové, Czechoslovak Socialist Republic

Abstract

BASIC CONSIDERATIONS FOR THE ESTABLISHMENT OF A BONE-MARROW BANK. The author discusses the problem of establishing bone-marrow banks in the most econom ic and effectiv e manner in sm all countries. The point of view o f m edical science is also mentioned. The author believes that it would be suitable to incorporate the bone-marrow bank as a unit within the framework of a tissue and/or organ bank.

Transplantation of bone marrow, like transplantation of other tissues and organs, is a very attractive idea. It was performed clinically for the first time by Mallarmé [1] 30 years ago. During the Second World War, however, attention was diverted from it, since it was necessary to solve more pressing problems. Nevertheless, this time was not completely lost for bone-marrow transplantation, because the immunological basis of modern grafting was laid. The results of post-war study of the action of ionizing radiation on man caused a renaissance of bone-marrow grafting, which both uses general rules of transplantation and constitutes a suitable study model of these radiation-induced processes. The vast set of experimental studies and a number of clinical experiences with bone-marrow preservation and transplantation made it possible to formulate the clinical application of stored bone-marrow transplantation in the present time, though these opinions are not universally accepted. Clinical application of bone-marrow transplantation and the technical equipment and methods involved form the basis of this paper on the establishment of a bone- marrow bank.

Every project in a health service should primarily contain a purely medical nucleus which, however, is enveloped by a solid coat of economy that determines the level and the extent of the care to be provided. From this point of view, the bone-marrow bank as an independent unit could exist only in large countries. Thus the small countries have two possibilities: first, to join together to form large groups, and secondly to find a model suitable for each of them individually. In the present paper the author deals with the establishment of a bone-marrow bank in a small country.

Although an attempt is made to preserve the integrity of the classical medical specialities and to avoid splitting them up, it is necessary to incorporate the various branches within a wider and more comprehensive specialization. Since the expansion is lateral as well as forwards, union of different spheres occurs not only with medicine but also with other sciences. In this way, surprising symbioses developed which were advantageous to all parties. These occupy an important place in our consideration.

Clinical application of preserved bone marrow is the first subject to be discussed. The decision on whether or not the necessity for bone-marrow

1 8 7

1 8 8 К LEN

transplantation is indicated lies with the clinician and, as has already been mentioned, there are differences of opinion in .this respect: There are three indications: first, the post-irradiation syndrome, second, treatment with cytostatics causing inhibition of the bone-marrow and, finally, aplastic anaemia. The post-irradiation syndrome may be a sequel of professional or war injury or of therapeutic irradiation which causes bone-marrow aplasia. The chemical equivalent of irradiation is the cytostatics, used-not only in the therapy of tumours but also for immunosuppression in transplantation. In the treatment of post-irradiation syndrome due to an injury in otherwise healthy subjects the most suitable method is to use autogenous bone-marrow withdrawn from the subject and preserved prior to the injury. The application of autogenous bone marrow in subjects with tumours, though examined very carefully, must be considered a hazard. In the author's opinion, every transplantation involving immunosuppression should have as a prerequisite a store of autogenous bone marrow. Aplastic anaemia can be cured only with homogenous bone marrow. With professionally incurred damage, the number of the injured is relatively small as a rule, and they can usually be transported without delay to a suitable hospital, preferably a hospital to which a bone-marrow bank is attached. Therapeutic treatment by irradiation or the administration of cytostatics leads to bone-marrow aplasia.These procedures can be planned ahead of time and with a known locality. Therefore patients may be transported to a suitable hospital except in wartime, when the number of patients would be too large to transport or treat. Since the transport of preserved bone marrow can also be easily performed (as is mentioned later on), the site or location of the bone- marrow bank does not seem to be a problem with respect to the location of the patient, although it should not be underestimated in some cases.The only reason for which the location is important is strategic.

Another aspect which must be considered is the technical equipment and the methods used for withdrawal, examination, preservation, and distribution of bone marrow.

Withdrawal of the marrow is the first problem. In autogenous bone- marrow transplantation the problem is confined mainly to the technical procedure. It can be carried out either in the blood bank or in the haematology department, or in another laboratory specialized in this work: Inhomogenous transplantation, the donor may be either living or dead. In the first case, the technical procedure is complicated by finding the donor and recompensing him for the bone marrow withdrawn, and in the other case by the permission for withdrawal, which is necessary in most countries. In living donors, the withdrawal may be carried out in the same departments as those for the withdrawal of autogenous bone marrow. F o r withdrawal from a dead donor, a blood bank or a haematology department is out of the question, but a tissue bank would be very suitable, especially if it also included in its activities the withdrawal and preservation of organs and/or preservation of cell strains, as is the case in the author's laboratory.

■The examinations constitute the second stage in the preparation of preserved bone marrow. They are relatively numerous, and come under several specialities with different criteria. F irs t, haematological examinations must be carried out, followed by immunological, bacteriological and, sometimes, biochemical examinations; before the preserved bone marrow is used it is also necessary to make sure of its viability. The haematological examinations consist primarily in counting the elements and in taking the

BONE-MARROW BANK 1 8 9

differential bone-marrow count. These methods are commonly used, fairly simple and thus easily accomplished. On the other hand, the immunological examinations, except for the examination of the basic blood groups, are highly specialized and precise, and so far are performed only in a few laboratories throughout the world. It seems likely that in future the establishment of such laboratories will be a decisive factor with regard to the establishment of organ banks. Bacteriological sterility controls are not exacting and can therefore be easily carried out. In some cases it is useful to perform the GPT and GOT examinations, which are the domain of biochemical laboratories. In cases where the transplantation of bone marrow is carried out in a hospital to which a bone-marrow bank is attached, the bank takes care of both thawing the marrow and removing the endocellular cryophylactic agents [2] as well as making a rapid and reliable examination of the viability of the preserved elements. Most of the methods of vital staining, which as a rule are not very difficult, are used for such tests [3], although opinions on these methods differ.

The third stage consists of short-term or long-term preservation.F o r short-term preservation, i . e. cooling, an ice-box will generally suffice. Long-term preservation with deep freezing of the impregnated elements is a substantially more complicated and expensive method, re quiring special apparatus for programmed lowering of temperature and a special deep freezer. Such apparatus is not commonly available but forms part of the equipment of cell-strain banks. The freezers either depend on motors or use compressed gases. Motor-powered devices that reach very low temperatures are rather complicated and depend on a continuous supply of electric current, so that a special power source is advantageous. In countries that produce enough compressed gases the motor equipment is less suitable, from the standpoint of both reliability and cost, as compared with devices using solid C 0 2 or liquid N2. Devices that use liquid N2 have been developed to a particularly high degree of perfection. Their cost does not substantially differ from the cost of the motor equipment, which by no means reaches such low temperatures. The rate of repair is much lower and the price of liquid N2, especially if it is locally supplied, is considerably lower than that of electric power. Bone marrow preserved on a long-term basis by deep freezing and impregnation with endocellular cryophylactic agents is thawed quickly immediately before application and the endocellular cryophylactic agents are removed.

Distribution is the last phase within the competence of the bank. This depends on how far the preserved bone marrow has to be transported. The blood banks and, especially, the tissue banks and cell-strain banks have experience in despatching deep-frozen material.

Indispensable to the work of a bone-marrow bank is efficient record keeping, which, together with the documentation of the department performing the bone-marrow transplantation, creates a basis for evaluation of the re sults, in which both the bank and the department carrying out the transplantation take part. This activity represents the first step in the research which should be an integral part of all progressive work. Experience shows that, in addition to development, applied and basic research can also be carried out in a department engaged in routine work. Such a department has all the prerequisites for taking part in the training of postgraduates, medical students and laboratory technicians.

1 9 0 KLEN

From the above analysis it follows that a bone-marrow bank can be established as a unit within the framework of some other department, as exemplified by a tissue bank. Since the tissue banks will in future also become organ banks, the extension of their activity to another tissue, i. e. bone marrow, is a logical continuation of the conception of tissue banking for transplantation purposes. From the point of view of efficiency, amalgamation with other departments working on similar principles, e.g. cell banks, should also be planned. Such an institute, built up step by step with the above-mentioned units, would definitely be the most suitable economically and would also ensure unity of the new border discipline, cryobiology.

A special and basic prerequisite is detailed calculations to obtain the optimal solution of given problems in individual countries. Detailed discussion of this factor has purposely been omitted from this paper because economic and organizational conditions vary from country to country. If, however, interest is shown in these calculations, we shall gladly undertake the task of performing them in co-operation with the Department of Social Medicine of the University. The task would consist, firstly, of the development of a general method of calculation, secondly, of the preparation of a program for the computer and, thirdly, in carrying out the calculation and its evaluation. The parties interested would only supply the input data, in the first instance the prices. By processing these data we can determine, for each participant, several solutions, from which the best one will be chosen. Another factor exists, however, which must not be forgotten; it is of a very individual character and has upset many a well prepared plan. This is the factor by which the exact economic calculation must be multiplied.Since it is very individual, the final result depends on it. It is a factor which we shall call "the human factor", and by this we mean mainly enthusiasm, devotion, ingeniousness and endurance.

R E F E R E N C E S[ 1 ] CONGDON, C . el a l . , J.n a tn .C an cer Inst, ^ (1 9 5 2 ) 73.[ 2 ] HUGGINS, C .E . , Fedn.Proc. Suppl. 15 (1965) SlflO.[3 ] PEGG, D .E ., Cryobiology 1 1 (1964) 64.

SUMMARY AND RECOMMENDATIONS OF THE PANEL

1. SUMMARYAccording to the Panel's evaluation, the Agency's interest in the topics

of bone-marrow transplantation and tissue transplantation is reaffirmed.Bone-marrow transplantation is important because of its potential use

in the treatment of persons who have suffered accidentad, or therapeutic radiation damage or who are suffering from diseases of the haemopoietic system.

Bone-marrow transplantations are being performed in a number of clinical institutions. So far, the marrow used has been fresh autologous or allogenic marrow aspirated from suitable donors and injected intravenously into selected recipients. In a number of cases, marrow, suspended in media such as glycerol or DMSO, has been stored at low and ultra-low temperatures and transfused after thawing. However, there is no method as yet that can be universally recommended for marrow collection, storage and transfusion. Nevertheless, research on these methods is being carried out intensively in a number of institutions. Because of the potential world-wide requirement for bone-marrow transplantation, for red blood-cell transfusions and for the treatment of radiation casualties and all states of aplastic disease, a close collaboration between the IAEA and WHO is urged in these efforts.

The Agency should keep a record of all the institutes in the world actively engaged in bone-marrow transplantation so as to maintain contact with them and to be able to use them as a source of human bone marrow when necessary. The Agency should also record the cases of patients who undergo bone- marrow transplantation and publish this information periodically for the benefit of the Member States.

The Agency should encourage scientific groups in the various Member States to establish bone-marrow banks and tissue banks on a national basis. These should be associated with competent tissue-typing laboratories as well as cryobiology and viability-testing laboratories.

The Agency should incorporate the fields of bone-marrow transplantation and tissue transplantation into its research contract program with emphasis on improving and developing methods for testing the viability of stored cells and for tissue compatibility.

Finally, it was considered that both the Agency's Manual on Radiation Haematology and this Panel Proceedings will contribute usefully towards implementing the recommendations of the Panel Meeting and will serve to further the existing program on radiosterilization of biomedical products, established in 1966.

2. RECOMMENDATIONS OF THE PANEL

(1) The panel considers bone-marrow transplantation to be one of the most challenging problems in modern immuno-haematology, and at the same time a subject of great promise since the steps in its future development are clearly demarcated and solutions proposed can be experimentally verified.(2) Several methods of preserving and storing bone-marrow and blood cells were discussed. However, more clinical and experimental work is necessary before a firm recommendation can be given on the efficacy of any one method.

1 9 1

1 9 2 SUMMARY AND RECOMMENDATIONS

(3) Since most'in-vitro tests to determine the viability of bone-marrow arid blood cells after storage do not show a direct proportionality between the vital sign investigated and the repopulating ability of bone-marrow cells and the functional capacity of red blood cells, new research appears essential to improve existing methods and to develop new ones. While there are experimental methods, such as ' spleen-colony counting' after transfusion of bone-marrow cells into irradiated mice, that do appear useful in studying viability, they are not yet applicable for clinical purposes. However, the further development of bone-marrow culture tests and of heterospecific marrow transplantation may be able to reduce the existing uncertainties in this field. Pre-clinical experiments with sub-human primates are recom mended before new techniques are introduced for clinical use. Methods of radioactively labelling blood cells before transfusion appear useful for investigating the retained viability of these cells.(4) There is no quantitative information on the length of time that bone- marrow or blood cells can be stored for clinical use. However, it appears that very low temperatures such as below -130°С are better for long-term storage than are higher temperatures. It is recommended that a worldwide list of potential living bone-marrow and blood leucocyte donors be established on the basis of histocompatibility testing and stored on computers for easy retrieval. This approach will be of great importance after it has been proven that haemopoietic stem cells form part of the circulating leucocytes and after suitable leucocyte separation methods have become widely available.(5) The Panel discussed extensively the problem of secondary disease after haemopoietic cell grafting. It was noted that no single method is able to control secondary disease in human and non-human primates. However,the better the histocompatibility of the donor to the recipient, the less severe is the secondary disease. At the present time there are physical, chemical and immunological methods to decrease the severity of secondary disease.It appears that an important task would be to improve the possibility of using histocompatibility typing for the specific purpose of bone-marrow and blood cell transplantation. The establishment of laboratories carrying this out routinely on a world-wide basis and as a chain of communicating centres that would serve all regions equally well is to be encouraged.(6) The Panel recommends that the Agency take the lead in establishing bone-marrow and blood-cell banks in association with existing blood banks and tissue-typing laboratories. Particular consideration should be given to regions where radiation accident victims are to be treated as well as persons suffering from different types of haemopoietic disease. This activity should be undertaken in association with other international and national organizations. This would further serve to solve the problem of organ transplantation in general through the development of organ storage procedures, of histocompatibility tests and of control of donor-host-graft reactions.(7) The Panel recommends the establishment of a committee of experts to formulate specific plans for the organization of a chain of interconnected 'transplantation centres' . These centres would consist of co-operative institutions and carry out the organization of training courses in tissue typing for scientific and technical personnel in the field of bone-marrow transplantation. ■ Furthermore, ad-hoc working groups should be appointed to consider problems in the field of bone-marrow, tissue and organ preservation, storage and viability as well as grafting reactions.

LIST OF PARTICIPANTS

M em b ers of the P a n e l

K .A . ANTONYAN

M .J . ASHW OOD-SMITH

J . L . CH ERTKO V

C . CONGDON

O . CO STA CH EL

H. E IB L

F . E . FA IN SH TEIN

A .G . FED O TEN K O V

T .M . FL IE D N E R

A. Y a . FR IE D E N SH TE IN

A rm enian Institu te of H aem atology and B lood T ra n sfu sio n ,

Y erev an , USSR

M ed ica l R e s e a rc h C ouncil, R ad io b io lo g ica l R e s e a rc h Unit, H arw ell, D idcot, B e r k s ,United Kingdom

C e n tra l Institu te of H aem atology and B lood T ran sfu sio n ,

M oscow , USSR

B io lo g y D iv ision ,O ak Ridge N ational L ab o ra to ry ,Oak R idge, T e n n .,United S ta tes of A m erica

O n co lo g ica l In stitu te ,B u c h a re s t 62, R om ania

Ö s te r r e ic h is c h e s In stitu t für H aem od erivate G es. m. b. H. ,

V ienna, A u stria

C e n tra l Institu te of H aem atology and B lood T ra n sfu s io n ,

M oscow , USSR

C e n tra l Institu te of H aem atology and B lood T ran sfu sio n ,

M oscow , USSR

A bteilung fü r K lin isch e P h y sio log ie d er U n iv ersitä t U lm ,

U lm , F e d e r a l R epublic of G erm any

In stitu te of M icrob io lo g y , n e a r G am alea,M oscow , USSR

1 9 3

1 9 4 LIST OF PARTICIPANTS

N. G. KARTASHEVSKY Leningrad Institute of Haematology and Blood Transfusion,

Leningrad, USSR

H. E. M. KAY Royal Marsden Hospital and Institute of Cancer Research,

London S. .W.3, .United Kingdom

A. E. KISELEV Central Institute of Haematology and Blood Transfusion,

Moscow, USSR

R. KLEN Píednosta tkáñové ústredny,Fakultní Nemocnice v Hradci Králové, Czechoslovak Socialist Republic

Ukraine Institute of Haematology and Blood Transfusion,

Kiev, USSR

D. METCALF Walter and Eliza Hall Institute,Royal Melbourne Hospital, Melbourne, Victoria, Australia

R. V. PETROV Institute of Biophysics,Ministry of Public Health of the USSR, Moscow, USSR

M. RADOTIC Boris' Kidric Institute of Nuclear Sciences Vinca, Belgrade, Yugoslavia

L. SCHWARZENBERG Institut de Cancérologie et d1 Immunogénétique,

Hôpital Paul Brousse, Villejuif, France

V. SERAPHIMOV-DIMITROV Institute of Haematology and Blood Transfusion,

Sofia, Bulgaria

D.W. VAN BEKKUM . Radiobiological Institute,Organization for Health Research TNO, Rijswijk, Z. H. ,The Netherlands

F . R. VINOGRAD-FINKEL Central Institute of Haematology and Blood Transfusion,

Moscow, USSR

LIST OF PARTICIPANTS 1 9 5

E . A . ZOTIKOV

Representatives of National and

W. SEELENTAG

W. R. BIBB

Central Institute of Haematology and Blood Transfusion,

Moscow, USSR

International Organizations

Radiation Health Unit,World Health Organization, Geneva, Switzerland

Medical Research Branch, Division of Biology and Medicine, US Atomic Energy Commission, Washington, D. C. ,United States of America

SECRETARIAT

Scientific G. KOZINETS Division of Life Sciences,Secretaries: V. AGRANENKO IAEA, Vienna,

Austria

Editor: E. DOYLE Division of Publications, IAEA, Vienna,Austria

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SUBJECT GROUP: Li fe Sciences

Bone-Marrow - IAEA

Documents

Transcript of Bone-Marrow - IAEA