Microprocessor–controlled vs. “dump–freezing”platelet and ... · combined with 6%...

Volumen 63, No 3 VOJNOSANITETSKI PREGLED Page 261

Correspondence to: Bela Balint, Military Medical Academy, Institute of Transfusiology, Crnotravska 17, 11 040 Belgrade. E-mail: [email protected]

O R I G I N A L A R T I C L E UDC: 615.387:641.437

Microprocessor–controlled vs. “dump–freezing”platelet andlymphocyte cryopreservation: a quantitative and qualitativecomparative study

Mikroprocesorski kontrolisana kriokonzervacija trombocitai limfocita nasuprot neprogramiranom zamrzavanju:

kvantitativna i kvalitativna komparativna studija

Bela Balint*/†, Dušan Vucetić*, Biljana Drašković‡, Danilo Vojvodić‡,Goran Brajušković║, Miodrag Čolić‡, Miroljub Trkuljić*

Military Medical Academy, *Institute of Transfusiology, ‡Institute for Medical Research,║Institute of Pathology, Belgrade; †Institute for Medical Research, Belgrade

Abstract

Background/Aim. Thermodynamical and cryobiological pa-rameters responsible for cell damages during cryopreservation(cryoinjuries) have not yet been completely explained. Thus,freezing procedures should be revised, exactly optimized toobtain an enhanced structural and functional recovery of fro-zen–thawed cells. The aim of this study was to compare mic-roprocessor–controlled (controlled–rate) with the compensati-on of the released fusion heat and “dump–freezing” (uncon-trolled–rate) of the platelet and lymphocyte cryopreservationefficacy. Methods. Platelet quantitative recovery (post–thawvs. unfrozen cell count), viability (using hypotonic shock res-ponse – HSR), morphological score (PMS), ultrastructural(electron microscopy) properties and expression of differentsurface antigens were investigated. In lymphocyte setting, cellrecovery and viability (using trypan blue exclusion test) as wellas functionality (by plant mitogens) were determined. Control-led–rate freezing and uncontrolled–rate cryopreservation werecombined with 6% (platelets) and 10% (lymphocytes) dimethylsulfoxide (DMSO). Results. Platelet recovery and functionalitywere superior in the controlled–rate system. The majority ofsurface antigen expression was reduced in both freezing groupsvs. unfrozen cells, but GP140/CD62p was significantly higherin controlled–rate vs. uncontrolled–rate setting. Controlled–rate freezing resulted with better lymphocyte recovery andviability (trypan blue–negative cell percentage). In mitogen–in-duced lymphocyte proliferative response no significant inter-group difference (controlled–rate vs. uncontrolled–rate) werefound. Conclusion. The data obtained in this study shownedthe dependence of cell response on the cryopreservation type.Controlled–rate freezing provided a superior platelet quantitativeand functional recovery. Lymphocyte recovery and viability werebetter in the controlled–rate group, although only a minor inter-group difference for cell proliferative response was obtained.

Key words:cryopreservation; blood platelets; lymphocytes;dimethyl sulfoxide; cell survival.

Abstrakt

Uvod/Cilj. Termodinamski i kriobiološki parametri, odgovorni zaoštećenje ćelija do kojih dolazi tokom kriokonzervacije (krio-oštećenja), još uvek nisu sasvim razjašnjeni. Stoga, postupke zamrz-vanja treba tako optimizirati da se poboljša i strukturni i funkcionalnioporavak zamrznuto-razmrznutnih ćelija. Cilj ove studije bio je upo-ređivanje efikasnosti kriokonzervacije trombocita i limfocita prime-nom programirane kriokonzervacije (mikroprocesorom kontrolisanabrzina hlađenja), uz kompenzaciju oslobođene toplote fuzije, i nep-rogramirane kriokonzervacije (bez kompjuterske kontrole brzinehlađenja). Metode. Istraživali smo kvantitativni oporavak trombo-cita (broj ćelija posle odmrzavanja nasuporot broju ćelija pre zamrza-vanja), vijabilnost (koristeći reakciju trombocita na hipotoni šok –HAR), morfološki skor trombocita (PMS), ultrastrukturne (elektro-mikroskopski) karakteristike i ekspresiju različitih površinskih antige-na. Kod limfocita odredili smo njihov kvantitativni oporavak i vija-bilnost (primenom testa isključivanja tripan plavog), kao i funkcio-nalnost (stimulacijom pomoću mitogena). Programirana i neprogra-mirana kriokonzervacija su kombinovane sa 6% (trombociti) i 10%(limfociti) dimetil-sulfidom (DMSO). Rezultati. Oporavak trombo-cita bio je bolji pri uslovima kontrolisane brzine. Većina ekspresijepovršinskih antigena snižena je bila u obe ispitivane grupe u odnosuna nezamrznute ćelije, dok je ekspresija GP140/CD62p bila znatnoveća pri uslovima programirane u odnosu na neprogramiranu krio-konzervaciju. Programirana kriokonzervacija je rezultovala boljimoporavkom i vijabilnošću limfocita (procenat tripan plavo – negativ-nih ćelija). Nismo utvrdili značajnu razliku među grupama (progra-mirana vs neprogramirana kriokonzervacija) u proliferativnoj reakcijilimfocita prilikom ispitivanja reakcije limfocita nakon indukcije mito-genom. Zaključak. Podaci dobijeni u ovoj studiji pokazali su zavis-nost oporavka ćelija od vrste kriokonzervacije. Zamrzavanje prikontrolisanoj brzini omogućilo je bolji kvantitativni i funkcionalnioporavak trombocita. Oporavak i vijabilnost limfocita bili su bolji ugrupi sa programiranom kriokonzervacijom, mada je konstatovanamala razlika u proliferativnoj reakciji ćelija među grupama.

Ključne reči:kriokonzervacija; trombociti; limfociti;dimetilsulfoksid; ćelija, preživljavanje.

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Balint B, et al. Vojnosanit Pregl 2006; 63(3): 261–270.

Introduction

Current increasing use of different cell–mediated thera-peutic approaches has resulted in increasing needs for bothblood cells and operating procedures in order to minimizecell damages during their collection, processing and storagein liquid or frozen state. Freezing of isolated cells by variouscryopreservation systems has been investigated for manyyears as a method of long–term storage at cryogenic (sub-zero) temperatures. Current freezing techniques are devel-oped from the initial report of the cryopreservation of bovinesperm 1. Cryothermal damages (cryoinjuries) may be the re-sult of extensive cell dehydration (“solution effect”) or intra-cellular ice crystallization (“mechanical damage”) and theyresult in cell destruction 2–8. Different freezing procedures(modifications of the Polge–Smith–Parkers method) are inroutine use for hematopoietic stem cell and progenitor, aswell as mature blood cell cryopreservation. The ability ofhuman stem cells to survive a freezing–thawing procedureresults in marrow repopulation and hematopoietic reconsti-tution after transplantation 9–21. Nowadays numerous freezingtechniques are used in the practice of platelet 22–31 and lym-phocyte 32–36 cryopreservation. However, the optimal cryo-preservation strategy with the greatest quantitative andqualitative cell recovery is still unsolved.

In this study, the influence of the different cryopreserva-tion approaches on the platelet and lymphocyte quantitativeand functional recover was compared. It is proposed that theuse of controlled–rate vs. uncontrolled–rate (“dump–freezing”)procedure will provide a superior cell recovery. Cryopreserva-tion was carried out using our own controlled–rate systemsdesignated for lymphocyte and platelet cryopreservation, aswell as by a standard "dump–freezing" technique under opti-mized dymethyl sulfoxide (DMSO) conditions.

Methods

Platelet and lymphocyte collection and testing

The study included 70 whole blood units collectedinto CPD SAGM plastic bags (Terumo, Japan), designatedfor platelet (n = 35) or lymphocyte (n = 35) harvest. Theprimary buffy–coat (BC–I) was obtained by centrifugation(3 890 × g for 10 min) (Hettich, Germany) and processingof whole blood (450±45 ml) collected from donors insteady–state hematopoiesis into a quadruple bag system(Terumo, Japan). Secondary buffy–coat (BC–II) and buffy–coat derived platelet concentrates (BC–PC) was preparedby centrifugation (377 × g for 5 min) and processing fromBC–I 12, 28. Lymphocytes were isolated from BC–II by cen-trifugation (320 × g for 35 min) using Ficoll–Paque (Phar-macia Fine Chemicals, Sweden) as a gradient agent. BC–PC samples (2 ml) were dropped by an automatic micropi-pette into thermo–stable plastic tubes for testing (controlgroup) or cryopreservation. Lymphocytes resuspended inRPMI 1640 culture medium (Serva Feinbiochemica, USA)were taken in the same quantities (2 ml) into plastic tubesfor in vitro investigations (control group) or cryopreserva-tion 28, 31, 34.

Platelet count was determined by flow cytometry(Technicon, USA). Platelet morphology was examined by aphase–contrast microscope (Polyvar, Austria) 31. The PMSwas calculated according to the numerical values (bal-loons = 0; dendrites = 1; spheres = 2; discs = 4) of differentplatelet shapes 28, 29. The platelet viability was assessed ac-cording to their hypotonic shock response (HSR) as de-scribed earlier 31, 32. Platelet ultrastructure was examined us-ing an electron microscope (Philips, The Netherlands).Platelet surface antigens (GPIb/CD42b, GPIIb–IIIa/CD41,GP140/CD62p, GP53/CD63, and GPIX/CD42a) were ex-amined using specific monoclonal antibodies (CLB, TheNetherlands), while afterward cells were analyzed by an Ep-ics XL (Coulter, USA) 25.

Lymphocyte count in the unfrozen (control) and fro-zen/thawed samples was quantified manually by a Neubauerchamber. Cell viability was investigated by the trypan blueexclusion test. Based on the quantity and viability of thawedlymphocytes, post–cryopreservation total trypan blue–nega-tive lymphocyte recovery (TBNCR) was determined. To as-sess functional statement of cryopreserved lymphocytes 0.5 ×106 cells were cultivated with different concentration of plantmitogens, phytohemaglutinin (PHA) (Sigma, USA) and con-canavalin–A (Con–A) (Sigma, USA), in complete RPMI 1640culture medium in 96–well microplates (Flow, Irvine, UK).Within the last 18 h cells were pulsed with 3H–thymidine(1 μCi/well) (Amersham, UK). Radioactivity was determinedon a beta scintillation counter (Beckman, Germany). Data areexpressed as counts per minute (CPM) ± SE and the stimula-tion index (SI) 33–37.

Cell cryopreservation

Cell samples (2 ml) were placed into plastic tubes forcryopreservation. Then, an equal volume (2 ml) of DMSO(Sigma, USA), diluted in autologous plasma (platelet setting)or RPMI 1640 culture medium (lymphocyte setting) wasadded to cell suspensions. DMSO in 6% (for platelets) and10% final concentration (for lymphocytes) with both con-trolled–rate and uncontrolled–rate freezing were combined.Thus, four cryopreservation protocols: platelet and lympho-cyte controlled–rate (Con−Plt and Con−Ly) and uncon-trolled–rate (Unc−Plt and Unc−Lymphocyte) were investi-gated.

The uncontrolled–rate freezing without the balancedcooling rate (around 1–2 °C/min) was performed after theequilibration period by simple placing of thecell/cryoprotectant suspension into a mechanical freezer at –90±5 °C 19–21. Our own controlled–rate procedures for plate-lets 28 and lymphocytes 12 were accomplished by a PlanerR203/200R and Planer 560–16 (Planer Products Ltd, Eng-land) with the equalized (1 °C/min) cooling velocity. Theplatelet controlled–rate freezing procedure consists of thefollowing stages: I = -5 °C/min, to 0 °C; II = 0 °C/min, for 5min (equilibration); III = -1 °C/min, for 5 min; IV = -2 °C/min, for 5 min; V = -1 °C/min, for 30 min and VI = -5 °C/min, for 5 min. The controlled–rate lymphocyte freezingmethod consisted of the following stages: I = -5° C/min, to 0° C;II = 0 °C/min, for 5 min (equilibration); III = -2 °C/min, for 5



min; IV = -1 °C/min, for 35 min and V = -5 °C, for 5 min. Dur-ing the phase transition period (liquid to solid state) from –5 °C to –15 °C (platelet setting) and from 0 °C to –10 °C(lymphocyte setting) an intensified (double over) coolingrate (2 °C/min) was used due to the compensation of the re-leased fusion heat 10, 28.

After the completion of controlled–rate freezing proce-dures, cells were placed into a mechanical freezer (Eurospi-tal, Italy) at the temperature of -90±5 °C till thawing (one totree months) and the structural and functional investigationsthat followed. The frozen cell samples were thawed rapidlyin a water bath at 37±3 °C.

Statistical analysis

The results were expressed as a mean value ± standarddeviation (SD) or mean value ± standard error (SE). Prolif-erative response of lymphocytes after controlled–rate vs. un-controlled–rate freezing was compared by the General LinearModel method and Paired Samples t–test. The results ob-tained before and after freezing of platelets were comparedby Student's t–test, using the Original PC Program. The dif-ferences were considered as significant at p < 0.05.

Results

The results related to platelet and lymphocyte recoveryand viability obtained in this study are presented in Table 1.

The intergroup (controlled–rate vs. uncontrolled–rate)differences showed that there was better platelet and lym-

phocyte quantitative recovery (p < 0.05) in the controlled–rate groups. Uncontrolled–rate freezing caused higherplatelet HSR reduction with the significant intergroupdifferences (p < 0.01). Regarding cell viability, there were nosignificant differences neither between the cryopreserved vs.control group, nor between lymphocytes frozen by bothfreezing methods. On the contrary, the TBNCR values forlymphocytes were significantly (p < 0.05) higher in thecontrolled–rate setting.

The PMS values were rapidly reduced after cryopreser-vation, regardless the applied procedure. For controlled–ratecryopreserved platelets, PMS was 81.8±2.8 %, while forthose frozen by the uncontrolled–rate technique PMS was75.7±3.9 % (Table 2).

The analysis of PMS recovery confirmed that the inter-group differences were significant (p < 0.05) in favor of thecontrolled–rate freezing.

The results of platelet surface marker investigations be-fore and after cryopreservation are presented in Table 3.

GPIb/CD42b expression was reduced, in both freezinggroups as compared to the control one, with no significantintergroup (controlled–rate vs. uncontrolled–rate) differ-ences. On the contrary, noticable intergroup differences(p < 0.05) for GP140/CD62p expression were observed, sug-gesting that controlled–rate freezing provided lower plateletactivation.

GP53/CD63 showed an increased expression as com-pared to the control group, but with no significant intergroup

Table 1The recovery and viability of the frozen/thawed cells

P l a t e l e t s L y m p h o c y t e sPltrecovery PltVB Lyrecovery LyVB LyTBNCR

Con−Plt* 91.0±5.5† 68.0±23.2‡ / / /Con−Ly* / / 89.7±5.2† 91.6±1.3 82.5±4.5†

Unc−Plt* 86.0±6.5† 51.6±17.5‡ / / /Unc−Ly* / / 71.2±16.1† 91.9±7.8 65.5±6.4†

Data are expressed as mean ± SD;*Protocols investigated: Con−Plt and Con−Ly = controlled–rate freezing with 6% and 10% DMSO;Unc−Plt and Unc−Ly = uncontrolled–rate freezing with 6% and 10% DMSO;Pltrecovery = cryopreserved/unfrozen (control) platelet count;PltVB = calculated accordingly of the hypotonic shock response – HSR.Lyrecovery = cryopreserved/unfrozen (control) lymphocyte count;LyVB = lymphocyte viability (determined by trypan blue exclusion test);TBNCR = post–cryopreservation total trypan blue–negative lymphocyte recovery;† Intergroup (controlled–rate vs. uncontrolled–rate) differences (p < 0.05);‡Intergroup (controlled–rate vs. uncontrolled–rate) differences (p < 0.01).

Table 2Platelets morphological score values

Absolute PMS Relative PMS[%]

Control group 356.5±10.3 100Con–Plt* 291.7±7.4†‡ 81.8±2.8†‡

Unc–Plt 269.9 ± 10.3† 75.7±3.9†

Data are expressed as mean ± SD;*Protocols investigated: Con−Plt = controlled–rate freezing with 6% DMSO;

Unc−Plt = uncontrolled–rate freezing with 6% DMSO;†Cryopreserved vs. unfrozen (control) (p < 0.01);‡Intergroup (controlled–rate vs. uncontrolled–rate) differences (p < 0.05).

VOJNOSANITETSKI PREGLED Volumen 63, No 3


differences. GPIIb–IIIa/CD41 values were lower than in thecontrol, but also without the significant intergroup differ-ences.

Figure 1a shows the normal unfrozen discoid platelet(control) with the typical ultrastructural properties. The elec-tron microscopy appearance of platelets cryopreserved bycontrolled–rate and uncontrolled–rate freezing are also pre-sented in Figure 1b–e.

Fig. 1a – The ultrastructural (electron microscopy) prop-erties of cryopreserved platelets

Unfrozen platelet.

Besides discoid and spheric shapes, there were severaldendritic platelets, in the controlled-rate freezing group (Fig-ure 1b and 1c). The discoid platelets had maintained intact

microtubules and an open canalicular system with a minimaldamage of the membrane. Platelets frozen by the uncon-trolled–rate procedure (Figures 1d and 1e) showed the mem-

brane damages and altered ultrastructural properties (den-dritic or balloon shapes). In addition, these platelets regularlyhad an unclear edge with numerous large pseudopodes.

Table 3Surface antigen expression of cryopreserved platelets

CD markers Control Con−Plt* Unc−Plt*GPIb/CD42b [%] 97.22 ± 2.6 83.2 ± 3.6† 86.08 ± 4.4†GPIIb–IIIa/CD41 [%] 98.45 ± 0.6 97.47 ± 1.3 96.16 ± 0.8 ‡GP140/CD62p [%] 8.88 ± 3.8 28.38 ± 5.2† 34.91 ± 8.3†‡GP53/CD63 [%] 0.79 ± 0.2 1.96 ± 0.9† 2.08 ± 1.2†*Protocols investigated:Con−Plt = controlled–rate freezing with 6% DMSO;

Unc−Plt = uncontrolled–rate freezing with 6% DMSO;†Cryopreserved vs. unfrozen (control) (p < 0.05);‡Intergroup (controlled–rate vs. uncontrolled–rate) differences (p < 0.05).

Fig. 1c – The ultrastructural (electron microscopy)properties of cryopreserved platelets

Controlled–rate cryopreserved platelets.

Fig. 1d – The ultrastructural (electron microscopy)properties of cryopreserved plateletsUncontrolled–rate cryopreserved platelets.

Fig. 1b – The ultrastructural (electron microscopy)properties of cryopreserved platelets

Controlled–rate cryopreserved platelets.



Finally, Table 4 shows the mitogen (PHA and Con–A)induced lymphocyte proliferative response in controlled–rateand uncontrolled–rate setting.

The analysis of the obtained results of the lymphocyteproliferative response demonstrated that the cryopreservationresulted in no significant intergroup differences, regardlessthe used freezing procedure.

Discussion

Our earlier cryoinvestigations 10, 12, 28 pointed out the im-portance of the kinetics of the programmed cooling for the to-tal nucleated cell (TNC), lymphocyte and platelet cryopreser-vation. In this study, we confirmed that the use of the con-trolled–rate method had significantly influenced the protectionof quantity and functionality of frozen/thawed platelets. Theplatelet postthaw recovery was exactly 91.0±5.5%, which wasbetter than in other studies (62 to 86%) reported 22, 23, 37, 38.

The results obtained in this research showed that thecontrolled–rate freezing yielded the improved platelet viabil-

ity, as projected across HSR. Although in this study thelower HSR values were observed for both freezing proce-dures (compared to the control one), the 32% reduction inthe controlled–rate setting is in accordance with other re-ports. However, HSR value depends on some additional pa-rameters, such as cell platelet age, cell membrane integrity,type of platelet shapes, etc 23, 31, 39. Finally, we believe thatHSR of cryopreserved platelets is not always a result of theunsuccessful response of all cells, but could rather representan unequal response of different platelet shapes.

The examination of PMS also confirmed clearly thatcontrolled–rate freezing was more efficient than the uncon-trolled–rate technique. Better PMS values obtained in the con-trolled–rate setting were primarily due to a greater proportionof platelets of disc shapes (around 70% in comparison with thecontrol). These data generally agree with those reported in theliterature on PMS values after freezing–thawing procedure. Itis generally accepted that the high–quality in vivo survival ofcells can be expected after transfusion if 50% or more ofplatelets keep their disc shape after cryopreservation 39–41.

The investigation of ultrastructural properties of plateletsconfirmed the advantage of the controlled–rate freezing, sinceit revealed that the discoid platelets cryopreserved by thisprotocol had kept intact microtubules and an open canalicularsystem, with the minimal membrane changes. The sphericshaped platelets showed the lower electron density of cyto-plasm, gradually changed the interior structure comparing toother shapes, which was manifested by the marginal locationof granules and other cytoplasmatic organelles. This finding isin accordance with our previous platelet cryoinvestigation 28.On the contrary, the samples cryopreserved by the uncon-trolled–rate technique showed an abundance of the damagedplatelets and cell detritus, along with the lower percentage ofplatelets with a partially protected membrane integrity. Theoccurrence of the regular dendritic and balloon shapes, as wellas rare spheric platelets indicated reduced morphologicalproperties and the subsequent functionality of thawed cells.

Fig. 1e – The ultrastructural (electron microscopy)properties of cryopreserved plateletsUncontrolled–rate cryopreserved platelets.

Table 4Mitogen–induced lymphocyte proliferative response

Controlled–rate freezing Uncontrolled–rate freezingCPM‡ SI║ CPM‡ SI║

Unstimulated 1196 ±315 / 893±179 /PHA I* 102404±16463 104±28 100638±13950 133±33PHA II* 111939±14852 113±27 106173±13036 139±31PHA III* 106167±11959 104±20 101005±10735 129±23PHA IV* 101770±7513 98±14 99222±9859 124±18Con–A I† 6445±949 6±1 6569±628 8±1Con–A II† 65919±18102 66±24 58158±12681 79±26Con–A III† 102994±11449 103±22 96190±10064 128±31Con–A IV† 98755±11092 96±18 101698±7977 134±29

Data are expressed as mean ± SE; (four lymphocyte samples per each group);‡CPM = counts per minute;║SI = stimulation index;*PHA concentrations used: I = 62.5 μg/ml;

II = 31.3 μg/ml;III = 15.6 μg/ml;IV = 7.8 μg/ml;

†Con–A concentrations used: I = 67.5 μg/ml;II = 33.8 μg/ml;III = 16.9 μg/ml;IV = 8.4 μg/ml.



In the present study, GPIIb–IIIa/CD41 platelet surfacemarker was investigated. In addition, platelet activation wasassessed by measuring the expression of GPIb/CD42b,GP53/CD63 and GP140/CD62p (P–selectin). The last one isexpressed on the cell surface after the release from α–gran-ules.

It is generally accepted that the cold–induced post–transfusion platelet clearance with following compromisedclinical effectiveness should be mediated by GPIb/CD42breorganization into “clusters” 42–47. Our preliminary cryoin-vestigation (29) of cold–induced GPIb/CD42b alterationswas in agreement with these studies, particularly when un-controlled–rate freezing was performed. We proposed thatthe reduction of the GPIb/CD42b expression in the presentstudy (from 11.1 to 14.0%) might be partially caused by theGPIbα/CD42b “relocation”. However, these values aremarkedly better than those reported by Lozano (reduction >50%) 44. The mentioned “relocation” could probably makesurface antigens less accessible to monoclonal antibodies,resulting in the obtained decrease of GPIb/CD42b expres-sion the of cryopreserved vs. the control platelets. How-ever, Hoffmeister et al. (personal communication) did notfind a reduced GPIbα/CD42b expression following coolingusing the monoclonal antibody for platelets, so they con-cluded that clustering did not lead to “pseudo hyporeactiv-ity”. In this context, GPIb/CD42b expression decreaseshould be mediated by other mechanisms such as proteoly-sis by plasmin, thrombin, neutrophil elastase or the activa-tion of the cell “death machinery”, etc 29, 46. The resultsobtained in our study are comparable with those publishedby Barnard et al., who also found a reduced GPIb/CD42bexpression after cryopreservation 46. They proposed thatdecreased antigen expression could be associated withGPIb/CD42b proteolysis, mediated by calcium–activatedprotease, or calpain.

It is generally accepted that the cold–inducedGP140/CD62p expression less than 40% could be an ade-quate threshold, because higher expression values ofGP140/CD62p have been shown to result in platelet in vivorecovery of less than 50% 48). As shown in Table 3, freezingpromoted a raise in the number of platelets expressingGP140/CD62p antigen. The percentage of platelets express-ing GP140/CD62p in the controlled–rate group was signifi-cantly lower than in the uncontrolled–rate sample. The ele-vated GP140/CD62p values for cryopreserved cells pointedto the platelet activation during the freezing/thawing proce-dure.

The expression of GP140/CD62p was associated with theclearance of platelets by the reticuloendothelial system 48. Morethan 50% of platelets stored at 22 °C expressed GP140/CD62pon their surface after 5 days, which indicated that the cryopre-served cells had the superior retention of intact granules, whichcorrelated with their in vivo functionality 49. With respect toGP53/CD63 antigen (activation marker of the externalizationof the contents from lysosomes), a considerable increase inthe expression of this antigen after freezing–thawing processwas observed. However, this increase in the GP53/CD63 ex-pression was not as high as for GP140/CD62p antigen. This

finding might reflect the more intensive activator stimulusnecessary for the release of lysosomes compared to that ofα–granules 50.

In this study, the GPIIb–IIIa/CD41 (the main plateletaggregation protein) examination showed the decrease incryopreserved platelets in both groups. However, the reduc-tion of GPIIb–IIIa/CD41 was not significant after con-trolled–rate freezing vs. the control group (opposite to un-controlled–rate freezing), which is in agreement with datafrom the literature 51, 52. GPIV/CD36 is an accelerator of theinteraction between platelets and collagen and it is a media-tor of the platelet adhesion to subendothelial surfaces 53.Cryopreserved platelets in the present investigation obtaineda significant reduction of the expression of GPIV/CD36 ascompared to unfrozen platelets, but without intergroup dif-ferences. These data are in accordance with data from the lit-erature 44.

For a successful cryopreservation not only the use ofappropriate freezing strategy, but also the selection of acryoprotectant in the optimized concentration is required.For platelet and lymphocyte freezing, DMSO and/or hy-droxyethyl starch (HES) dissolved in a cell–specific“freezing medium” is commonly used, but with differentconcentrations of cryoprotectant 54, 55. In the present study,6% DMSO in autologous plasma (platelet setting), and 10%DMSO in RPMI 1640 (lymphocyte setting) were used. Ourearlier cryoinvestigation showed that the TNC and thecommitted progenitor recovery was better when the lower(5%) DMSO and controlled–rate freezing were used. Incontrast, pluripotent Makrow Repopulating Ability (MRA)cells could only be well cryopreserved in the presence of10% DMSO 10. These data implied a different “cryobi-ological request” of MRA cells in comparison to the com-mitted progenitors or TNCs. However, as recently pre-sented, the postthaw cell quality is better when rHu–DNase,membrane stabilizers or bioantioxidants are added to theConventional Freezing Medium (CFM) 56–58. Accordingly,it is possible that the use of these additives is more success-ful than the reduction of DMSO concentration. We alsoshowed that the differences in the cell recovery and func-tionality rates were not related to the changes in the totalnumber of frozen/thawed cells 10, 11.

In this research, the obtained lymphocyte recovery wassignificantly better in the controlled–rate vs. uncontrolled–rate settings. These results are in agreement with our earlierfindings for TNCs and lymphocytes 11, 35, and they are also inaccordance with the data from other authors 36, 37, 59. On the otherhand, we obtained no significant differences in mitogen inducedproliferation of lymphocytes after the controlled–rate vs. uncon-trolled–rate procedure. Proliferative response to mitogens or mi-crobial antigens is often used as a measure of lymphocyte func-tionality. Weinberg et al. 60 showed that the results of functionalassays on cryopreserved peripheral blood MNCs are associatedwith cell viability. Although the obtained TBNCR values weremarkedly higher in the controlled–rate group, the proportionof viable cells was high (≈ 90%) for both procedures (Table 1)which can explain a similar proliferative response of lymphocytesafter different freezing methods.



Conclusion

In conclusion, the results presented here point undoubt-edly out the controlled–rate system (with the compensatedfusion heat) which resulted in a superior platelet recovery ofthe tested parameters. A simultaneous analysis of plateletsurface antigens and cell micro–integrity markers could pre-dict certain in vivo properties of the cooled, especially cryo-

preserved platelets, however these postulates should be con-firmed. The lymphocyte recovery and TBNCR values wereobviously lower in the uncontrolled–rate setting. The ob-tained high–level postthaw cell viability can explain theirsimilar proliferative response regardless the used freezing.Further researches in various blood–derived cell settings arerecommended to define the optimal cell–specific cryopreser-vation strategy with the best cell recovery.

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The paper was received on November 8, 2005.

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