Vol. 19, No. 2, 2003

ImmunohematologyJ O U R N A L O F B L O O D G R O U P S E R O L O G Y A N D E D U C A T I O N

V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3

ImmunohematologyJ O U R N A L O F B L O O D G R O U P S E R O L O G Y A N D E D U C A T I O N

V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3

C O N T E N T S

33Review: biochemistry of carbohydrate blood group antigens

L.G. GILLIVER AND S.M. HENRY

43Neonatal alloimmune thrombocytopenia due to anti-HPA-2b (anti-Koa)

M. GOLDMAN, E.TRUDEL, L. RICHARD, S. KHALIFE,AND G.M. SPURLL

47Quantitation of red cell–bound IgG, IgA, and IgM in patients with autoimmune hemolytic anemia and

blood donors by enzyme-linked immunosorbent assayA.A. BENCOMO, M. DIAZ,Y.ALFONSO, O.VALDÉS,AND M.E.ALFONSO

54ABO and Rh(D) blood typing on the PK 7200 with ready-to-use kits

P. MONCHARMONT,A. PLANTIER,V. CHIRAT,AND D. RIGAL

57Jk and Mi.III phenotype frequencies in North Vietnam

NGUYEN THI HUYNH, DEREK S. FORD,TRAN THI DUYEN,AND MAI THANH HUONG

59C O M M U N I C A T I O N S

Letter to the EditorsVel– donors in Yugoslavia

M. DJOKIC,A. KUZMANOVIC, Z. BUDISIN, S. SRZENTIC, J. POOLE,AND R. STEVANOVIC

60B O O K R E V I E W S

HENRY M. RINDER, MDJOHN D. ROBACK, MD, PHD

63L I T E R A T U R E R E V I E W

General (2000–2003)

68 68 69A N N O U N C E M E N T S C L A S S I F I E D A D A D V E R T I S E M E N T S

71I N S T R U C T I O N S F O R A U T H O R S

EDITOR-IN-CHIEF MANAGING EDITORDelores Mallory, MT(ASCP)SBB Mary H. McGinniss,AB, (ASCP)SBB

Rockville, Maryland Bethesda, Maryland

TECHNICAL EDITOR MEDICAL EDITORChristine Lomas-Francis, MSc S. Gerald Sandler, MD

New York, New York Washington, District of Columbia

EDITORIAL BOARD

EDITORIAL ASSISTANTLinda Berenato

PRODUCTION ASSISTANTMarge Manigly

COPY EDITORLucy Oppenheim

ELECTRONIC PUBLISHERPaul Duquette

Immunohematology is published quarterly (March, June, September, and December) by the American Red Cross, National Headquarters,Washington, DC 20006.

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Copyright 2003 by The American National Red CrossISSN 0894-203X

Patricia Arndt, MT(ASCP)SBBLos Angeles, California

James P.AuBuchon, MDLebanon, New Hampshire

Malcolm L. Beck, FIBMS, MIBiolKansas City, Missouri

Richard Davey, MDNew York, New York

Geoffrey Daniels, PhDBristol, United Kingdom

Sandra Ellisor, MS, MT(ASCP)SBBAnaheim, California

George Garratty, PhD, FRCPathLos Angeles, California

Brenda J. Grossman, MDSt. Louis, Missouri

Christine Lomas-Francis, MScNew York, New York

Gary Moroff, PhDRockville, Maryland

Ruth Mougey, MT(ASCP)SBBCarrollton, Kentucky

John J. Moulds, MT(ASCP)SBBRaritan, New Jersey

Marilyn K. Moulds, MT(ASCP)SBBHouston, Texas

Scott Murphy, MDPhiladelphia, Pennsylvania

Paul M. Ness, MDBaltimore, Maryland

Mark Popovsky, MDBraintree, Massachusetts

Marion E. Reid, PhD, FIBMSNew York, New York

Susan Rolih, MS, MT(ASCP)SBBCincinnati, Ohio

David F. Stroncek, MDBethesda, Maryland

Marilyn J.Telen, MDDurham, North Carolina

This review presents the basics of the structuralchemistry of blood group glycoconjugates,with specialreference to red cell serology. Its aim is to create anappreciation of the inherent subtleties of thecarbohydrate blood group antigens, which arecurrently poorly understood within the field of bloodtransfusion. It is hoped that a better understanding ofthe intricacies of the carbohydrate blood groupsystems will lead to further contributions to the bodyof knowledge within this growing field.

IntroductionIn 1900, Karl Landsteiner discovered the

carbohydrate blood group system of ABO after mixingthe cells and serum of his colleagues. Over the next100 years many other blood group systems werediscovered and later resolved at both the molecular andthe genetic level. It is now clearly recognized that mostblood group systems are either carbohydrate, protein,or protein-carbohydrate in nature, although they mayassociate with other components and complexes to berecognized as specific blood group determinants.Essentially, a blood group antigen is any polymorphicthree-dimensional conformation expressed on theouter membrane of a RBC against which an antibody isavailable. The concept of the three-dimensional shapeis very important because this is what antibodiesrecognize, and the number of potential shapes that canbe found in carbohydrate chains is enormous. Thevariability in characteristics such as sugar type, linkagetype, branch positioning, shape, charge, ring size, andepimeric (pairs of monosaccharides which differ onlyin the configuration about one carbon atom areepimers of each other e.g., D-glucose and D-mannoseare epimers with respect to the C2 carbon atom) andanomeric configuration results in more than 1.05 × 1012

possible structural combinations from a six-sugar

glycan (the largest size expected for a carbohydrateepitope). This is compared with only 46,656combinations possible with a chain made up of sixamino acids, more than seven orders of magnitudelower than the diversity possible in glycans.1

Fortunately,despite the vast potential for complexity incarbohydrate chains, there is generally only a limitedrange of structures representing the carbohydrateblood group epitopes (glycotopes), although many ofthe structural differences are subtle and some epitopesurfaces may be common. These subtleties mayrepresent different blood group antigens and in somecases, specific antibodies may recognize the commonsurfaces. For example, anti-A,B recognizes thesimilarities between A and B antigens,not the subtletieswhich distinguish them.2 Unfortunately, this potentialvariation makes structural determination of carbo-hydrate antigens complex, requiring combinations ofpowerful techniques such as nuclear magneticresonance spectroscopy and mass spectrometry toelucidate the structures of carbohydrate antigenswithout ambiguity.3,4 Progress in determining thestructures of carbohydrate blood group antigens hasbeen slow, hindered by a combination of factors, suchas the fact that carbohydrate blood group antigens are not the primary product of a gene—being rathersecondary biosynthetic products—and that theisolation and purification of carbohydrate blood groupantigens in requisite quantities is difficult. As a result,we know only the basic structures of the majorglycotopes and antigens expressed in the carbohydrateblood group systems. We have not yet characterizedmany of the phenotypic subtleties, such as those thatdetermine ABO subgroups.

Because carbohydrate blood group antigens areimportant to transfusion medicine and are becomingmore recognized in transplantation (especially xeno-

I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3 33

Review: Biochemistry ofcarbohydrate blood groupantigensL.G. GILLIVER AND S.M. HENRY

transplantation5) and studies of micoorganism bindingand interactions,6 it is becoming more important forserologists to understand the biochemistry ofcarbohydrate blood group antigens.

We presently recognize several blood groupsystems and collections7 where the blood groupglycotopes are comprised entirely of carbohydrate,such as ABO, Lewis, H, Ii, P, Sd, Cad, etc.; or others, suchas MN, where carbohydrate moieties are essential tothe structure of the epitope; or even others, wherecarbohydrates are associated with the character of theantigen—e.g., GPI-linked membrane proteins such as Cromer, Dombrock, Cartwright, and John MiltonHagen, etc.

This review is not intended to cover thecarbohydrate blood group systems, rather it is intendedto provide a basic understanding of carbohydrateblood group chemistry, and in this regard we will useABO antigens as examples.

MonosaccharidesThe common blood group–related sugars, namely

β-D-glucose (Glc), N-acetyl-β-D-glucosamine (GlcNAc),β-D-galactose (Gal), N-acetyl-β-D-galactosamine(GalNAc),α-L-fucose (Fuc), and D-mannose (Man) are allsix-carbon sugars (hexoses). The carbonyl group(C=O) of each residue is an aldehyde (HC=O), and thusthey are all classified as aldohexoses. Another specialmonosaccharide which contains nine carbons is alsocommon in blood group antigens, with the mostcommon blood group variation being N-acetyl-α-D-neuraminic acid (also referred to as NeuAc or sialicacid). There are various forms of sialic acid,8,9 but thesewill not be reviewed here.

Sugar molecules can have configurational isomerswhich differ in their arrangement in space even thoughthey have identical composition. The variation in theconfiguration of individual carbohydrates was firstcharacterized in relation to the smallest chiralcarbohydrate, glyceraldehyde, by German chemist EmilFischer in the late 19th century. In extremely simpleterms, chirality is “handedness”—that is, the existenceof left/right apposition. For example, your left handand right hand are mirror images and therefore “chiral.”

The enantiomeric (mirror image) configurationsFischer described were designated D and L by Rosanoffin 1906.10 Today, all monosaccharides are classifiedaccording to this principle, which depends on theorientation of the hydroxyl group attached to thehighest numbered chiral carbon (C(n-1), where n is the

total number of carbons in the structure). If thishydroxyl group points to the right when the carbonylgroup is at the top, the monosaccharide is said to be inthe D configuration. The mirror image of this structure,with the hydroxyl group pointing to the left, is the L

configuration. The D and L configurations of glucoseare shown as open chain Fischer projection formulas(Fig. 1). In blood group glycoconjugates, sugars existalmost exclusively in the D configuration, exceptfucose, which is found in the L configuration. Thechiral centers of saccharides can also be designated asR or S under the Cahn-Ingold-Prelog system. This is amore modern notation; however, it is not often used incarbohydrate chemistry because it causes confusion inthe classification of some monosaccharides.

The seven common blood group sugars almostexclusively form pyran rings—six atoms forming thering, compared with furan, which is a five-atom ring(Fig. 2).

Numbering of the carbons in the pyranose ringformation of hexose and nonose sugars is shown inFigure 3.

The pyran ring comes about through the formationof an oxygen bridge between a carbon (carbon 1 in thecommon hexose blood group sugars, or carbon 2 insialic acid) and the oxygen of the hydroxyl group fivecarbons away.10 In hexoses, carbon 5 rotates so that itshydroxyl group apposes carbon 1. The double-bondedoxygen on carbon 1 is reduced—taking the hydrogen

34 I M M U N O H E M A T O L O G Y, V O L U M E 1 9 , N U M B E R 2 , 2 0 0 3


Carbon numbering D-glucose L-glucose

Fig. 1. Carbon numbering of linear hexose sugars and the D- and L-structures of glucose. The carbon numbering of a generic 6-car-bon sugar is shown on the left (with the chiral center hydro-gens and hydroxyl groups not shown). Carbons 2, 3, 4, and 5are the chiral centers of 6-carbon sugars. The enantiomericform is designated by the direction of the hydroxyl group onthe highest numbered chiral carbon (arrows)—in the case of 6-carbon sugars, this is carbon 5. In D-glucose (middle), thehydroxyl group of carbon 5 points to the right, and in L-glucose(right), this hydroxyl group points to the left.

from the hydroxyl group of carbon 5, in the processoxidizing the hydroxyl group to an oxygen radical withonly one bond. This leaves carbon 1 as a radical also,with only three bonds. This is one less than thepreferred complement of bonds in the case of bothatoms, enabling the formation of a bond between them(see Fig. 4 for the scheme of this reaction, using D-galactose as an example). The scheme is basically thesame for sialic acid, except that the bridge is formedbetween carbon 2 and the oxygen on carbon 6.

The two ring formations possible for each sugar areeither the α or β anomeric forms. These are twoisomeric forms that differ only in the configuration atthe carbon involved in the formation of the oxygenbridge, which is carbon 1 in the common blood group

hexose sugars and carbon 2 in sialic acid. This carbonis termed the anomeric carbon, and the configurationaldifference occurs in the orientation of its hydroxylgroup in relation to the oxygen on the anomeric-reference atom, which is carbon 5 in most hexoses.


Review: carbohydrate blood group antigens

Fig. 2. Furan and pyran rings of D-galactose. The open chain formationof D-galactose is shown in the middle of the figure. The furanose(lower left) and pyranose (lower right) forms of D-galactose (β isused in this example) are formed from the open chain structure.The generic furan (upper left) and pyran (upper right) ring for-mations are also shown.

Generic furan ring Generic pyran ring

β-D-galactofuranose β-D-galactopyranoseOpen chainD-galactose

Fig. 4. Scheme showing the formation of the pyran ring. Step 1—Carbon 5 (square) of the open chain form of D-galactose rotatesso that its hydroxyl group apposes carbon 1 (circle). Step 2—Thedouble-bonded oxygen on carbon 1 is reduced and the hydroxylgroup of carbon 5 is oxidized, leaving carbon 1 and the oxygenon carbon 5 each with one less than their preferred number ofbonds. Step 3—A bond forms between carbon 1 and the oxygenof carbon 5 to yield β-D-galactopyranose.

D-galactose

1

2

3

β-D-galactopyranose

Fig. 5. Fischer linear ring projection showing the configurational differ-ence at the anomeric carbon hydroxyl group (top arrows) of α-D-galactose (left) and β-D-galactose (right) in relation to the anomer-ic reference oxygen (bottom arrows). The anomeric carbonhydroxyl group and the anomeric reference oxygen are on thesame side of the carbon chain in the α-form, and on oppositesides of the carbon chain in the β-form.

α-D-galactose β-D-galactose

Fig. 3. Carbon numbering in pyranose rings. Most of the common bloodgroup sugars are 6-carbon (hexoses), except for sialic acid, whichhas 9 carbons (nonose). Carbons are numbered in a clockwisedirection from the oxygen bridge.

Generic hexose Nonose sugar, i.e., sialic acid

When in the Fischer linear representation of the ring(Fig. 5), the α form occurs when the anomeric carbonhydroxyl is on the same side of the carbon chain as theoxygen on carbon 5. Accordingly, in the β form, theanomeric carbon hydroxyl and the oxygen on carbon 5are on opposite sides. Due to the rotation of carbon 5in hexoses (carbon 6 in sialic acid) and hence itshydroxyl group (Fig. 4), this rule cannot be applied tothe Haworth projection of the ring. However, theanomeric form can be deduced in the Haworthprojection—it is α when the anomeric hydroxyl groupis down, and β when the anomeric hydroxyl is up.

The Haworth projections of the α and β forms ofmannose and galactose are shown in Figure 6. The twocyclic α and β anomeric forms exist in equilibriumwith the open chain structure,11 however, the openchain configuration is present only in minute amounts.Because these anomers are interchangeable, theanomerity of the sugar is really only of interest once itis made permanent by bonding with anothersaccharide.

Figure 6 also shows the other saccharidescommonly found in blood group antigens. D-glucoseand D-galactose are most often found in the β form,while L-fucose, which is almost identical in structure togalactose (except that fucose does not have a hydroxylgroup on its sixth carbon, thus it is also known as6-deoxygalactose) is found in the α form. D-mannose isa sugar residue commonly found in glycoproteins(exclusively N-linked oligosaccharides) and exists inboth the α and β configuration.

N-acetyl-D-glucosamine and N-acetyl-D-galactosa-mine are examples of amino sugars (Fig. 6). The

hydroxyl group of carbon two is replaced by anacetamido group, which is an amino group with anacetyl group substituted for one of the hydrogens (Fig. 7). This important substitution differentiatesblood group A from blood group B. Blood group A hasN-acetyl-α-D-galactosamine, while blood group B has α-D-galactose. Furthermore, modification of the blood group A sugar N-acetyl-α-D-galactosamine bybacterial deacetylase enzymes can generate theacquired B antigen (GalNAc to GalNH2—Fig 6). This iswhere N-acetyl-α-D-galactosamine loses its acetyl group but retains the nitrogen—thus, the acquired B antigen is similar but not identical to the B

determinant α-D-galactose.N-acetyl-D-glucosamine mostcommonly adopts the βform in blood group glyco-conjugates, while N-acetyl-D-galactosamine most commonlyadopts the α configuration—although the β form is alsoseen. Sialic acid is unlike the other sugar residuesdiscussed here in that it has nine carbons and theanomeric carbon is carbon 2(Fig. 6). It is substituted atcarbon 5 with an acetamidogroup as described for carbon2 of N-acetyl-D-glucosamineand N-acetyl-D-galactosamine.

The carboxyl group (COOH) of carbon 1 gives sialicacid its acid characteristics, and because of this,glycoconjugates incorporating it are known asgangliosides, which are a subset of acidic glyco-sphingolipids. The acidic glycosphingolipid group alsoencompasses uronoglycosphingolipids (containinguronic acid residues), sulfoglycosphingolipids(containing sulfate ester groups), phosphoglyco-sphingolipids (containing phosphate mono- or diester groups), and phosphonoglycosphingolipids(containing [2-aminoethyl]hydro-xyphosphorylgroups).



Fig. 6. Haworth projection formulae of the sugar residues commonly found in blood group–related glycoconjugates.

α-L-fucose α-D-galactose N-acetyl-α-D-galactosamine

Acquired Bdeterminant

β-D-galactose

β-D-glucose N-acetyl-β-D-glucosamine

α-D-mannose β-D-mannose Sialic acid (N-acetyl-α-D-neuraminic acid)

Fig. 7. The acetamido group of N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and sialic acid. The acetamido group is made up ofthe amino group and the acetyl group.

Amino group Acetyl group Acetamido group

Up to this point, the pyranose-forming mono-saccharides have all been represented as having planarrings with functional groups projecting above orbelow. In reality this is not the case—the electronorbitals of the carbons and the oxygen in the ring andthe large functional groups attached to the carbonsprevent the rings from existing in a planar form. Theresult is that the ring contorts into one of three three-dimensional shapes, two of which are suggestive of achair and one of which looks like a boat (Fig. 8). The

functional groups attached to the ring carbons are ableto adopt either an axial position—perpendicular to thegeneral plane of the ring—or an equatorial position—in line with the general plane of the ring. In this way asingle saccharide can potentially assume three differentthree-dimensional shapes, although in practice thelowest energy conformation is preferred, i.e., the onewhich minimizes the proximity of large functionalgroups. The functional groups in the equatorialorientation are less crowded together than those in theaxial position, and so the lowest energy conformationoccurs when as many as possible of the large hydroxylgroups, rather than the relatively small hydrogens, aresituated in the equatorial orientation. Only saccharideswith alternating chirality, such as β-D-glucose (Fig. 1),can position all the ring hydroxyl groups in theequatorial position, because crowding in the axialorientation is reduced due to the ring hydrogens beingable to alternate above and below the plane of the ring(Fig. 9).

Bonds and LinkagesSugar residues can be joined into chains by the

synthesis of glycosidic bonds. As previouslymentioned, the two monosaccharide cyclic forms—αand β—are in equilibrium with the open chainformation when in aqueous solution. However, whenjoined together by glycosidic linkages, the residuesbecome fixed in ring formation except for the lastsugar in the chain.12

Glycosidic bonds are the result of a condensationreaction between the hydroxyl group of carbons 2, 3,4, 6, or 8 of one residue and the anomeric carbonhydroxyl group of a second sugar. The bond isconsidered to belong to the sugar whose anomericcarbon is involved, which for hexoses is carbon 1, andis expressed as 1-2, 1-3, 1-4, or 1-6 depending on thepoint of attachment. Sialic acid, whose anomericcarbon is carbon 2, forms 2-3, 2-6, or 2-8 bonds.

Two types of glycosidic bond are possible; thisdepends on the configuration of the anomeric carbonhydroxyl group involved in the bond. The bond

formed when the anomeric hydroxyl group is in the αconfiguration is designated an α bond. Likewise, a βbond will result when the anomeric hydroxyl is in theβ configuration. The A type 1 structure shown inFigure 10 has two α bonds and three β bonds.

Gangliosides contain sialic acid residues. Asdiscussed above, the anomeric carbon of sialic acid iscarbon 2 (Figs. 3 and 6), and thus the glycosidic bondoccurs at the hydroxyl group of this carbon. Sialic acidcan be added to a hexose residue at carbons 3 or 6;α2-3 and α2-6 bonds between sialic acid and galactoseare shown in Figure 11. Sialic acid usually occupies a



Fig. 8. Chair and boat conformations of pyranose rings. There are twovariants of the chair conformation, which differ mainly in theaxial versus equatorial orientations of the ring functional groups(X and Y). In the boat conformation, the functional groupslabeled A are in an axial position, while those labeled E are equatorial.

Chair Chair Boat

Fig. 10. Example of α and β linkages in the A type 1 molecule. This dia-gram shows A type 1 in two schematic forms; one structural andthe other alphanumeric—both a simple form (lower right) andan expanded form associated with the structural diagram to iden-tify the appropriate sugars and bonds. R = the remainder of themolecule.

Fig. 9. β-D-glucopyranose in itspreferred chair conforma-tion. All the hydroxylgroups are in the equatori-al orientation. Note thatthe hydrogens alternateabove and below theplane of the ring in theaxial positions.



terminal (and sometimes also a sub-terminal) position.A terminal sialic acid residue prevents furtherelongation of the chain except for the addition ofanother sialic acid residue through an α2-8 linkage (Fig. 11).

Different glycoconjugate precursor chainstructures have been identified and categorized into sixgroups based on the identity of the terminaldisaccharide sugar residues and the type of glycosidiclinkage that joins them together.13 These terminaldisaccharide structures of the six groups are shown inTable 1.

Biosynthesis of Saccharide ChainsAccording to carbohydrate terminology, an

oligosaccharide consists of 3 to 10 sugar residues and apolysaccharide contains more than 10 residues.11 Theblood group structures can exist in both linear andbranched forms and range from a few saccharide unitsup to more than 60 sugar residues. Irrespective of thesize of any given polysaccharide, it is generallyaccepted that the actual surface recognized byantibody (glycotope) is usually no larger than 2–3sugars. However, sometimes glycotopes may lookbigger (up to six sugars) because some extra sugars areneeded to stabilize the three-dimensional configurationof the sugars actually recognized by the antibody

(R. Oriol, personal communi-cation). The actual structureand size of the poly-saccharide is determined byinteractions of various poly-morphic blood group glyco-syltransferases and othermonomorphic glycosyltrans-ferases. Glycosyltransferasesare gene–encoded proteins(enzymes), and are respon-sible for the stepwiseaddition of substrate residuesto acceptor structures. It is

therefore clear that the biosynthesis of carbohydrateblood group antigens is not a straightforward geneequals antigen process. Instead, a gene must encode afunctional protein (enzyme),which must be retained inthe Golgi apparatus,and in the presence of appropriateprecursors and substrates a carbohydrate blood groupantigen can be synthesized (Fig. 12). Even this is overly

simplistic, because the formation of a carbohydrateblood group antigen requires the interaction of severalglycosyltransferases unrelated to blood groups beforethe blood group determinant(s) can be added.Furthermore, competition and sometimes cooperationoccur between the various glycosyltransferases, someof which show redundancy and degeneration.14

Redundancy is said to occur when the samecarbohydrate structure can be made by two separateenzymes. For example, Se is capable of transferring afucose to the terminal β-D-galactose of both type 1 and2 chains, while H can only utilize type 2 chains. Thus,H type 2 can be made by two genetically differentenzymes. The presence of Se-formed H type 2 antigenson Bombay RBCs is the basis of some para-Bombayphenotypes. Degeneration refers to the situation

Conceptual pathway Biosynthesis of A antigengenetic message (DNA, mRNA) A gene

↓ ↓enzyme (glycosyltransferase) N-acetyl-α-D-galactosaminyl

transferase↓ ↓

precursor + activated sugar H antigen + UDP-GalNAc↓ ↓

carbohydrate antigen A antigen■■ membrane/antigen presentation ■■

↑ ↑recognition by antibodies/lectins/receptors

Fig. 11. Sialic acid linkages. Left: N-acetyl-α-D-neuraminic acid (left) bonded to β-D-galactose (right) throughan (α2-3) linkage. Center:N-acetyl-α-D-neuraminic acid (left) bonded to β-D-galactose (right) through an (α2-6) linkage. Right: two N-acetyl-α-D-neuraminic acid residues joined together through an (α2-8) link-age. R = the remainder of the molecule.

Sialic acid (αα2-3) galactose Sialic acid (αα2-6) galactose Sialic acid (αα2-8) sialic acid

Table 1. Six terminal disaccharide chain types.

Terminal Disaccharide Structure

Type 1 Galβ1-3GlcNAcβ1-R

Type 2 Galβ1-4GlcNAcβ1-R

Type 3 Galβ1-3GalNAcα1-R

Type 4 Galβ1-3GalNAcβ1-R

Type 5 Galβ1-3Galβ1-R

Type 6 Galβ1-4Glcβ1-R

Fig. 12. Conceptual scheme for the formation of carbohydrate bloodgroup antigens.



where one enzyme can make two different structures.Using the same example,Se shows degeneration in thatit can make type 1 and type 2 H structures. Thus, it canbe seen that the occurrence of specific glyco-conjugates is indirectly controlled by genes throughthe occurrence and subsequent synthetic actions ofenzymes. After glycoconjugates are synthesized, theyare transported to various destinations in and out of thecell,as well as being incorporated in the cell membranewhere they can be detected by various antibodies. Amore comprehensive review of the pathways leadingto the expression of the blood group–related glyco-sphingolipids can be found elsewhere.13

Glycolipids vs. GlycoproteinsGlycans can be conjugated to protein or lipid

molecules in biological systems; such molecules arecalled glycoproteins or glycolipids, respectively. Theglycosylation pathways followed by glycoproteins andglycolipids are not always identical. This possiblyreflects the different biological functions ofmembrane–bound glycoconjugates (glycolipids andglycoproteins) versus soluble structures (usuallyglycoproteins).15,16

GlycolipidsGlycosphingolipids are a class of glycolipids which

are composed of a carbohydrate chain and asphingolipid tail, the trivial name of which is ceramide.Sphingolipids are common components of cellmembrane lipid bilayers and are composed of a long-chain base (LCB) amino linked to a fatty acid (FA). Dueto the long hydrophobic hydrocarbon chains presentin LCBs and the FAs, ceramides are able to insert intothe lipid bilayer of RBCs. The hydroxyl group oncarbon 1 of the sphingolipid is oxidized to form the β-linkage with theanomeric carbon of β-D-glucose to form a mono-glycosyl sphingolipid (Fig.13). This linkage is desig-nated β1-1 because thetwo hydroxyl groupsinvolved belong to carbon1 of their respectivestructures.

There is considerableheterogeneity in ceramides,mainly due to variations inhydrocarbon chain length,

level of saturation (presence of double bonds), and thenumber of hydroxyl groups—in fact, there are over 60different variations of LCBs,17,18 each of which can becombined with one of 20 common variants of FAs.10 Itis speculated that some of the variants may beimportant for presentation of the antigen.

GlycoproteinsGlycans can be attached to proteins via nitrogen or

oxygen—N– or O-glycosidically linked, respectively.N-glycosidically linked glycoproteins are foundprimarily in serum and cell membranes, while O-glycosidically linked glycoproteins are located inexocrine gland secretions and mucins, as well as in cellmembranes, although this is less frequent.7

O-linked glycoproteins An O-linkage occurs between the hydroxyl groups

of serine or threonine amino acid residues and theanomeric carbon hydroxyl group of N-acetyl-α-D-galactosamine, with the linkage type being α1 (Fig. 14). Glycolipids are also O-linked, but the sugar

Fig. 13. Structure of a sphingolipid (ceramide) and a monoglycosylsphigolipid. Top: the sphingolipid is composed of a FA (nervonicacid C24:115—upper chain) and a LCB (4-sphingenine d18:14—lower chain). Below: β-D-glucose is joined to the sphingolipid viaa β-linkage between carbon 1 of the saccharide and ceramide.

Fig. 14. Structure of glycoprotein O- and N-linkages. Left and center: O-linked glycoproteins where N-acetyl-D-galactosamine is linked through an α bond to the oxygen of the amino acids serine or threonine.Right: N-linked glycoprotein where N-acetyl-D-glucosamine is linked through a β bond to the nitrogen ofthe amino acid asparagine. R1 and R2 represent the remainder of the polypeptide chain.

Serine Threonine Asparagine



involved is β-D-glucose. Unlike the N-linked glyco-proteins, there is no single common oligosaccharidecore structure for O-linked glycoproteins. Insteadthere are six distinct di- and trisaccharide corestructure types and a variety of terminal structures.7

O-linked glycoproteins range from the very simplemono-glycosylated structures to highly complexmolecules containing large amounts of galactose andN-acetylglucosamine, along with lesser amounts ofN-acetylgalactosamine, fucose, and sialic acid. O-linkedglycoproteins do not contain mannose residues.

N-linked glycoproteins An N-linkage occurs between the nitrogen of the

amino acid asparagine and the hydroxyl group ofN-acetyl-β-D-glucosamine, with the linkage type beingβ1 (Fig. 14).

N-linked glycoproteins have a commonpentasaccharide structure consisting of Man3GlcNAc2.

7

There are three main groups into which N-linkedglycoproteins can be divided: high mannose type,complex type, and hybrid type. A comprehensivedescription of these types can be found elsewhere.7

Red Cell Glycosylation and Serology The outside of the human RBC is

highly glycosylated, incorporatingboth glycoproteins and glycolipids.The major function of theglycoconjugate coat of the RBC isprobably to make them morehydrophilic and to provide a “hard”but flexible protective coating.However, if this was the onlyfunction of red cell glycosylation,then it could be achieved by theexpression of large amounts ofsimple carbohydrate chains, whichdoes not account for the complexpattern of glycosylation that actuallyexists. Some of the glycoconjugatesare very complex and beargenetically defined polymorphisms,for example ABH, Lewis, and P. Whythese polymorphisms exist isunknown, but there is emergingevidence of evolutionary andbiological function.15,16,19,20

The intention of this review is topresent basic concepts of

carbohydrate chemistry in order to develop anunderstanding of the complexities that exist withincarbohydrate blood group systems. Traditionally, theantigens of these systems have been viewedgenerically, without appreciation of the subtledifferences between them or the impact this may haveon phenotyping or biological function. Not only isthere a variety of glycotopes within an antigendesignation, but there also is extensive variation in thecarbohydrate molecules which carry the glycotopes(Fig. 15).

This range of variants can be further expanded bythe polymorphic proteins and lipids which thensupport them. The presentation of the carbohydratechain at the membrane surface may differ significantlyamong the different types, as shown by molecularmodeling.21 The membrane orientation of saccharidechains may vary from almost perpendicular (type 1), tonearly parallel (types 2, 3, and 4). These differentconformations of the carbohydrate chains areimportant for antibody binding and for theimmunogenic and microbial-interaction characteristics.Some may actually determine phenotypecharacteristics, although this has not yet beendetermined. In addition, the carrier molecule mayinfluence the positioning of the carbohydrate chain on

Fig. 15. Structure of nine examples of A glycolipid structures in the group A erythrocyte membrane.Shorthand nomenclature according to Holgersson.23

A type 1; A-6-1 A type 2; A-6-2

ALeb; A-7-2 ALey; A-7-2

A type 4; Globo-A; A-7-4 A type 2; A-8-2

A type 3; Repetitive A; A-9-3

A-14-2

A-12-2



the cell membrane in relation to other membranecomponents.22

From the perspective of blood transfusion, thecurrent lack of depth of understanding of carbohydrateblood group antigens has been accepted as adequate.However, as can be seen from other blood groupsystems (e.g.,Rh and HLA), there is value to be found inunraveling their intricacies. It is hoped that byinvestigating the subtleties of the carbohydrate bloodgroup systems, important new biological information,in particular microbial interactions, will be discovered.

References1. Laine RA. Invited commentary. Glycobiology

1994;4:759-67.2. Oriol R, Samuelsson BE, Messeter L. Workshop 1.

ABO antibodies. Serological behaviour andimmuno-chemical characterization. J Immuno-genet 1990;17:279-99.

3. Samuelsson BE, Pimlott W, Karlsson KA. Massspectrometry of mixtures of intact glycosphingo-lipids. Methods Enzymol 1990; 193:623-46.

4. Karlsson H, Johansson L, Miller-Podraza H, KarlssonKA. Fingerprinting of large oligosaccharides linkedto ceramide by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Highlyheterogeneous polyglycosylceramides of humanerythrocytes with receptor activity forHelicobacter pylori. Glycobiology 1999;9:765-78.

5. Oriol R, Barthod F, Bergemer AM, Ye Y, Koren E,Cooper DK. Monomorphic and polymorphiccarbohydrate antigens on pig tissues: implicationsfor organ xenotransplantation in the pig-to-humanmodel. Transpl Int 1994;7:405-13.

6. Henry SM, Samuelsson BE. ABO polymorphismsand their putative biological relationships withdisease. In: King M-J, ed. Human blood cells:consequences of genetic polymorphisms andvariations. Imperial College Press, 2000:15-103.

7. Schenkel-Brunner H. Human blood groups:chemical and biochemical basis of antigenspecificity. Revised Edition. New York: SpringerWien, 2000.

8. Reuter G, Gabius HJ. Sialic acids structure-analysis-metabolism-occurrence-recognition. Biol ChemHoppe Seyler 1996;377:325-42.

9. Traving C, Schauer R. Structure, function andmetabolism of sialic acids. Cell Mol Life Sci 1998;54:1330-49.

10. Voet D,Voet JG. Biochemistry. New York:Wiley andSons, 1998.

11. Atkins RC, Carey FA. Organic chemistry: a briefcourse. New York: McGraw-Hill, 1990.

12. Pazur JH. Neutral polysaccharides. In: Chaplin MF,Kennedy JF, eds. Carbohydrate analysis: a practicalapproach. 2nd ed. Oxford: IRL Press, 1994:100.

13. Oriol R. ABO, Hh, Lewis, and secretion: serology,genetics, and tissue distribution. In: Cartron J,Rouger P, eds. Blood cell biochemistry: molecularbasis of human blood cell antigens. New York:Plenum Press, 1995:37-73.

14. Oriol R, Le Pendu J, Mollicone R. Genetics of ABO,H, Lewis, X and related antigens. Vox Sang1986;51:161-71.

15. Karlsson KA. Meaning and therapeutic potential ofmicrobial recognition of host glycosphingolipids.Mol Microbiol 1998;29:1-11.

16. Karlsson KA. The human gastric colonizerHelicobacter pylori: a challenge for host-parasiteglycobiology. Glycobiology 2000;10:761-71.

17. Karlsson KA. On the chemistry and occurrence ofsphingolipid long-chain bases. Chem Phys Lipids1970;5:6-43.

18. Leray C. Cyberlipid Center:Your www site for fatsand oils. Available at: http://www.cyberlipid.org/amines/amin0001.htm#1. Accessed September 26,2001.

19. Henry SM. Review: phenotyping for Lewis andsecretor histo-blood group antigens.Immunohematology 1996;12:51-61.

20. Moulds JM, Moulds JJ. Blood group associationswith parasites, bacteria, and viruses. Transfus MedRev 2000;14:302-11.

21. Nyholm P-G, Samuelsson BE, Breimer M, Pascher I.Conformational analysis of blood group A-activeglycosphingolipids using HSEA-calculations. Thepossible significance of the core oligosaccharidechain for the presentation and recognition of the A-determinant. J Mol Recognit 1989;2:103-13.



22. Hakomori S, Handa K, Iwabuchi K, Yamamura S,Prinetti A. New insights in glycosphingolipidfunction: “glycosignalling domain,” a cell surfaceassembly of glycosphingolipids with signaltransducer molecules, involved in cell adhesioncoupled with signalling. Glycobiology 1998;8:11-9.

23. Holgersson J, Breimer ME, Samuelsson BE. Basicbiochemistry of cell surface carbohydrates andaspects of the tissue distribution of histo-bloodgroup ABH and related glycosphingolipids. APMISSuppl 1992;27:18-27.

Lissa G. Gilliver, BSc, BAppSc(Hons), Project Leader,Kiwi Ingenuity Ltd., Auckland, New Zealand, andPhD student, Auckland University of Technology, NewZealand. Stephen M. Henry, PhD, Director, KiwiIngenuity Ltd., 19 Mount St., Auckland, New Zealand,and Director, Glycoscience Research Centre, AucklandUniversity of Technology, Private Bag 92006,Auckland 1020, New Zealand, email: [email protected](corresponding author).

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Most severe cases of neonatal alloimmune thrombocytopenia(NAIT) are due to anti-HPA-1a (anti-PlA1) antibodies. We report acase of NAIT due to anti-HPA-2b that resulted in in utero intracranialhemorrhage.A 33-year-old G2P1A0 Caucasian woman had a routineultrasound at 34 weeks. The fetus appeared to have a lefthemispheric hematoma. IVIG, 1g/kg, was started immediately andadministered weekly until delivery. One day after receiving the firstdose of IVIG, fetal platelet count was 18 × 109/L, and Hb was 116g/L. Eleven mL of matched platelets compatible by monoclonalantibody immobilization of platelet antigens (MAIPA) assay weretransfused in utero, raising the platelet count to 62 × 109/L. Repeattransfusions were done later that week and 1 week later, withpretransfusion counts of 19 × 109/L and 16 × 109/L, respectively.Delivery by C section was done at 35.5 weeks, after the thirdplatelet transfusion. Platelet count at birth was 77 × 109/L.Drainage of the hematoma was performed after transfusion. Testingwith a solid phase ELISA revealed reactivity against GP1b/IX.MAIPA testing after platelet treatment with the protease inhibitorleupeptin demonstrated the presence of anti-HPA-2b. On PCR-SSPthe mother was HPA-2a homozygous, the father was HPA-2a/2b.Antibodies against the HPA-2b antigen located on the GP1b/IXcomplex have been reported in rare cases of NAIT. Testing iscomplicated by proteolytic degradation of the antigen-bearingfragment. Compatible platelets are easily found sinceapproximately 85 percent of donors are HPA-2a/2a.Immunohematology 2003;19:43–46.

Key Words: neonatal thrombocytopenia purpura,NAIT, anti-HPA-2b, anti-Koa

Neonatal alloimmune thrombocytopenia (NAIT) iscaused by maternal antibodies directed against anantigen on paternal and fetal platelets. NAIT can resultin intracranial hemorrhage and death or severeneurologic sequelae.1 Unlike its RBC counterpart,erythroblastosis fetalis, NAIT may affect the firstborninfant. In the Caucasian population, most severe casesof NAIT are due to anti-HPA-1a (anti-PlA1). Anti-HPA-5b(anti-Bra) antibodies account for approximately 20percent of serologically proven NAIT. They usually areassociated with milder thrombocytopenia andtherefore lead to less severe clinical manifestations.HPA-2 (Ko, Sib) platelet alloantigens are located onGPIbα.2 Anti-HPA-2b antibodies have been found in

multitransfused patients and in rare cases of NAIT.3–5

We report an additional case of severe NAIT due toanti-HPA-2b. The case illustrates the importance ofperforming sufficient laboratory investigations todetect antibodies other than anti-HPA-1a in a clinicalsetting suggestive of NAIT, in order to supplyappropriate phenotyped platelets for transfusionsupport.

Case ReportThe patient, a 33-year-old Caucasian woman of

Greek origin, G2P1A0, had an unremarkable firstpregnancy. During her second pregnancy, a routineultrasound of the fetus at 34 weeks’ gestation showed aleft hemispheric hematoma. She was taking nomedication and had no other medical problems. IVIG(1g/kg/week) was started. Percutaneous umbilicalcord blood sampling (PUBS) done 1 day post IVIGadministration showed a fetal Hb of 116 g/L and a fetalplatelet count of 18 × 109/L. Microplate ELISA testingdemonstrated strong reactivity against GPIb/IX in the

Neonatal alloimmunethrombocytopenia due to anti-HPA-2b (anti-Koa)M. GOLDMAN, E.TRUDEL, L. RICHARD, S. KHALIFE, AND G.M. SPURLL

Fig. 1. Perinatal transfusions and the fetal platelet count are illustrated.


maternal serum as well as against HLA Class I antigens.Three intrauterine transfusions of 11 mL each weregiven using platelets that were crossmatch compatiblein the monoclonal antibody immobilization of plateletantigens (MAIPA) assay. Perinatal transfusions and thefetal platelet count are illustrated in Figure 1. AfterCesarean delivery, a fourth crossmatch-compatibletransfusion was given and the hematoma, reconfirmedon ultrasound, was evacuated. No petechiae or othersigns of bleeding were noted at delivery. The baby wasdischarged home and is developmentally normal todate, although a seizure disorder is present.

Material and Methods

GTI PAK™ 12 assayIn the ELISA assay,patient’s serum was added to the

microwells of the GTI PAK™ 12 platelet antibodyscreening kit (GTI, Brookfield,WI) coated with plateletglycoproteins and HLA class I antigens, allowing forantibody to bind when present. After washing awayunbound immunoglobulins, an alkaline phosphatase–labeled anti-human IgG was added. Unbound anti-IgGwas washed away and the enzyme substrate P-nitrophenyl phosphate was added. After incubation,the reaction was stopped by a sodium hydroxidesolution and the optical density was measured in aspectrophotometer at 405 nm.

MAIPAThe MAIPA assay was slightly modified from the

method described by Kiefel et al.6 Platelets (20 × 106)isolated from EDTA-anticoagulated blood from knowndonors were washed, suspended in 50 µL of PBS-BSA2%, and incubated with 50 µL of human serum at 37°Cfor 30 minutes. Platelets were then washed in PBS-BSA2% and incubated with 0.2 µg of the glycoprotein-specific monoclonal antibody (anti-GP1b/IX,Immunotech, Marseille, France) at 37°C for another 30minutes. Platelets were washed in 100 µL of isotonicsaline and centrifuged × 3 at 12,000–14,000 g for 2minutes. After the last centrifugation, the plateletswere suspended in 100 µL solubilization buffer(containing 1% leupeptin) and allowed to lyse at 4°Cfor 30 minutes. The lysates were then centrifuged at12,000–14,000 g for 30 minutes at 4°C and 50 µL of thesupernatant was diluted in 200 µL of Tris buffer saline(TBS). One hundred µL of the dilution was distributedinto wells of a microtiter plate coated with goat anti-mouse IgG (1 in 500). After overnight incubation at4°C the microplate was washed × 5 with TBS and

incubated 120 minutes at 4°C with 100 µL of goat anti-human IgG labeled with peroxydase (1 in 500). Thewells were then washed × 5 and filled with 100 µL oforthophenylene diamine (OPD) substrate solution.After a 15-minute incubation at room temperature inthe dark, the enzyme reaction was measured in aspectrophotometer at 490 nm.

Allele-specific PCR (PCR-SSP)Amplification was run using 25 µM each of the

specific primers, as described by Skogen et al.7 Theprimers are designed as follows:

HPA-2a specific primer: 5'-CCCCCAGGGCTCCTGAC-3'

HPA-2b specific primer: 5'-CCCCCAGGGCTCCTGAT-3'

HPA-2c common primer: 5'-GCAGCCAGCGACGAAAATA-3'

The cycling protocol used includes one cycle of94°C for 5 minutes; 30 cycles of 94°C for 1 minute,65°C for 2 minutes, and 72°C for 1 minute; and onecycle of 72°C for 10 minutes. The mixture is madeusing 1.5 mM MgCl2 10 X reaction buffer, 25 µM dNTPsmix, 5 U/µL Taq polymerase (2.5 U/amplification), andthe human growth hormone gene as an internalcontrol.

ResultsOn GTI PAK™ 12 testing, strong reactivity was

demonstrated against GPIb/IX in the maternal serum(OD = 0.919 vs. OD = 0.036 for negative control), aswell as against HLA Class I (OD = 1.741 vs. OD = 0.056for negative control).

MAIPA testing using leupeptin-treated platelets andmonoclonal antibody anti-GPIb/IX demonstratedreactivity against paternal and known HPA-2b(+)platelets, as demonstrated in Table 1.

In PCR-SSP, the mother was found to be HPA-2a/2a,while the father was HPA-2a/2b.

DiscussionThe HPA-2 (Ko) alloantigen system was first

described by van der Weerdt et al. in 1961.8 The genefrequency of HPA-2b is 0.09 in Caucasians in the United

M. GOLDMAN ET AL.

Table 1. MAIPA testing of maternal serum: optical density

Platelet Father Panel 1 Panel 2 Panel 3 Panel 4 Panel 5Genotypes HPA-2ab HPA-2ab HPA-2ab HPA-2ab HPA-2aa HPA-2aa

Control serum 0.121 0.009 0.026 0.009 0.028 0.009

Maternal serum 0.365 0.269 0.298 0.319 0.029 0.019


NAIT due to anti-HPA-2b

States, 0.18 in African Americans, and 0.13 in Koreans.9

The GPIb/IX/V complex is the platelet receptor for vonWillebrand Factor (vWF). The HPA-2 alloantigens arelocated in the N-terminal globular portion of themolecule, as is the vWF binding site. In vitro, anti-HPA-2b antibodies have been shown to inhibit vWFbinding.7 It is therefore possible that the antibody mayaffect platelet function as well as platelet survival.

The N-terminal globular portion of the GPIbmolecule is sensitive to proteases.7 Therefore, it ispreferable to use a protease inhibitor, such asleupeptin,during MAIPA testing to avoid degradation ofthe antigen. In our protocols, 1% leupeptin is added toplatelet suspensions before testing, and also at the stepof solubilization in the lysing buffer.

The GTI PAK™ 12 microplate testing systempermits relatively rapid identification of reactivityagainst a given platelet glycoprotein. However, we andothers have found that alloantibodies may beundetectable in PAK™ 12 testing or may givenonspecific reactions.10,11 Therefore, although the GTIkit is useful as a screening test, it must becomplemented by other laboratory investigations. TheMAIPA assay, first described by Kiefel et al.,6 is used inmany platelet serology laboratories. At the 10th ISBTInternational Platelet Genotyping and SerologyWorkshop, in 2000, the MAIPA assay was reported to beused alone, or in combination with other methods, by28 of 38 laboratories.12 Although this assay may bemore sensitive and specific than the GTI kit, it alsorequires a minimum of 10 hours of technical time andan overnight incubation period. In addition, amonoclonal antibody against the glycoprotein complexof interest must be added. MAIPA testing usingmonoclonal antibodies solely against GPIIb/IIIa andGPIa/IIa in the expectation of finding an anti-HPA-1a oranti-HPA-5b antibody, respectively, would have given anegative result in this case. In addition, the use of afrozen platelet panel or of platelets that have beenstored without protection from proteases may lead toantigen degradation and a negative result. Finally,allele-specific PCR confirms the alloantigen incompatibilitybetween the two parents. However, it does not provethat an alloantibody is actually present. Becausealloantisera are extremely rare, genotyping permits thelaboratory to develop a panel of platelets of knownHPA-2 specificities and to establish a bank of HPA-2a/2adonors.

There have been several other reported cases ofNAIT due to anti-HPA-2b. Kroll et al.13 reported two

cases in Germany. In the first case, the third child ofhealthy parents was found to have a postpartumplatelet count of 53 × 109/L at delivery, whichdecreased to 11 × 109/L on day 4. Three maternalplatelet transfusions were necessary to preventbleeding. In the second case, a second pregnancy wascomplicated by erythroblastosis fetalis due to anti-CDtreated by intrauterine transfusion. Fetal deathoccurred, and a platelet count of 3 × 109/L was found.Because of the small numbers of cases reported andthe laboratory diagnostic difficulties discussed, it isdifficult to know if these cases represent the true rangeof severity of NAIT due to anti-HPA-2b.

The frequency of NAIT due to anti-HPA-2b isdifficult to determine. In a large prospective study byde Moerloose et al.,14 of 8388 newborns in Switzerland,40 newborns had NAIT; anti-HPA-1a and anti-HPA-5bantibodies were detected in three and two mothers,respectively, and no anti-HPA-2 antibodies werepresent. In a study by Hohlfeld et al.,15 of 5194neonates in France undergoing fetal blood sampling forcytogenetic analysis and various infections andhematologic disorders, anti-HPA-1a and anti-HPA-5bwere detected in 23 and 2 mothers, respectively,but noanti-HPA-2 antibodies were present. In a series of 975cases of NAIT reported by McFarland et al.16 spanning11 years of testing by a tertiary referral center, onlythree cases of anti-HPA-2b were detected. In all threecases, anti-HPA-2b was found in the presence of otheralloantibodies (anti-HPA-3a in one, anti-HPA-3b inanother, and anti-HPA-1a and anti-HPA-3a in the third).

Although anti-HPA-2b antibodies are a rare cause ofNAIT, they should be considered in a clinical settingthat is suggestive of NAIT, when more commonly seenantibodies have not been found. The detection of theseantibodies in the MAIPA assay requires fresh platelets,treatment with leupeptin, and the use of an anti-GPIb/IX monoclonal antibody. The GTI PAK™ 12 assaymay be useful in screening for these antibodies.

AcknowledgmentsWe would like to thank Mona Banville,Sonia Grand-

Maison, and Madeleine Malette for performing thelaboratory studies and Sylvain Chiroux for assistancewith the manuscript. We would also like to thank Dr. S.Santoso and Dr. H. Kroll, Institute for ClinicalImmunology and Transfusion Medicine, Giessen,Germany, for confirming the laboratory results.


M. GOLDMAN ET AL.

References1. Kaplan C, Forestier F, Daffos F, et al. Management of

fetal and neonatal alloimmune thrombocytopenia.Transfus Med Rev 1996;10:233-40.

2. Kuijpers R, Ouwehand W, Bleeker P, et al.Localization of the platelet-specific HPA-2(Ko)alloantigens on the N-terminal globular fragment ofplatelet glycoprotein Ibα. Blood 1992;79:283-8.

3. Marcelli-Barge A, Poirier JC, Daussett J. Alloantigensand alloantibodies of the Ko system.Serological andgenetic study. Vox Sang 1973;24:1-11.

4. Bizarro N, Dianese G. Neonatal alloimmuneamegakaryocytosis.Vox Sang 1988;54:112-4.

5. Saji H, Maruya E, Fuji H, et al. New platelet antigen,Siba involved in platelet transfusion refractorinessin a Japanese man. Vox Sang 1989;56:283-7.

6. Kiefel V, Santoso S,Weisheit M, Müeller-Eckhardt C.Monoclonal antibody-specific immobilization ofplatelet antigens (MAIPA): A new tool for theidentification of platelet-reactive antibodies. Blood1987;70:1722-6.

7. Skogen B, Bellissimo DB, Hessner MJ, et al. Rapiddetermination of platelet alloantigen phenotypesby polymerase chain reaction using allele-specificprimers. Transfusion 1994;34:169-76.

8. van der Weerdt CM, van der Wiel H, Engelfriet CP, etal. A new platelet antigen. Proceedings of the 8thCongress of the European Society of Hematology.Basel, Switzerland: Karger, 1961:379.

9. Kim HO, Jin Y,Kickler TS, et al. Gene frequencies ofthe five major human platelet antigens in AfricanAmerican, white, and Korean populations.Transfusion 1995;35:863-7.

10. Lucas GF, Rogers SE. Evaluation of an enzyme-linked immunosorbent assay kit (GTI PakPlus‚) forthe detection of antibodies against human plateletantigens. Transfus Med 1999;9:63-7.

11. Chiroux S, Malette M, Décary F, Roy G, Goldman M.Evaluation of the GTI PAK™ 12 Elisa kit for plateletalloantibody detection (abstract). Transfusion1999;39(Suppl):37S.

12. Panzer S. Report on the Tenth International PlateletGenotyping and Serology Workshop on behalf ofthe International Society of Blood Transfusion. VoxSang 2001;80:72-8.

13. Kroll H, Kiefel V, Muntean W, Mueller-Eckhardt C.Anti-Koa as the cause of neonatal alloimmunethrombocytopenia (abstract). Vox Sang 1994;(Suppl):12.

14. de Moerloose P, Boehlen F, Extermann P, Hohlfeld P.Neonatal thrombocytopenia: incidence andcharacterization of maternal antiplatelet antibodiesby MAIPA assay. Br J Haematol 1998;100:735-40.

15. Hohlfeld P, Forestier F, Kaplan C,Tissot J-D, Daffos F.Fetal thrombocytopenia: A retrospective survey of5,194 fetal blood samplings. Blood 1994;84:1851-6.

16. McFarland J, Curtis B, Friedman K,Aster H. Plateletalloantigen incompatibilities implicated in 975cases of suspected neonatal alloimmunethrombocytopenia (abstract). Blood 2001;98(Suppl):449a.

Mindy Goldman, MD, Associate Senior Director,Medical Affairs, Hematology, Héma-Québec, 4045Côte-Vertu, Montreal, Quebec, Canada, H4R 2W7;Élise Trudel, Service Chief, Hospital ServicesLaboratory and Lucie Richard, PhD, Hospital ServicesLaboratory Director, Héma–Québec, Quebec, Canada;Samir Khalife, MD, Department of Obstetric-Gynecology, Royal Victoria Hospital, 687, Pine AveWest Montreal, Quebec, Canada, H3A 1A1; andGwendoline M. Spurll, MD, Blood Bank Director,Royal Victoria Hospital, Montreal, Quebec, Canada.


This paper describes an enzyme immunoassay for the quantitativedetermination of IgG, IgA, and IgM immunoglobulins on RBCs.Ether eluates made from RBCs were followed by an enzyme-linkedimmunosorbent assay of immunoglobulin concentration.Calibration curves were derived from immunoglobulin standardsand the number of molecules of each isotype per RBC wascalculated. The assay was carried out in 200 healthy blood donorsand 62 patients with warm autoimmune hemolytic anemia (AIHA),two of them with a negative DAT. For healthy blood donors, meanvalues were 58 IgG, 16 IgA, and 3 IgM molecules per RBC. Forpatients with a positive DAT, the mean values were 3435 IgG, 157IgA, and 69 IgM molecules per RBC. An increased level of IgA wasfound in 12 patients without IgA autoantibodies demonstrable inRBC eluates. Increased IgG levels were also observed in patientswith a negative DAT and, in one case, an increased level of IgA wasalso found. The enzyme-linked immunosorbent assay using ethereluates is a sensitive method for quantitating RBC autoantibodies inpatients with AIHA as well as immunoglobulins bound to RBCs inhealthy individuals. Immunohematology 2003;19:47–53.

Key Words: enzyme-linked immunosorbent assay,autoimmune hemolytic anemia, IgG, IgA, and IgMimmunoglobulins, RBCs, quantitative method

Autoimmune hemolytic anemia (AIHA) ischaracterized by immune-mediated hemolysis associatedwith the presence of IgG, IgA, or IgM immunoglobulinson the RBC membrane.1 The most common type ofAIHA is associated with warm-reactive antibodies and inmost cases they are detectable by the DAT.2 Thepresence of immunoglobulins can also be revealed onthe surface of RBCs of DAT-negative healthy individualsby more sensitive methods.3 Several methods toquantitate RBC-bound immunoglobulins have beenreported, including an immunoradiometric assay,autoanalyzer assays, antiglobulin consumption assays,

and enzyme-linked immunosorbent assay (ELISA).4–10

Quantitation of immunoglobulins in a patient with AIHAwould allow a more precise method of monitoring thedisease or defining the synergistic effect of the differentimmunoproteins in causing RBC destruction.11

We have developed an enzyme immunoassay forthe quantitative determination of immunoglobulinisotypes in ether eluates made from RBCs.This methodcan be used to study RBC-associated immunoglobulinsin patients with AIHA and in healthy individuals.

Materials and Methods

Blood samples and ether elutionAfter informed consent had been obtained from

patients and blood donors, blood samples werecollected from venous blood in EDTA (Merck). Thesamples were centrifuged at 270 × g for 8 minutes atroom temperature, and the plasma and buffy coat wereremoved.

Ether eluates were prepared after washing 1 mL ofpacked RBCs × 10 using 10 volumes of saline. Elutionwas performed by adding 1 mL of PBS, pH 7.2,containing 0.4% BSA (Sigma Chemical Co.) and 1 mL ofdiethyl ether (Merck) to 1 mL of RBCs (an average of 1.1× 1010 RBCs). The mixture was incubated in a 37°Cwater bath for 10 minutes, with frequent mixing. Aftercentrifugation, the upper layer of ether was discardedand the hemoglobin-stained eluate was transfered into atest tube. The residual ether was evaporated at 37°C for15 minutes.12 An average of 1 mL of eluate wasrecovered.Eluates were kept frozen at –30°C until used.

Quantitation of red cell–boundIgG, IgA, and IgM in patients withautoimmune hemolytic anemiaand blood donors by enzyme-linked immunosorbent assayA.A. BENCOMO, M. DIAZ,Y.ALFONSO, O.VALDÉS,AND M. E.ALFONSO


ELISA for the quantitation of IgG, IgA, and IgM onRBCs

IgG, IgA, and IgM standard curves were preparedfrom reference sera (Nor-Partigen, Behring) inconcentration ranges from 0.0025 to 0.1 µg/mL in PBS,0.05% Tween (Tw) 20 (Merck), and 0.4% BSA(PBS/Tw/BSA). Flat-bottomed 96-well plates(Maxisorp,Nunce Immunoplates) were precoated with100 µL per well of 5 µg/mL goat anti-human IgG, IgA,orIgM (Sigma) in 0.05M carbonate buffer, pH 9.6, at 4°Ctemperature overnight. After washing × 4 in PBS/Tw,0.2%, 100 µL per well of standards or diluted eluates inPBS/Tw/BSA were incubated at 37°C for 60 minutes.The plates were washed × 4 in PBS/Tw, 0.05%, beforeaddition of 100 µL per well of peroxidase conjugated(Sigma) goat anti-human IgG or IgM diluted 1 in 5000or goat anti-human IgA diluted 1 in 1000. The plateswere incubated with the conjugate at 37°C for 60minutes and then washed as above. A substratesolution (100 µL per well) of 0.24 mg/mL of O-phenylenediamine (Merck) in 0.05M phosphate citratebuffer, pH 5.0, with 0.024% of hydrogen peroxide(Merck) was added. Color development was stoppedafter 15 minutes by adding 100 µL per well of 3MH2SO4 (BDH) and absorbance was read at 492 nm onthe Titertek Multiscan MC plate reader. Absorbances ofduplicate determinations were plotted against theconcentration of the standard points (linear scale). Theamount of IgG, IgA, and IgM was calculated and theresults were expressed as the approximate number ofmolecules of immunoglobulin per RBC, usingAvogadro’s constant and the molecular weights of IgGor IgA (160,000 daltons) and IgM (970,000 daltons).According to Avogadro’s constant, 1 µg contains 376 ×1010 molecules of IgG or IgA and 62 × 1010 molecules ofIgM. The number of molecules of immunoglobulins onRBCs is equal to the concentration in µg/mL of Igs ineluates, multiplied by the molecules of Igs in 1µg, anddivided among the number of RBCs in 1 mL.

Effect of test sample milieu on IgG, IgA, and IgMquantitation by ELISA

We obtained free hemoglobin from human RBCs asfollows:To 6 mL × 10 washed RBCs an equal volume ofhypotonic lytic solution (5M sodium phosphate buffer,pH 7.4) was added. We placed the mixture in a 37°Cwater bath for 10 minutes. The hemoglobin solutionwas collected by centrifugation at 370 × g for 30minutes at room temperature and dialyzed overnightagainst 20 volumes of PBS. IgG, IgA, and IgM standard

curves from reference serum (Nor-Partigen, Behring)were prepared in the hemoglobin solution and onesample without immunoglobulins was used as a blank.To 1 volume of each point of standard curves preparedin the hemoglobin solution and the blank, an equalvolume of diethyl ether was added and mixed. Themixture was incubated at 37°C for 10 minutes. Theupper layer of ether was carefully discarded and theresidual evaporated at 37°C for 15 minutes. BSA and Tw20 were added to each sample to obtain a concentrationof 0.4% and 0.05%, respectively. The ELISA was carriedout with standard curves with this treatment and withstandard curves prepared in PBS/Tw/BSA. The slopes ofthe curves were compared with the t test.

Analytical evaluation of the ELISAThe limit of detection was calculated as the

concentration of the dilution of the first point of thecurves that gave an absorbance more than the meanabsorbance of the blank, plus 3 standard deviations(SDs). The undiluted and diluted 1 in 5, 1 in 10, 1 in100, and 1 in 500 in PBS/Tw/BSA of hemoglobinsolution treated with ether and the PBS/Tw/BSA wereused as blanks and also as a control for nonspecificbinding by the assay. The mean and SD were calculatedon 20 repeat estimation assays.

The working range of the assay was determined bytesting in replicates of 10 at each point of the standardcurves of IgG, IgA, and IgM and the coefficient ofvariation (CV) was calculated. The working range was accepted as that over which CV was no greaterthan 10%.

Parallelism was investigated after serial dilution inPBS/Tw/BSA of seven eluates made from RBCs withreactions of 4+ (IgG, IgA) or 3+ (IgM), 1+, and negativein the DAT with anti-IgG (BPL; Elstree, UK), -IgA, and -IgM (CLB;Amsterdam).

The eluate of a pool of ten DAT-negative RBCs aswell as two eluates with IgG, IgA, and IgMautoantibodies, and a standard human serum withknown concentration of immunoglobulins(Immunotrol, Bio-Merieux, France) adjusted to aconcentration of 0.052 µg/mL of IgG, IgA, and IgM,were used to determine the precision of the ELISA. Theintra-assay and the inter-assay coefficient of variation onseparate days were calculated on 20 determinations.

Test samplesThe ELISA was carried out in duplicate in ether

eluates from 200 healthy blood donors with a negative

A.A. BENCOMO ET AL.


Quantitation of red cell immunoglobulins

DAT, 60 DAT-positive patients with warm AIHA, andtwo patients with AIHA and a negative DAT. Hemolyticanemia was documented by a low hemoglobinconcentration, increased reticulocytes, increasedunconjugated bilirubin concentration, the presence ofmarrow erythroid hyperplasia, and the exclusion ofother hemolytic disorders. Thirty-nine patients hadidiopathic AIHA; the hemolytic anemia in theremainder was associated with other conditions: fourpatients had systemic lupus erythematosus, four hadunspecified respiratory infections, four had chroniclymphocytic leukemia, three were on methyldopa,three had chronic active hepatitis, two had multiplemyeloma, two had acute lymphocytic leukemia, andone had non-Hodgkin’s lymphoma. Patients’ agesranged from 1 to 84 years (median 45 years) and 41were female and 21 were male. The eluates from blooddonors were tested undiluted and diluted 1 in 5; thosefor patients were tested after serial dilution inPBS/Tw/BSA to ensure that the tests were carried outwithin the working ranges for the assay.

Immunohematologic studiesThe immunoglobulin classes of the autoantibodies

were determined by the DAT13 using monospecific anti-IgG (BPL), -IgM, and -IgA reagents (CLB, Amsterdam)and in the eluates by a microplate test.

The microplate test was performed usingmicrotiter plates with V-shaped bottom wells (Greiner)as follows: 10 µL of the mixture of three kinds ofpacked group O RBCs (R1R1, R2R2, and rr), wereincubated with 100 µL of the eluate at 37°C for 1 hourin a tube. The RBCs were washed × 4 and 0.5% of RBCsuspensions in saline were made. Anti-IgG, -IgA, and -IgM were diluted 1 in 2 , 1 in 5, 1 in 10, and 1 in 20 inPBS with 5% of fetal calf serum (Gibco, UK). Then 25µL of the antiserum dilutions and the diluent (asnegative control) were transferred into appropriatemicrotiter wells and an equal volume of the sensitizedRBC suspension was added. After overnight incubationat 4°C,the microtiter plate was placed at a 60° angle for10 minutes, then inverted for 5–10 minutes and read.When the result was positive, the cells remainedtogether in a tight button, when negative they randown in a smear.

All the patients with a positive DAT had IgGautoantibodies. In 11 and six patients, IgA and IgMautoantibodies, respectively, were also present. Similarpatterns of immunoglobulin classes were found in theeluates and with the DAT. Antibodies were not

detected in eluates from patients with AIHA and anegative DAT.

ResultsCurves of IgG, IgA, and IgM represent the mean of

ten consecutive standard curves, each performed induplicate. Immunoglobulin standards made up inhemoglobin and treated with ether did not significantlyaffect the standard curves (p > 0.49) (Fig. 1). Thehemoglobin solution treated with ether used as a blankgave values of optical density from 0.053 to 0.061similar to the PBS/Tw/BSA (0.051). The working rangesfor the ELISA were found to be 0.0025 to 0.07 µg/mLfor IgG and IgM and 0.0025 to 0.06 µg/mL for IgA. Thelimit of detection for IgG corresponded to 0.00062

Fig. 1. Curves for quantitating IgG (Fig. 1A) and IgA (Fig. 1B) withimmunoglobulin standards prepared in PBS,Tween 20 (0.05%),and BSA (0.4%) (●●) and in hemoglobin treated with ether (●).The curve for IgM is similar to that for IgG.

Op

tica

l den

sity

IgG, µg/mL

Op

tica

l den

sity

IgA, µg/mL


A.A. BENCOMO ET AL.

µg/mL, for IgA to 0.0011 µg/ml, and for IgM to 0.0068µg/ml (approximately 1 molecule of Igs per RBC). TheELISA showed excellent parallelism for all the samplesdiluted in the measuring ranges with interdilutionalcoefficent of variation from 5.9% to 16% for IgGsamples, from 2.9% to 13% for IgA samples, and from2.4% to 8.6% for IgM samples (Fig. 2). The intra-assayvariation ranged from 3.6% to 6.7% for IgG samples,from 3.1% to 6.5% for IgA samples, and from 5.5% to6.7% for IgM samples. The inter-assay variation rangedfrom 9.2% to 10% for IgG samples, from 7.5% to 9.5%for IgA samples, and from 8.2% to 9.3% for IgMsamples.

It was established that the immunoglobulinspresent in the eluates were not contaminated by serumimmunoglobulins after ten washes of the RBCs (as

described in Materials and Methods). The supernatantof the last wash gave values of optical density similar tothose of the blank in the ELISA.

The ELISA on healthy blood donors gave mean andstandard deviation values of 58 ± 35 molecules of IgGper RBC (range from 2 to 146), 16 ± 11 molecules ofIgA per RBC (range from 1 to 81), and 3 ± 2 moleculesof IgM per RBC (range from 1 to 11).

In AIHA patients with a positive DAT, the range ofresults for IgG was from 206 to 20,000 molecules perRBC, with a mean of 3435 molecules. In the group ofpatients with IgA autoantibodies the range of resultswas from 90 to 250 molecules per RBC, with a mean of157 molecules. An increased level of IgA, with a rangefrom 94 to 385 molecules per RBC, was found in 12patients with a negative DAT and in the test of eluatesby microplate with anti-IgA. In the remainder of cases,the values of molecules of IgA per RBC were within therange found in blood donors. A mean value of 69 witha range from 26 to 109 molecules per RBC wasobtained in patients with IgM autoantibodies andresults were within the normal range in the rest of thepatients.The results are shown in Figure 3.

Increased IgG levels of 370 and 460 molecules perRBC were observed in two patients with AIHA and anegative DAT. In one case an increased level of IgA(119 molecules per RBC) was also found.

DiscussionThe sandwich ELISA described provides a method

for the quantitation of RBC-bound IgG, IgA, and IgMimmunoglobulins. The assay was carried out in ethereluates because our laboratory routinely performselution in the investigation of autoantibodies in AIHAand because the testing of eluates is a more sensitivemethod to detect immunoglobulins than direct testingof the RBCs.14

Differences in the milieu in test samples andimmunoglobulin standards can produce nonspecificeffects, which modify the kinetics of antigen-antibodyreactions and could affect the estimation of Igconcentration from the standard curve.11 However, thepresence of hemoglobin and the ether treatment ofimmunoglobulins did not affect standard curves.Therefore, all the test samples were investigated withimmunoglobulin standards prepared in PBS/Tw/BSA.Hemoglobin has a peroxidase activity and obscures theantibody assay if a peroxidase conjugate is used.15 Inthis ELISA the hemoglobin in eluates was removed bywashing and did not interfere with the peroxidase

Fig. 2. The relationship between the diluted samples and the amount ofIgG detected in the eluates with reactions of 4+ (●) (Fig.2A), and1+ (■) and negative reactions (▲) (Fig. 2B) in the DAT with anti-IgG. Similar results were obtained in the assays for estimation ofIgA and IgM concentration after serial dilutions of the eluates.

IgG

,µg

/mL

Dilutions

A

IgG

,µg

/mL

Dilution factor

Strengthreaction

1+

Strengthreaction

4+

B



conjugate. The unaffected immunoglobulin detectionafter ether treatment was not surprising in view of theknown use of this elution method for recoveringantibody attached to RBCs,16 although Dumaswala et al.17 found decreased detectability of IgG aftertreatment with another organic solvent such as xylene,using an immunoblotting technique with peroxidase-labeled anti-IgG.

The ELISA showed a wide measuring range; thelower limit corresponded approximately to onemolecule of immunoglobulin per RBC. Accordingly,this assay is suitable for the quantitation ofimmunoglobulins on RBC eluates from DAT-positivepatients and from normal subjects. Although theworking range had an upper limit of 0.07 µg/mL forIgG and IgM, and 0.06 µg/mL for IgA, it could beextended by dilution of high concentration sampleswith PBS/Tw/BSA. This was in agreement with theexcellent dilution parallelism over the measuring rangeshowed by assay with all sample types tested.

We found the mean amount of 58 IgG, 16 IgA, and3 IgM molecules per RBC by using the eluates fromhealthy donors. Other methods gave similar results forIgG and IgM.4,5,8–10 There has been only one previousreport of the number of RBC-associated IgA moleculeson normal human RBCs.10 That study reported amedian of < 29 molecules per RBC, which iscomparable to the amount quantitated by our assay.10

There was considerable variation in the number ofIgG, IgA, and IgM molecules per RBC in patients withAIHA,demonstrating the ability of the assay to measure

widely different degrees of sensitization in clinicalsamples. The results are comparable with previousstudies where the reported ranges in patients withAIHA were from 230 to about 30,000 IgG molecules,from 20 to 168 IgM molecules, and from < 29 to 4500IgA molecules per RBC.8,9,18,19

We encountered IgA autoantibodies in 18 percentof DAT-positive patients, which is similar to the 14percent reported previously by Sokol et al.14 Anincrease of RBC-bound IgA by ELISA was also found in12 patients without IgA autoantibodies demonstrableon RBCs by the DAT and in the eluates by a microplatetest. The clinical significance of antibodies onlydetected by ELISA should be further elucidatedbecause an increased amount of cell-boundimmunoglobulins may also be due to immunecomplexes and nonspecific adsorption of Igs ontoRBCs rather than as a result of autoantibodies.3

Similar considerations are applicable to the resultsfound in two patients with AIHA and a negative DATwithout antibodies demonstrated in eluates. However,in these cases there were findings to suggest that IgGand IgA (in one case) antibodies detected by ELISAwere responsible for the hemolysis. The hemolyticanemia in these patients was associated with chroniclymphocytic leukemia and non-Hodgkin’s lymphoma,respectively. It is known that the incidence of AIHA is higher in patients with these hematologicalmalignancies than in the general population.20

Previous investigators have demonstrated IgG and IgAautoantibodies on RBCs with more sensitive methods

Fig. 3. The number of IgG (A), IgA (B), and IgM (C) molecules per RBC in patients with AIHA. The increased values for IgA and IgM (●) were found inpatients with positive DATs with anti-IgA and -IgM, respectively. An increased level of IgA was also found in 12 patients without IgA autoantibod-ies demonstrable on their RBCs. A heavy horizontal line inside Figure 3 (A, B, and C) shows the highest value of molecule per red cell obtained inhealthy blood donors.

Mo

lecu

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per

red

cel

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Mo

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per

red

cel

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Mo

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IgG IgA IgM

A B C


A.A. BENCOMO ET AL.

than the DAT in patients with a DAT-negative AIHA.21–25

The patients were diagnosed by clinical manifestationsincluding the exclusion of other hemolytic disordersand a positive clinical course following administrationof corticosteroid.

These results showed that the quantitativedetermination of immunoglobulins on eluates by ELISAappears to be useful for the serologic diagnosis of DAT-negative AIHA, although the assay should be evaluatedwith a larger number of cases.

Previous investigation identified RBC autoanti-bodies of the IgM class in approximately 30 percent ofpatients with warm AIHA by enzyme-linked DAT.26 Wefound a frequency (around 10%) of warm IgMautoantibodies that is similar to those found in earlierstudies20 in both the DAT and the ELISA. Thisdifference may be attributable to multiple washingrequired by our assay, which carries the possibility forloss of small quantities of IgM autoantibodies prior toelution. In addition, it can be difficult to obtain IgMautoantibodies in eluates. An investigation showed thatheat elution was the better method compared to acidstromal, chloroform-trichlorethylene, and freeze-thawmethods.27 We detected IgM antibodies in ether eluatesof only three of the six patients with IgMautoantibodies, when a conventional IAT with anti-IgMwas used (data not shown). However, all the IgMautoantibodies revealed by the DAT were detected inthe eluates by the microplate test and ELISA. Asdescribed previously, ELISA also quantitated a verysmall quantity of IgM on RBCs of healthy blood donors.We have no explanation for our different findings froma previous report.26

In essence, the ELISA, using ether eluates, is asensitive method to follow patients with RBCautoantibodies as well as immunoglobulins bound toRBCs in healthy individuals. The assay can be alsoextended to measure IgG subclasses or C3 by usingappropriate specific antiserum.

References1. Engelfriet CP, Overbecke MAM, von dem Borne

AEGKr. Autoimmune hemolytic anemia. SeminHematol 1992;29:3-12.

2. Mollison PL, Engelfriet CP, Contreras M. Bloodtransfusion in clinical medicine. 10th ed. Oxford:Blackwell Science, 1997.

3. Garratty G. Effect of cell-bound proteins on the invivo survival of circulating blood cells.Gerontology1991;37:68-94.

4. Merry AH, Thomson EE, Rawlinson VI, Stratton F. Aquantitative antiglobulin test for IgG for use inblood transfusion serology. Clin Lab Haematol1982;4:393-402.

5. Jeje MO, Blajchman MA, Steeves K, Horsewood P,Kelton JG. Quantitation of red cell-associated IgGusing an immunoradiometric assay. Transfusion1984;24:473-6.

6. Szymanski IO, Huff SR, Delsignore R. Anautoanalyzer test to determine immunoglobulinclass and IgG subclass of blood group antibodies.Transfusion 1982;22:90-5.

7. Gilliland BC, Leddy JP,Vaughan JH.The detection ofcell-bound antibody on complement-coated humanred cells. J Clin Invest 1970;49:898-906.

8. Kiruba R, Han P. Quantitation of red cell-boundimmunoglobulin and complement using enzyme-linked antiglobulin consumption assay.Transfusion1988;28:519-24.

9. Dubarry M,Charron C,Habibi B,Bretagne Y,LambinP. Quantitation of immunoglobulin classes andsubclasses of autoantibodies bound to red cells inpatients with and without hemolysis. Transfusion1993;33:466-71.

10. Hazlehurst DD, Hudson G, Sokol RJ. A quantitativeELISA for measuring red cell-boundimmunoglobulins.Acta Haematol 1996;95:112-6.

11. Sokol RJ, Hudson G. Quantitation of red cell boundimmunoprotein.Transfusion 1998;38:782-95.



12. Rubin H. Antibody elution from red blood cells. JClin Pathol 1963;16:70-3.

13. Vengeler-Tyler V, ed. Technical manual. 12th ed.Bethesda, MD: American Association of BloodBanks, 1996.

14. Sokol RJ, Hewitt S, Booker DJ, Bailey A. Erythrocyteautoantibodies, subclasses of IgG and autoimmunehaemolysis.Autoimmunity 1990;6:99-104.

15. Scott ML. The principles and application of solid-phase blood group serology. Transfus Med Rev1991;5:60-72.

16. Issitt PD. Applied blood group serology. 3rd ed.Miami: Montgomery Scientific Publications, 1985.

17. Dumaswala UJ, Sukati H, Greenwalt TJ.The proteincomposition of red cell eluates. Transfusion1995;35:33-6.

18. Giles CM, Davies KA, Loizou S, Moulds JJ, WalportMJ. Quantification of IgG on erythrocytes ofpatients and normals by radio-ligand-binding assay.Transfus Med 1991;1:223-8.

19. Merry AH, Thomson EE. The role of quantitativeantiglobulin test in immunohaematology;determination of the significance of erythrocyte-bound IgG: Investigation of some parametersaffecting the sensitivity of the antiglobulin test.Biotest Bull 1984;2:137-44.

20. Petz LD, Garratty G. Acquired immune hemolyticanemias. New York: Churchill Livingstone, 1980.

21. Petz LD, Branch DR. Serological test for thediagnosis of immune haemolytic anaemias. In:McMillan R, ed. Methods in haematology: immunecytopenias. New York: Churchill Livingstone,1983:9-48.

22. Salama A, Mueller-Eckhardt C, Bhakdi S.A two stageimmunoradiometric assay with 125I-staphylococcalprotein A for the detection of antibodies andcomplement on human blood cells. Vox Sang1985;48:239-45.

23. Sokol RJ, Hewitt S, Booker DJ, Stamps R. Smallquantities of erythrocyte bound immunoglobulinsand autoimmune haemolysis. J Clin Pathol1987;40:254-7.

24. Meyer F, Garin L, Smati C, Gaspard M, Giannoli C,Rigal D. Application du gel test utilisant uneantiglobuline anti-IgA au diagnostic immuno-logique d’anémie hémolytique auto-immune à testde Coombs direct négatif. Transfus Clin Biol 1999;6:221-6.

25. Garratty G, Postoway N, Nance S, Brunt D. Thedetection of IgG on the red cells of “Coombs-negative” autoimmune hemolytic anemia(abstract). Transfusion 1982;22:430.

26. Sokol RJ, Hewitt S, Booker DJ, Bailey A. Red cellautoantibodies, multiple immunoglobulin classesand autoimmune hemolysis. Transfusion 1990;30:714-7.

27. Ellis JP, Sokol RJ. Detection of IgM autoantibodies ineluates from red blood cells. Clin Lab Haematol1990;6:99-104.

Antonio A.Bencomo, BSc, Senior Investigator, Head ofthe Department of Immunohematology, Institute ofHematology and Immunology, P.O. Box 8070, Ciudadde la Habana, CP 10800, Cuba; and Martha Díaz,MD;Yalile Alfonso, MS; Odalys Valdés, MD; and MaríaE. Alfonso, MD, Senior Investigator, Department ofImmunohematology, Ciudad de la Habana, Cuba.

Attention: State Blood Bank Meeting OrganizersIf you are planning a state meeting and would like copies of Immunohematology for distribution, pleasecontact Mary McGinniss, Managing Editor, 4 months in advance, by phone or fax at (301) 299-7443.


The performance of ready-to-use kits was evaluated on the PK 7200blood grouping system. The Olymp Group (kit 1) and OlympGroup II (kit 2) containing anti-A, -B, -AB, and -D reagents weretested for first and second determinations of A, B, and D antigens.More than 500 RBC samples, including several variant ABO and Dphenotypes, were evaluated for specificity, repeatability,reproducibility, and sensitivity. Specificity was tested with well-characterized reagent RBCs. Repeatability was established by atleast 12 assays per run with three reagent RBCs, and reproducibilitywas established on one run per day for 5 days. No discrepancy was observed in ABO and D determinations with either kit. Inrepeatability, three discrepancies were found with group A and BRBCs with kit 1. In reproducibility, no discrepancies wereobserved. The kit 1 anti-A reagent detected A3 but not Ax RBCs andanti-AB detected both. A B3 RBC was detected by both kits. Amongeight weak D phenotypes, six were positive with kit 1. With kit 2,only one of five weak D phenotypes was detected.Immunohematology 2003;19:54–56.

Key Words: ABO, Rh(D), PK 7200 automated bloodgrouping system

Improvements in ABO and D typing may be relatedto the introduction of new technologies1 or to changesin reagents.2 New technologies include microplatetesting, i.e., the gel test,3 and solid phase systems.4

Blood typing reagents manufactured from human oranimal plasma or serum have been replaced with newmonoclonal reagents. With new reagents andtechnologies, automation of blood typing usingcomputerized systems has been tested and introducedinto the laboratories. Recently, ready-to-use reagentslinked to one automated system were offered by themanufacturer. We tested their ready-to-use kits for ABOand D blood typing on a fully automated bloodgrouping system, the PK 7200, and detected certainproblems regarding identification of weak Dphenotypes.

Materials and Methods

SamplesWhole blood was collected, in tubes containing

EDTA (Vacutainer, Becton Dickinson, Le Pont de Claix,France), from healthy blood donors whose ABO and D

blood groups had been determined on at least twoprior donations. Samples were processed immediatelyafter collection (ABO and D, an IAT alloantibodyscreening, anti-A and -B, and Hct). At a first donationeach sample had been evaluated on the automated PK7200 (Olympus Biology, Rungis, France) for ABO and Dblood groups with monoclonal anti-A, -B, and -ABreagents (first kit, AbiO+, Lyon, France) and onemonoclonal anti-D reagent (IgM like + IgG Bioclone®,Ortho-Clinical Diagnostics, Issy, les Moulineaux,France). In a second donation, the samples had beentested with other monoclonal anti-A, -B, and -ABreagents (AbiO+) and one other monoclonal anti-Dreagent (Totem®, IgG + IgM, Diagast Laboratories, Loos,France). When a weak A or B phenotype wassuspected, a gel test (DiaMed, Cressier sur Morat,Switzerland)3 was used, and if necessary, an elution wasperformed using anti-A or -B reagents. For a weak D,the sample was evaluated by an IAT in LISS and by a geltest (DiaMed).

MethodsTwo ready-to-use kits, the Olymp Group (kit 1) and

Olymp Group II (kit 2) (Diagast Laboratories, Loos,France) were evaluated on the PK 7200 (OlympusBiology, Rungis, France). Monoclonal anti-A, -B, -AB, and-D reagents were used in these kits. Clones for each kitwere different (see Table 1). Specific microplates areused on the PK 7200. In each well, hemagglutinationreading is enhanced by a charge-coupled device (CCD)

ABO and Rh(D) blood typing onthe PK 7200 with ready-to-use kitsP. MONCHARMONT, A. PLANTIER,V. CHIRAT, AND D. RIGAL

Table 1. Clones included in each ready-to-use kit

Olymp group Olymp group IIReagent (kit 1) (kit 2)

Specificity Clones Clones

Anti-A 2521 B 8 + 16243 G 2 9113 D 10

Anti-B 9621 A 8 164 B 5 G 10 + 7821 D 9

Anti-AB 2521 B 8 + 16243 G 2 152 D 12 + 9113 D 10

+ 16247 E 10 + 7821 D 9

Anti-D P3x61 + P3x21223 B 10 HM 10

P3x290 + P3x35


ABO and D typing on the PK 7200

camera with a high-resolution image. Results arereported as positive, negative, or indeterminate.Control samples: group A, B, AB, O, D+, and D– weretested before and after each run.

Four parameters were evaluated: specificity,repeatability, reproducibility, and sensitivity. For eachantibody specificity,at least 100 RBC samples known tobe positive for the antigen and 100 RBC samplesknown to be negative for the antigen were tested.Repeatability was established on at least 12 assays perrun, with three well-characterized RBC samples perreagent. Reproducibility was determined in one runper day for 5 successive days with the same RBCsamples, which were stored at 4°C. For sensitivity, thefollowing weak phenotypes were tested: A3, Ax, and B3

for the ABO system and weak D RBCs. When a falselynegative test was obtained with a reagent, the variantRBCs were retested.

ResultsThree hundred samples of A, B,AB, and O RBCs and

240 D+ and D– RBCs (122 D+, 118 D–) were testedversus kit 1. Two hundred sixty-nine samples of A, B,AB, and O RBCs and 329 samples of D+ and D– RBCs(226 D+ and 103 D–) were tested versus kit 2. Nodiscrepancies were observed in A, B, or Ddeterminations. However, in repeatability testing, threediscrepancies were detected with group A and B RBCswith kit 1. Two were group A and one was group B. Nodiscrepancy was found with kit 2. In reproducibilitytesting, no discrepancy was observed with kit 1 or kit2 with RBCs tested for 5 successive days (Table 2).

Kit 1 anti-A reagent detected A3 RBCs but not the Ax

RBCs, and the anti-B reagent gave a positive result withthe B3 RBCs. These three variant RBCs were detectedalso by the anti-AB reagent. Kit 2 detected the A3 andB3 RBCs. Unfortunately, no fresh Ax RBCs were availableat the time we tested kit 2.

With kit 1, eight known weak D RBC samples weretested. Four were positive, two gave indeterminateresults, and two gave a falsely negative result. With kit

2, of the five weak D RBC samples tested, only one waspositive, none gave indeterminate results, but four gavefalsely negative reactions (Table 3).

DiscussionOur findings indicate that using the PK 7200 and

ready-to-use kits, difficulties remain, particularly intyping blood donors with variant RBCs (i.e., A3 andweak D). With the first-generation Olympus bloodgrouping system, the PK 7100, sensitive monoclonalanti-D reagents did not guarantee detection of weak Dphenotypes.5 Confirmation with another sensitivetechnology (e.g., a gel test) was needed. The detectionsystem for the PK 7200 includes a CCD camera toenhance the detection of hemagglutination in thereaction wells and, therefore, increase the effectivenessof the tests. Nevertheless, to avoid falsely positive orfalsely negative reactions, reagents must be adapted tothe automated system in use. Recently, somemanufacturers have offered ready-to-use reagents.

Our study shows that two ready-to-use kits failed todetect weak D phenotypes. Despite improvements inthe PK 7200, kit 2 failed to detect four of five weak Dphenotypes. The anti-D reagents in kits 1 and 2 aredifferent. Kit 1 anti-D reagent contains a mix of fourmonoclonal antibodies, while kit 2 has only one. Theanti-D reagents in both kits include an IgM monoclonalantibody (P3x61 clone for kit 1, and HM 10 for kit 2).In fact, Jones et al.6 recommend the use of an IgMmonoclonal anti-D with high avidity in saline fordetection of weak D phenotypes. Williams7 observedgood detection of weak D phenotypes with both theIgM monoclonal antibody clone P3x61 (84%) andclone HM 10 (74%). In our short study, kit 1 anti-D(P3x61) detected 75 percent (6 of 8) of the weak Dphenotypes, whereas with kit 2 (HM 10) anti-Dreagent, which contains one monoclonal antibody, only20 percent (1 of 5) were detected. Futhermore, Scottand Voak8 state that, for detecting all weak Dphenotypes, a single monoclonal anti-D is notadequate. To increase the efficiency in blood donortyping, particularly for weak D phenotypes, kit 2 mustbe used with another kit for the same automatedsystem or with another technology (e.g., a gel test).

Table 2. ABO discrepancies (repeatability and reproducibility)

Repeatability* Reproducibility†RBCs Kit 1 Kit 2 Kit 1 Kit 2

Group A 2 0 0 0

Group B 1 0 0 0

Group AB 0 0 0 0

*Established on ≥ 12 assays per run vs. three well-characterized RBCs per reagent†Determined by one run per day for 5 successive days with the same RBCs

Table 3. Detection of weak D phenotypes

Kit 1 Kit 2(8)* (5)*

positive indeterminate negative positive indeterminate negative

4 2 2 1 0 4

*Number of samples tested


P. MONCHARMONT ET AL.

Improvement in kit 2, i.e., addition of one or more anti-D monoclonal antibodies, is needed. Lastly,detection ofAx RBCs is increased by using an anti-AB reagent.

Several hypotheses could explain the discrepanciesobserved with kit 1 anti-A and anti-B reagents with therepeatability assay. First, a lower volume of RBCsand/or reagent could have been dispensed by the PK7200 in the wells of the microplate. Nevertheless, atthe time of the assay, no other grouping difficulty wasobserved. Secondly, a wrong reading was possible, butno problem was noted with the CCD camera. Eventhough the assay was performed according to themanufacturer’s directions, these discrepancies are notclearly explained. Nevertheless, discrepancies with kit1 anti-A and anti-B reagents were only observed withthe repeatability assay. The other tests for ABO bloodgrouping gave good results with kit 1.

ConclusionThe ready-to-use kits for ABO and D blood typing

on the PK 7200 blood grouping system are efficient inspecificity, repeatability, and reproducibility for routinetesting of RBCs with normal ABO and D antigenexpression. However, numerous discrepancies, namelyfalsely negative reactions, remain with kit 2 for weak Dphenotypes. We recommend the use of kit 2 inassociation with kit 1 or with another ready-to-usereagent or technology for weak D typing.

References 1. Chiaroni J,Mannessier L.Methodological evolutions

in immunohematology. Transfus Clin Biol 2000;7(Suppl 1):44-50.

2. Le Pennec P Y. Evolution of reagents in erythrocyteimmunohematology. Transfus Clin Biol 2000;7(Suppl 1):40-3.

3. Lapierre Y, Rigal D,Adam J, et al.The gel-test : a newway to detect red cell antigen-antibody reactions.Transfusion 1990;30:109-13.

4. Beck ML, Rachel JM, Sinor LT, Plapk FV. Semi-automated solid phase adherence assays for pre-transfusion testing. Med Lab Sci 1984;41:374-81.

5. Moncharmont P, Giannoli C, Chirat V, et al. Weakrhesus D (RH 1) grouping on the Olympus PK 7100blood grouping system.Transfus Med 1997;7:66-7.

6. Jones J, Scott ML,Voak D. Monoclonal anti-Dspecificity and RH D structure:criteria for selectionof monoclonal anti-D reagents for routine typing ofpatients and donors.Transfus Med 1995;5:171-84.

7. Williams M. Monoclonal reagents for Rhesus-Dtyping of Irish patients and donors: a review. Br JBiomed Sci 2000;57:142-9.

8. Scott ML,Voak D. Monoclonal antibodies to Rh D—development and uses. Vox Sang 2000;78(Suppl 2):79-82.

Pierre Moncharmont, MD, PhD, Chief of office,Department of Immunology, E.F.S.Rhône-Alpes Site deLyon, 1-3 rue du Vercors, 69364 Lyon cedex 07 France;Annick Plantier, Technician, Véronique Chirat,Technician, and Dominique Rigal, MD, PhD, Director,E.F.S. Rhône-Alpes Site de Lyon, Lyon, France.

Notice to Readers: Immunohematology, Journalof Blood Group Serology and Education, is printed on acid-free paper.


One hundred voluntary blood donors in Hanoi were typed forantigens in the MNS, Rh, Kell, Duffy, and Kidd blood group systems.They were also tested for the presence of the Mi.III (GP.Mur)phenotype and Lewis system antigens. The Jk phenotypefrequencies were markedly different from those previouslyreported. The frequency of the Mi.III phenotype was similar to thatreported in Chinese and Taiwanese. Immunohematology 2003;19:57–58.

Key Words: blood groups, phenotype frequencies,Vietnam, Jk, Miltenberger

There is minimal information on blood groupfrequencies in Vietnam in English-language scientificjournals. The few reports have not included theMiltenberger (Mi) phenotypes, of which Mi.III(GP.Mur) is present with a relatively high frequency inChinese,Thai, and Taiwanese populations. There is alsoa likelihood of erroneous information on Jkphenotypes in previous publications.1,2

Materials and MethodsTo investigate the frequency of the Mi.III (GP.Mur)

phenotype and to reevaluate Jk phenotypefrequencies,100 volunteer blood donors in Hanoi weretyped for antigens of the MNS, Rh, Kell, Duffy, and Kiddblood group systems and antigens in the Lewis system.ABO groups were not performed, as previous reportsfrom different areas of Vietnam were in generalagreement. A total of 160 donors were tested for Mi.III,using anti-Mur and anti-Mur/Hop sera.

Blood group antisera weredonated by CSL Biosciences(Australia) and Gamma Biologicals(USA). Anti-Jka and anti-Jkb were IgMmonoclonal reagents. Human anti-Mur and anti-Mur/Hop sera werekindly supplied by Mr M.K. Mak ofthe Hong Kong Red Cross BloodTransfusion Service. All bloodtypings were performed manuallyby the tube method according to

manufacturers’ instructions. Blood samples werecollected randomly from volunteer blood donors at theblood bank at Thanh Nhan Hospital, Hanoi.

ResultsMost of the blood group phenotype frequencies

were similar to those in other Asian populations (Table1). However, the Jk phenotype frequencies weremarkedly different from those previously reported inVietnam (Table 2). The Mi.III phenotype was present ata level similar to that reported in the Chinese and theTaiwanese (6%), but testing of an additional 60 donorsfor Mi.III raised the frequency to 6.5 percent (Table 1).

DiscussionThe phenotype frequencies reported here should

be considered to be approximate and someamendments may be expected when larger numbers ofVietnamese are tested.

For the majority of the phenotypes, the resultscorresponded closely to those obtained on blooddonors in Ho Chi Minh City (South Vietnam) by TranVan Bé,1 but significant differences were noted in theJk system.

The discrepancies between our Jk typings andthose reported in earlier studies of the Vietnamesepopulation are striking.1,2 The earlier studies reporteda high frequency of the Jk(a–b–) phenotype, while the

Jk and Mi.III phenotypefrequencies in North VietnamNGUYEN THI HUYNH, DEREK S. FORD,TRAN THI DUYEN,AND MAI THANH HUONG

Table 1. Approximate phenotype frequencies of Hanoi donors based on testing 100 donors

MNSs Rh Fy Jk Kell Le

Ms 58% CDe 57% Fy(a+b–) 88% Jk(a+b–) 25% K–k+ 100% Le(a+b–) 0%

MSs 7% CcDe 13% Fy(a+b+) 11% Jk(a+b+) 53% K+k+ 0% Le(a–b+) 62%

MNs 18% cDEe 6% Fy(a–b+) 1% Jk(a–b+) 22% K+k– 0% Le(a+b+) 8%

Ns 17% cDE 3% Le(a–b–) 30%

CcDEe 19%

cDe 1%

CDEe 1%

Mi.III (GP.Mur) 6.0%*

*Testing of an additional 60 donors raised the incidence to 6.5%


NGUYEN THI HUYNH ET AL.

Jk(a+b+) phenotype was completely absent in onestudy1 and present at only 5.26 percent in the other.2

Our results show an absence of the Jk(a–b–)phenotype, with frequencies much closer to thosereported in other Asian populations. It is possible thatthe Jk typings in the earlier Vietnamese studies wereerroneous due to the use of low-potency antisera thatfailed to detect the Jk(a+b+) phenotype.

The presence of the Mi.III phenotype in theVietnamese population had not been reportedpreviously, but its presence was expected as it hadbeen reported in Thailand,3 Hong Kong,4,5 China,6

Taiwan,7 and in Chinese in Australia.8 Mi.III waspresent in 6.5 percent of 160 donors. Variation inreactions with two reagents labeled as anti-Mi.IIIsuggests that Mi.VI (Bun) may also be present in NorthVietnam as it is in Thailand. Only those samples thatreacted with both reagents were classified as Mi.III inthe tables. King et al.9 have shown the value ofimmunoblotting in determination of Mi phenotypes,but molecular biological and immunoblottingtechniques could not be performed in this study. It ishoped that such tests will be performed in the futureto more accurately identify the Mi phenotypes present.

Antibodies to the antigens represented in the Mi.IIIphenotype have previously been reported as causingboth hemolytic transfusion reactions10 and hemolyticdisease of the newborn11 in Asians. Given a 6.5 percentfrequency of the Mi.III phenotype in the Vietnamese, itis possible that its presence has resulted in some of thecases of transfusion reactions and hemolytic disease ofthe newborn that have been reported to be due tounidentified antibodies. It becomes important thatscreening panels used in Vietnam include RBCs withthe Mi.III phenotype, as do those used in Hong Kong.

Further studies to determine whether thedifferences in JK phenotype frequencies between testson Hanoi (North Vietnam) donors and on donors fromHo Chi Minh City (South Vietnam) represent a truevariation in the population are warranted. Testing ofethnic minorities found in the mountain regions ofNorthwest Vietnam would also be of anthropologicalinterest.

References1. Tran Van Bé. Blood groups on red cells. J Haematol

Blood Tx Soc Viet Nam, 1994;3:1-4.2. Do Trung Phan, Tran Hong Thuy, Bui Mai An, et al.

Haematology indexes in mature people of VietNam. In:Memoirs of scientific researches of Ha NoiMedical College 1998;1:162-9.

3. Chandanyingyong D,Pejrachandra S. Studies on theMiltenberger complex frequency in Thailand andfamily studies. Vox Sang 1975;28(2):152-5.

4. Lin CK, Mak KH, Cheng G, et al. Serologiccharacteristics and clinical significance ofMiltenberger antibodies among Chinese patients inHong Kong. Vox Sang 1998;74:59-60.

5. Poole J. The Mi.III phenotype among Chinesedonors in Hong Kong: immunochemical andserological studies. Transfus Med 1991;1:169-75.

6. Metaxas-Buehler M, Metaxas MN, Sturgeon P. MNSand Miltenberger frequencies in 211 Chinese. VoxSang 1975;29:394-9.

7. Broadberry RE, Lin M. The distribution of the Mi.III(GP.Mur) phenotype among the population ofTaiwan. Transfus Med 1996;6:145-8.

8. Winkler IG, Chi-Shen Wu, Woodland N, Flower R.Detection of Miltenberger blood grouppolymorphisms: should cells carrying Miltenbergerantigens be included in screening panels? Topics inTransfus Med 1999;6(1):9-13.

9. King MJ, Poole J, Anstee DJ. An application ofimmunoblotting in the classification of theMiltenberger series of blood group antigens.Transfusion 1989;29(2):106-12.

10. Lin M, Broadberry RE. An intravascular haemolytictransfusion reaction due to anti-Mi(a) in Taiwan.Vox Sang 1994;67:320.

11. Lin CK, Mak KH, Szeto SC, et al. First case ofhaemolytic disease of the newborn due to anti-Murin Hong Kong. Clin Lab Haematol 1996;18:19-22.

Nguyen Thi Huynh, Tran Thi Duyen, Mai ThanhHuong, Hematology/Blood Bank, Thanh NhanHospital, Hanoi, Vietnam; and Derek S. Ford, BAppSci(corresponding author), ImmunohematologyConsultation and Education Services, PO Box 5436,Port Macquarie, NSW 2444, Australia.

Table 2. Different phenotype frequencies in the Jk system, according tothree studies

Jk Jk Jk JkReferences (a+b–) (a+b+) (a–b+) (a–b–)

This study 25% 53% 22% 0%

Tran Van Bé1 23.85% 0% 21.10% 55.05%

Do Trung Phan et al.2 13.68% 5.26% 62.12% 18.94%


Letter to the Editors

Vel– donors in YugoslaviaThe Vel antigen belongs to the high-frequency antigen series (901), ISBT number 901.001.1 It was described for

the first time in 1952 when it was discovered in the serum of a previously transfused patient,2 and the secondexample was described in 1955.3 The Vel– phenotype appears to be inherited as a recessive character.4 Since thefrequency of the antigen is > 99 percent and the antibody usually causes hemolysis,4 Vel– donors have found a placeon many rare blood group donor lists. Anti-Vel is sometimes difficult to identify. In 1994, a misidentified anti-Vel,which caused a fatal hemolytic transfusion reaction due to inappropriate use of serologic techniques,was described.5

Anti-Vel was detected in the serum of a group A patient during routine selection of blood for transfusion. Shehad a history of two pregnancies and had been transfused with four units of blood 4 years before.

Serum prepared from the patient’s plasma,obtained by manual plasmapheresis,was used to type donors’bloodfor the Vel antigen. We typed 3108 group A donors and 3633 group O donors.

The serum demonstrated strongly positive reactions at room temperature, by IAT, and versus enzyme-treatedRBCs. Antibody specificity, anti-Vel, was determined by the IBGRL in Bristol. Further investigation of the patient’sRBCs confirmed the following phenotype: group A, CcDEe, NNss, Lu(a–b+), kk, Le (a–b+), Fy(a–b+), Jk(a–b+), P1 +,and Vel–. The antibody not only reacted in saline and by IAT, but also demonstrated hemolytic activity (IBGRL).The titer of anti-Vel was 256 at room temperature and by IAT. The IAT was also performed using monospecificreagents. Anti-IgG and -IgA reacted 2+ by IAT. Anti-IgM, -C3, and -C4 reacted 4+ by IAT.

Out of 6741 donors typed for Vel, two Vel– donors (0.02966%) were identified. The calculated frequency ofVel was 0.9997, which is in accordance with the results obtained in other Caucasian populations.

This case points out the necessity of establishing a list of rare donors in our country and listing them with aninternational blood exchange.

References1. Reid ME, Lomas-Francis C. The blood group antigen facts book. San Diego, CA: Academic Press, 1997:373.2. Sussman LN, Miller EB. Un nouveau facteur sanguin ‘Vel’. Rev Hématol 1952;7:368.3. Levine P, Robinson EA, Herrington LB, Sussman LN. Second example of the antibody for the high-incidence

blood factor Vel. Am J Clin Pathol 1955;25:751.4. Issitt PD,Austee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery Scientific Publications,

1998:801-4.5. Storry JR, Mallory DM. Misidentification of anti-Vel due to inappropriate use of prewarming and adsorption

techniques. Immunohematology 1994;10:84-6.

Milan Djokic, MD, PhD National Blood Transfusion Institute, Immunohematological Reference Laboratory,

St. Sava 39, PO Box 13, 11000, Belgrade, Yugoslavia

Andrija Kuzmanovic, MD Clinical Center of Kraguzevac, Yugoslavia

Zivko Budisin, MDNational Blood Transfusion Institute, Belgrade

Snezana Srzentic, MD National Blood Transfusion Institute, Belgrade

Joyce Poole, FIBMS, IBGRL, SW Blood Transfusion Center, Bristol, UK

Radica Stevanovic National Blood Transfusion Institute, Belgrade, Yugoslavia

C O M M U N I C A T I O N S


Platelets in Thrombotic and Non-thromboticDisorders: Pathophysiology, Pharmacology, andTherapeutics. Paolo Gresele, Clive P. Page, ValentinFuster, and Jos Vermylen, eds. Cambridge UK:Cambridge University Press, 2002. 1124 pp. List Price:$300.00 (£245.00). ISBN 052180261X. On-lineordering: http://titles.cambridge.org/catalogue.asp?isbn=052180261X

Platelets in Thrombotic and Non-thromboticDisorders is an informative and extensively referencedtextbook that is highly recommended to biomedicalscientists and clinical hematologists with stronginterests in platelet physiology and disease states. Theeditors have assembled an outstanding group ofcontributors to provide overviews of plateletphysiology, pathologic platelet disorders, and thepharmacology of platelet-dependent disease. Thistextbook will be most useful to scientists studying thepathophysiology of platelet disorders and the basicscience of platelet function; many of the chapters,especially the 29 physiology chapters, include therelevant historical context and hard-to-find seminalreferences that are essential for methodology,manuscript preparation, and general background. Thetextbook was published in 2002; therefore, the mostup-to-date references are from 2000–2001, includingseveral from 2001 that are listed as in press.

The scope of this textbook encompasses a largenumber of separate chapters, necessitating relativelyconcise presentation of data and discussions; thisenables the reader to quickly focus on a particulartopic, although more cross-referencing betweenchapters would be helpful for future editions. Thechapters on platelet physiology are highly focused toprovide the reader with state-of-the-art knowledge ondistinct aspects of platelet function. For example,platelet signaling is discussed over four separatechapters, including the roles of cAMP and cGMP,tyrosine kinases, protein kinase C, and calcium. Thesebriefs include important methodological detail that iswell referenced for benchwork. There is a strong groupof platelet pathology chapters that discussthrombocytopenia; a more comprehensive backgroundin earlier chapters on platelet circulation kineticsmight better serve these chapters. One novel aspect ofthis textbook is the number of chapters that explore

the interactions of platelets with heterotypic cells inboth normal physiology and disease. These includevascular control of platelet function, leukocyte-plateletconjugate formation in vascular disease, the role ofplatelets in tumor invasiveness and metastaticpotential, and the interaction of platelets with variouspathogens, both in vivo and during storage. Severalother chapters are highlighted below to provide asnapshot of some of the specific strengths of this text.

A concise, highly relevant overview of the mostcritical issues and controversies in platelet storage andtransfusion is provided by Scott Murphy. This chapteris valuable for hematology and transfusion medicinefellows and includes an excellent discussion of plateletpreparation and storage methodologies andcomprehensive tables for quick reference. The clinicalfocus also encompasses the relevant indications forplatelet transfusion and includes a succinct outline ofalloimmunization and strategies for finding compatibleplatelets. Thomas Kunicki authors a readable overviewof gene regulation of platelet function. This chapterincludes details on the regulation of megakaryocyte-specific genes found in hematopoietic-derived celllines and of transcription factors, including the GATAand Ets families. Recent studies that have examinedplatelet polymorphisms as risk factors for clinicallyrelevant atherothrombosis are also discussed, includingclearly presented tables on α2β1 density andpolymorphisms of GPIbα,VNTR, and αIIbβ3.

The chapter on platelet procoagulant function byHemker is broadly written to give a concise overviewof the interactions between platelets and soluble/plasma coagulation factors. There has been anexplosion of new information in this field since thischapter was prepared, but the general principlesrelated to platelet-associated thrombin generation areclearly written and serve as a firm background forfurther reading and research. Several chapters areincluded on the evaluation of trials of antiplatelettherapy for cardiovascular, cerebrovascular, andperipheral vascular disease. All of these includecomprehensive tables and detailed analyses ofantiplatelet efficacy and outcomes, as well as summaryrecommendations on current therapeutic regimens.

Future scenarios for antiplatelet therapy arediscussed in the chapter on pharmacogenetics. Thischapter highlights some of the possible targets of

B O O K R E V I E W


pharmacogenomic therapy, including those that areapplicable to general risk, e.g., high C-reactive proteinpopulation subsets, and platelet-derived risk factors,including polymorphisms of GPIIIa (PlA1/2),differential thienopyridine metabolism, and the novelP2Y12 receptor; the latter two are relevant to plateletADP-receptor antagonist therapy. There is also aninteresting discussion of the possibilities for futurescreening of atherothrombotic risk factors, and thesubsequent use of targeted antiplatelet therapy.

Platelets in Thrombotic and Non-thromboticDisorders emphasizes strongly focused chapters andcomprehensive referencing on platelet physiology and

function; this extensive framework for up-to-dateplatelet knowledge makes this text essential forhematologists and blood bankers in training, and forresearchers and veteran clinicians with an interest inhemostasis.

Henry M. Rinder, MDAssociate Professor of Laboratory Medicine

and Internal Medicine (Hematology)Director, Coagulation Laboratory

Yale University School of MedicineNew Haven, CT 06520-8035

Masters (MSc) in Transfusion and Transplantation SciencesAt

The University of Bristol, England

Applications are invited from medical or science graduates for the Master of Science (MSc) degree inTransfusion and Transplantation Sciences at the University of Bristol. The course starts in October 2003 and willlast for one year. A part-time option lasting three years is also available. There may also be opportunities tocontinue studies for PhD or MD following MSc. The syllabus is organised jointly by The Bristol Institute forTransfusion Sciences and the University of Bristol, Department of Transplantation Sciences. It includes:

• Scientific principles underlying transfusion and transplantation• Clinical applications of these principles• Practical techniques in transfusion and transplantation• Principles of study design and biostatistics• An original research project

Applications can also be made for Diploma in Transfusion and Transplantation Science or a Certificate inTransfusion and Transplantation Science.

The course is accredited by the Institute of Biomedical Sciences.

Further information can be obtained from the website:

http://www.blood.co.uk/ibgrl/MscHome.htm

For further details and application forms please contact:

Professor Ben Bradley

University of Bristol, Department of Transplantation Sciences

Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, England

FAX +44 1179 595 342, TELEPHONE +44 1779 595 455, E-MAIL: [email protected]


Cellular Therapy: New Frontiers in TransfusionMedicine. Ronald A. Sacher, MD, ed. Bethesda, MD:American Association of Blood Banks (AABB) Press,2002. Stock #023030; softcover, 130 pages; list price:$80.00; member price $50.00. To order: call (866) 222-2498 or fax (301) 951-7150.

This reviewer has a research interest in adoptiveimmunotherapy, and gladly agreed to review CellularTherapy: New Frontiers in Transfusion Medicine. Iwas not disappointed. Although the book is relativelyshort, the chapters are detailed and well-referenced andpresent an appropriate spectrum of currently practicedcellular therapy techniques and more esotericapplications.

The first chapter is a concise yet thoroughhistorical perspective on cellular therapy written byHarvey Klein, MD, and originally delivered as the 2001Emily Cooley Memorial Lecture. Covering topicsincluding the development of the blood cell separator,initial observations supporting the clinical feasibility ofadoptive immunotherapy, somatic cell gene therapy,and the cellular vaccines, this chapter nicely covers thecontinuing evolution of transfusion medicine in thearea of cellular therapy.

There are comprehensive chapters on establishedcellular therapy methods that many blood banks arealready performing. Mobilization and collection ofperipheral blood progenitor cells, increasingly used forhematopoietic cell transplantation, is discussed byDavid Stroncek, MD, and Susan Leitman, MD. Includedare mobilization protocols, expected results, and donorissues. As a companion chapter, Elizabeth Read, MD,and Charles Carter describe methods for T-celldepletion of hematopoietic progenitor cell products

prior to allogeneic transplantation, and the NIH clinicalexperience with this procedure. While not quite “how-to” guides, these chapters would nonetheless serve asexcellent introductions for practitioners whoseclinicians may request these services.

An evolving methodology with particularlyexciting potential is pancreatic islet cell transplantationfor type I diabetes, as discussed by Elizabeth Read, MD,Janet Lee, MT(ASCP), and David Harlan, MD. Theauthors cover pancreas procurement, processing toisolate islets, and the current status of clinical trials.After reading this chapter, it seems increasingly likelythat islet cell processing will become a techniquelargely performed and overseen by transfusionmedicine specialists, and this chapter is a good primeron the subject.

The remaining three chapters are more forward-looking, covering areas that very few physicians andtechnologists currently have experience with. Forexample, David Williams, MD, discusses geneticmodification of stem cells, with an emphasis onretroviral and other vectors currently in use. Ena Wang,MD, and Francesco Marincola, MD, discuss immuno-therapies for solid tumors. Finally, Catherine Verfaillie,MD, and colleagues give an overview on tissue-residentstem cells, including mesenchymal stem cells.

In summary, I recommend this well-written bookfor anyone who wants an introduction to wheretransfusion medicine may be heading, and whatcomponents we may be asked to prepare in the future.

John D. Roback, MD PhDEmory University School of Medicine

WMB 2307, 1639 Pierce DriveAtlanta, GA 30322

B O O K R E V I E W

Attention SBB and BB Students: You are eligible for a free 1-year subscription to Immunohematology. Askyour education supervisor to submit the name and complete address for each student and the inclusive datesof the training period to Immunohematology, P.O. Box 40325, Philadelphia, PA 19106.


General (2000–2003)

Blood group antigens1. Cao Y, Merling A, Karsten U, Schwartz-Albiez R.

The fucosylated histo-blood group antigens H type2 (blood group O CD173) and Lewis Y (CD174)are expressed on CD34+ hematopoieticprogenitors but absent on mature lymphocytes.Glycobiology 2001;11:677-83.

2. Cartron JP, Colin Y. Structural and functionaldiversity of blood group antigens. Transfus ClinBiol 2001;8:163-99.

3. Cooling LL, Kelly K. Inverse expression of Pk andLuke blood group antigens on human RBCs.Transfusion 2001;41:898-907.

4. Grahn A, Elmgren A,Akerg L, et al. Determinationof Lewis FUT3 gene mutations by PCR usingsequence-specific primers enables efficientgenotyping of clinical samples. Hum Mutat2001;18:358-9.

5. Hosoi E, Hirose M, Hamano S. Expression levels ofH-type α (1,2)-fucosyltransferase gene and histo-blood group ABO gene corresponding tohematopoietic cell differentiation. Transfusion2003;43:65-71.

6. Iwamoto S, Kamesaki T, Oyamada T, et al.Reactivity of autoantibodies of autoimmunehemolytic anemia with recombinant rhesus bloodgroup antigens or anion transporter band 3. Am JHematol 2001;68:106-14.

7. Jaskiewiez E, Gerwinski M, Culin Y, Lisowska E.Recombinant forms of Gerbich blood groupantigens: expression and purification. TransfusClin Biol 2002;9:121-9.

8. Martine GHM Tax,Van der Schoot CE, van DoornR, et al. RHC and RHc genotyping in differentethnic groups. Transfusion 2002;42:634-44.

9. Nathalang O, Kuvanout S, Punyaprasiddhi P,Tasaniyanonda C, Sriphaisal T. A preliminary studyof the distribution of blood group systems in Thaiblood donors determined by the gel system. SEAsian J Trop Med Pub Health 2001;32:204-7.

10. Noizat-Pirenne F, LePennec P-Y, Mouro I, et al.Molecular background of D(c)(e) haplotypeswithin the white population. Transfusion2002;42:627-33.

11. Olsson ML, Irshaid NM, Hosseini-Maaf B, et al.Genomic analysis of clinical samples withserologic ABO blood grouping discrepancies:identification of 15 novel A and B subgroupalleles. Blood 2001;98:1585-93.

12. Reid ME. The Dombrock blood group system: areview. Transfusion 2003;42:107-114.

13. Rios M, Hue-Roye K, Øyen R, Miller J, Reid ME.Insights into the Holley– and Joseph– phenotypes.Transfusion 2002;42:52-8.

14. Rios M, Hue-Roye K, Lee AH, Chiofolo JT, Miller JL,Reid ME. DNA analysis for the Dombrockpolymorphism. Transfusion 2001;41:1143-6.

15. Roubinet F, Janvier D, Blancher A. A novel cis ABallele derived from a B allele through a singlepoint mutation. Transfusion 2002;42:239-46.

16. Scott M. Section 1A: Rh serology. Coordinator’sreport. Transfus Clin Biol 2002;9:23-9.

17. Seltsam A, Hallensleben M, Eiz-Vesper B, Leuhard V,Heymann G, Blasczyk R. A weak blood group Aphenotype caused by a new mutation at the ABOlocus. Transfusion 2002;42:294-301.

18. Wu GG, Jin SZ, Deng ZH, Zhao TM. Polymerasechain reaction with sequence-specific primers-based genotyping of the human Dombrock bloodgroup DO1 and DO2 alleles and the DO genefrequencies in Chinese blood donors. Vox Sang2001;81:49-51.

19. Yahalom V, Ellis MH, Poole J, et al. The rare Lu:–6phenotype in Israel and the clinical significance ofanti-Lu6. Transfusion 2002;42:247-50.

Blood group antibodies1. Boctor FN,Ali NM, Mohandas K, Uehlinger J.

Absence of D-alloimmunization in AIDS patientsreceiving D-mismatched RBCs. Transfusion2003;43:173-6.

2. Burnette RE, Couter K. A positive antibodyscreen—an encounter with the Augustineantibody. J Natl Med Assoc 2002;94:166-70.

3. Collinet P, Subtil D, Puech F, et al. Successfultreatment of extreme fetal anemia due to Kellalloimmunoization. Obstet Gynecol2002;100:1102-5.

4. Daniels G, Hadley A, Green CA. Causes of fetalanemia in hemolytic disease due to anti-K (letter).Transfusion 2003;43:115.

L I T E R A T U R E R E V I E W


5. Daniels G. Section 4:Antibodies to other bloodgroup antigens. Coordinator’s report. TransfusClin Biol 2002;9:75-80.

6. Fernandez-Jimenez MC, Jimenez-Marco MT,Hernandez D, Gonzalez A, Omenaca F, de laCamara C. Treatment with plasmapheresis andintravenous immune globulin in pregnanciescomplicated with anti-PP1Pk or anti-Kimmunization: a report of two patients. Vox Sang2001;80:117-20.

7. Hareuveni M, Merchav H,Austerlitz N, Rahimi-Leveen N, Ben-Tal O. Donor anti-Jka causinghemolysis in a liver transplant recipient.Transfusion 2002;42:363-7.

8. Joshi SR. Streptomycin-dependent panagglutinincausing error in forward blood grouping. VoxSang 2001;81:55.

9. Levin M-D, de Veld JC, vander Holt B,Van’t VeerMB. Screening for alloantibodies in the serum ofpatients receiving platelet transfusions: acomparison of the ELISA, lymphocytotoxicity, andthe indirect immunofluorescence method.Transfusion 2003;42:72-7.

10. Lynen R, Krone O, Legler TJ, Köhler M, Mayr WR. Anewly developed gel centrifugation test forquantification of RBC-bound IgG antibodies andtheir subclasses IgG1 and IgG3: comparison withflow cytometry. Transfusion 2002;42:612-8.

11. Maley M, Babb R, Chapman CE, Fitzgerald J,Cavanagh G. Identification and quantification ofanti-D, -C, and -G in alloimmunized pregnantwomen. Transfus Med 2001;11:443-6.

12. Sarci SU,Alpay F, Yesilkaya E, et al. Hemolyticdisease of the newborn due to isoimmunizationwith anti-E antibodies: a case report.Turkish JPediatr 2002;44:248-50.

13. Strobel E. Comparison of monoclonal andpolyclonal anti-P1 reagents. Clin Lab 2001;47:249-55.

14. Yahalom V, Ellis MH, Poole J, et al. The rare Lu:–6phenotype in Israel and the clinical significance ofanti-Lu6. Transfusion 2002;42:247-50.

15. Yasuda H, Ohto H,Yamaguchi O, et al. Threeepisodes of delayed hemolytic transfusion reactiondue to multiple red cell antibodies, anti-Di, anti-Jk,and anti-E. Transfus Sci 2000;23:107-12.

Blood group genetics1. Cartron JP, Colin Y. Structural and functional

diversity of blood group antigens. Transfus ClinBiol 2001;8:163-99.

2. Darke C, Street J, Guttridge MG. Sequence,genetics and serology of a new HLA-B alleleB*4903. Tissue Antigens 2001;57:478-80.

3. Grahn A, Elmgren A,Aberg L, et al. Determinationof Lewis FUT3 gene mutations by PCR usingsequence-specific primers enables efficientgenotyping of clinical samples. Hum Mutat2001;18:358-9.

4. Hosoi E, Hirose M, Hamano S. Expression levels ofH-type α (1,2)-fucosyltransferase gene and histoblood group ABO gene corresponding tohematopoietic cell differentiation. Transfusion2003;43:65-71.

5. Jaskiewiez E, Czerwinski M, Colin Y, Lisowska E.Recombinant forms of Gerbich blood groupantigens: expression and purification. TransfusClin Biol 2002;9:121-9.

6. LePendu J, Henry S. Section 2: Immunochemical,immunohistological and serological analysis ofmonoclonal antibodies with carbohydrates.Coordinator’s report. Transfus Clin Biol 2002;9:55-60.

7. Lo YM. Fetal DNA in maternal plasma: applicationto non-invasive blood group genotyping of thefetus. Transfus Clin Biol 2001;8:306-10.

8. Martine GHM Tax,Van der Schoot CE, van DoornR, et al. RHC and RHc genotyping in differentethnic groups. Transfusion 2002;42:634-44.

9. Noizat-Pirenne F, LePennec P-Y, Mouro I, et al.Molecular background of D(c)(e) haplotypeswithin the white population. Transfusion2002;42:627-33.

10. Olsson ML, Irshaid NM, Hosseini-Maaf B, et al.Genomic analysis of clinical samples withserologic ABO blood grouping discrepancies:identification of 15 novel A and B subgroupalleles. Blood 2001;98:1585-93.

11. Reid ME, Lisowska E, Blanchard D. Section 3:Epitope determination of monoclonal antibodiesto glycophorin A and glycophorin B.Coordinator’s report. Antibodies to antigenslocated on glycophorins and band 3. Transfus ClinBiol 2002;9:63-72.

12. Rios M, Hue-Roye K, Lee AH, Chiofolo JT, Miller JL,Reid ME. DNA analysis for the Dombrockpolymorphism. Transfusion 2001;41:1143-6.

13. Roubinet F, Janvier D, Blancher A. A novel cis ABallele derived from a B allele through a singlepoint mutation. Transfusion 2002;42:239-46.

14. Seltsam A, Hallensleben M, Eiz-Vesper B, Lenhard V,Heymann G, Blasczyk R. A weak blood group A


phenotype caused by a new mutation at the ABOlocus. Transfusion 2002;42:294-301.

15. Sobel E, Papp JC, Lange K. Detection andintegration of genotyping errors in statisticalgenetics. Am J Hum Genet 2002;70:496-508.

16. Yan L, Zhu F, Fu Q. Jk(a–b–) and Kidd blood groupgenotypes in Chinese people (letter). Transfusion2003;42:289:90.

Blood group biochemistry1. Cao Y, Merling A, Karsten U, Schwartz-Albiez R.

The fucosylated histo-blood group antigens H type2 (blood group O CD173) and Lewis Y (CD174)are expressed on CD34+ hematopoieticprogenitors but absent on mature lymphocytes.Glycobiology 2001;11:677-83.

Monoclonal antibodies1. Kumpel BM. In vivo studies of monoclonal anti-D

and the mechanisms of immune suppression.Transfus Clin Biol 2002;9:9-14.

2. Reid ME, Lisowska E, Blanchard D. Section 3:Epitope determination of monoclonal antibodiesto glycophorin A and glycophorin B.Coordinator’s report. Antibodies to antigenslocated on glycophorins and band 3. Transfus ClinBiol 2002;9:63-72.

3. LePendu J, Henry S. Section 2: Immunochemical,immunohistological and serological analysis ofmonoclonal antibodies with carbohydrates.Coordinator’s report. Transfus Clin Biol 2002;9:55-60.

4. Strobel E. Comparison of monoclonal andpolyclonal anti-P1 reagents. Clin Lab 2001;47:249-55.

Red cell serology/methods1. Brumit MC, Stubbs JR, et al. Conventional tube

agglutination with polyethylene glycol versus redcell affinity column technology (ReACT): acomparison of antibody detection methods. AnnClin Lab Sci 2002;32:155-8.

2. Engelfreit CP, Reesink HW, Krusius T, et al. The useof the computer cross-match.Vox Sang2001;80:184-92.

3. Kopko PM, Paglieroni TG, Popovsky MA, Muto KN,MacKenzie MR, Holland PV. TRALI: correlation ofantigen-antibody and monocyte activation indonor-recipient pairs. Transfusion 2003;43:177-84.

4. Levin M-D, de Veld JC, vander Holt B,Van’t VeerMB. Screening for alloantibodies in the serum ofpatients receiving platelet transfusions: acomparison of the ELISA, lymphocytotoxicity, and

the indirect immunofluorescence method.Transfusion 2003;42:72-7.

5. Lynen R, Krone O, Legler TJ, Köhler M, Mayr WR.A newly developed gel centrifugation test forquantification of RBC-bound IgG antibodies andtheir subclasses IgG1 and IgG3: comparison withflow cytometry. Transfusion 2002;42:612-8.

6. Scott M. Section 1A: Rh serology. Coordinator’sreport. Transfus Clin Biol 2002;9:23-9.

7. Shulman IA, Downes KA, Sazzma K, Maffei LM.Pretransfusion compatibility testing for red bloodcell administration. Curr Opin Hematol 2001;8:397-404.

8. Toyohiro T,Yasuyuk I,Toshio M. IgM alloantibodydetection by M-MPHA, a new solid-phase assaysystem. Transfus Sc 2002;23:47-54.

9. Wu GG, Jin SZ, Deng ZH, Zhao TM. Polymerasechain reaction with sequence-specific primers-based genotyping of the human Dombrock bloodgroup DO1 and DO2 alleles and the DO genefrequencies in Chinese blood donors. Vox Sang2001;81:49–51.

White cell/platelet serology/methods1. Blumberg N, Phipps RP, Kaufman J, Heal JM. The

causes and treatment of reactions to platelettransfusions (letter). Transfusion 2003;43:291.(Reply: Heddle N):292.

2. Darke C, Street J, Guttridge MG. Sequence,genetics and serology of a new HLA-B alleleB*4903. Tissue Antigens 2001;57:478–80.

3. Kao GS,Wood IG, Dorfman DM, Milford EL,Benjamin RI. Investigations into the role of anti-HLA class II antibodies in TRALI. Transfusion2003;42:185–91.

4. Levin M-D, de Veld JC, vander Holt B,Van’t VeerMB. Screening for alloantibodies in the serum ofpatients receiving platelet transfusions: acomparison of the ELISA, lymphocytotoxicity, andthe indirect immunofluorescence method.Transfusion 2003;42:72–7.

5. Lim J, Kim Y, Kyungja H, et al. Flow cytometricmonocyte phagocytic assay for predicting platelettransfusion outcome.Transfusion 2002;42:309–16.

6. Torio A, Moya-Quiles MR, Muro M, et al.Discrepancies in HLA-C typing in transplantation:comparison of PCR-SSP and serology results.Transplant Proc 2002;34:419–20.

Hemolytic anemias1. Frohn C, Jabs WJ, Fricke L, Goerg S. Hemolytic

anemia after kidney transplantation: case report


and differential diagnosis. Ann Hematol2002;81:158–60.

2. Iwamoto S, Kamesaki T, Oyamada T, et al.Reactivity of autoantibodies of autoimmunehemolytic anemia with recombinant rhesus bloodgroup antigens or anion transporter band 3. Am JHematol 2001;68:106–14.

3. Kondo H, Oyamada T, Mori A, et al. Direct-antiglobulin-test-negative immune hemolyticanaemia and thombocytopenia in a patient withHodgkin’s disease.Acta Haematol 2001;105:233–6.

4. Maheshwari VD, Kumar R, Singh S. Coomb’snegative autoimmune hemolytic anemia withthrombocytopenia (Evan’s syndrome). J AssocPhysicians India 2002;50:457–8.

5. Shnaider A, Busok A, Rogachev BV, Zlutnik M,Tomer A. Severe Coombs-negative autoimmunehemolytic anemia in a kidney transplant patient.Am J Nephrol 2001;21:494–7.

Hemolytic disease of the newborn1. Anderson AS, Praetorius L, Jorgensen HL, Lylloff K,

Larsen KT. Prognostic value of screening forirregular antibodies late in pregnancy in rhesuspositive women. Acta Obstet Gynecol Scand2002;81;407–11.

2. Chandrasekar A, Morris KG,Tubman TR,Tharma S,McClelland WM. The clinical outcome of non-RhDantibody affected pregnancies in NorthernIreland. Ulster Med J 2001;70:89–94.

3. Collinet P, Subtil D, Puech F, et al. Successfultreatment of extreme fetal anemia due to Kellalloimmunoization. Obstet Gynecol2002;100:1102–5.

4. Cotorruelo C, Biondi C, Garcia BS. Early detectionof RhD status in pregnancies at risk of hemolyticdisease of the newborn. Clin Exp Med 2002;2:77–81.

5. Daniele G, Hadley A, Green CA. Causes of fetalanemia in hemolytic disease due to anti-K (letter).Transfusion 2003;43:115.

6. Fernandez-Jimenez MC, Jimenez-Marco MT,Hernandez D, Gonzalez A, Omenaca F, de laCamara C. Treatment with plasmapheresis andintravenous immune globulin in pregnanciescomplicated with anti-PP1P

k or anti-Kimmunization: a report of two patients. Vox Sang2001;80:117–20.

7. Lo YM. Fetal DNA in maternal plasma: applicationto non-invasive blood group genotyping of thefetus. Transfus Clin Biol 2001;8:306–10.

8. Mair DC, Scofield TI. HDN in a motherundergoing in vitro fertilization with donor ova(letter). Transfusion 2003;43:288.

9. Maley M, Babb R, Chapman CE, Fitzgerald J,Cavanagh G. Identification and quantification ofanti-D, -C, and -G in alloimmunized pregnantwomen. Transfus Med 2001;11:443–6.

10. Patel RK, Nicolaides K, Mijovic A. Severehemolytic disease of the fetus following in vitrofertilization with anonymously donated oocytes(letter). Transfusion 2003;43:119–21.

11. Sarci SU,Alpay F,Yesilkaya E, et al. Hemolyticdisease of the newborn due to isoimmunizationwith anti-E antibodies: a case report.Turkish JPediatr 2002;44:248–50.

12. Sarci SU,Yurdakok M, Serdar MA, et al. An early(sixth hour) serum bilirubin measurement isuseful in predicting the development ofsignificant hyperbilirubinemia and severe ABOhemolytic disease of the newborn with ABOincompatibility. Pediatrics 2002;109:53.

13. Stanworth S, Fleetwood P, deSilva M. Severehaemolytic disease of the newborn due to anti-Jsb.Vox Sang 2001;81:134–5.

14. Stockman JA. Overview of the state of the art ofRh disease: history, current clinical management,and recent progress. J Pediatr Hematol/Oncol2001;23:554–62.

Transfusion/transplantation1. Atterbury C,Wilkinson J. Blood transfusion. Nurs

Stand 2000;14:47–52.2. Blumberg N, Phipps RP, Kaufman J, Heal JM. The

causes and treatment of reactions to platelettransfusions (letter). Transfusion 2003;43:291.(Reply: Heddle N):292.

3. Brown M,Whalen PK. Red blood cell transfusionin critically ill patients. Emerging risks andalternatives. Crit Care Nurse 2000(Suppl):1–14.

4. Frohn C, Jabs WJ, Fricke L, Goerg S. Hemolyticanemia after kidney transplantation: case reportand differential diagnosis. Ann Hematol 2002;81:158–60.

5. Gryn J, Zeigler ZR, Shadduck RK,Thomas C.Clearance of erythrocyte allo-antibodies usingRituximab. Bone Marrow Transplant 2002;29:631–2.

6. Hareuveni M, Merchav H,Austerlitz N, Rahimi-Leveen N, Ben-Tal O. Donor anti-Jka causinghemolysis in a liver transplant recipient.Transfusion 2002;42:363–7.


7. Howie JC,Tansey PJ, et al. Blood transfusion insurgical practice—matching supply to demand. BrJ Anaesth 2002;89:214–6.

8. Krombach J, Kampe S, Gathof BS, Diefenbach C,Kasper S-M. Human error: the persistent risk ofblood transfusion: a report of five cases.AnesthAnalg 2002;94:154–6.

9. Maxwell EL, Metz J, Haeusler MN, et al. Use of redblood cell transfusions in surgery. ANZ J Surg2002;72:561–6.

10. Reed W,Vichinsky EP. Transfusion therapy: acoming-of-age treatment for patients with sicklecell disease. J Pediatr Hematol/Oncol 2001;23:197–202.

11. Shulman IA, Downes KA, Sazzman K, Maffei LM.Pretransfusion compatibility testing for red bloodcell administration. Curr Opin Hematol2001;8:397–404.

12. Torio A, Moya-Quiles MR, Muro M, et al.Discrepancies in HLA-C typing in transplantation:comparison of PCR-SSP and serology results.Transplant Proc 2002;34:419–20.

13. Yasuda H, Ohto H,Yamaguchi O, et al. Threeepisodes of delayed hemolytic transfusion reactiondue to multiple red cell antibodies, anti-Di, anti-Jk,and anti-E. Transfus Sci 2000;23:107–12.

Disease associations1. Boctor FN,Ali NM, Mohandes K, Uehlinger J.

Absence of D– alloimmunization in AIDS patientsreceiving d-mismatched RBCs. Transfusion2003;43:177–6.

2. Joshi SR. Streptomycin-dependent panagglutinincausing error in forward blood grouping. VoxSang 2001;81:55.

3. Kao GS,Wood IG, Dorfman DM, Milford EL,Benjamin RI. Investigations into the role of anti-HLA class II antibodies in TRALI. Transfusion2003;42:185–91.

4. Kondo H, Oyamada T, Mori A, et al. Direct-antiglobulin-test-negative immune hemolyticanaemia and thombocytopenia in a patient withHodgkin’s disease.Acta Haematol 2001;105:233–6.

5. Kopko PM, Paglieroni TG, Popovsky MA, Muto KN,MacKenzie MR, Holland PV. TRALI: correlation ofantigen-antibody and monocyte activation indonor-recipient pairs.Transfusion 2003;43:177–84.

6. Maheshwari VD, Kumar R, Singh S. Coomb’snegative autoimmune hemolytic anemia withthrombocytopenia (Evan’s syndrome). J AssocPhysicians India 2002;50:457–8.

7. Reed W,Vichinsky EP. Transfusion therapy: acoming-of-age treatment for patients with sicklecell disease. J Pediatr Hematol/Oncol 2001;23:197–202.

8. Shnaider A, Busok A, Rogachev BV, Zlutnik M,Tomer A. Severe Coombs-negative autoimmunehemolytic anemia in a kidney transplant patient.Am J Nephrol 2001;21:494–7.

Miscellaneous1. Hoppe B, Stibbe W, Bielefeld A, Pruss A, Salama A.

Increased RBC autoantibody production inpregnancy. Transfusion 2001;41:1559–61.

2. Krombach J, Kampe S, Gathof BS, Diefenbach C,Kasper S-M. Human error: the persistent risk ofblood transfusion: a report of five cases.AnesthAnalg 2002;94:154–6.

3. Kumpel BM. On the mechanism of tolerance tothe RhD antigen medicated by passive anti-D(RhD prophylaxis). Immunol Lett 2002;82:67–73.

4. Mair DC, Scofield TI. HDN in a motherundergoing in vitro fertilization with donor ova(letter). Transfusion 2003;43:288.

5. Nathalang O, Kuvanout S, Punyaprasiddhi P,Tasaniyanonda C, Sriphaisal T. A preliminary studyof the distribution of blood group systems in Thaiblood donors determined by the gel system. SEAsian J Trop Med Pub Health 2001;32:204–7.

6. Patel RK, Nicolaides K, Mijovic A. Severehemolytic disease of the fetus following in vitrofertilization with anonymously donated oocytes(letter). Transfusion 2003;43:119–21.

7. Stockman JA. Overview of the state of the art ofRh disease: history, current clinical management,and recent progress. J Pediatr Hematol/Oncol2001;23:554–62.


Phone, Fax, and Internet Information: If you have any questions concerning Immunohematology,Journal of Blood Group Serology and Education, or the Immunohematology Methods and Proceduresmanual,contact us by e-mail at [email protected] information concerning the National ReferenceLaboratory for Blood Group Serology, including the American Rare Donor Program, please contact SandraNance, by phone at (215) 451-4362, by fax at (215) 451-2538, or by e-mail at [email protected]

C L A S S I F I E D A D

SBB Program. The Department of Transfusion Medicine, National Institutes of Health, is acceptingapplications for its 1-year Specialist in Blood Bank Technology Program. Students are federal employeeswho work 32 hours per week. This program introduces students to all areas of transfusion medicine,including reference serology, cell processing, and HLA and infectious disease testing. Students also designand conduct a research project. NIH is an Equal Opportunity Employer. Application deadline is June 30, 2003, for the January 2004 class. Contact: Karen M. Byrne, NIH/CC/DTM, Bldg. 10/Rm. 1C711,10 Center Drive, MSC 1184, Bethesda, MD 20892-1184; phone: (301) 496-8335 or e-mail:[email protected].

Monoclonal antibodies available. The New YorkBlood Center has developed murine monoclonalantibodies that are useful for donor screening and fortyping red cells with a positive DAT. Anti-Rh17 is adirect agglutinating monoclonal antibody. Anti-Fya, anti-K, anti-Jsb, and anti-Kpa are indirect agglutinatingantibodies that require anti-mouse IgG for detection.These antibodies are available in limited quantities atno charge to anyone who requests them. Contact:Marion Reid,New York Blood Center,310 E.67th Street,New York, NY 10021; e-mail: [email protected]

Workshop on Blood Group Genotyping. TheISBT/ICSH Expert Panel in Molecular Biology hasrecommended that a workshop be held on bloodgroup genotyping by molecular techniques. The resultsof the meeting would culminate in a report at the ISBTCongress in 2004 in Edinburgh. It was decided thatonly laboratories that provide a reference service inblood group genotyping would be included in theworkshop. One of the aims of the workshop would beto establish an external quality assurance plan. If youhave any suggestions as to how the workshop shouldbe organized, we would be grateful for your opinions.If you are interested in taking part in such a workshop, please contact Geoff Daniels([email protected]). Offer presented by GeoffDaniels, Martin L. Olsson, and Ellen van der Schoot.

Annual Symposium. The National Institutes ofHealth, Department of Transfusion Medicine, will holdtheir annual symposium. Immunohematology andBlood Transfusion, on October 2 and October 3,2003. The symposium will be co-hosted by theGreaater Chesapeake and Potomac Region of theAmerican Red Cross and is free of charge. Advanceregistration is encouraged. For more information andregistration, Contact: Karen Byrne, NIH/CC/DTM,Bldg. 10/Rm. 1C711, 10 Center Drive, MSC 1184,Bethesda,MD 20892;e-mail: [email protected] orvisit our Web site: www.cc.nih.gov/dtm.

A N N O U N C E M E N T S

NATIONAL REFERENCE LABORATORY FORBLOOD GROUP SEROLOGY

Immunohematology ReferenceLaboratory

AABB,ARC, New York State, and CLIA licensed(215) 451-4901—24-hr. phone number

(215) 451-2538—Fax

American Rare Donor Program(215) 451-4900—24-hr. phone number

(215) 451-2538—[email protected]

Immunohematology(215) 451-4902—Phone, business hours

(215) 451-2538—[email protected]

Quality Control of Cryoprecipitated-AHF(215) 451-4903—Phone, business hours

(215) 451-2538—Fax

Granulocyte Antibody Detection and Typing

• Specializing in granulocyte antibody detectionand granulocyte antigen typing

• Patients with granulocytopenia can be classifiedthrough the following tests for proper therapyand monitoring:—Granulocyte agglutination (GA)—Granulocyte immunofluorescence (GIF)—Monoclonal Antibody Immobilization of

Granulocyte Antigens (MAIGA)

For information regarding services, call Gail Eiberat: (651) 291-6797, e-mail: [email protected],

or write to:Neutrophil Serology Reference Laboratory

American Red CrossSt. Paul Regional Blood Services

100 South Robert StreetSt. Paul, MN 55107

CLIA LICENSED

National Platelet Serology ReferenceLaboratory

Diagnostic testing for:

• Neonatal alloimmune thrombocytopenia (NAIT)

• Posttransfusion purpura (PTP)

• Refractoriness to platelet transfusion

• Heparin-induced thrombocytopenia (HIT)

• Alloimmune idiopathic thrombocytopenia

purpura (AITP)

• Medical consultation available

Test methods:

• GTI systems tests

—detection of glycoprotein-specific platelet

antibodies

—detection of heparin-induced antibodies (PF4

ELISA)

• Platelet suspension immunofluorescence test

(PSIFT)

• Solid phase red cell adherence (SPRCA) assay

• Monoclonal antibody immobilization of platelet

antigens (MAIPA)

For information, e-mail: [email protected]

or call:

Maryann Keashen-Schnell

(215) 451-4041 office

(215) 451-4205 laboratory

Sandra Nance

(215) 451-4362

Scott Murphy, MD

(215) 451-4877

American Red Cross Blood ServicesMusser Blood Center

700 Spring Garden StreetPhiladelphia, PA 19123-3594

CLIA LICENSED


A D V E R T I S E M E N T S


A D V E R T I S E M E N T S C O N T ’ D

IgA/Anti-IgA Testing

IgA and anti-IgA testing is available to do the following:• Monitor known IgA-deficient patients• Investigate anaphylactic reactions• Confirm IgA-deficient donors

Our ELISA assay for IgA detects antigen to 0.05 mg/dL.

For information on charges and sample requirements, call (215) 451-4351, e-mail:

[email protected],or write to:

American Red Cross Blood ServicesMusser Blood Center

700 Spring Garden StreetPhiladelphia, PA 19123-3594

ATTN: Ann Church

CLIA LICENSED

Reference and Consultation Services

Red cell antibody identification and problem resolution

HLA-A, B, C, and DR typing

HLA-disease association typing

Paternity testing/DNA

For information regarding our services, contact

Zahra Mehdizadehkashi at (503) 280-0210, or

write to:

Pacific Northwest Regional Blood Services

ATTENTION: Tissue Typing Laboratory

American Red Cross

3131 North Vancouver

Portland, OR 97227

CLIA LICENSED,ASHI ACCREDITED

IMPORTANT NOTICE ABOUT MANUSCRIPTSFOR

IMMUNOHEMATOLOGY

Mail all manuscripts (original and 2 copies) to Mary H. McGinniss, Managing Editor, 10262 Arizona Circle,Bethesda, MD 20817. In addition, please e-mail a copy to Marge Manigly at [email protected]

IMMUNOHEMATOLOGY IS ON THE WEB!

www.redcross.org/pubs/immuno

Password “2000”

For more information or to send an e-mail

message “To the editor”

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Notice to Readers: All articles published,including communications and book reviews,reflect the opinions of the authors and do notnecessarily reflect the official policy of theAmerican Red Cross.


SCIENTIFIC ARTICLES, REVIEWS, AND CASE REPORTSBefore submitting a manuscript, consult current issues of

Immunohematology for style. Type the manuscript on white bondpaper (8.5" × 11") and double-space throughout. Number the pagesconsecutively in the upper right-hand corner, beginning with thetitle page. Each component of the manuscript must start on a newpage in the following order:1. Title page2. Abstract3. Text4. Acknowledgments5. References6. Author information7. Tables—see 6 under Preparation8. Figures—see 7 under Preparation

Preparation1. Title page

A.Full title of manuscript with only first letter of first word capitalized

B. Initials and last name of each author (no degrees; all CAPS),e.g., M.T. JONES

C.Running title of ≤ 40 characters, including spacesD.3 to 10 key words

2. AbstractA.1 paragraph, no longer than 200 wordsB. Purpose, methods, findings, and conclusions of studyC.Abstracts not required for reviews

3. Text (serial pages)Most manuscripts can usually, but not necessarily, be divided intosections (as described below). Results of surveys and reviewpapers are examples that may need individualized sections.A. IntroductionPurpose and rationale for study, including pertinent backgroundreferences.B. Case Report (if study calls for one)Clinical and/or hematologic data and background serology.C.Materials and MethodsSelection and number of subjects, samples, items,etc. studied anddescription of appropriate controls, procedures, methods, equip-ment, reagents, etc. Equipment and reagents should be identifiedin parentheses by model or lot and manufacturer’s name,city,andstate. Do not use patients’ names or hospital numbers.D.ResultsPresentation of concise and sequential results, referring to perti-nent tables and/or figures, if applicable.

E. DiscussionImplications and limitations of the study, links to other studies;if appropriate, link conclusions to purpose of study as stated inintroduction.

4. AcknowledgmentsAcknowledge those who have made substantial contributions tothe study, including secretarial assistance.

5. ReferencesA. In text, use superscript, arabic numbers.B. Number references consecutively in the order they occur in

the text.C.Use inclusive pages of cited references, e.g., 1431–7.D.Refer to current issues of Immunohematology for style.

6. TablesA.Number consecutively, head each with a brief title, capitalize

first letter of first word (e.g., Table 1. Results of ...), and use nopunctuation at the end.

B. Use short headings for each column, and capitalize first letterof first word.

C.Place explanations in footnotes (sequence: *, †, ‡, §, ¶, **, ††).7. Figures

A.Figures can be submitted either drawn or photographed (5″ × 7″ glossy).

B. Place caption for a figure on a separate page (e.g.,Fig.1.Resultsof ...), ending with a period. If figure is submitted as a glossy,put title of paper and figure number on back of each glossy submitted.

C.When plotting points on a figure, use the following symbolswhen possible: ●● ● ▲▲ ▲ ■■ ■.

8. Author informationA.List first name, middle initial, last name, highest academic

degree, position held, institution and department, and complete address (including zip code) for all authors. Listcountry when applicable.

SCIENTIFIC ARTICLES AND CASE REPORTS SUBMITTED AS LETTERS TO THE EDITOR

Preparation1. Heading—To the Editor:2. Under heading—title with first letter capitalized.3. Text—write in letter format (paragraphs).4. Author(s)—type flush right; for first author: name, degree,

institution, address (including city, state, and zip code); for otherauthors: name, degree, institution, city, and state.

5. References—limited to ten.6. One table and/or figure allowed.

ImmunohematologyJOURNAL OF BLOOD GROUP SEROLOGY AND EDUCATION

Instructions for Authors

Send all submissions (original and two copies) to:Mary H. McGinniss, Managing Editor, Immunohematology,10262 Arizona Circle, Bethesda, MD 20817.

In addition, please e-mail your manuscript to MargeManigly at [email protected]

What is a certified Specialist in Blood Banking (SBB)?• Someone with educational and work experience qualifications that successfully passes the American Society for

Clinical Pathology (ASCP) board of registry (BOR) examination for the Specialist in Blood Banking.• This person will have advanced knowledge, skills and abilities in the field of transfusion medicine and blood banking.

Individuals who have an SBB certification serve in many areas of transfusion medicine:• Serve as regulatory, technical, procedural and research advisors• Perform and direct administrative functions • Develop, validate, implement, and perform laboratory procedures• Analyze quality issues preparing and implementing corrective actions to prevent and document issues• Design and present educational programs• Provide technical and scientific training in blood transfusion medicine• Conduct research in transfusion medicine

Who are SBBs?Supervisors of Transfusion Services Managers of Blood Centers LIS Coordinators EducatorsSupervisors of Reference Laboratories Research Scientists Consumer Safety OfficersQuality Assurance Officers Technical Representatives Reference Lab Specialist

Why be an SBB?Professional growth Job placement Job satisfaction Career advancement

How does one become an SBB?• Attend a CAAHEP-accredited Specialist in Blood Bank Technology Program OR• Sit for the examination based on criteria established by ASCP for education and experience

Fact #1: In recent years, the average SBB exam pass rate is only 38% Fact #2: In recent years, greater than 73% of people who graduate from CAAHEP-accredited programs pass the SBB

exam.

Conclusion:The BEST route for obtaining an SBB certification is to attend a CAAHEP-accredited Specialist in Blood BankTechnology Program

Becoming a Specialist in Blood Banking (SBB)

Contact the following programs for more information:PROGRAM CONTACT NAME CONTACT INFORMATION

Walter Reed Army Medical Center William Turcan 202-782-6210;[email protected]

Transfusion Medicine Center at Florida Blood Services Marjorie Doty 727-568-5433 x 1514; [email protected]

Univ. of Illinois at Chicago Veronica Lewis 312-996-6721; [email protected]

Medical Center of Louisiana Karen Kirkley 504-903-2466; [email protected]

NIH Clinical Center Department of Transfusion Medicine Karen Byrne 301-496-8335; [email protected]

Johns Hopkins Hospital Christine Beritela 410-955-6580; [email protected]

ARC-Central OH Region, OSU Medical Center Joanne Kosanke 614-253-2740 x 2270; [email protected]

Hoxworth Blood Center/Univ. of Cincinnati Medical Center Catherine Beiting 513-558-1275; [email protected]

Gulf Coast School of Blood Bank Technology Clare Wong 713-791-6201; [email protected]

Univ. of Texas SW Medical Center Barbara Laird-Fryer 214-648-1785; [email protected]

Univ. of Texas Medical Branch at Galveston Janet Vincent 409-772-4866; [email protected]

Univ. of Texas Health Science Center at San Antonio Bonnie Fodermaier SBB Program: 210-358-2807,Linda Smith [email protected]

MS Program : 210-567-8869; [email protected]

Blood Center of Southeastern Wisconsin Lynne LeMense 414-937-6403; [email protected]

Additional Information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org, and www.aabb.org

Musser Blood Center700 Spring Garden StreetPhiladelphia, PA 19123-3594

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Vol. 19, No. 2, 2003

Documents

Transcript of Vol. 19, No. 2, 2003