Quantitative determination of platelet surface alloantigens using a monoclonal probe

Quantitative Determination of Platelet Surface Alloantigens Using a Monoclonal Probe* Marleen Janson, Janice McFarland,* and Richard H. Aster

ABSTRACT: A monoclonal antibody with specificity for the Fc portion of lgG was used to determine the number of lgG alloantibody molecules bound at saturation to alloantigens of the P1Al and HLA systems on normal human platelets. In preliminary studies, it was found that the number of cell-bound IgG molecules recognized by this probe correlates well with the number measured by electroimmunoassay, an independent measure of alloantibody binding. PlAl-positive platelets could be divided into two groups binding 34,000-43,000 or 19,000-24,000 alloantibody molecules. Family studies and studies with a cytolytic assay showed that the former group is homozygous and the latter heterozygous for Pl^1. Because the number of glycoprotein IIIa (GPIIIa) molecules carrying the P1^1 determinant on the surface of normal platelets is thought to be about 40,000, these findings suggest that each GPIHa molecule carries one PIAI determinant. The number of class I HLA molecules expressed on normal platelets was considerably smaller than the number of Pl^1 determinants, ranging from 4400 to 10,000 (HLA-A2), 870 to 8400 (Bw4), and 1300 to 5800 (Bw6). Preliminary analysis indicates that stronger or weaker expression of Bw4 and of Bw6 correlates with certain "private" HLA-B determinants carried on the HLA-B molecule as found in previous studies using an indirect method to measure alloantigen density. These findings appear to explain why antibodies reactive with platelet-specific antigens such as Pl^l react more strongly with platelets than HLA-specific antibodies in most serologic tests. The weak expression of HLA determinants on platelets of some subjects may account for the less than perfect correlation between in vitro compatibility tests and post-transfusion platelet survivals observed in most studies. INTRODUCTION Several alloantigens systems are expressed on the surface of normal human platelets [1]. Some, such as the P1A (or Zw) [2,3] and Bak [4] systems, appear to be platelet specific. Others, such as the class I H L A antigens, are shared with lymphocytes and other tissues. Erythrocyte alloantigens carried on the platelet appear to be limited to those o f the A B O [ 1,5] Ii [6], and Lewis [7] systems. Sensitization to platelet-specific antigens is seen in two thrombocytopenic disorders, posttransfusion purpura [2,8] and neonatal alloimmune thrombocytopenic purpura From The Blood Center of Southeastern Wisconsin; and The Departments of Medicine and Pathology, Medical Collegeof Wisconsin, Milwaukee, Wisconsin. *Supported by Grant HL-13629 from the National Heart, Lung and BloodInstitute, and, in part, by a Fogarty International Fellowship AwardJWO0 915-01 (Dr. Aster) and the Department of Medicine, Monash Medical School, Melbourne, Australia. ~Dr. McFarland is the recipient of Clinical Investigator Award HL-01376 from the NHLBL Address reprint requeststo Richard H. Aster, M.D., The BloodCenterof Southeastern Wisconsin, 1701 West Wisconsin Avenue, Milwaukee, WI 53233. ReceivedApril 26, 1985; acceptedJuly 1, 1985.

Human Immunology15, 251-262 (1986) © ElsevierSciencePublishingCo., Inc., 1986 52 VanderbiltAve., New York, NY 10017

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M. Janson et al. [8,9]. Immunization to class I HLA antigens appears to be a major cause of refractoriness to platelet transfusions in multiply transfused patients [10,11]. ABO antigens are of relatively minor importance in platelet transfusion therapy [12]. Numerous qualitative studies of platelet surface alloantigens have been carried out, but little is known about the quantity of these substances expressed on the surface of normal platelets. Using quantitative complement fixation, it is possible to differentiate between normal persons homozygous and heterozygous for the platelet-specific alloantigen, Pl ^~ [2], and platelets from donors homozygous for HLA-A2 can be distinguished from heterozygotes by inhibition of the cytotoxic action of an anti-HLA-A2 antibody [13]. These methods are cumbersome, however, and do not provide an absolute measure of the number of antigenic determinants expressed on the cell surface. A method for quantitating platelet surface alloantigens would facilitate determination of zygosity for platelet-specific markers such as P1A~ in husbands of PIA~-negative women who have given birth to infants with neonatal alloimmune thrombocytopenic purpura as an aid to genetic counselling. Quantitation of the class I HLA molecules of platelets might suggest ways to treat alloimmunized thrombocytopenic patients in view of evidence that expression of these markers varies widely on platelets from different normal individuals [ 14,15]. LoBuglio and his colleagues recently described the use ofa murine monoclonal antibody specific for the Fc portion of human IgG to quantitate platelet-associated IgG in idiopathic (autoimmune) thrombocytopenic purpura [16,17]. They presented evidence that this probe binds to the Fc portion of each of the subclasses of human IgG in a 1:1 ratio and obtained extremely low values for surface IgG in normal, unsensitized platelets. Given these two properties, it seemed possible that this probe might be useful for quantitation of alloantibodies bound to platelet surface alloantigens and for determination of the number of such antigens. Presented here are the results of preliminary studies confirming the usefulness of this probe for measurement of HLA and Pl AI antigens on the platelet surface.

MATERIALS A N D M E T H O D S P1A~-specific antibodies were obtained from two pIAl-negative patients during the acute stage of post-transfusion purpura and were shown to contain only weakly reactive HLA antibodies of limited specificity by testing in lymphocytotoxicity against a panel of lymphocytes from 100 donors of known HLA phenotypes. The sera were used at dilutions beyond the limits at which the HLA-specific antibodies were reactive with platelets in the monoclonal binding assay (below). HLA-specific antisera were obtained from multiparous blood donors and were determined to be monospecific by testing in lymphocytotoxicity against the same panel and against a panel of platelets using the monoclonal binding assay. Platelet donors were normal subjects known to be positive or negative for the P1A1 antigen on the basis of previous testing. Platelets were isolated from blood collected in ethylenediamine tetraacetic acid (EDTA), washed, and suspended in phosphate-buffered saline-EDTA, containing 1% bovine serum albumin (PBSEDTA-BSA) at pH 6.8 [17]. The final preparations never contained more than one white blood cell per thousand platelets. Assays were done on the same day blood was drawn or after storage overnight at 4°C. Studies with anti-HLA-A2, anti-HLA-Bw4, and anti-HLA-Bw6 antibodies were performed using platelets from normal subjects known to be heterozygous for these markers on the basis of lymphocyte phenotyping. The 5~Cr release assay was performed with papain-treated, 5~Cr-labeled plate-

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lets, cytotoxic antibody specific for PIA1, and human complement as previously described [18]. Mouse monoclonal antibody specific for the Fc portion of human IgG was purchased from Bethesda Research Laboratories, Gaithersburg, MD, as ascites fluid (BRL 9474-SA). Properties of the antibody and evidence that it binds equally well to all four IgG subclasses on a 1:1 basis have been described by LoBuglio and his colleagues [16,17]. IgG was isolated from the ascites fluid by absorption onto Staphylococcal protein A-Sepharose (Pharmacia) in 0.1 M phosphate buffer, pH 8.0, followed by elution with 0.1 M citrate buffer, pH 6.0 [19], and dialysis against 0.1 M phosphate buffered saline, pH 7.4. The purified IgG was radiolabeled with 1/.~Ci ~5I by the iodogen method using Iodobeads (Pierce Chemical Co., Rockville, IL) according to the manufacturer's instructions except that only 0.1 mg protein, rather than the recommended 1.0 mg, was used with each bead. The 1~5I in the final labeled product was more than 95% precipitable with trichloroacetic acid. Absorbtion with red cells sensitized with anti-Rho(D) antibody demonstrated that 70-80% of labeled protein represented antibody specific for IgG. By Scatchard analysis [20], the antibody was found to be homogenous with a binding constant (Ka) of 2.9 × 108 l/mol, a value similar to that reported by LoBuglio [ 16]. In preliminary studies, the amounts of anti-Pl A~ and anti-HLA antibodies required to saturate target platelets were determined by incubating fixed quantities of platelets with increasing amounts of antisera. The quantity of monoclonal anti-Fc antibody needed to achieve saturation of platelet-bound IgG was similarly determined. In experimental studies, alloantisera were used in at least threefold excess and labeled monoclonal antibody was used in at least twofold excess of the amount needed to achieve saturation. Alloantibody bound to target platelets was quantified by a modification of the technique described by LoBuglio [16,17]. Platelets were sensitized with antiP1A* by incubating 0.1 ml of antibody with 0.1 ml of plasma containing platelets at a concentration of 1.25 x 108/ml (total of 2.5 × 107 platelets) for 30 min at room temperature. The platelets were then washed three times in 1 ml PBSEDTA-BSA, pH 6.8, centrifuging in a Serofuge (Clay-Adams) for 7 min at 10,000g. The final platelet button was suspended in 0.2 ml PBS-EDTA-BSA. Two micrograms radiolabeled monoclonal antibody in a 0.05 ml volume was then added and the mixture was incubated for an additional 30 min at room temperature. PBS-EDTA-BSA (0.25 ml) was then added and 0.1 ml aliquots of this mixture were layered on 0.2 m125% Percoll (Pharmacia Fine Chemicals) in 0.4 ml Sarstedt tubes (Sarstedt, Inc.) previously washed with PBS-EDTA-BSA. The tubes were centrifuged at 10,000 x g for 4 min in a Beckman Microfuge II centrifuge using a Beckman horizontal rotor. Each tube was clamped with a hemostat in the center of the Percoll gradient, the supernatant was aspirated and discarded, and the buttons containing the sedimented platelets and bound monoclonal probe were severed with a wire cutter into a tube for determination of 125I content in a gamma scintillation counter. The number of molecules of radiolabeled probe bound to each target platelet was determined from the number of counts bound/platelet and the specific radioactivity (cpm//~g of protein) of the probe as described by LoBuglio [ 16,17]. A correction was applied for the small quantities of radioactive probe that bound to control platelets treated with autologous plasma rather than antibody. In preliminary studies using 5LCr_labeled platelets, it was found that more than 98% of the platelets added were recovered in the final button and that comparable results were obtained with platelets washed twice or four times in PBS-EDTA-BSA, rather than three times prior to addition of radiolabeled monoclonal antibody. Validation of the assay as a measure of the

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quantity of alloantibody bound to target antigens was obtained by determining platelet-associated IgG in parallel by electroimmunoassay as described in Results. With anti-HLA antibodies, variable results were obtained when platelets were sensitized in platelet-rich-plasma, a finding we attribute to soluble, HLA-bearing proteins known to be present in normal plasma [13]. For this reason, platelets were sensitized with HLA antibodies by resuspending 2.5 × 107 washed platelets in 0.2 ml of antibody-containing plasma. RESULTS Expression of Pl A* A n t i g e n on Platelets

Binding of radiolabeled monoclonal antibody to platelets from 21 normal subjects treated wtih saturating amounts of anti-Pl AI is shown in Figure 1. Three of the donors were known to be pIA*-negative from previous typing and seven were relatives of a pIA~-negative woman who gave birth previously to a pIA~-positive infant with neonatal alloimmune thrombocytopenia due to maternal-fetal incompatibility for P1A1. From the established mode of inheritance of the PIA~ antigen [2], the child and the p1Al-positive parents of this woman are obligate heterozygotes for P1A1 (Figure 2). As shown in Figure 1, the monoclonal binding assay permitted platelets from the 21 subjects to be classified into three discrete groups: Group 1 (34,000-43,000 molecules monoclonal antibody bound/platelet), Group 2 (19,800-23,800 molecules monoclonal antibody bound/platelet), and Group 3 consisting of the three p1A~-negative donors, whose platelets bound 400-1200 molecules of monoclonal antibody. Platelets from each of the three obligate heterozygote donors fell into Group 2. Platelets from two of the group 1 donors and two of the group 2 donors were also studied using anti-Pl A1 antibody obtained from a second patient with posttransfusion purpura with essentially identical results. To corroborate the monoclonal assay, we measured the amount of anti-P1 A* IgG bound to homozygous target platelets from two donors, by an independent but less precise method, electroimmunoassay [21], and obtained values of 8.5 and 11.0 fg, corresponding to 34,000 and 44,000 molecules of IgG, respectively. Release of 5~Cr induced by different quantities of anti-Pl A~ antibody was determined using radiolabeled platelets from nine of the donors. As shown in Figure 3, Group 1 platelets were significantly more sensitive to anti-P1A~ than Group 2 platelets. As expected, no significant 5~Cr release occurred with P1A*negative platelets. Expression of HLA Antigens on Platelets

Binding of the monoclonal probe to heterozygote platelets maximally sensitized with antibodies specific for HLA-A2, HLA-Bw4, and HLA-Bw6 is illustrated in Figure 4. The number of monoclonal antibody molecules bound to platelets sensitized with anti-HLA-A2 ranged from 4400 to 10,000 (average 6416 molecules/platelet). Platelets sensitized with anti-Bw4 bound 870-8400 monoclonal molecules (average 3633). Platelets sensitized with anti-Bw6 bound 1300-5800 monoclonal molecules (average 3769). The values of platelet-bound, antigenspecific IgG measured on four occasions by electroimmunoassay following sensitization with anti-HLA-A2 were 0.8, 1.6, 2.1, and 2.2 fg, equivalent to 3200, 6400, 8400, and 8800, molecules of IgG, respectively. This method of assay is inherently inaccurate because normal platelet-associated IgG (3-5 fg, 12,000-20,000 molecules) is also detected and must be subtracted from values obtained for

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sensitized platelets to determine net I g G bound. Considering this source of error, the values obtained are consistent with those measured with the monoclonal probe. T h e small n u m b e r o f H L A determinants relative to Pl A1 determinants detected on the platelet preparations studied led us to investigate whether H L A antigens or a n t i g e n - a n t i b o d y complexes might be lost from platelets when they were

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FIGURE 2 Familial relationship of eight of the donors whose platelets were used in the studies shown in Figure 1. The arrow indicates a PIA~-negative woman who gave birth previously to a Pl ^ 1-positive infant with neonatal alloimmune thrombocytopenia associated with maternal anti-Pl^1 antibody. Numbers indicate the groups (Figure 1) into which platelets of family members fell. Platelets from each of the obligate heterozygotes (child and both parents of the propositus) fell into Group 2.

washed prior to or after sensitization. It was found that platelets washed only once did not differ signficantly in measured HLA content from those washed three times prior to sensitization and that platelets washed four times after sensitization bound 9 5 - 1 0 0 % as much monoclonal antibody as platelets washed only two or three times (data not shown). These findings argue against the possibility that significant numbers o f HLA determinants or bound alloantibody molecules were lost from platelets during the washing procedures. It has been reported previously that HLA determinants are stable on platelets stored in buffer for prolonged periods [14,22]. Using an indirect assay (inhibition of 51Cr release from lableled Bw4 and Bw6 positive platelets), we previously showed that expression of the "public" antigens HLA-Bw4 and Bw6 on platelets of normal subjects is variable and is related to the "private" HLA-B determinant carried on the same HLA-B molecule [15]. The number o f Bw4 and Bw6 molecules detected on platelets of different donors grouped according to "private" HLA-Bw4 and Bw6-associated specificities is shown in Figure 5. Because of the limited number of donors studied to date, these data can be considered only preliminary, but the findings are in agreement with our earlier studies [ 15]. For example, Bw4 was expressed relatively strongly on platelets from the five donors positive for B5 (Bw51, Bw52). Similarly, Bw6 was strongly expressed on platelets of four B7-positive donors. Probable bimodal distribution of Bw4 on platelets from the seven Bw44-positive donors, found in our previous studies [14,15], was also seen. On the basis of cell-mediated reactions, T e k o l f et al. have shown that "strong" and "weak" expression of Bw44 on platelets correlates with a "split" of Bw44 not yet recognizable by serologic means [23]. The relatively strong expression of Bw4 on platelets from two of the four donors positive for B 13 is a possible exception to the pattern observed in our earlier studies in which weak expression of Bw4 was found on platelets from five B13-positive donors [15]. The two B13-positive donors with weak platelet Bw4 shown in Figure 5 were among those found to have this phenotype in the previous study, however.

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DISCUSSION E x p r e s s i o n o f PI A~ From Figure 1, it is apparent that platelets from pIAl-positive donors can be separated into two groups capable o f binding an average o f 38,900 and 21,900 molecules o f monoclonal anti-Fc antibody, respectively, when maximally sensitized with anti-Pl A1. In contrast, P1Al-negative platelets bound only about 1000 molecules o f this probe. Platelets from each of three donors who were obligate heterozygotes for P1A1 (Figure 2) fell into Group 2. Platelets from G r o u p 1

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FIGURE 5 Binding of radiolabeled monoclonal antibody to platelets from heterozygote donors sensitized with anti-Bw4 or anti-Bw6 plotted according to "private" B-locus HLA determinants associated with Bw4 and Bw6 in these individuals. Bw4 was more strongly expressed on platelets of HLA-B5 (Bw51 and Bw52)-positive subjects and Bw6 was more strongly expressed on platelets of HLA-B7-positive donors. Expression of HLA-Bw4 on platelets of donors positive for Bw44 appears to be bimodal as shown in previous studies using a different method of quantitation [ 14,15]. donors could also be distinguished from those of Group 2 on the basis of their sensitivity to anti-P1 ^~ in a cytotoxic assay (Figure 3). Together, these findings indicate that persons whose platelets fall into Group 1 on the basis of the monoclonal binding assay are homozygous for P1^1 and that those in Group 2 are heterozygous. LoBuglio [ 17] and Rosse et al. [24], each of whom studied a single PIA~-positive donor o f unspecified zygosity, obtained values of 41,000 and 36,000 molecules, respectively, for binding of the BRL-9474 SA probe to platelets saturated with anti-Pl ^~. We previously showed that the Pl AI determinant is carried on platelet membrane glycoprotein IIIa [25]. Newman et al., utilizing a monoclonal probe (AP-3) specific for a determinant on GPIIIa other than Pl A~, found that resting human platelets normally express an average of 40,200 GPIIIa molecules [26], a value similar to that determined by us for expression o f Pl A~ on platelets of homozygous subjects (Figure 1). Together, these findings suggest that each GPIIIa molecule carries one P1^~ determinant. The clear distinction between G r o u p 1 and Group 2 platelets achieved with

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M. Janson et al. the monoclonal assay suggests that this method can be used routinely to determine Pl A~ zygosity in husbands of P1Al-negative women who have given birth to an infant with neonatal alloimmune thrombocytopenic purpura (NATP) to assess the probability that a subsequent infant will be at risk for NATP. We have, in fact, been able to make this distinction without difficulty on blood samples sent to us from other centers.

Expression of H L A As noted in Figure 4, the number of HLA determinants on the surface of normal platelets measured with a monoclonal probe is considerably fewer than the number of P1A~ determinants on a gene-dose basis. The average number of HLA-A2, Bw4, and Bw6 molecules detected on platelets of the donors sampled were 6416, 3633, and 3769, respectively. Platelets from seven of the 21 Bw4-positive donors and four of 21 Bw6-positive donors expressed fewer than 2000 of these determinants. A second feature of platelet HLA expression is its variability, which is in contrast to the uniform expression of class I HLA determinants on lymphocytes demonstrated in a previous study [15]. Variable expression of class I HLA antigens on platelets is genetically determined [ 14,15] and expression of the "public" determinants Bw4 and Bw6 appears to be related to the "private" HLA-B determinant carried on the same HLA-B molecule [15] (Figure 5). The basis for this interesting relationship is not yet known. Our data indicate that the antigen HLA-A2 varies two to three fold in its expression on normal platelets. This is consistent with earlier results obtained with different methods [14,27]. The interesting suggestion has recently been made that class I HLA antigens of platelets may be acquired passively by absorption from plasma [28,29]. Our data do not provide specific evidence for or against this possibility.

Implications for Detection of Platelet-Reactive Antibodies It has been our experience (Collins J, Aster RH, personal observations), that platelets sensitized with anti-Pl A* give much stronger reactions than those sensitized with anti-HLA when tested by indirect immunofluorescence. The finding that there are far more P1A~ determinants on the platelet surface than any single HLA determinant provides an explanation for this observation. Our data also provide a tentative explanation for the difficulties experienced by some groups in developing reliable in vitro assays to predict the effectiveness of platelets transfused to alloimmunized, thrombocytopenic patients ("platelet crossmatches"). It is generally agreed that normal platelets each contain 3-5 fg of IgG, or about 12,000-20,000 molecules [21,30] and that 1-2 fg (4000-8000 molecules) of IgG is recognized on the platelet surface by most probes [31-33]. If there are only a few thousand copies of the individual HLA determinants on each platelet, HLA-specific alloantibodies bound to these determinants could be very difficult to detect using assays that measure total platelet-associated IgG. Even using methods that measure only platelet surface IgG, it could be difficult to discriminate between specifically bound, monospecific HLA antibody and normal platelet-associated IgG. ACKNOWLEDGMENTS

A portion of these studies and preparation of this manuscript were completed during the period of a Visiting Fellowship in the Department of Medicine, Monash University Medical School, Melbourne, Australia arranged by Dr. Barry Firkin. Dr. Sharon Pfueller of the

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Department kindly made her laboratory available to Dr. Aster for this work. The services of the Word Processing Department of The Blood Center are greatly appreciated.

REFERENCES 1. Aster RH: Platelet antigen systems. In: CP Engelfriet, JJ van Loghem, AEGK yon dem Borne, Eds., Immunohematology, Amsterdam, Elsevier, 1984, pp. 23-32. 2. Shulman NR, Aster RH, Leimer A, Hiller MD: Post-transfusion purpura due to a complement-rising antibody against a genetically-controlled platelet antigen: A proposed mechanism for thrombocytopenia and its relevance in "autoimmunity." J Clin Invest 40:1597, 1961. 3. van der Weerdt CM, Veenhoven-von Riesz LE, Nijenhuis JE, van Loghem JJ: The Zw blood group system in platelets. Vox Sang 8:513, 1963. 4. yon dem Borne AEGK, yon Riesz E, Verheugt FWA, TenCate JW, Koppe JG, Engelfriet CP, Nijenhuis LE: Bak ~a new platelet-specific antigen involved in neonatal alloimmune thrombocytopenia. Vox Sang 39:113, 1980. 5. Kelton JG, Hamid C, Acer S, Blajchman M: The amount of blood group A substance on platelets is proportional to the amount in plasma. Blood 59:980, 1982. 6. Dunstan RA, Simpson MB, Rosse WF: The presence of the Ii blood group system on human platelets. Am J Clin Pathol 82:74, 1984. 7. Dunstan RA, Simpson MB, Roose WF: Lea blood group antigen on human platelets. A m J Clin Pathol 83:90, 1985. 8. Aster RH: Clinical significance of platelet-specific antigens and antibodies. In: J McCullough, Ed., Advances in immunobiology: blood cell antigens and bone marrow transplantation. New York, AR Liss, 1984, pp. 103-118. 9. Shulman NR, Aster RH, Pearson HA, Hiller MC: Reactions of maternal isoantibodies responsible for neonatal purpura. Differentiation of a second platelet antigen system. J Clin Invest 41:1059, 1962. 10. Yankee RA, Grumet FC, Rogentine GN: Platelet transfusion therapy. The selection of compatible platelet donors for refractory patients by lymphocyte HL-A typing. N EnglJ Med 281:1208, 1969. 11. Lohrmann H-P, Ball MI, Decter JA, Yankee RA, Graw RG: Platelet transfusions from HL-A compatible unrelated donors to alloimmunized patients. Ann Int Med 80:9, 1974. 12. Duquesnoy RJ, Andersen AJ, Tomasulo PA, Aster RH: Influence of ABO compatibility on effectiveness of platelet transfusions in alloimmunized thrombocytopenic patients. Blood 54:595. 13. Aster RH, Miskovich BH, Rodey GE: Histocompatibility antigens of human plasma. Localization to the HDL-3 lipoprotein fraction. Transplantation 16:205, 1973. 14. Liebert M, Aster RH: Expression of HLA-B12 on platelets, on lymphocytes and in serum: A quantitative study. Tissue Antigens 9:199, 1977. 15. Szatkowski NS, Aster RH: HLA antigens of platelets. IV. Influence of "private" HLA-B locus specificities on the expression of Bw4 and Bw6 on human platelets. Tissue Antigens 15:361, 1980. 16. LoBuglio AF, Court WS, Vincour L, Maglott G, Shaw GM: Immune thrombocytopenic purpura. Use of a 125I-labeled anti-human IgG monoclonal antibody to quantify platelet-bound IgG. N Engl J Med 309:459, 1983. 17. LoBuglio AF, Viocur L, Maglott JG, Court WS: Measurement of cell-associated IgG

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M. Janson et al. using monoclonal antibody. In: R McMillan, Ed., Immune cytopenias (methods in hematology). New York, Churchill-Livingston, 1983, pp. 214-223. 18. Kunicki TJ, Aster RH: Qualitative and quantitative tests for platelet alloantibodies and drug-dependent antibodies. In: R McMillan, Ed., Immune cytopenias (methods in hematology). New York, Churchill-Livingston, 1983, pp. 49-67. 19. Ey PL, Prowse SJ, Jenkin CR: Isolation of pure IgG-1, IgG-2a and IgG-2b immunoglobulins from a mouse serum using protein A-Sepharose. Immunochemistry 15:429, 1978. 20. Scatchard G: The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660, 1949. 21. Kunicki TJ, Aster RH: Direct quantitation of platelet-associated IgG by electroimmunoassay. Blood 60:54, 1982. 22. ColombaniJ, Colombani M: Serologic recognition ofhistocompatibility antigens using complement fixation. Sem Hematol 9:273, 1974. 23. Tekolf WA, Biddison WE, Aster RH, Shaw S: Two subgroups of HLA-Bw44 defined by cell-mediated lympholysis which differ in Bw44 expression of genetic linkage disequilibrium. J Immunol 129:1474, 1982. 24. Rosse WF, Devine DV, Ware R: Reactions of immunoglobulin G-binding ligands with platelets and platelet-associated immunoglobulin G. J Clin Invest 73:489, 1984. 25. Kunicki TJ, Aster RH: Isolation and immunologic characterization of the human platelet alloantigen, P1A1. Mol Immunol 16:353, 1979. 26. Newman PJ, Allen RW, Kahn RA, Kunicki TJ: Quantitation of membrane glycoprorein IIIa on intact human platelets using the monoclonal antibody AP-3. Blood 65:227, 1985. 27. Svejgaard A: Complement-fixing platelet iso-antibodies. III. Quantitation of the HLAA2 antigen on platelets. Vox Sang 17:112, 1969. 28. Lalezari P, Driscoll AM: Ability of thrombocytes to acquire HLA specificty from plasma. Blood 59:167, 1982. 29. Blumberg N, Masel D, Mayer T, Horan P, Heal J: Acquisition of new HLA-A,B phenotypes by human platelets. Blood 60:276, 1982. 30. Morse BS, Guiliani D: Measurement of platelet-associated antibody with immunodiffusion and nephelometry. In: R McMillan, Ed. Immune (cytopenias, methods in hematology). New York, Churchill, Livingston, 1983, pp. 142-157. 31. Kelton JG, Denomme G, LucareUi A, Garvey J, Powers P, Carter C: Comparison of the measurement of surface or total platelet-associated IgG in the diagnosis of immune thrombocytopenia. Am J Hematol 18:1, 1985. 32. Luiken GA, McMillan R, Lightsey AL, Gordon P, Zevely S, Shulmann I, Gibble J, Longmire RL: Platelet-associated IgG in immune thrombocytopenic purpura. Blood 50:317, 1977. 33. Sugiura K, Steiner M, Baldini MG: Platelet antibodies in idiopathic thrombocytopenic purpura and other thrombocytopenias. J Lab Clin Med 96:640, 1980.