Vox Sanguinis (2005) 89, 39–43 © 2005 Blackwell Publishing DOI: 10.1111/j.1423-0410.2005.00662.x ORIGINAL PAPER Frequencies of maternal platelet alloantibodies and autoantibodies in suspected fetal/neonatal alloimmune thrombocytopenia, with emphasis on human platelet antigen-15 alloimmunization Blackwell Publishing, Ltd. M. Mandelbaum,1 D. Koren,1 B. Eichelberger,1 L. Auerbach2 & S. Panzer1 1 Clinic for Blood Group Serology, Medical University Vienna, Vienna, Austria Department for Obstetrics and Gynecology, Division of Special Gynecology, Medical University Vienna, Vienna, Austria 2 Background and Objectives Serological evaluation of maternal sera for platelet antibodies in suspected fetal/neonatal alloimmune thrombocytopenia (FNAITP) discloses in only ≈ 30% of individuals a platelet-specific antibody. Transfusion-induced alloimmunization against human platelet antigen-15 (HPA-15) has been reported to be about as common as against HPA-5, the second most common platelet antibody. Thus, anti-HPA-15 may also contribute significantly to yet-unclear cases of FNAITP. Materials and Methods In this retrospective analysis, we provide data on maternal platelet antibodies from 309 mothers who delivered an offspring with suspected FNAITP. Results Genotyping maternal and paternal samples (together n = 573) revealed a gene frequency of 0·496 for HPA-15a and a gene frequency of 0·504 for HPA-15b. HPA-15 antibodies were detected in 2% of all samples. Anti-HPA-15a and -15b were detected in two and three samples, respectively. One serum reacted equally with HPA-15a and -15b platelets. The most frequent platelet-specific antibodies were anti-HPA-1a (22%), but anti-HPA-5b (8·4%) were more frequent than anti-HPA-15. In addition, panreactive (5·5%) or autoreactive (5·2%) anti-GPIIb/IIIa or anti-GPIb/IX were detectable in maternal samples. Received: 17 February 2005, revised 11 April 2005, accepted 14 April 2005, published online 25 May 2005 Conclusions These data indicate that HPA-15 alloimmunization needs only to be considered in subjects with suspected FNAITP if no other platelet-specific antibody is detectable. The presence of panreactive or autoreactive antibodies should also be considered in neonatal thrombocytopenia. Key words: HPA-15, neonatal alloimmune thrombocytopenia, platelet antibodies. Introduction Fetal/neonatal alloimmune thrombocytopenia (FNAITP) is a clinical syndrome that resembles haemolytic disease of the newborn as a result of maternal anti-D formation, but differs Correspondence: Simon Panzer, MD, Clinic for Blood Group Serology, Medical University Vienna, Waehringer Guertel 18–20, 1090 Vienna, Austria E-mail: simon.panzer@meduniwien.ac.at in that the fetal/neonate’s platelets, and not red cells, are affected. The maternal immunoglobulin G (IgG) alloantibodies, which react with an alloantigen on the fetal platelets that is inherited from the father, but the mother is lacking, cross the placenta and induce platelet destruction in the fetus. The low platelet counts predispose the fetus towards bleeding. In contrast to Rh incompatibility induced by maternal anti-D, which requires a previous alloimmunization, the first offspring can be affected and suffer from FNAITP [1]. The most common alloantigen involved in FNAITP is human platelet antigen (HPA)-1a [1]. Other alloantigens, which can 39 40 M. Mandelbaum et al. give rise to FNAITP, have been described and are compiled in the nomenclature for these platelet antigens [2]. However, a platelet-specific alloantibody is only detectable in ≈ 25–30% of sera from mothers who have delivered a thrombocytopenic offspring with suspected FNAITP [1,3], even though these sera are investigated by specialized institutions. It is therefore assumed that alloantigens, which have not been characterized so far, could be responsible for maternal–fetal incompatibilities and thus for some of the unclear cases of neonatal thrombocytopenias. In 1990, Kelton et al. [4] described a new alloantigen system, the Gova/b system, that they discovered in association with transfusion incompatibilities and later on in association with FNAITP [5]. These authors localized this alloantigen to CD109 [6]. The molecular characterization of the Gov system [7] facilitated the genotyping of large cohorts and definition of the gene frequency among these individuals, which was found to be similar to that seen by serological investigation on the initially small number of individuals [4]. The Gov system was incorporated, in 2003, into the platelet alloantigen nomenclature as HPA-15 [2]. Only a few years ago, Berry et al. [8] investigated, in a retrospective analysis, the occurrence of HPA-15 antibodies in sera from patients refractory to HLA-matched platelet concentrates and maternal sera from individuals with suspected FNAITP. Overall, the frequency of HPA-15 antibodies was found to be similar to that of HPA-5 antibodies. These findings suggest that some cases of FNAITP, which could not be attributed to a specific alloimmunization in the past, might have resulted from HPA-15 alloantibodies. Based on these data, we decided, in 2004, to include, in our serological investigations for suspected FNAITP, the detection of anti-HPA-15. During this time-period we detected one HPA-15 antibody in 36 samples, the same number as an HPA-5 antibody, while anti-HPA-1a was detected in 10 samples. These findings prompted us to investigate all our stored samples for HPA-15 alloantibodies retrospectively, and to compare these with the frequency of other antibodies, which we have seen in the maternal sera. The data from this study show that anti HPA-15 is rarely associated with FNAITP in our population. Interestingly, autoantibodies and panreactive antibodies were found in ≈ 5% of the maternal samples. Materials and methods Patients For this retrospective analysis, we used 309 sera from mothers (between 1997 and 2004) who had delivered an offspring with suspected FNAITP. Samples from the respective fathers and the newborns were available in 297 and 64 cases, respectively. Serological investigations All samples had been investigated for HPA-1, -2, -3 and -5 alloantibodies by using the modified indirect monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay [9,10] with a platelet panel from blood group O donors who were homozygous positive for HPA-1, -2, -3, and -5 a or b, and for human leucocyte antigen (HLA) class I alloantibodies [11]. Furthermore, maternal sera were tested for reactivities against glycoprotein (GP)IIb/ IIIa, GPIb/ IX, GPIa/ IIa and HLA class I of the fathers’ platelets, if available. All maternal autologous platelets were also evaluated for antibodies coating their GPIIb/IIIa and GPIb/ IX to exclude maternal autoantibodies. Antibodies that reacted with all panel platelets and the paternal, but not the maternal platelets, are termed ‘panreactive’. Antibodies free in serum, which reacted also with the maternal platelets, are considered ‘autoantibodies’. The MAIPA assay was further modified for the detection of HPA-15 alloantibodies. In brief, EDTA-anticoagulated blood was obtained from genotyped blood group O donors for platelet separation. Platelets from five different homozygous HPA-15a or -15b donors were pooled for analysis in the MAIPA assay. Pelleted, washed platelets (4 × 107) of both allotypes were incubated with 50 µl of serum for 30 min at 37 °C. After washing twice, platelets were resuspended in 30 µl of phosphate-buffered saline (PBS) /EDTA/bovine serum albumin (BSA) and incubated with 40 µg of the monoclonal antibody TEA 2/16 (Pharmingen, Heidelberg, Germany), for 30 min. Cells were washed twice and solubilized with 130 µl of 10 mM Tris-buffered saline (TBS) containing 145 mM NaCl and 0·5% Triton X-100. After 30 min of incubation at 4 °C, the lysates were centrifuged (13 000 g, 4 °C, 30 min), 104 µl of the lysate /supernatants were diluted with 130 µl of TBS washing buffer containing 145 mM NaCl, 0·05% Tween 20, 0·5 mM CaCl2, 0·5% Triton X-100, 0·02% BSA and 0·01% gelatine. One-hundred microlitres of the diluted platelet lysate was transferred in duplicate to microtitre wells and the MAIPA assay was performed as described. Genotyping by polymerase chain reaction– sequence-specific primers (PCR–SSP) DNA was extracted from EDTA-anticoagulated blood by using the Puregene DNA Purification Kit (Gentra Systems, Minneapolis, MN). HPA-1, -2, -3 and -5 genotypes were determined as published [12]. HPA-15 genotyping followed this earlier protocol with some modifications [13]: PCR experiments were carried out in thin-walled tubes in a total volume of 20 µl. For each PCR assay, a mastermix of 10 µl was prepared, consisting of 4·4 µl of primers HPA-15a or HPA-15b, 4·4 µl of HPA-15 common [7], and 0·2 µl each of the two human growth hormone internal control primers © 2005 Blackwell Publishing Ltd. Vox Sanguinis (2005) 89, 39–43 41 Platelet antibodies in fetal/neonatal alloimmune thrombocytopenia and 0·8 µl of aqua bidest. A DNA mastermix containing 8·8 µl of 10× PCR buffer (GeneAmp 10× PCR buffer; Applied Biosystems, Branchburg, NJ), 0·7 µl of dNTPs (25 mM each), 16·9 µl of aqua bidest, 0·8 µl of AmpliTaq (Applied Biosystems) and 8·8 µl of DNA was prepared, and 10 µl thereof was added to the corresponding tubes of HPA-15a and HPA-15b. After an initial denaturation at 94 °C for 2 min, amplification was performed in a GeneAmp 9600 thermocycler (Perkin Elmer, Norwalk, CT) comprising 10 cycles of denaturation (94 °C for 10 s; 65 °C for 1 min) and 20 cycles of annealing (94 °C for 10 s; 61 °C for 50 s) and extension (72 °C for 30 s). Twenty microlitres of the PCR products and 4 µl of 5× loading buffer were subjected to gel electrophoresis on a 1·5% agarose gel. After ethidium bromide staining, the typing results were examined under ultraviolet-light transillumination and documented by a digital camera. Results The genotype frequency of HPA-15a (0·496) and HPA-15b (0·504) in our population (in Hardy–Weinberg equilibrium, χ2 = 0·9) was similar to that published in other Caucasoid populations [4,8,14]; of note, HPA-15b was slightly more frequent than HPA-15a, which is in accordance with the findings from others who presented to the 12th International Workshop on Platelet Immunology [15]. Of the submitted samples, we were able to genotype the mothers in 292 cases, the fathers in 281 cases, and in 64 cases we also had the material to type the offspring. A compilation of mismatches is shown in Table 1. We were able to type the fathers and the neonates in five and eight cases, respectively, without having the corresponding maternal DNA. In four cases we only had maternal sera. Maternal sera from all HPA-15 maternal-paternal or maternal-offspring mismatches were evaluated for anti-HPA-15 by using the modified MAIPA assay. In addition, maternal sera were tested if the mother’s genotype was unknown (17 cases). The results are summarized in Table 2. In addition, we listed the other platelet antibodies that were detected in these maternal sera. The most frequent platelet-specific alloantibodies were directed against HPA-1a; anti-HPA-15 alloantibodies were rarer than HPA-5 antibodies. Of note, Table 1 Human platelet antigen (HPA)-15 match between the mothers and the corresponding fathers and neonates Father (n) Neonate (n) Mother Aa ab bb aa ab bb aa ab bb 17 34 24 33 67 35 10 38 18 5 3 0 8 16 8 0 7 9 © 2005 Blackwell Publishing Ltd. Vox Sanguinis (2005) 89, 39–43 Table 2 Specificities of platelet-reactive antibodies in 309 mothers who delivered a child with suspected fetal/neonatal alloimmune thrombocytopenia (FNAITP) Specificity Solitary HPA-15a HPA-15b HPA-1a HPA-1b HPA-5b HLA Panreactive CD109 Panreactive GPIb /IX Panreactive GPIIb /IIIa Autoreactive GPIIb /IIIa Autoreactive GPIIb/IIIa, free in serum Combined HPA-15a + HLA HPA-15b + HLA HPA-1a + HLA HPA-1a + HPA-5b HPA-5b + HLA Panreactive GPIb/IX + HLA Panreactive GPIIb/IIIa + HLA Autoreactive GPIb /IX + HLA Autoreactive GPIIb /IIIa + HLA Autoreactive GPIIb/IIIa + HLA, free in serum n 0 3 44 2 16 126 1 1 5 3 1 2 0 22 2 8 4 6 3 8 1 GP, glycoprotein; HLA, human leucocyte antigen; HPA, human platelet antigen. panreactive or autoantibodies were detected at a rate of ≈ 5% each. Out of the autoreactive antibodies, two sera also reacted with all panel platelets and the paternal platelets (0·7% of available fathers). Discussion The data from this retrospective study show that anti-HPA15 are rather rare in clinically suspected FNAITP. However, panreactive antibodies, which are not linked to any known platelet-specific alloantigen, and autoantibodies, were rather frequent. Our primary aim was to determine the frequency of HPA15 antibodies in sera from mothers who had delivered a thrombocytopenic offspring. The data from this retrospective analysis revealed the frequency of panreactive and autoantibodies, which has not been disclosed in previous studies with an emphasis on alloantibodies [1,3]. Panreactive antibodies, which react with the paternal platelets as well as with all HPA-characterized donor platelets, but not with the mothers’ autologous platelets, may be responsible, in some cases, for FNAITP. The reoccurrence of such antibodies during 42 M. Mandelbaum et al. a subsequent pregnancy with another thrombocytopenic offspring would strongly suggest that these antibodies are indeed responsible for the neonates’ thrombocytopenia. Unfortunately, we do not have data to support this suggestion. Second, we identified autoantibodies in a number of mothers who gave birth to a child with suspected FNAITP. Some of these antibodies were only detectable on the maternal platelets, while others were also free in serum and reacted with the fathers’ as well as with all donors’ platelets. The difference from the aforementioned antibodies is that the maternal platelets were also IgG coated, stongly suggesting the presence of autoantibodies, even though the mother was not thrombocytopenic. It is known that pregnancy-associated autoimmune thrombocytopenia occurs in ≈ 5% of pregnant women, but thrombocytopenia is moderate in most of the neonates [16,17]. Furthermore, autoantibodies are even detectable at delivery in women who give birth to neonates without thrombocytopenia, which raises the question of the clinical significance of such antibodies [18,19]. Thus, in addition to HPA-specific alloantibodies, two distinct reactions are seen in close association with delivery, namely the generation of panreactive ‘alloantibodies’ and of autoantibodies. These maternal immune reactions may well be responsible for moderate and transient thrombocytopenia in the newborns. Some mothers may have been thrombocytopenic themselves, but this was not reported by the referring institutions. Third, our data confirm the previous results of Berry et al. [8], that FNAITP, as a result of HPA-15 alloimmunization, is rare in comparison to HPA-1a, or even HPA-5, antibodies. Of note, based on the genotype frequency, a very high frequency of HPA-15 alloimmunization can be expected, but many additional factors, such as the density of the alloantigen per platelet, conformation of the protein, antigen presentation, and possibly the presence of a specific immune-response gene, determines the immunogenicity of the antigen. Thus, the HPA-15 antibody frequency in suspected FNAITP is ≤ 2% [8, and this study]. We have approached our question by genotyping the mother and, if possible, the father of the affected offspring. In ≈ 25% of cases we also had a DNA sample from the newborn. We were therefore able to restrict the serological evaluation to those mothers who, based on their genotype and that of the father or newborn, were able to form an alloantibody. All maternal sera were tested if the mothers’ genotype could not be determined or if the father was not available. We may have missed a few samples that needed to be tested if a falsely submitted parental sample was not from the biological father. However, we had DNA from the newborns in a substantial number of cases to guide our laboratory procedure. In all cases we tested for antibodies against both allotypes using homozygous platelets, to have an additional negative control. Thereby we detected one serum that reacted with both allotypes. As a result of the retrospective nature of this study, we could not test the maternal autologous platelets for CD109 autoantibodies, and thus distinguish between these CD109 autoantibodies and panreactive antibodies. Owing to the rather low expression of CD109 (only 2000 ± 400 copies per platelet) [20], which varies from one individual to the next, an antibody can easily be missed. In order to circumvent this variability of CD109 expression, we pooled the platelets from five donors who are homozygous for HPA-15a and -15b, respectively. Thereby, with the positive controls we obtained, by using the MAIPA assay, absorbance values that were equivalent to those obtained in the past when using the same donors, and we successfully identified all HPA-15 antibodies in samples from the International Workshops on Platelet Immunology in 2000 [21], 2002 [22], and 2004 [15]. In summary, our data confirm the previously published results [8] that anti-HPA-15 are detectable in ≈ 2% of sera from mothers who have delivered an offspring with suspected FNAITP. Furthermore, these data emphazise the need, in the serological evaluation for suspected FNAITP, to focus not only on alloantibodies, which react with known platelet antigen specificities, but also to consider a maternal state of increased immunoresponsiveness around the time of delivery, which prompts the formation of auto- or panreactive platelet antibodies. These antibodies may just be transient and missed if the maternal sample is not obtained early after delivery. Whether or not these platelet antibodies are indeed responsible for the offspring’s thrombocytopenia remains to be demonstrated. References 1 Mueller-Eckhardt C, Kiefel V, Grubert A, Kroll H, Weisheit M, Schmidt S, Mueller-Eckhardt G, Santoso S: 348 cases of suspected neonatal alloimmune thrombocytopenia. Lancet 1989; 1:363 –366 2 Metcalfe P, Watkins NA, Ouwehand WH, Kaplan C, Newman P, Kekomaki R, De Haas M, Aster R, Shibata Y, Smith J, Kiefel V, Santoso S: Nomenclature of human platelet antigens. Vox Sang 2003; 85:240 –245 3 Davoren A, Curtis BR, Aster RH, McFarland JG: Human platelet antigen-specific alloantibodies implicated in 1162 cases of neonatal alloimmune thrombocytopenia. Transfusion 2004; 44:1220–1225 4 Kelton JG, Smith JW, Horsewood P, Humbert JR, Hayward CP, Warkentin TE: Gova /b alloantigen system on human platelets. Blood 1990; 75:2172 –2176 5 Bordin JO, Kelton JG, Warner MN, Smith JW, Denomme GA, Warkentin TE, McGrath K, Minchinton R, Hayward CP: Maternal immunization to Gov system alloantigens on human platelets. Transfusion 1997; 37:823 –828 6 Smith JW, Hayward CP, Horsewood P, Warkentin TE, Denomme GA, Kelton JG: Characterization and localization of the Gova /b alloantigens to the glycosylphosphatidylinositol-anchored protein CDw109 on human platelets. Blood 1995; 86 :2807–2814 © 2005 Blackwell Publishing Ltd. Vox Sanguinis (2005) 89, 39–43 Platelet antibodies in fetal/neonatal alloimmune thrombocytopenia 7 Schuh AC, Watkins NA, Nguyen Q, Harmer NJ, Lin M, Prosper JY, Campbell K, Sutherland DR, Metcalfe P, Horsfall W, Ouwehand WH: A tyrosine 703 serine polymorphism of CD109 defines the Gov platelet alloantigens. Blood 2002; 99:1692–1698 8 Berry JE, Murphy CM, Smith GA, Ranasinghe E, Finberg R, Walton J, Brown J, Navarrete C, Metcalfe P, Ouwehand WH: Detection of Gov system antibodies by MAIPA reveals an immunogenicity similar to the HPA-5 alloantigens. Br J Haematol 2000; 110:735 –742 9 Kiefel V, Santoso S, Weisheit M, Mueller-Eckhardt C: Monoclonal antibody-specific immobilization of platelet antigens (MAIPA): a new tool for the identification of platelet-reactive antibodies. Blood 1987; 70:1722–1726 10 Leitner GC, Stiegler G, Kalhs P, Greinix HT, Rabitsch W, Sillaber C, Hoecker P, Panzer S: The influence of human platelet antigen match on the success of allogeneic peripheral blood progenitor cell transplantation following a reduced-intensity conditioning regimen. Transfusion 2005; 45:195 –201 11 Kurz M, Knobl P, Kalhs P, Greinix HT, Hocker P, Panzer S: Platelet-reactive HLA antibodies associated with low posttransfusion platelet increments: a comparison between the monoclonal antibody-specific immobilization of platelet antigens assay and the lymphocytotoxicity test. Transfusion 2001; 41:771–774 12 Kluter H, Fehlau K, Panzer S, Kirchner H, Bein G: Rapid typing for human platelet antigen systems-1-2-3 and -5 by PCR amplification with sequence-specific primers. Vox Sang 1996; 71:121– 125 13 Bugert P, Lese A, Meckies J, Zieger W, Eichler H, Kluter H: Optimized sensitivity of allele-specific PCR for prenatal typing © 2005 Blackwell Publishing Ltd. Vox Sanguinis (2005) 89, 39–43 14 15 16 17 18 19 20 21 22 43 of human platelet alloantigen single nucleotide polymorphisms. Biotechniques 2003; 35:170–174 Randen I, Sorensen K, Killie MK, Kjeldsen-Kragh J: Rapid and reliable genotyping of human platelet antigen (HPA)-1-2-3-4, and -5 a/b and Gov a/b by melting curve analysis. Transfusion 2003; 43:445 –450 12th ISBT Platelet Immunology Workshop Report. Available from: http://www.isbt2004.com/platelet Burrows RF, Kelton JG. Fetal thrombocytopenia and its relation to maternal thrombocytopenia. N Engl J Med 1993; 329:1463–1466 Dreyfus M, Kaplan C, Verdy E, Schlegel N, Durand-Zaleski I, Tchernia G. Frequency of immune thrombocytopenia in newborns: a prospective study. Immune Thrombocytopenia Working Group. Blood 1997; 89:4402–4406 Tchernia G, Morel-Kopp MC, Yvart J, Kaplan C: Neonatal thrombocytopenia and hidden maternal autoimmunity. Br J Haematol 1993; 84:457 –463 de Moerloose P, Boehlen F, Extermann P, Hohfeld P: Neonatal thrombocytopenia: incidence and characterization of maternal antiplatelet antibodies by MAIPA assay. Br J Haematol 1998; 100:735 –740 Sutherland DR, Yeo E, Ryan A, Mills GB, Bailey D, Baker MA: Identification of a cell-surface antigen associated with activated T lymphoblasts and activated platelets. Blood 1991; 77:84 – 93 Panzer S: Report on the tenth international platelet genotyping and serology workshop on behalf of the international society of blood transfusion. Vox Sang 2001; 80:72 – 78 Goldman M, Trudel E, Richard L: Report on the eleventh international society of blood transfusion platelet genotyping and serology workshop. Vox Sang 2003; 85:149 –155