Monoclonal Antibody-specific Immobilization Of Platelet Antigens Research Articles

The relevance of HPA-15 antigen expression for anti-HPA-15 antibody detection

The HPA-15 antigen system is characterized by a low antigen expression on platelets. The antibodies against this antigen are implied in fetal/neonatal alloimmune thrombocytopenia (F/NAIT), post-transfusion purpura, and refractoriness to platelet transfusions. Detection of these antibodies appears to be related to the level of HPA-15 expression on the platelets used in the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay. We performed genotyping of 300 healthy blood donors for HPA-15 by TaqMan real-time PCR technology, and the HPA-15 antigen expression was investigated in 13 HPA-15aa and 19 HPA-15bb individuals. We also investigated the relevance of HPA-15 antigen expression on donor platelets used in MAIPA for antibody detection in 223 multitransfused hematological patients and 271 women with suspected F/NAIT. In Polish donors, the HPA-15a allele frequencies were lower than the HPA-15b (0.480 vs. 0.515). We identified three HPA-15 expression groups: high (36.7 ± 8.36 MFI - eight cases), medium (19.5 ± 6.2 MFI - 21 cases), and low (6.5 ± 5.9 MFI - three cases). The HPA-15 expression was stable over time. The HPA-15aa and HPA-15bb platelets with high antigen expression were used for anti-HPA-15 antibody detection; anti-HPA-15 antibodies were detected in 4/223 (1.8%) patients receiving multiple transfusions but in none of the 271 women with suspected F/NAIT. Further examination of the four sera by MAIPA with various platelets revealed the optical density in the assay to be closely related to the level of HPA-15 antigen expression. Anti-HPA-15 antibody detection should be based on carefully selected platelets with high HPA-15 expression level.

Maternal antibody titration as a predictive parameter for fetal status and therapy effectiveness in pregnancies associated with alloimmune thrombocytopenia

Alloimmune thrombocytopenia is the most common cause of severe isolated fetal and neonatal thrombocytopenia. In view of the recurrence of thrombocytopenia in subsequent pregnancies with incompatible fetuses, antenatal management has been developed. Until recently the only possibility of assessing the fetal status both before and during therapy was to perform fetal blood samplings (FBS). In view of the risks involved in the procedure, FBS has been restricted and non‐invasive strategies have been developed. The determination of maternal parameters predictive of severe fetal thrombocytopenia is crucial for tailored intervention. A first retrospective study was designed to analyse the predictive value of the maternal anti‐HPA‐1a antibody concentration. With this in view, we developed a quantitative method based on the monoclonal antibody‐specific immobilization of platelet antigens (MAIPA) technique, which is the gold standard method for serological investigations in platelet immunology [1]. Maternal anti‐HPA‐1a antibody concentrations were determined at the same time as the FBS carried out as a part of the antenatal management prior to therapy. A statistically significant correlation was observed between the high antibody concentration [≥ 28 international units (IU)/ml] and severe fetal thrombocytopenia (< 50 × 109/l; P = 0·0021 [2]). A larger retrospective study was subsequently performed to search for additional maternal predictive parameters during managed pregnancies, taking into account maternal anti‐HPA‐1a antibody concentrations and maternal genetic background (ABO blood group and HLA‐DRB3 allele). We confirmed the predictive value of the maternal antibody concentration before 28 weeks of gestation (wg) and before treatment. The follow‐up of the concentration during pregnancy allowed the measurement of the area under the curve, weighted by the weeks between the first and the last quantification. This new parameter was predictive of the therapy effectiveness: under 24 IU/ml/wg, a majority of women delivered a severely thrombocytopenic newborn (< 50 × 109/l; P = 0·0153 [3]). In conclusion, our work gives new insights into maternal predictive parameters for fetal status and therapy effectiveness, allowing non‐invasive strategies. Follow‐up of the antibody concentration during pregnancy could help to predict the outcome of the pregnancy, so as to prevent severe hemorrhagic disorders in the neonate.

Determination of platelet-bound glycoprotein-specific autoantibodies by flow cytometric immunobead assay in primary immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by premature platelet destruction induced by autoantibodies directed against platelet glycoproteins (GPs). Despite being a clinically important disorder, ITP lacks a feasible diagnostic assay for routine clinical use. This study was meant to evaluate a newly developed flow cytometric immunobead assay for determination of platelet-bound GP-specific autoantibodies in comparison with indirect monoclonal antibody-specific immobilization of platelet antigen (MAIPA) in the diagnosis of ITP. Platelet-bound and plasma GPIIb/IIIa and GPIb/IX autoantibodies were determined by flow cytometric immunobead assay and indirect modified MAIPA, respectively. The average fluorescence level for platelet-bound, GP-specific autoantibodies was given as a ratio to three normal controls tested simultaneously. The median value of platelet-bound GPIIb/IIIa and GPIb/IX autoantibodies in ITP group were 3.09 (range 0.78, 30.2) and 3.09 (range 0.72, 19.2), respectively, which were significantly higher than non-ITP group [1.01 (0.67, 5.59) and 1.01 (0.79, 5.56), respectively, P<0.001] and normal controls [1.02 (0.72, 1.76) and 1.03 (0.79, 1.73), respectively, P<0.001]. The receiver-operating characteristics curve analysis showed an area under the curve of 0.895 for GPIIb/IIIa autoantibody and 0.859 for GPIb/IX autoantibody, respectively. Combined detection of GPIIb/IIIa or GPIb/IX autoantibodies by flow cytometric immunobead assay showed a sensitivity of 82.11% for ITP diagnosis. This study demonstrated that determination of platelet-bound, GP-specific autoantibodies by flow cytometric immunobead assay was a convenient, sensitive, and specific test for the differential diagnosis of thrombocytopenic patients.

Establishment of a cell line panel for the detection of antibodies against human platelet antigen 4b

Antibodies against human platelet antigens (HPAs) play important roles in thrombocytopenia. In Japan, HPA-4b antibody is frequently responsible for HPA-related neonatal alloimmune thrombocytopenia. A highly sensitive assay using platelets has been developed for the detection of antibodies against HPAs. However, it is difficult to obtain the platelets expressing specific HPAs required for the assay. Therefore, an alternative method not requiring platelets would be helpful to detect antibodies against HPAs. Glycoprotein IIIa (GPIIIa) cDNA encoding HPA-4b was individually co-transduced with that of wild-type GPIIb in K562 cells, which is a non-adherent cell line, using a retroviral vector. The expression of transgene products in cultured cells were observed for over 6months. Next, to evaluating the sensitivity and specificity of this cell line panel, we performed monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay with a serum previously identified by another method. All HPA-4b antibodies in serum samples were positive, and all serum samples, including normal serum and serum containing HLA antibodies were negative. No difference was observed in the specificity and sensitivity between our method and conventional MAIPA using platelets. The present results indicate that this established cell line panel permits highly sensitive detection of specific antibodies against HPA-4b.

Two cases of platelet transfusion refractoriness associated with anti‐CD36

Antibodies to platelet (PLT) glycoprotein (GP) IV (CD36) have been implicated in rare cases of PLT refractoriness, particularly in non-Caucasians. We report two cases of PLT transfusion refractoriness linked to anti-CD36. A 5-year-old female of Lebanese descent and a 70-year-old male of Chinese descent both failed to respond to HLA-matched PLT transfusions during acute myelogenous leukemia induction therapy. Antibody screening was performed using a PLT antibody solid-phase kit (PAKPLUS, GTI Diagnostics), followed by the monoclonal antibody-specific immobilization of PLT antigen (MAIPA) test and, for the second case, the modified antigen capture enzyme-linked immunosorbent assay (MACE). Both patients demonstrated antibody to GP IV (CD36) on the PAKPLUS assay. On MAIPA testing, both phenotyped as CD36 negative. Anti-CD36 was demonstrated by MAIPA in the first case. In the second case, antibodies were not detected by MAIPA and variably detectable by MACE, depending on the mouse monoclonal antibody (MoAb) used. Because no Canadian CD36-negative donors were available, antigen-negative plateletpheresis units from the BloodCenter of Wisconsin were successfully transfused. Two cases of clinically significant CD36 antibodies are reported. Investigation of one case was complicated by steric inhibition of binding in the MAIPA and MACE assays with certain MoAbs. The cases demonstrate the importance of maintaining an ethnically diverse pool of rare donors and the value of international cooperation in the management of these patients.

Platelet-Associated IgG May Be Associated with Thrombocytopenia In Patients with Myelodysplastic Syndromes

AbstractAbstract 2943 Background.Myelodysplastic Syndromes (MDS) are a heterogeneous group of bone marrow disorders characterized by ineffective and dysplastic hematopoiesis, progressive bone marrow failure, cytopenias and a high risk of transformation into acute leukemia. Thrombocytopenia is detected in up to two thirds of patients with MDS and severe thrombocytopenia is present in approximately 10%. Besides ineffective thrombopoiesis, immune destruction of platelets could be an additional factor in the genesis of thrombocytopenia, since immunological abnormalities are also frequent in patients with MDS. The detection of platelet associated IgG (PAIgG) by immunofluorescence (platelet immunofluorescence test or PIFT) is a highly sensitive assay. In addition, some morphological platelet indices (PDW and MPV) are correlated with the occurrence of immune thrombocytopenia. We prospectively analysed platelet-bound IgG and platelet indices (PDW and MPV) in 35 patients with MDS. Methods:Thirty-five patients with MDS (mean age ± SD: 63 ± 19 yo; range 21–89 yo; 15female/20male) were evaluated. According to FAB, 27 patients were classified as RA, 5 as RARS and 3 as RAEB. Clinical manifestations of immunological disorder were not present in this population. Blood samples were analyzed by PIFT, in order to detect platelet associated IgG and results were expressed as a ratio of patient fluorescence/negative control fluorescence (R). Cell-dyn Sapphire blood cell analyzer (Abbott, Illinois, USA) was used to measure platelet count, mean platelet volume (MPV) and platelet size deviation width (PDW). Thrombocytopenia was defined as a platelet count <100 × 109/L. All samples were analyzed on the day of collection. Results.Platelet counts of the entire population ranged from 6.7 to 708 ×109/L, with median of 95.4 × 109/L. Eighteen patients (51.43%) had platelet count <100 × 109/L. A strong association between thrombocytopenia and PAIgG measured by PIFT (R) was demonstrated taking into account three analysis: an inverse correlation between the number of platelets and the fluorescence ratio (p=0.01, r=-0.39, Spearman test), the higher positivity of PIFT in patients with platelet count <100 × 109/L (p=0.007, Wilcoxon rank sum test) and a lower platelet count in patients with positive PIFT (p=0.059, Wilcoxon rank sum test). MPV was significantly higher in patients with platelet count <100 × 109/L (median, min-max: 9.38, 6.9–23.1 vs 8.46, 4.88–12.83; p<0.001, Wilcoxon rank sum test). PDW showed no statistical difference between these groups. Discussion: Immune thrombocytopenia is a relatively frequent hematological disorder of unknown origin and until today depends mainly upon clinical diagnosis. Assays for the detection of glycoprotein-specific antibodies such as MAIPA (monoclonal antibody-specific immobilization of platelet antigens) are highly specific but less sensitive than PIFT. MAIPA is laborious and require a certain amount of platelets, which is not always available in thrombocytopenia Thus, PIFT could be recommended as a screening test to discriminate patients with MDS in whom the hyperdestructive component of thrombocytopenia is important. Our results also showed a higher MPV in patients with platelet count <100 × 109/L, suggesting that this index may be of interest for detection of immune-mediated hyperdestructive thrombocytopenia in MDS. Conclusion:Thus, we propose that a combination of a simple index as MPV and a highly sensitive and easy to perform screening test for PAIgG as PIFT could be applied to select a subset of MDS patients in which we would be able to prevent the overuse of unnecessary platelet transfusions and who could be candidates for an immunosuppressive therapeutic approach. Supported by INCTS, FAPESP, CNPq. Disclosures:No relevant conflicts of interest to declare.

The detection of platelet antibodies by simultaneous analysis of specific platelet antibodies and the monoclonal antibody–specific immobilization of platelet antigens: an interlaboratory comparison

Glycoprotein (GP)-specific platelet (PLT) antibodies can cause allo- or autoimmune thrombocytopenia. Their detection is of high diagnostic value. The simultaneous analysis of specific PLT antibodies (SASPA) assay is based on simultaneous detection of various PLT-specific antibodies by flow cytometry and has entered routine use in our Mannheim institution. In this study, we performed an interlaboratory comparison investigation of PLT-specific antibodies using SASPA versus the "gold standard," the monoclonal antibody-specific immobilization of PLT antigen (MAIPA) assay. Sera from 194 patients with suspected PLT allo- or autoantibodies were tested against GPIIb/IIIa, IX, Ia/IIa, IV, and HLA Class I by SASPA (in Mannheim) and MAIPA (in Vienna). All data were reported blinded to those from the respective other method. Sensitivity studies included dilution studies with known antibodies against HPA-1a, -1b, -3b, -5b, and -15b and HLA Class I. Overall, results were concordant in 78.9%. The specificity and sensitivity of SASPA, based on the MAIPA results, were 97.3 and 86.3%, respectively, for the detection of alloantibodies. The respective results for the detection of autoantibodies were 95.3 and 44.9%. Serial dilution experiments with sera containing anti-HPA1a, -1b, -3b, -5b, and -15b and anti-HLA Class I revealed a higher sensitivity of the SASPA assay with all alloantibodies. In this first blind interlaboratory comparison, SASPA yielded similar results to those of MAIPA. The SASPA assay may be superior to the MAIPA assay for the detection of weak alloantibodies while simultaneous detection of a variety of antibody specificities or immunoglobulin classes and the need of fewer PLTs are obvious advantages.

Prenatal treatment of fetomaternal thrombocytopenia

A 34-year old Caucasian woman (gravida 2, para 1) with a history of fetomaternal alloimmune thrombocytopenia presented for antenatal care in her second pregnancy at 12 weeks' gestation. Her first baby was severely affected by fetomaternal alloimmune thrombocytopenia (platelet count &lt; 3×109/litre) and presented with neonatal purpura and fits caused by intracranial haemorrhage. Fetomaternal platelet incompatibility and presence of maternal anti-HPA-1b antibodies was demonstrated (Table 1) and the baby treated successfully, with platelet transfusions (HPA-1 negative). During her second pregnancy, the mother's booking platelet count was 250 × 109/litre. Fetal platelet typing via amniocentesis at 16 weeks showed fetal platelet genotype HPA 1a1b. The modified direct monoclonal antibody-specific immobilization of platelet antigens (MAIPA) test showed that the mother was still positive for anti-HPA-1b antibodies (Table 1), indicating a high risk of fetomaternal alloimmune thrombocytopenia in this pregnancy. Starting at 16 weeks, she received weekly intravenous immunoglobulin 1 g/kg. The anomaly scan at 20 weeks was unremarkable. Further monitoring included weekly cranial scans to detect intracranial haemorrhage, and monthly growth scans. These were all normal. The mother declined fetal blood sampling to assess the fetal platelet count. Steroids were given at 33 weeks and baby delivered by planned caesarean section at 34 weeks. The boy weighed 2.75 kg and had no signs of bleeding. His platelet count was 100 × 109/litre and he has been well since.

Flow cytometry detection of platelets autoantibodies in children with idiopathic thrombocytopenic purpura

Immune thrombocytopenic purpura is an acquired disorder, in which accelerated platelet consumption is due to platelet autoantibodies. The aim of this study was to investigate the clinical value of platelet autoantibodies assay in children with ITP and to evaluate flow cytometry in the detection of platelet autoantibodies in comparison with monoclonal antibody specific immobilization of platelet antigen (MAIPA) assay. We measured platelet autoantibodies by flow cytometry and MAIPA in 18 children with ITP (6 acute, 7 chronic and 5 in remission), in addition to 5 healthy children with matched age and sex as a control group. Significant elevation of platelet-associated immunoglobulin G (PAIgG), PAIgM and PAIgA was demonstrated in children with acute ITP compared to controls and children with chronic ITP (P < 0.05). There was significant elevation of PAIgG and PAIgM in children with acute ITP compared to children with ITP in remission (P < 0.05). There was significant negative correlation between platelet count and PAIgG levels in ITP children (r = -0.717; P = 0.001). Flow cytometry found PAIgG in 94.4% of ITP children. MAIPA has detected platelet specific IgG autoantibodies in 83.3% of ITP children. ROC analysis revealed sensitivity of 94%, specificity of 57% with overall accuracy of 83% for detection of PAIgG by flow cytometry compared to MAIPA. Platelet autoantibodies testing in ITP can discriminate acute from chronic forms of the disease and is helpful in follow up of patients. Determination of PAIgM in combination of PAIgG could be of interest in the investigation of ITP. Flow cytometry is a sensitive method of detection of platelet autoantibodies that could be used in screening of suspected ITP and should be followed by MAIPA assay in positive cases to establish the diagnosis.

Antibodies to platelet glycoproteins in haemophiliacs infected with HIV.

The techniques of Western blotting and the monoclonal antibody specific immobilization of platelet antigen (MAIPA) assay were used to detect antibodies to platelet glycoproteins in 43 samples of serum from 23 anti-HIV positive haemophiliacs (8 with severe thrombocytopenia, 6 with moderate thrombocytopenia, and 9 with a normal platelet count), six anti-HIV negative haemophiliacs and ten controls. Antibodies were present in the majority of anti-HIV positive patients' sera even before the onset of thrombocytopenia. Thrombocytopenia was associated with an increase in the incidence of antibodies to GPIIIa and GPIb, whereas the antigen most frequently recognized in patients without thrombocytopenia was GPIIb. Anti-GPIIb and/or GPIIIa reactivity was also seen in three out of the six anti-HIV negative patients. There was no correlation between the absolute platelet count and the detection of antibodies in either assay. Effective therapy for thrombocytopenia with zidovudine, interferon or splenectomy did not influence the presence of antibody. Eight of nine patients with AIDS were negative in the MAIPA assay, consistent with their depressed immune status. It is concluded that the production of antibodies to platelet membrane glycoprotein in anti-HIV positive haemophiliacs is influenced by factors other than HIV. The presence of such antibodies is independent of the platelet count and is therefore unlikely to play a causative role in HIV-related thrombocytopenia.

Is flow cytometry accurate enough to screen platelet autoantibodies?

The diagnosis of immune thrombocytopenic purpura (ITP) is a diagnosis of exclusion, as stated by international guidelines. Nevertheless, the assessment of platelet (PLT) antibodies has been reported as helpful for the diagnosis and the follow-up of ITP patients. PLT antibodies are detected by highly specialized assays, such as monoclonal antibody-specific immobilization of PLT antigen (MAIPA) test. Flow cytometry for PLT-associated immunoglobulin G (PAIgG) detection has been described more recently. This study was meant to evaluate the utility of flow cytometry to screen accurately patients needing further MAIPA testing. PAIgG, PAIgM, and PAIgA were determined in 107 consecutive patients and in 147 healthy controls in parallel. MAIPA testing was performed in all patients. The accuracy of flow cytometry was assessed with a receiver operating characteristics (ROC) curve analysis versus MAIPA. MAIPA assay found PLT-specific IgG in 27 patients (25%). The ROC curve analysis showed that no false-negative result in flow cytometry was obtained for a mean fluorescence intensity (MFI) cutoff of 0.2. With this cutoff, PAIgG were positive in 61 patients (57%). In this series, MAIPA was unnecessary in 42 percent of patients (corresponding to true-negative results). When MAIPA was positive, PAIgM values ranged from 0.1 to 1.0, and PAIgA from 0.1 to 2. Flow cytometry for PAIgG assessment may be used to accurately decide whether or not MAIPA must be subsequently performed. In this series, MAIPA was unnecessary in 42 percent of patients. Moreover, PAIgM results suggested that its determination combined with PAIgG may be of interest in ITP investigation.

A modified rapid monoclonal antibody‐specific immobilization of platelet antigen assay for the detection of human platelet antigen (HPA) antibodies: a multicentre evaluation

The monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay is the cornerstone technique for the detection and identification of human platelet antigen (HPA) antibodies. However, the original technique described by Kiefel and colleagues requires approximately 8 h adding to diagnostic delay. Moreover, proficiency exercises indicate that there are substantial variations in the MAIPA protocol, and that these may account for interlaboratory differences in sensitivity and specificity. A review of current MAIPA assay protocols from six laboratories together with performance in quality-assessment schemes identified several key variables potentially affecting the assay results. An optimized protocol was derived and assay time reduced to 5 h. The modified rapid MAIPA (MR-MAIPA) assay was evaluated using 61 samples with a range of HPA antibodies typically encountered in cases of fetomaternal alloimmune thrombocytopenia (n = 22), post-transfusion purpura (n = 8), platelet refractoriness (n = 7) and other platelet immune conditions (n = 24). The sensitivity of the assay was assessed using three international standards and the recombinant HPA-1a antibody CamTran007. The results obtained were compared with the original findings obtained with the local MAIPA assays. In addition, four different glycoprotein IIb/IIIa capture monoclonal antibodies were evaluated for their effect on assay sensitivity. Complete concordance was found between the original MAIPA results and those obtained with the new assay when testing a selected panel of clinical samples. The modified assay had nanogram level sensitivity for the detection of HPA-1a antibodies and titration of HPA-1a and HPA-5b antibody sensitivity standards yielded end-points equal to or greater than the mean recorded in international workshops. The MR-MAIPA assay offers improved turnaround for the detection of HPA antibodies without loss of sensitivity.

Neonatal alloimmune thrombocytopenia caused by human leucocyte antigen‐B27 antibody

Neonatal alloimmune thrombocytopenia (NAIT) occurs when maternal alloantibodies to antigens presented on foetal platelets cause their immune destruction. Whether human leucocyte antigen (HLA) antibodies can cause NAIT is controversial. Here, a patient was described who suffered from a NAIT caused by an HLA-B27 antibody. Sera from the mother and the newborn were tested for human platelet antigen antibodies and HLA antibodies by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay, solid phase-linked immunosorbent assay (ELISA), lymphocytotoxicity assay (LCT) and flow cytometric analysis. No antibodies against cluster designation (CD)109 and platelet glycoproteins of the father were found in patient's and mother's serum. However, HLA ELISA was used to identify HLA antibody in both sera. The antibody was specified as HLA-B27 antibody. Typing results showed that the father descended HLA-B27 antigen on patient and his brother. The mother was HLA-B27 negative. It is most conceivable that the previous pregnancy of the mother induced the production of anti-HLA-B27 antibody, which crossed the placenta and subsequently caused an NAIT in the case presented.

Light chain phenotypes of HLA antibodies in cases with suspected neonatal alloimmune thrombocytopenia

Platelet-specific antibodies are detectable in only about 30% in suspected neonatal alloimmune thrombocytopenia (NAIT). Human leucocyte antigen (HLA) class I antibodies are often detectable, and as platelets express the corresponding antigens, it has been suggested that these antibodies can be responsible for NAIT. Light chain phenotyping may assist in the diagnosis of HLA antibodies-induced NAIT. We determined light chain phenotypes of platelet reactive HLA antibodies in 17 sera from mothers who delivered offspring with suspected NAIT. Ten sera also contained platelet-specific antibodies: HPA-1a (n = 5, one with HPA-15b), HPA-5b (n = 4) and autoantibodies (n = 1). Sera were tested for kappa or lambda restriction by monoclonal antibody-specific immobilization of platelet antigens (MAIPA). The cross-match with paternal platelets was positive in all cases due to HLA antibodies. We identified 5, 3 and 3 sera to contain lymphocytotoxic antibodies of single, two or multiple specificities, respectively. In six cases, HLA antibodies were only detectable by MAIPA. Light chain restriction (n = 9) was not associated with HPA containing antibodies or to any pattern of the HLA antibodies. Similarly, polyclonal antibodies (n = 8) were seen in all categories. We show that pregnancy-associated HLA antibodies can be clonal or polyclonal, irrespective of a diagnosis of NAIT. ONE-SENTENCE SUMMARY: Pregnancy associated HLA antibodies can be clonal or polyclonal, irrespective a diagnosis of NAIT.

Severe thrombocytopenia due to host‐derived anti‐HPA‐1a after non‐myeloablative allogeneic haematopoietic stem cell transplantation for multiple myeloma: a case report

Host- or donor-derived alloimmune thrombocytopenia can develop after non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT). We report the first case of host-derived HPA-1a antibodies. A 52-year-old male patient received HSCT from his human leucocyte antigen (HLA)-A, -B, -C, -DR identical brother after reduced intensity conditioning. Bilinear engraftment around day 12 was accompanied by a continuous decrease of platelet counts. We investigated for platelet antibodies because of a progressive decline of platelet counts and refractoriness to platelet transfusions. The patient's serum was tested by enzyme-linked immunosorbent assay (ELISA), a solid phase assay and monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay. Recipient's DNA from the time before HSCT and donor's DNA were genotyped for human platelet antigens. Serum obtained on day 15 after HSCT reacted strongly with the donor's platelets due to host-derived anti-HPA-1a- and anti-HLA I antibodies. Serum samples from days 39, 45 and 65 after HSCT contained only anti-HLA I; no antibodies were detectable on day 149. Platelet counts increased on day 20 spontaneously. The decrease of the antibodies accompanied by the increase of the platelet counts suggests progressive elimination of residual host cells. The HPA-1a antibodies affected thrombopoietic engraftment and the success of platelet transfusions.

Anti‐HPA‐9bw (Maxa) fetomaternal alloimmunization, a clinically severe neonatal thrombocytopenia: difficulties in diagnosis and therapy and report on eight families

Fetal or neonatal alloimmune thrombocytopenia (FMAIT) results from a maternal alloimmunization against fetal platelet (PLT) antigens. In Caucasian persons, HPA-1a is the most frequently implicated antigen. During the past few years, FMAIT has been reported associated with rare or private antigens. Since the first documented case of FMAIT due to anti-HPA-9bw (Max(a)), no additional cases have been reported. Here a retrospective analysis is presented of the cases referred to our laboratories in recent years. The diagnosis was performed by genotyping and identification of the maternal alloantibody by the monoclonal antibody-specific immobilization of PLT antigens (MAIPA) technique. Parental genotyping showed HPA-9bw (Max(a)) mismatch as the sole antigenic incompatibility in seven of eight families. Because the father was found to be HPA-9bw (Max(a)) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. The maternal alloantibody was identified in the MAIPA technique. These data strongly suggest, however, that recognition of the HPA-9bw (Max(a)) epitope is not uniform. The neonatal thrombocytopenia was severe in most cases with bleeding. The outcome was good in all the cases but one. This analysis confirms that anti-HPA-9bw (Max(a)) FMAIT is not uncommon and was found to be approximately 2 percent of our confirmed FMAIT cases. It is a clinically severe syndrome that requires prompt diagnosis, albeit difficult, and maternal PLT transfusion therapy. Laboratory investigation of a suspected FMAIT case should be carried out in a specialist laboratory well-experienced in optimal testing. Appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding.

A novel method for simultaneous analysis of specific platelet antibodies: SASPA

Glycoprotein (GP)-specific platelet antibodies can cause allo-immune and auto-immune thrombocytopenia. The specific detection of relevant antibodies is a prerequisite for diagnosis and treatment. Here, we describe an improved method based on simultaneous detection of various platelet-specific immunoglobulin G (IgG) and IgM antibodies. Bead populations with distinct fluorescence intensities, coated with monoclonal antibodies specific for mouse heavy chain isotypes, were used for the simultaneous immobilization of platelet-GP [IIb/IIIa, Ib/IX, human leucocyte antigen (HLA) class I, or Ia/IIa, CD32, GPIV or CD109, Ib/IX, HLA class I]. In order to detect human IgG and IgM antibodies simultaneously, phycoerythrin- and fluorescein isotiocyanate-conjugated goat anti-human IgG and IgM were added. On this basis, the abundance of six distinct antibodies (three anti-GP, each with subclasses IgG and IgM) were simultaneously analysed without cross-reaction by flow cytometry. For evaluation, sera and platelets from 169 patients with platelet-binding and/or platelet-associated antibodies were investigated. The monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay was performed in parallel as reference test. The simultaneous analysis of platelet-specific antibodies (SASPA) assay was able to detect all platelet-specific IgG and IgM that were also recognized by MAIPA with a comparable sensitivity. SASPA proved to be a rapid and reliable assay that required less platelets than other methods. This method has the potential to pave the way for new investigations of platelet-specific antibodies.

La recherche des anticorps anti-plaquettes: étude de 701 dépistages positifs aspects pratiques

Platelet-specific immunization screening is a more and more in demand laboratory test. A study of 701 samples, positive on screening for platelet-specific antibodies was performed. During the sample study, some practical difficulties appeared. Platelet antibodies were screened with an immuno-capture test and identified by the monoclonal antibody specific immobilization of platelet antigens (MAIPA) technique. Both bound and circulating platelet antibodies were screened. At the screening stage, of the 701 positive samples, 95 were incomplete (13.6 %) — inadequate for study due to a poor quality or quantity. Nevertheless, of these 95 samples, platelet-specific antibodies were detected in 28 cases (29.5 %). Of the 606 remaining samples, identification was not possible in 21 cases (3.5 %). A total of 192 (32.8 %) of the 585 complete samples turned out to have platelet-specific antibodies - 84 (43.8 were allo-antibodies and 108 (56.8 %) were auto-antibodies. Among the platelet allo-antibodies, anti-HPA 5b was the most frequent whereas among the auto-antibodies the anti-GP IIb IIIa specificity was the most prevalent. However, it is worth noting that as so few clinical data on patients were received, together with the request for tests, the laboratory has not been in a position to give details on the possible origin of the anti-platelet immunization. Within the context of platelet-specific antibody researches this study shows that progress must be made both as regards the collection of samples and of clinical data, that the specificity of screening tests has to be estimated and improved and that new platelet antigenic systems mut be introduced into the MAIPA tests.

Platelet autoantibodies and lupus-associated thrombocytopenia.

To characterize the antigenic targets of anti-platelet antibodies (APA) found in systemic lupus erythematosus (SLE)-associated thrombocytopenia, 48 patients with immune thrombocytopenia and SLE were compared with 20 patients with SLE who had never been thrombocytopenic. Both cases and controls were tested for circulating APA by an indirect platelet suspension immunofluorescence assay (PSIIFT) and by indirect monoclonal antibody specific immobilization of platelet antigens (MAIPA). A direct platelet suspension immunofluorescence assay (PSIFT) was also used for antibodies bound to platelets in vivo in thrombocytopenic patients; 13 of them with high titres of platelets-bound APA were investigated by direct and indirect MAIPA and platelet eluate analysis. Circulating APA were detected by PSIIFT in 88% of cases and 55% of controls (P = 0.0066) and platelet-bound antibodies were detected by PSIFT in 90% of cases. Indirect MAIPA detected specific APA (mainly directed against GpIIbIIIa) in 36% of cases and only 5% of the controls (P = 0.0076). Nine out of the 13 fully investigated thrombocytopenic patients (69%) had a positive direct MAIPA and/or APA detected in platelet eluates. In conclusion, the production of specific anti-platelet autoantibodies, mainly directed against GpIIb/IIIa, and their binding to platelet membrane plays an important role in the pathogenesis of SLE-associated thrombocytopenia.

Semiquantitative measurement of IgG subclasses and IgM of platelet‐specific antibodies in a glycoprotein‐specific platelet–antigen capture assay

The detection of platelet-specific antibodies is of high clinical interest in diseases with immune thrombocytopenia. The glycoprotein-specific platelet-antigen capture (PAC)-assay developed in this study is especially suited to the differentiation of platelet-specific immunoglobulin (Ig) G subclasses and the determination of platelet-specific IgM in serum or on platelets. The problems with unspecific signals or low sensitivity usually seen with the detector antibodies available are effectively overcome, as unbound detector antibodies are removed at an early stage in the assay. We investigated 14 maternal alloantisera from cases of neonatal alloimmune thrombocytopenia (NAITP) and six sera from patients with autoimmune thrombocytopenic purpura (AITP). In NAITP sera, we found IgG1 alone in 57%, IgG1 + IgG3 in 21% and IgG1 + IgG2 in 14% of cases. One serum contained IgG1 + IgG2 + IgG3. In AITP, three out of the six sera contained IgG1 alone. One serum contained IgG1 + IgG2. One patient, with highly refractory AITP, had platelet-specific IgG1 + IgG2 + IgG3 in his serum. A patient with AITP in remission and normal platelet counts only showed platelet-bound IgG2. The detection of platelet-specific 'whole IgG' is possible too. However, at this time the commonly used monoclonal antibody-specific immobilization of platelet antigens (MAIPA) method should not be replaced for this purpose, as it is well standardized and used with similar results in many laboratories. The PAC assay sensitively detects the subclasses of platelet-specific IgG and human leucocyte antigen (HLA)-antibodies independently. It is easy to perform and takes less time than other platelet glycoprotein-specific methods.