Monoclonal Antibody Immobilization Of Platelet Antigens Assay Research Articles

Circulating antiplatelet antibodies in pregnant women with immune thrombocytopenic purpura as predictors of thrombocytopenia in the newborns

Newborns from mothers with immune thrombocytopenic purpura (ITP) have a risk of thrombocytopenia due to passage of maternal antiplatelet antibodies into fetal/neonatal circulation. We looked for predictors of neonatal thrombocytopenia (nTP) in pregnant women with ITP. One hundred pregnant women with platelet count <100 × 109/l, no non-immune causes of thrombocytopenia and increased platelet associated IgG (PA-IgG) were included in the study. Thirty seven and 63 of them gave birth to babies with and without nTP, respectively (nTP+ and nTP− groups). Platelet count, mean platelet volume, PA-IgG, antiplatelet circulating antibodies (cAB), time of ITP onset (before or during pregnancy), and frequency of corticosteroid treatment were compared in these groups. There were no differences in all test parameters between nTP+ and nTP− groups except cAB. These antibodies were detected in 33 out of 37 in nTP+ group and in 2 out of 63 mothers in nTP− group (p < 0.001). The sensitivity of this test was 89% and its specificity was 97%. A strong reverse correlation (r = −0.749, p < 0.001) was established between maternal cAB titer and neonatal platelet count. Antibodies against glycoproteins IIb–IIIa and/or Ib were identified in antigen specific MAIPA (Monoclonal Antibody Immobilization of Platelet Antigen) assay only in 10 out of 19 (53%) test sera with cAB. Antiplatelet cAB in pregnant women with ITP could serve as reliable predictors of nTP in their babies.

Detection of platelet autoantibodies to identify immune thrombocytopenia: state of the art.

Immune Thrombocytopenia (ITP) is diagnosed by exclusion of other causes for thrombocytopenia. Reliable detection of platelet autoantibodies would support the clinical diagnosis of ITP and prevent misdiagnosis. We optimized our diagnostic algorithm for suspected ITP using the direct monoclonal antibody immobilization of platelet antigens assay (MAIPA), which evaluates the presence of platelet autoantibodies on the glycoproteins (GP) IIb/IIIa, Ib/IX and V bound on the patient platelets. The direct MAIPA was shown to be a valuable technique for the detection of platelet autoantibodies and could possibly become a guide for optimizing therapy towards a more personalized treatment of ITP.

Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients

BackgroundPlatelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation.MethodsCapture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs.ResultsThe coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%.ConclusionsA quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.

Analysis of Genotype of HLA/HPA and Antibody Specificity in Platelet Transfusion Refractoriness Patients

Objective To analyze the genotype of human leukocyte antigen (HLA)/human platelet antigen (HPA) and the antibody specificity in platelet transfusion refractoriness (PTR) patients caused by HLA antibodies combined with HPA antibodies.Methods From January 2010 to June 2012,a total of 6 patients who had HLA class Ⅰ antibodies and HPA antibodies were selected as research subjects from PTR patients who accepted platelet (PLT) crossmatch in Guangzhou Blood Center.HLA antibodies and antibody of membrane glycoprotein (GP) were measured by enzyme-linked immunosorbent assay (ELISA),and the HLA and HPA1-17 genotype were analyzed by sequence specific primers-polymerase chain reaction (PCR-SSP) method.Then the antibodies specificity was confirmed according to HLA/HPA genotype and the antibodies detecting results using monoclonal antibody immobilization of platelet antigens assay (MAIPA).The study protocol was approved by the Ethical Review Board of Investigation in Human Being of Guangzhou Blood Center.Informed consent was obtained from all participants.Results In these 6 PTR patients,anti-HLA-A * 2,-A * 11 and anti-HLA-B * 40 of HLA-Ⅰ antibodies were common.The highest expression of PLT GP specific antibody was GP Ⅱ b/Ⅲ a,accounting for 83.3％ (5/6).3 patients (50.0％,3/6) had anti-HPA-3a antibody; 2 patients (33.3％,2/6) had anti-HPA-3b antibody; only 1 case (16.7％,1/6) had anti-HPA-5b antibody.Conclusions In clinically,the analysis of HLA/HPA genotype and antibody specificity in PTR patients could help us to avoid PLT transfusion which expresses HLA/HPA antibodies against corresponding antigen,to find the blood donors who have the same HLA/HPA genotypes with patients,to improve the efficacy of PLT transfusion and avoid blood waste. Key words: Platelet antibodies; Platelet transfusion; HLA antigens; Antigens, human platelet

Detection of autoantibodies against platelet glycoproteins in patients with immune thrombocytopenic purpura by flow cytometric immunobead array

BackgroundThe goal of this study is to develop a flow cytometric immunobead array (FCIA) assay to detect platelet autoantibodies commonly present in bleeding patients with immune thrombocytopenic purpura (ITP). MethodsPolystyrene microbeads coated with antibodies against human platelet glycoproteins (GPs) IX (SZ1), Ib (SZ2), IIIa (SZ21), IIb (SZ22), and P-selectin (SZ51) were incubated with platelet lysate from 50 ITP patients and 86 controls. The platelet antigen–autoantibody complexes were detected by flow cytometry using an FITC-labeled antibody. The results were compared with that of a monoclonal antibody immobilization of platelet antigen (MAIPA) assay. ResultsBy FCIA, platelet autoantibodies against GPIb, GPIIb, GPIIIa, GPIX and P-selectin were detected in ITP patients. Mean fluorescent intensity values with antibodies SZ1, SZ2, SZ21, SZ22 and SZ51 were all higher in ITP patients than controls (p values<0.01). In ROC analysis, values of the area under the curve were 0.89, 0.82, 0.93, 0.94 and 0.95, respectively. In ITP diagnosis, the FCIA assay with these five antibodies had better sensitivity and accuracy than the MAIPA assay (96% vs. 44% in sensitivity; 80.9% vs. 64.7% in accuracy, p<0.01). ConclusionFCIA assays with multiple antibodies against platelet GPs may be used to improve the diagnosis of ITP in hospitals.

The clinical significance of circulating B cells secreting anti-glycoprotein IIb/IIIa antibody and platelet glycoprotein IIb/IIIa in patients with primary immune thrombocytopenia

ObjectivesThe objective of the study is to evaluate the possible roles of the detection of circulating B cells secreting anti-glycoprotein IIb/IIIa antibody, platelet glycoprotein IIb/IIIa, and anti-glycoprotein IIb/IIIa antibody in the diagnosis of primary immune thrombocytopenia (ITP) patients.MethodsCirculating B cells secreting anti-glycoprotein IIb/IIIa antibody, platelet glycoprotein IIb/IIIa and anti-glycoprotein IIb/IIIa antibody in 64 patients with ITP, 33 non-ITP patients, and 32 controls were measured with enzyme-linked immunospot assay (ELISPOT), monoclonal antibody immobilization of platelet antigens assay (MAIPA) and flow cytometic analysis (FCM), respectively.ResultsCompared with the controls and non-ITP patients, the frequency of circulating B cells secreting anti-glycoprotein IIb/IIIa antibody was significantly increased, whereas the positive rate of platelet glycoprotein IIb/IIIa was significantly decreased (P < 0.05) in ITP patients, respectively. The sensitivities for the diagnosis of ITP of ELISPOT and FCM were 68.8% and 57.8%, and the specificities of 90.9% and 90.9%, respectively. The sensitivities of ELISPOT and FCM were higher than MAIPA's sensitivity (39.1%) (P < 0.05). However, there was no apparent difference of the sensitivities of ELISPOT and FCM and the specificities of those three detections (MAIPA's specificity was 81.8%) (P > 0.05).DiscussionELISPOT and FCM for detecting the circulating B cells secreting anti-glycoprotein IIb/IIIa antibody and the platelet glycoprotein IIb/IIIa were as specific as that of MAIPA for assay of anti-glycoprotein IIb/IIIa antibody, but ELISPOT and FCM had higher sensitivities. So ELISPOT and FCM were sensitive and specific for identifying patients with autoantibody-mediated thrombocytopenia and these should be used as diagnostic tests in clinic.

Relative Efficacy of Steroid Therapy in Ameliorating Autoimmune Thrombocytopenia Mediated by Anti-Platelet GPIIbIIIa Versus GPIbα Antibodies.

Abstract 1323Poster Board I-345 BackgroundIdiopathic thrombocytopenic purpura (ITP) is a common autoimmune disease characterized by autoantibody-induced platelet destruction and consequent bleeding disorders. Despite considerable investigation, the pathogenesis of ITP remains incompletely understood, and we currently lack a consensus for therapy. The major targeted platelet autoantigens are located on two distinctive platelet receptors; the GPIIbIIIa and GPIb complexes. It has been reported that thrombocytopenia induced by anti-GPIba antibodies, which may differ from that induced by anti-GPIIbIIIa, can be mediated via an Fc-independent pathway. We further demonstrated in a murine model that anti-GPIba-mediated-ITP is less responsive to IVIG treatment (Blood. 2006; 108(3):943-6), which is consistent with a subsequent retrospective study in human ITP patients. Since IVIG is expensive and steroid therapy is the first line of treatment for most patients, particularly in developing countries, we tested whether gluococorticosteroid therapy has equal efficacy in ITP patients caused by these two different antibodies. Patients and MethodsA total of 107 ITP patients (71 females and 36 males) were recruited for the present study. Patients were first treated with Dexamethasone (DXM, 20mg/day x 3-5 days), then changed to oral administration of Prednisone (1mg/kg/day). The dose of Prednisone was gradually decreased and maintained for = 30 days. The patients were considered as responders to the therapy if no obvious haemorrhage was detected and their platelet counts were raised to = 50 × 109/L or increased by = 30 × 109/L from their original platelet count before treatment. All patients' plasma samples were collected before therapy and specific autoantibodies against GPIIbIIIa and/or the GPIb complex were measured with a monoclonal antibody immobilization of platelet antigen assay (MAIPA). ResultsAmong the 107 patients, 23% (25/107) had antibodies against GPIIbIIIa, 18% (19/107) had antibodies against the GPIb complex, 34% (36/107) had antibodies against both the GPIIbIIIa and GPIb complex, and 25% (27/107) had no detectable antibody to either of these two antigens. We found that a majority of the patients with antibodies against GPIIbIIIa (15/22) were sensitive to the steroid therapy, which is significantly different from those with antibodies against the GPIb complex (5/19) (X2 = 7.15, P <0.01) or those with antibodies against both the GPIb complex and GPIIbIIIa (6/34) (X2 = 14.5, P <0.01). Although no statistically significant difference was observed between patients with anti-GPIb antibodies and those with both anti-GPIb and anti-GPIIbIIIa antibodies (X2 = 0.55, P >0.05), there is a tendency that patients with double positive antibodies were less responsive to steroid therapy therapy. The most sensitive patients for this therapy were those without detectable antibodies to either of these two autoantigens (21/27). ConclusionsOur data suggest that anti-GPIb-mediated ITP may be less responsive to both IVIG and steroid therapies when compared to anti-GPIIbIIIa-mediated ITP. The pathogenesis and treatment of anti-GPIb-mediated ITP merits further investigation. DisclosuresNo relevant conflicts of interest to declare.

Rituximab‐associated acute thrombocytopenia: An under‐diagnosed phenomenon

Acute infusion reactions are the most common documented adverse reactions reported with rituximab, with overt cytokine release syndrome, and hematological adverse events being much rarer. The clinical course of a patient with mantle cell lymphoma, who developed acute thrombocytopenia and leukopenia following rituximab administration, is described and the literature reviewed. Serum complement and the levels of three cytokines--TNF-alpha, IL-6, and IL-1, were measured 2 days after the infusion of rituximab by using ELISA assay. Drug-dependent antibodies against platelets were evaluated by two procedures as follows: an immunofluorescence test applying flow cytometry and Monoclonal Antibody Immobilization of Platelet Antigen (MAIPA). Serum levels of TNF-a were significantly increased compared with normal, whereas those of IL-6 and IL-1 were not increased significantly. Flow cytometry assay and the MAIPA assay failed to detect rituximab-dependent antibodies against platelets. Complement levels were decreased compared with normal. Literature search yielded 10 publications reporting on another 15 patients. The most common type of lymphoma was mantle cell lymphoma, six patients had bone marrow involvement, and 10 patients had splenomegaly. In 10 patients, acute cytopenia was preceded by cytokine release syndrome or infusion-related symptoms. Usually, thrombocytopenia was not associated with bleeding manifestations. Thrombocytopenia was the most commonly acute cytopenia reported. The postulated pathogenesis is associated with cytokine release syndrome and complement activation. Patients with potential risk factors like splenomegaly and bone marrow involvement, who develop clinical manifestations compatible with cytokine release syndrome, should be closely monitored for rituximab-associated cytopenia.

External quality assessment of platelet serology and human platelet antigen genotyping: a 10‐year review

In this review, the results of an external quality assessment (EQA) over 10 years of platelet (PLT) serology and of human platelet antigen (HPA) polymorphisms genotyping are shown. The detection and identification of PLT antibodies and the distinction between PLT-specific antibodies and HLA Class I antibodies are evaluated. Each year, serum samples from five patients and four donor blood samples for DNA typing were distributed. Laboratories could participate as a screening laboratory (SL; n = 7) or as an identification laboratory (IL; n = 8). SLs scored 57 to 100 percent correct positive and 91 to 100 percent correct negative results in the detection of PLT-specific antibodies. SLs only using a PLT immunofluorescence test (PIFT) scored less well than those using a PLT glycoprotein-based antibody detection method. ILs scored 70 to 100 percent correct positive and 87 to 100 percent correct negative results for, respectively, the detection and identification of PLT-specific antibodies. Both the specificity and the sensitivity for the detection and identification of PLT-specific antibodies were not as good in ILs using solid-phase enzyme-linked immunosorbent assay methods as in those using the monoclonal antibody immobilization of PLT antigens (MAIPA) assay. For HPA-1, -2, -3, and -5 genotyping, incorrect results were observed only twice in 280 genotyping assays. The data underscore the necessity of using the most accurate methods with a high level of knowledge, experience, and technical training. For screening purposes, it is not sufficient to use only the PIFT, whereas for identification of PLT-specific antibodies, the MAIPA assay is the superior assay.

Differentiation of anti-platelet antibodies using microspheres and flow cytometry Einsatz von Mikrosphären und Durchflusszytometrie für die Differenzierung von Antithrombozyten-Antikörpern

Abstract Background: Allo- and autoantibodies against membrane structures of human platelets cause different types of immune thrombocytopenic disorders and require a sensitive and specific assay. The well-established monoclonal antibody immobilization of platelet antigens (MAIPA) test is able to distinguish between specifically bound immunoglobulins and coating of membrane-associated immune complexes; but MAIPA is rather time consuming and requires 2×107 platelets or more. For patients with very low platelet count and for little children with small blood volume, this may present a serious limitation. Methods: The advantages of flow cytometry and of modern microsphere technology were combined with the selectivity of MAIPA to overcome the disadvantages of this assay. In order to compare different types of beads, i) goat anti-mouse Ig-labelled magnetic beads, ii) dyed polymeric beads designed for multiplexed assays, and iii) blanc polymeric beads have been studied. After loading with glycoprotein-specific or anti-β2-microglobulin-specific antibodies, these beads could be stored for months at 4°C without substantial loss of sensitivity. Glycoproteins from platelet membranes, loaded with platelet-specific auto-/alloantibodies, have been immobilized on the bead surfaces during detergent solubilization of platelets prior to labelling with anti-human IgG-Biotin and Extravidin® R-Phycoerythrin. Results: Magnetic beads are very easy to handle, show strong signals and low nonspecific binding, but have stronger autofluorescence compared to polymeric beads. Polymeric beads have low autofluorescence but stronger tendency to nonspecific binding. All types of beads tested in this study can be used to detect antibodies against platelet antigens, even against glycoproteins with low frequency such as anti-HPA-5 autoantibodies and with as few as 106 platelets or less. In multiplexed settings, they are suitable for simultaneous detection of multiple antibodies. Conclusions: Flow cytometry is a fast and sensitive method for a bead-based MAIPA assay and is especially useful for low platelet counts and for pediatric patients. It is further improved by the use of multiplexed beads.

Report on the 12th International Society of Blood Transfusion platelet immunology workshop

The aims of the 12th International Society of Blood Transfusion (ISBT) Platelet Immunology Workshop were to evaluate the proficiency of molecular human platelet antigen (HPA) genotyping and detection of platelet antibodies of unusual specificity or reactivity, to assess whether quantification of anti-HPA-1a is practicable, and to determine the variability of reagents and components used in the monoclonal antibody immobilization of platelet antigens assay (MAIPA). Forty participants from 23 countries were sent 10 samples for DNA typing, five samples for antibody detection, a freeze-dried anti-HPA-1a standard, three samples for anti-HPA-1a quantification and a MAIPA method questionnaire. The detection and identification of HPA antibodies varied from 2.7 to 95% of participants. The number of HPA genotyping errors per sample ranged from 0 to 3.96% per HPA loci. The majority of laboratories were able to assign an arbitrary number of units/ml of anti-HPA-1a activity to the unknown samples. The MAIPA questionnaire indicated a wide variation among participants, both in method and in reagents used. The results obtained from this workshop highlighted deficiencies in testing regimes and identified a need for internationally available reference materials.

The dilemma of screening for antibodies against low‐frequency human platelet antigens

The dilemma of screening for antibodies against low‐frequency human platelet antigens

Delayed immunologic thrombocytopenia induced by abciximab.

Abciximab is an anti-GPIIb-IIIa drug widely used to prevent thrombotic complications during percutaneous coronary intervention. We now report on the immunologic origin of thrombocytopenia developing between 7 and 12 days after the onset of abciximab infusion. Antibodies directed against abciximabcoated platelets were located in 5 patients with delayed thrombocytopenia, just as they were present in a patient whose platelet count fell within a few hours after receiving the drug. Abciximab-dependent IgG antibody was revealed in serum using control platelets in the monoclonal antibody immobilization of platelet antigens assay (MAIPA) performed with SZ22, a MoAb to GPIIb. The presence of IgG antibodies specific for platelets sensitized with abciximab was confirmed by flow cytometry. They were not located in 13 patients receiving abciximab but whose platelet counts remained stable. For three patients, antibodies were transient and their presence related to the extent of the thrombocytopenia. Surprisingly, antibodycontaining plasma from three patients induced abciximabdependent activation and aggregation of normal platelets, a finding confirmed by electron microscopy. Immunogold labeling revealed that abciximab was associated with platelets in the aggregate, suggesting that its inhibitory effect was overcome by the platelet stimulation. In summary, these results show that abciximab-dependent thrombocytopenia can be delayed and potentially prothrombotic.

Evaluation of an enzyme-linked immunosorbent assay kit (GTI PakPlus) for the detection of antibodies against human platelet antigens.

Twenty-six serum samples from 24 patients were investigated for the presence of platelet-specific antibodies in a partly retrospective (n = 15) and partly prospective (n = 9) study. The sera contained either alloantibodies to human platelet antigens (HPA) (n = 23) or were from clinically suspected cases of fetomaternal alloimmune thrombocytopenia (FMAITP) in which platelet-specific antibodies had not been detected (n = 3). Three techniques were used to detect platelet antibodies: the platelet immunofluorescence test, the monoclonal antibody immobilization of platelet antigens (MAIPA) assay and a commercially available enzyme-linked immunosorbent assay--GTI PakPlus (GTI kit). Two alkaline phosphatase-conjugated antiglobulin reagents provided by the manufacturer were used in the GTI kit: an antihuman IgG/IgA/IgM (IgGAM) conjugate and an antihuman IgG conjugate. The GTI kit with the anti-IgGAM conjugate failed to detect eight antibody specificities in seven sera (anti-HPA-1a [n = 3], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1] and anti-HPA-5b [n = 3]). Greater signal-to-background ratios were achieved in the GTI kit with the anti-IgG conjugate but five antibody specificities (anti-HPA-1a [n = 1], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1], anti-HPA-5b [n = 2]) remained undetectable. All the sera were detected by MAIPA assay and, furthermore, the MAIPA assay achieved the greatest signal-to-background ratio in the majority of sera tested. These findings re-emphasize the value of the MAIPA assay in reference laboratories and illustrate that the GTI kit may either fail to detect or incorrectly identify clinically significant HPA antibodies.

Platelet autoantibodies (IgG, IgM, IgA) against glycoproteins IIb/IIIa and Ib/IX in patients with thrombocytopenia.

Autoimmune thrombocytopenic purpura (AITP) is most frequently induced by platelet-specific autoantibodies against epitopes on platelet GP Ib/IX or GP IIb/IIIa. These antibodies are reliably detected on the patients' autologous platelets. So far, studies on the characterization of platelet autoantibodies have been restricted to IgG antibodies. We used the monoclonal antibody immobilization of platelet antigens assay (MAIPA) in a modified version to detect GP-specific IgG, IgM, and IgA antibodies. Platelets of 46.2% of patients carried elevated amounts of IgG antibodies. IgM and IgA antibodies were observed less frequently and showed only weak OD signals in the MAIPA assay. Circulating IgG antibodies in serum were found in 11.5% of patients. Circulating IgM autoantibodies were observed in 8.9% and IgA antibodies in no patient with AITP. Results of direct MAIPA assay were compared with the reactivity of eluates in the platelet adhesion immunofluorescence assay and were found to be highly concordant. Patients with AITP in remission carried high percentages of anti-GP IIb/IIIa. Findings made in this study suggest that autoantibodies of the IgM and IgA classes play only a minor role in the pathogenesis of AITP.

A close spatial relationship between GP IIb—IIIa complexes and CD9 antigen as demonstrated by the MAIPA technique

CD9 is a well-defined component of the platelet plasma membrane and has a copy number almost equivalent to that of glycoprotein (GP) IIb-IIIa complexes, the aggregation receptor on platelets. It has an apparent molecular mass of 24 kD and is otherwise known as p24. Stimulation of p24 by monoclonal antibodies (MAb) induces platelet aggregation and granule release, involves FcγRII, and is mainly mediated through the stimulation of phospholipase C. In accordance with a signalling function, p24 has been reported to associate with small GTP-binding proteins and to GP IIb-IIIa complexes upon activation. We now report further evidence of a strong relationship between p24 and GP IIb-IIIa in platelets. Using the MAIPA (monoclonal antibody immobilization of platelet antigens) assay in the screening of human antibodies to platelet glycoproteins, we found that GP IIb-IIIa-antibody complexes were almost invariably associated with p24 in the harvested detergent-soluble fraction of platelet lysates. Thus, associated human antibodies were detected following the targeting of either GP IIb-IIIa or p24 by monospecific murine monoclonal antibodies (MAbs). This is a point to bear in mind when assessing for antibodies to p24 or GP IIb-IIIa in immune thrombocytopenias.

Evidence for a light chain restriction of glycoprotein Ib/IX and IIb/IIIa reactive antibodies in chronic idiopathic thrombocytopenic purpura (ITP).

To address the assumption of clonally restricted antibodies in immune thrombocytopenias we studied sera from 19 patients with chronic ITP known to possess antibodies reactive with glycoprotein (GP) Ib/IX and/or GPIIb/IIIa. These sera were re-analysed using the standard monoclonal antibody immobilization of platelet antigens (MAIPA) assay and 16 patients exhibited IgG antibodies reactive with GPIIb/IIIa; seven patients showed also a reactivity with GPIb/IX. Employing a light-chain-specific MAIPA assay, 75% (12/16) of these sera displayed GPIIb/IIIa-specific antibodies that were light chain restricted; only 13% (2/16) of the GPIIb/IIIa reactive sera showed a mixed kappa and lambda phenotype. A light-chain-restricted phenotype was also seen for the GPIb/IX reactive antibodies. To further substantiate these findings, the MAIPA assay was modified in order to avoid interference from human anti-mouse antibodies. A high frequency of light-chain restricted platelet antibodies was also found using the modified MAIPA technique. These results support the hypothesis of a clonal B-cell expansion in immune thrombocytopenias, producing antibodies with a restricted idiotype repertoire and reacting with a limited number of epitopes.

The MAIPA assay and its applications in immunohaematology.

The monoclonal antibody immobilization of platelet antigens (MAIPA) assay has proved to be a reliable and useful tool in platelet immunology. After immunochemical localization of the most important platelet allo- and autoantigens on platelet glycoproteins (GPs) IIb/IIIa, Ia/IIa, Ib/IX, the corresponding antibodies can easily be detected with this technique because mAbs against these membrane constituents are now readily available. The assay also allows reliable typing of platelet alloantigens. Detection of GP-specific PAIgG (by direct MAIPA) has proved to be a specific diagnostic sign of AITP. The MAIPA assay allowed identification of 'new' alloantibody specificities including alloantibodies against 'low-frequency' antigens. The principle of this technique has successfully been applied to characterize antibodies against lymphocytes, endothelial cells and granulocytes.