Immobilization Of Platelet Antigens Research Articles

Establishment of a Multivariate Analysis Based on Photonic Crystal Hydrogel Bead Arrays and Its Application for Detecting Platelet-specific Antibodies

ABSTRACTPhotonic crystals are nanomaterials with unique structural coloration, and they have been used in multiple analyses, gene function analyses, and nanoarray biosensors. Here, we used agarose hydrogel microcarriers with characteristic reflection peak codes to improve the biocompatibility of the material and the efficiency of detection, which may be helpful for further clinical applications. The paper has two parts: condition optimization and product application. Goat antihuman immune globulin G, human immune globulin G, and fluorescein isothiocyanate-labeled goat antihuman immune globulin G are commonly used materials in double-antibody sandwich techniques, which are used for optimization in the application of advanced composite materials. Goat antihuman immune globulin G was immobilized on the surfaces of photonic crystal hydrogel beads through chemical bonding. When the target immune globulin G was captured, the fluorescence-labeled antibody could combine with photonic crystal hydrogel beads. The reaction conditions were optimized using the gray-scale value of fluorescent intensities. Photonic crystal hydrogel beads with different reflection spectra were coated with purified platelet glycoprotein IIb/IIIa and glycoprotein Ib/IX. A total of 64 clinical samples including sera from patients with immune thrombocytopenia (n = 32) and healthy human sera (n = 32) were tested using photonic crystal hydrogel beads and monoclonal antibody immobilization of platelet antigen (MAIPA). Photonic crystal hydrogel bead array reaction conditions were established for multiple analysis, showing similar sensitivity and specificity with MAIPA in testing different platelet antibodies and shortening the analysis time. The features of these composite materials make them suitable for potential applications in clinical screening assays.

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.

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.

L’allo-immunisation plaquettaire par anticorps anti-HPA-15 : prévalence et difficultés du dépistage et de l’identification

Summary HPA-15a and -15b-specific alloantibodies (Ab) may induce refractoriness to platelet (PLT) transfusion and fetal neonatal alloimmune thrombocytopenia (FNAIT) due to maternal alloimmunization. In order to establish the prevalence of these Ab, a study was performed in non-selected patients with a “Monoclonal Antibody-specific Immobilization of Platelet Antigen”-adapted assay. Of 7000 samples, 279 were found initial reactive (IR) (4.0 %). Five were not retested because of insufficient volume of serum. Among the 274 remaining samples, 20 were found to be repeat reactive (7.3 %). After characterization, anti-HPA-15b were found in three sera, one of which from a patient with a severe FNAIT; 17 were anti-HPA-15a, however the interpretation of results was delicate with those. Anti-HPA-15 Ab reactivity depends on the type of PLT used and on the level of expression of the antigens present on the PLT membrane. HPA-15-specific Ab are uncommon and their detection remains difficult in practice.

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 < 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.

The effect of platelet autoantibodies on the course of the disease and clinical response of patients with idiopathic thrombocytopenic purpura

In this study, we evaluated the response to treatment of 409 idiopathic thrombocytopenic purpura (ITP) patients who were tested for the presence of platelet-associated autoantibodies by direct-platelet immunofluorescence test (PIFT) and for the presence of plasma antibodies directed against the GPIIb/IIIa, GPIb and GPIa/IIa by monoclonal antibody immobilization of platelet antigens (MAIPA). In patients with platelet autoantibodies in comparison with patients without antibodies more frequently were observed the chronic form of disease (83.5%vs. 68.5%) and severe symptoms of haemorrhage diathesis (17.3%vs. 6.9%). Evaluation of the treatment response (to corticosteroids, immunosuppressive drugs and splenectomy) referred to patients with complete response, e.g. complete remission defined as platelet count of >100 x 10(9)/l for at least 2 years. The percentage of complete response in the whole population of ITP patients, both with and without autoantibodies regardless of the method of treatment, was similar (about 54%). However, the presence of platelet autoantibodies had effect on patients treated with corticosteroids: complete response approximately 71% (36/51) of patients with autoantibodies and in 60% (72/120) of patients without antibodies, as well as in patients treated with immunosuppressive drugs (cyclophosphamide, azathioprine, vincristin and vinblastin); complete response approximately 51% (11/21) of patients with autoantibodies and in 34.8% (6/17) of patients without autoantibodies. The presence of autoantibodies had no effect on the response of splenectomy patients.

Report on the 13th International Society of Blood Transfusion Platelet Immunology Workshop

The aim of the 13th International Society of Blood Transfusion Platelet Immunology Workshop was to compare the sensitivity and specificity of the in-house method for the detection of human platelet antigen (HPA) antibodies currently used in participating laboratories with a modified rapid protocol for the monoclonal antibody (mAb) immobilization of platelet antigen (MR-MAIPA) assay. Twenty-eight laboratories from 15 countries participated. A set of four freeze-dried minimum potency reference reagents with known single-specificity HPA antibodies were supplied for testing by titration with both assays and two coded freeze-dried plasma samples were provided for antibody specificity testing. Critical reagents and materials for the MR-MAIPA were provided including lyophilized panel platelets and five capture mAbs. Titration of the reference standards showed that the sensitivity of the MR-MAIPA was the same as the in-house methods. The proposed replacement anti-HPA-1a reference reagent 05/106 gave results that did not differ significantly from the current reference reagent 93/710. The results with the two blinded samples showed that in the first sample, 27 out of the 28 laboratories were able to correctly identify the anti-HPA-1a present when using their respective in-house methods, but only 23 correctly identified the antibody when using the rapid MAIPA method. The results from the second sample, which contained multispecificities, showed that only 50% of the participants correctly identified all five antibodies present using their in-house method. The results for the rapid MAIPA were lower, with only 32% identifying all specificities. The variability in the reconstitution of the freeze-dried platelets may have been one of the contributing factors to the poorer results. The sensitivity of the MR-MAIPA compared favourably with that of the in-house methods. Most laboratories were able to identify anti-HPA-1a alone in Sample 1 but more than half of the participants were not able to correctly assign the specificity of all HPA antibodies present in the second sample. The usefulness of the panel of freeze-dried platelets varied considerably between laboratories.

Pregnancy-Associated Autoimmune Neonatal Thrombocytopenia: Role of Maternal HLA Genotype

In a prospective study between 1993 and 1998, data was collected from 46 pregnant women and subsequently from their babies. 25 pregnant women with active autoimmune thrombocytopenic purpura (AITP) or a history of AITP (group A) and 21 pregnant women with isolated thrombocytopenia and identification of specific platelet autoantibodies detected by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay (group B) were evaluated for platelet-associated immunoglobulin-G (PAIgG), MAIPA assay and HLA genotype. Neonatal platelet counts were performed at least three times in the first week. 11 neonates were thrombocytopenic (23.9%). No severe haemorrhage occurred. There were no significant differences regarding the values of PAIgG or positive MAIPA tests between mothers of thrombocytopenic or healthy newborns. A significant difference, however, regarding the HLA DRB3* allele was found, with a high incidence in the subgroup of mothers of healthy newborns (P = 0.005). A similar trend was found among mothers with anti-GPIIbIIIa antibodies (P = 0.06). In contrast, HLA DRB5* allele appeared to be present especially in mothers of thrombocytopenic newborns (not significant). Our data suggest that mothers with AITP who have the HLA DRB3* genotype are unlikely to give birth to a thrombocytopenic baby. This study provides a preliminary report on a noninvasive test to identify infants who are likely to be affected.

Assay of antigens of antiplatelet antibodies in patients with various thrombocytopenias

Sandwich enzyme-linked immunosorbent assay was developed for evaluation of target antigens reacting with antiplatelet auto- and alloantibodies. The method based on specific immobilization of platelet antigens via monoclonal antibodies was applied to identify antigens of platelet-associated and serum autoantibodies in patients with different thrombocytopenias and to determine platelet HPA-1 alloantigens.

Severe Neonatal Alloimmune Thrombocytopenia due to Anti‐HPA‐3a

Abstract Background and Objectives: Neonatal alloimmune thrombocytopenia is usually attributable to HPA‐la antibodies. We report a case of parental platelet antigen incompatibility associated with a severe neonatal thrombocytopenia secondary to alloimmunization to HPA‐3a. Materials and Methods: Platelet antibodies were detected by the monoclonal antibody‐specific immobilization of platelet antigens, genotyping of the platelets by PCR, and HLA typing by serologic procedures and PCR. Results: Genotyping of maternal and paternal platelets confirmed the incompatibility in the HPA‐3a system. It is noteworthy that the mother is of the HLA type DRB3*0101, is ABO‐incompatible with her husband, and also has HLA class I antibodies. Conclusion: Severe neonatal thrombocytopenia associated with HPA‐3a alloimmunization is infrequent and all the factors mentioned above could have played a role in the severity of the disease.

Incidence of anti-mouse antibodies in thrombocytopenic patients with autoimmune disorders

Idiopathic thrombotycopenic purpura (ITP) is an autoimmune disorder in which circulating autoantibodies react with target antigens on the platelet membrane. In order to identify the autoimmune response in ITP, two MAIPA (Monoclonal Antibody (MAb) Immobilization of Platelet Antigen) assays (MAIPA I and MAIPA II) were performed on sera from thrombocytopenic patients. In the classic MAIPA assay (MAIPA I), control platelets were incubated simultaneously with human serum and a mouse MAb to a platelet glycoprotein. In MAIPA II, the control platelets were incubated first with the human serum and then, after washing, with the selected mouse MAb. A positive MAIPA I test but a negative MAIPA II has been shown to result from the presence of serum antibodies recognizing mouse MAb to platelet glycoproteins used in the assay. We compared the frequency of such 'anti-mouse' antibodies in patients with thrombocytopenia associated or not with other autoimmune states and in healthy donors with a normal platelet count. Statistically significant differences were found in the incidence of anti-mouse antibodies between patients and healthy donors. Furthermore, the identity of the targeted mouse MAbs varied in sera from the patients. The detected anti-mouse antibodies may include anti-idiotypic antibodies produced against cross-reactive idiotypes shared by human and mouse anti-platelet antibodies.

Autoantibodies and anti-mouse antibodies in thrombocytopenic patients as assessed by different MAIPA assays.

Two MAIPA (monoclonal antibody [MAb] immobilization of platelet antigen) assays were performed to determine (a) autoantibodies to platelet glycoproteins (GP) and (b) serum antibodies recognizing mouse MAbs used in the assay. In MAIPA I, control platelets were incubated simultaneously with human serum and a mouse MAb to a platelet glycoprotein (GP IIb-IIIa, Ib-IX, Ia-IIa, IV and p24). In MAIPA II, the control platelets were incubated first with the human serum and then, after washing, with the selected mouse MAb. A series of 25 patients with autoimmune thrombocytopenic purpura (ATP) associated or not with other autoimmune states were examined. Autoantibodies (both MAIPA I and MAIPA II positive) or anti-mouse Abs (MAIPA I positive and MAIPA II negative) were frequent in both groups of patients. Statistically significant differences existed in the incidence of anti-mouse Abs between patients (56.5%) and healthy donors (10%). This suggests that their production may be related to thrombocytopenias associated with autoimmune disease. We speculate that the presence of anti-mouse antibodies could reflect an abnormality in the immunological modulation of the idiotypic network.

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.

A Patient with Autoimmune Thrombocytopenic Purpura Demonstrating Serum Antibodies Reactive with Mouse Cross-Reactive Idiotypes

Autoimmune thrombocytopenic purpura (ATP) is a syndrome of destructive thrombocytopenia due to platelet-binding antibodies. We report the case of a young woman (C.V.) who has a history of chronic ATP with severe but transient bouts of thrombocytopenia. Using the classic monoclonal antibody (MAb) immobilization of platelet antigens (MAIPA) assay to screen serum antibody specificity, results were strongly positive with MAbs to glycoproteins (GPs) Ib-IX, Ia-IIa, IV, and p24, but weakly positive or negative for GP IIb-IIIa. In contrast, a two-step incubation assay (MAIPA II), in which platelets were incubated sequentially with C.V. serum and the murine MAb, gave negative results for all GPs. Affinity chromatography performed using Bx-1, a MAb to GP Ib, showed that the patient's serum contained antibodies to determinants expressed by mouse immunoglobulins. These were present on Fab fragments of Bx-1. A survey of sera from other patients with thrombocytopenia of immune origin revealed that antibodies reactive with selected idiotypes of mouse MAbs were not infrequent and raises the question of their role in the thrombocytopenia.

Neonatal Alloimmune Thrombocytopenia in Taiwan Due to an Antibody against a Labile Component of HPA-3a (Bak^a)

We report on two siblings who developed severe neonatal alloimmune thrombocytopenia (NAIT) due to an alloantibody against a labile component or components of the HPA-3a (Bak^a) antigen. The antibody reacted only with fresh, unfixed platelets by the solid-phase red cell adherence test, immunofluorescence test and mixed passive haemagglutination test. In the latter method, weakly fixed platelets also gave a weak positive reaction. Monoclonal-antibody-specific immobilization of platelet antigens and immunoblotting tests gave negative results. Our findings may possibly help to explain why in some cases of NAIT no platelet-specific antibody is demonstrable in tests with fixed or solubilized platelets.

Genotypisierung von HPA-1 bis HPA-5 mit der allelspezifischen Ligasekettenreaktion nach Amplifikation von genomischer DNA in einer Multiplex-PCR

Platelet-specific antibodies are involved in refractoriness to platelet transfusions, neonatal alloimmune thrombocytopenia, and posttransfusion purpura. Binding to different epitopes on platelet glycoproteins, human platelet antigens (HPA), they can cause severe thrombocytopenia. The polymorphic DNA regions of the best known HPA systems HPA-1 to HPA-5 were amplified from genomic DNA in a multiplex polymerase chain reaction (PCR). An automatable ligation-based assay (LCR) using allele-specific oligonucleotide probes was developed for rapid genotyping. The results were correlated with the well established allele-specific restriction enzyme assay (ASRA), and the phenotype was determined by the monoclonal antibody-specific immobilization of platelet antigens (MAIPA). We found a 100% concordance of genotypes between the HPA-LCR in 54 cases of NAIT, PTP and normal controls with the established methods. The gene frequencies calculated from 216 German and 55 Japanese random blood donors were similar to those determined by other time-consuming typing methods in a Caucasian and Japanese population, respectively. Multiplex PCR and the allele-specific HPA ligation assay provide a reliable tool for typing both small series and all platelet donors of a transfusion center in order to identify rare genotypes necessary for the treatment of patients suffering from NAIT, PTP or refractoriness to platelet transfusions.

Human platelet alloantigen typing: PCR analysis is not a substitute for serological methods.

Currently, five platelet alloantigen (alloAg) systems have been established (HPA-1, -2, -3, -4, -5). Three of these are expressed on the glycoprotein (GP) IIb-IIIa complex, HPA-1, HPA-3 and HPA-4, inherited in an autosomal codominant mode. Recent investigations of the molecular basis of these platelet alloantigen systems have shown that only one nucleic acid base substitution in the genes encoding for GP IIb and GP IIIa is responsible for the polymorphism. This substitution is reflected in a difference in restriction enzyme recognition allowing platelet alloantigen typing by restriction fragment length polymorphism (RFLP) analysis of DNA amplified by the polymerase chain reaction (PCR). To validate the PCR technology for platelet typing, we have compared PCR-RFLP with monoclonal-antibody-specific immobilization of platelet antigens (MAIPA). For this purpose, we have studied different Glanzmann thrombasthenic families and particularly heterozygous individuals, who are not lacking GP IIb-IIIa, as a model to detect the occurrence of discrepancies between these two technologies. In two families, we have found differences between molecular biology and serological methods with the lack of expression of one antigen on the platelet membrane surface. In the first family, the abnormality is related to the HPA-1 alloantigen system with three informative members; in the second, the HPA-3 alloantigen system is concerned with two informative members. Considering these results, there may not always be a perfect correlation between molecular biology and serological methods, as an unknown molecular defect could interfere with the PCR results and lead to false platelet typing.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the Br Polymorphism on a 144 bp Exon of the GPIa Gene and Its Application in Platelet DNA Typing

Alloimmunization against the human platelet alloantigen system Br (HPA-5) is the second most common cause of neonatal alloimmune thrombocytopenia (NAIT) in Caucasian populations. We have recently shown that a single base polymorphism at position 1648 on platelet mRNA coding for GPIa results in an aminoacid substitution at position 505 on the mature GPIa which is associated with the two serological defined Br phenotypes. Since DNA-typing of platelet alloantigens offers possibilities for useful clinical applications, we designed genomic DNA-based restriction fragment length polymorphism (RFLP) typing for Br alloantigens. To establish this technique we analyzed the genomic organization of GPIa adjacent to the polymorphic base. Using the polymerase chain reaction (PCR) of blood cell DNA we have identified two introns (approximately 1.7 and 1.9 kb) flanking a 144 bp coding sequence of the GPIa gene encompassing the polymorphic base 1648. Based on the intron sequence, a PCR primer was constructed to amplify a 274 bp fragment which was used for allele-specific RFLP to determine the Br genotypes. The results of RFLP analysis using MnlI endonuclease obtained from 15 donors (2 Bra/a, 2 Bra/b and 11 Brb/b) correlate perfectly with serological typing by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay.

Brb, a Platelet Alloantigen Involved in Neonatal Alloimmune Thrombocytopenia

Serum from a pregnant woman with the May-Hegglin anomaly contained a platelet-specific antibody. The serum reacted in the platelet indirect immunofluorescence test (PIIFT) with 97.6% of random donor platelets and those of the father but not with the mother's own platelets. This antibody induced a moderate thrombocytopenia in the infant that responded to infusion of intravenous immunoglobulin concentrates. The platelet phenotypes were PLA1+, Baka+, Bra+/Brb- for the mother, PLA1+, Baka+, Bra-/Brb+ for the father, and PLA1+, Bra+/Brb+ for the neonate. Analysis of the maternal serum with an immunoassay based on monoclonal antibody immobilization of platelet antigens (MAIPA) and immunoprecipitation techniques demonstrated the absence of antibodies directed against HLA class I antigens and that the antigen recognized was located on the platelet-GpIa/IIa complex. This antigen was present on 113/115 random donor platelets, in 7 of the 7 unrelated May-Hegglin platelets, and only absent in 3/24 Bra+ individuals, including the mother. No platelet-specific antibody was present in the serum of the 7 unrelated May-Hegglin subjects. The antigen recognized by this platelet-specific antibody thus meets the criteria defining the antithetic allele of Bra, i.e. the Brb alloantigen.