Rapid detection of HPA-1 alloantibodies by platelet antigens immobilized onto microbeads (original) (raw)
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Vox Sanguinis, 1995
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 (Baka) 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.
Transfusion, 2009
BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is mostly caused by maternal alloantibodies directed against the human platelet alloantigen (HPA)-1a. Currently, the serologic diagnosis of FNAIT is based on the characterization of the HPA alloantibodies in monoclonal antibody-based antigencapture assays (e.g., MAIPA assay). Accumulated current evidence indicated that such assays may overlook some HPA-1a antibodies. STUDY DESIGN AND METHODS: This study employed surface plasmon resonance (SPR) technology using immunoaffinity-purified glycoprotein IIb/IIIa isoforms immobilized on biosensor chips to study the binding kinetics of HPA-1a alloantibodies from different FNAIT cases in real time. RESULTS: Analysis of HPA-1a alloantibodies from FNAIT cases (n = 9) in SPR showed a moderate relative response (22.2-69.7 resonance units [RU]) and slow antibody dissociation. After the dissociation phase, varying amounts of bound antibodies (41%-79%) remained on the chip. In contrast in HPA-1a alloantibodies from a patient suffering from posttransfusion purpura, a high relative response (~490 RU) was observed at the end of the association phase and no dissociation of antibody binding was detectable. Of particular relevance, by the use of this SPR technique, HPA-1a alloantibodies were detected in two severe FNAIT cases that had determined as false negative by MAIPA assay. In SPR, these HPA-1a alloantibodies showed low-avidity nature characterized by gradual dissociation of antibody during the association phase and complete detachment of antibody binding after the dissociation phase. This high "off-rate" character of lowavidity HPA-1a alloantibodies indicates that such antibody binding is easily detachable by the extensive washing procedure of the MAIPA. CONCLUSIONS: Our results demonstrated that the SPR method can facilitate the diagnosis of clinically relevant low-avidity HPA-1a antibodies. F etal and neonatal alloimmune thrombocytopenia (FNAIT) is induced by maternal immunization against a fetal human platelet alloantigen (HPA). 1 In Caucasians, FNAIT has an incidence of 1:1000 to 1:2000 births. 2,3 Approximately 75 percent of serologically verified cases of alloantibodies against HPA-1a are responsible for the disease. 4 Antibodies against HPA-1a react with the Leu33 but not Pro33 isoform of platelet (PLT) glycoprotein (GP)IIIa or integrin b3 subunit. 5 Recently, the three-dimensional structure of the b3 integrin has been elucidated; 6,7 however, the precise binding site(s) of HPA-1a alloantibodies is not known. A previous study suggested that the amino terminal PSI domain containing the polymorphic residue 33 is sufficient to form HPA-1a epitopes. 8 However, various studies have shown that other residues located in the hybrid and epidermal growth factor domains of the b3 ABBREVIATIONS: D-PBS = Dulbecco's phosphate-buffered saline; FNAIT = fetal and neonatal alloimmune thrombocytopenia; GP(s) = glycoprotein(s); MAIPA assay = monoclonal antibody-based antigen-capture assay; PAIFT = platelet adhesion immunofluorescence test; PTP = posttransfusion purpura; RU = resonance units; SPR = surface plasmon resonance.
Vox Sanguinis, 2005
Background and Objectives Serological evaluation of maternal sera for platelet antibodies in suspected fetal/neonatal alloimmune thrombocytopenia (FNAITP) discloses in only ≈ 30% of individuals a platelet-specific antibody. Transfusion-induced alloimmunization against human platelet antigen-15 (HPA-15) has been reported to be about as common as against HPA-5, the second most common platelet antibody. Thus, anti-HPA-15 may also contribute significantly to yet-unclear cases of FNAITP. Materials and Methods In this retrospective analysis, we provide data on maternal platelet antibodies from 309 mothers who delivered an offspring with suspected FNAITP. Results Genotyping maternal and paternal samples (together n = 573) revealed a gene frequency of 0•496 for HPA-15a and a gene frequency of 0•504 for HPA-15b. HPA-15 antibodies were detected in 2% of all samples. Anti-HPA-15a and-15b were detected in two and three samples, respectively. One serum reacted equally with HPA-15a and-15b platelets. The most frequent platelet-specific antibodies were anti-HPA-1a (22%), but anti-HPA-5b (8•4%) were more frequent than anti-HPA-15. In addition, panreactive (5•5%) or autoreactive (5•2%) anti-GPIIb /IIIa or anti-GPIb /IX were detectable in maternal samples. Conclusions These data indicate that HPA-15 alloimmunization needs only to be considered in subjects with suspected FNAITP if no other platelet-specific antibody is detectable. The presence of panreactive or autoreactive antibodies should also be considered in neonatal thrombocytopenia.
British Journal of Haematology, 1994
We analysed the titre and isotype composition of antibodies produced by mothers giving birth to babies with or without neonatal alloimmune thrombocytopenic purpura (NAITP) and patients with post-transfusion purpura (PTP). All these individuals produced an antibody specific for the HPA-1a allotype present on the platelet glycoprotein IIb-IIIa (GPIIb-IIIa). Sera from mothers who gave birth to thrombocytopenic babies (group 1, n = 36), non-thrombo-cytopenic babies (group 2, n = 4) or from PTP patients (group 3, n = 3) were tested by an indirect-ELISA. Results indicated no evident differences in the isotype composition or titre of the antibodies from the three groups of sera. The antibody titre ranged from 1: 120 to 1: 3500. Antibodies with the IgGl subclass were present in all sera. Most sera contained IgGl alone (24/43 sera tested) or in combination with IgG3 (10/43). IgG2 was never present and only three sera showed intermediate reactivity with anti-IgG4 MAb. Few sera (nine sera from groups 1 and 2) were weakly positive when tested with the anti-IgM antibodies. These results suggest that neither the titre nor the isotype composition can be used to predict the severity or the occurrence of thrombocytopenia in newborns.
Thrombosis Research, 1998
is a relatively rare but important cause of severe neonatal thrombocytopenia. Its inci-The monoclonal antibody-specific immobilization dence is about 1:1000-1:2000 births [1]. In Caucasian of platelet antigens (MAIPA) assay is considered population, platelet antigens the most frequently as a possible reference method for the detection involved are HPA-1a, HPA-5b, and HPA-3a [2-6]. of maternal alloantibodies when a foetomaternal Since detection of alloantibodies is very important alloimmunization is suspected. However, this method for the diagnosis and treatment of the newborn, is tedious. In this study, we have compared the the diagnosis has to be done quickly. Immunobead MAIPA results of 54 samples of mothers with assay [7] and monoclonal antibody-specific immobi-(nϭ34) or without (nϭ20) alloantibodies with those lization of platelet antigens (MAIPA) assay [8] are obtained with a new antigen capture ELISA. Using considered as possible reference methods. However, the cutoff of 2.0 given by the manufacturer, the new these assays are time-consuming, not adapted for kit had a sensitivity of 88.2% (95% CI 72.6-96.7) routine use, and not easily available. In recent years, and a specificity of 100% (95% CI 98.0-100). From a other sensitive and specific tests to detect platelet receiver-operating characteristic curve analysis, the antibodies have been developed, but they are still more appropriate cutoff for a screening assay would rather tedious [9-13]. A new commercial test, albe 1.6, which gives a sensitivity of 100% (95% CI ready used by some laboratories, has been recently 89.7-100) with a specificity of 94.0% (95% CI 89.4proposed but, to the best of our knowledge, no 96.9). In conclusion, this new simple assay appears extensive comparisons between this new test and promising and could be used, with the modified other reference methods have been published. The cutoff, as a screening assay for the detection of plateaim of our study was to compare the performances of let alloantibodies.
Transfusion, 2011
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disorder caused by transplacental passage of maternal antibodies to fetuses whose platelets (PLTs) express the corresponding human PLT antigen (HPA). We observed a fetus with FNAIT who died from a severe intracranial hemorrhage. Analysis of maternal serum in antigen capture assay with paternal PLTs showed reactivity with PLT glycoprotein (GP)IIb/IIIa (α(IIb) β(3) ) and GPIa/IIa (α(2) β(1) integrin), indicating the presence of anti-HPA-1a and an additional alloantibody against GPIa (termed anti-Swi(a) ). By immunochemical studies, the localization of the Swi(a) antigen on GPIa/IIa could be confirmed. Analysis of paternal GPIa full-length cDNA showed a single-nucleotide substitution C(3347) T in Exon 28 resulting in a Thr(1087) Met amino acid substitution. Testing of family members by polymerase chain reaction-restriction fragment length polymorphism using MslI endonuclease showed perfect correlation with phenotyping...
…, 2008
Background Neonatal alloimmune thrombocytopenia is most commonly due to transplacental passage of maternal anti-HPA 1a antibodies. A prospective study was carried out to evaluate the pattern and quantity of maternal anti-HPA 1a antibodies in order to predict the level of thrombocytopenia in the neonates. Design and Methods A monoclonal antibody immobilization of platelet antigen assay was used to detect antibodies in maternal samples from 1,990 HPA 1bb women. HLA DRB3*0101 typing was performed in all immunized women by sequencing the HLA DRB3 gene when present. Results Primary immunization more often took place in connection with delivery than during the first pregnancy. There was a strong correlation between maternal antibody levels and the platelet counts in the newborn (R 2 =0.49, p<0.001). A maternal antibody level above 3.0 IU/mL measured in gestational week 22 or 34 had a diagnostic sensitivity and specificity of 93% and 63%, respectively, for predicting the grade of neonatal thrombocytopenia. The women who were negative for HLA DRB3*0101 had significantly lower anti-HPA 1a antibody levels than those who were HLA DRB3*0101 positive (p<0.007). In contrast to primigravida, in whom anti-HPA 1a antibody levels increased during pregnancy, the antibody level decreased in 92 of 147 women who had been pregnant previously (P92 or more of 147 = 0.003). The anti-HPA 1a antibody level regularly increased after delivery. Conclusions Maternal anti-HPA 1a antibody levels in weeks 22 and 34 of pregnancy are good predictors of the degree of thrombocytopenia in the newborn both in the first and subsequent pregnancies. Most mothers became immunized at the time of delivery.