A novel assay for the detection of anti-human platelet antigen antibodies (HPA-1a) based on peptide aptamer technology

Background

Neonatal alloimmune thrombocytopenia is mostly due to the presence of maternal antibodies against the fetal platelet antigen HPA-1a on the platelet integrin GPIIb-IIIa. Accurate detection of anti-HPA-1a antibodies in the mother is, therefore, critical. Current diagnostic assays rely on the availability of pools of human platelets that vary according to donors and blood centers. There is still no satisfactory standardization of these assays.

Design and Methods

Peptide aptamer was used to detect and identify HPA-1a-specific antibodies in human serum that do not require human platelets. A peptide aptamer library was screened using an anti-HPA-1a human monoclonal antibody as a bait to isolate an aptamer that mimics the human platelet antigen HPA-1a.

Results

This is the first report in platelet immunology of the use of a peptide aptamer for diagnostic purposes. This assay gives better results than the MAIPA currently in use, detecting around 90% of the expected alloantibodies.

Conclusions

This assay could help define a standard for the quantitation of anti-HPA antibodies. This report also demonstrates that peptide aptamers can potentially detect a variety of biomarkers in body fluids; this is of particular interest for diagnostic purposes.     

One-tube method for complete HPA-1 genotyping by PCR using sequence-specific primers

Human platelet antigens (HPA) can be targets for antibody responses that cause life-threatening thrombocytopenia following platelet transfusions or pregnancy. As an aid to diagnosis and prevention, serologic and DNA-based methods have been developed to type HPA. Of the DNA-based strategies, those using the polymerase chain reaction (PCR) are very sensitive, but often require processing of amplification products. Sequence-specific primers (SSP) in the PCR eliminate the need for extensive handling of reaction products beyond gel electrophoresis. However, current methods require a separate reaction for each allele being typed. In this report we describe a method to simultaneously and completely genotype both alleles of HPA-1 in a single PCR. In addition, because the absence of an amplification product might also show the failure of a SSP, we introduced a recombinant template that can only be amplified by the SSP, thus ensuring primer performance and the identified genotype.     

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.     

Frequencies of maternal platelet alloantibodies and autoantibodies in suspected fetal/neonatal alloimmune thrombocytopenia, with emphasis on human platelet antigen-15 alloimmunization

BACKGROUND AND OBJECTIVES: Serological evaluation of maternal sera for platelet antibodies in suspected fetal/neonatal alloimmune thrombocytopenia (FNAITP) discloses in only approximately 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.     

Predicting risk severity and response of fetal neonatal alloimmune thrombocytopenia

Fetal neonatal alloimmune thrombocytopenia (FNAIT) is a devastating bleeding disorder in the fetus or neonate caused by transplacental transport of maternal alloantibodies to paternal‐derived antigen on fetal platelets. In Caucasians, up to 80% of FNAIT cases result from maternal immunization to human platelet antigen (HPA)‐1a. New methods have developed facilitating detection of common and private antibodies against HPAs triggering FNAIT. Understanding the pathogenesis of FNAIT made it possible to develop a novel strategy to treat this disorder. To date, recombinant monoclonal antibodies directed against the β3 integrin and Fc receptors have been tested in a mouse model of FNAIT, and seem to be promising. Whether those novel treatments will eventually replace the conventional high dose immunoglobulin G in women with FNAIT is yet unknown.     

Interlaboratory variation in the detection of clinically significant alloantibodies against human platelet alloantigens

This report describes the results of eight workshop exercises which were designed to test the proficiency of laboratories in the detection of antibodies to human platelet antigens (HPA). Detection of the most clinically significant alloantibody, anti-HPA-1a, is adequate. However, despite improvements in consistency of test results between laboratories over the last 3 years, there is still a high probability that clinically significant antibodies against other HPA alloantigens will not be detected.     

European collaborative study of the antenatal management of feto-maternal alloimmune thrombocytopenia

The aims of this study were to determine whether the severity of fetomaternal alloimmune thrombocytopenia (FMAIT) in the current pregnancy could be predicted from the history of FMAIT in previous pregnancies, and to assess the effects of different types of antenatal intervention. Fifty-six fetuses were studied that all had a sibling affected by FMAIT due to human platelet antigen 1a (HPA-1a) alloimmunization. Cases with a sibling history of antenatal intracranial haemorrhage (ICH) or severe thrombocytopenia (platelet counts of < 20 x 109/l) had significantly lower pretreatment platelet counts than cases whose siblings had less severe thrombocytopenia or postnatal ICH. Maternal therapy resulted in a platelet count exceeding 50 x 109/l in 67% of cases. None of the fetuses managed by serial platelet intrauterine transfusions (IUT) suffered ICH following treatment. However, several serious complications arose with fetal blood sampling (FBS). Overall, intervention improved outcome, as three study cases suffered from antenatal ICH and three others died whereas 15 study cases had a sibling with an ICH, eight of whom died. The results of this study suggest that the start of therapy can be stratified on the basis of the sibling history of FMAIT, and support the use of maternal therapy as first-line treatment.     

Genetic typing of human platelet antigen 1 (HPA-1) by oligonucleotide ligation assay in a specific and reliable semi-automated system

Genotyping of platelet alloantigens with the possibility of using any type of cellular material as a source of DNA has become a preferred procedure, particularly in thrombocytopenic patients when platelet counts are too low for phenotyping. Recently human platelet antigen 1 (HPA-1) has been identified as an inherited risk factor for coronary thrombosis. The different detection methods currently used have disadvantages for large-scale DNA diagnosis, including the need for electrophoresis (allele-specific restriction enzyme analysis, amplification with sequence-specific primers) or the potential risk of reduced specificity (allele-specific oligonucleotide hybridization). In this report we describe the adaptation of an automated oligonucleotide ligation assay to genotype HPA-1 in polymerase chain reaction (PCR)-amplified DNA samples. HPA-1a and HPA-1b phenotypes corresponded to the results of the different genotyping assays. The genotypes determined with the ELISA-based PCR-oligonucleotide ligation assay were in 100% concordance with the results obtained by conventional allele-specific restriction enzyme site analysis and PCR amplification with sequence-specific primers. The automated oligonucleotide ligation assay provides a rapid, reliable, nonisotopic method to genotype human platelet antigens that can rapidly be applied to large population screening.     

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

Introduction: 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. Methods: 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. Results: 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. Conclusion: Anti‐HPA‐15 antibody detection should be based on carefully selected platelets with high HPA‐15 expression level.     

A simplified [3H] serotonin release assay for the detection of platelet antibodies.

The [3H] platelet serotonin release assay is a sensitive means for detecting antiplatelet antibodies. We have found that by using prelabeled frozen platelets which have been treated with the cryoprotective agent dimethyl sulfoxide (DMSO) the performance of this assay can be facilitated without interfering with its sensitivity. There was 100% correlation between the standard [3H] platelet serotonin release assay using fresh platelets and the modified assay using prelabeled, frozen, cryopreserved platelets when sera from 12 patients with known antiplatelet antibodies were tested. When normal serum samples (n = 111) were tested against ten platelet donors, a false-positive rate of 3.9% was observed. This modification provides a simple means for quickly screening large numbers of potential platelet donors at one time.     

Evidence of heterogeneity in the antibody response against the platelet antigen 3a; recognition of an 11-mer peptide carrying the HPA-3a polymorphic determinant

Background  The immune processes involved in the development of alloantibodies against the human platelet antigens in alloimmune disorders remain unclear. Antibody recognition of the platelet antigens on their respective platelet glycoproteins has been shown to be dependent on glycoprotein conformation. Furthermore, the post-translational modification of glycoproteins adds complexity to the alloantigenic determinants.
Methods  Nine anti-HPA-3a sera along with several control sera were tested for reactivity to an 11-mer peptide straddling the HPA-3a/b polymorphism. Sera found to specifically recognize the 3a peptide were further assessed by platelet pre-exposure and immunoblotting.
Results  Three of the nine antisera were found to specifically recognize an 11-mer synthetic 3a peptide by ELISA. Further analysis of all anti-HPA-3a sera by Western blot showed that only those reactive to the 3a peptide were able to bind both reduced and non-reduced GPIIb.
Conclusion  The results presented in this study provide the first known evidence for the identification of an antibody population capable of recognizing a linear and non-glycosylated form of the HPA-3a epitope.     

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.     

Idiopathic autoimmune thrombocytopenia: evidence for redistribution of platelet antibodies into the circulation after immunoadsorption treatment

Platelet antibodies are detectable in only about 50% of patients with chronic autoimmune thrombocytopenia (AITP). We determined platelet antibodies against GPIa/IIa, GPIb/IX, GPIIb/IIIa, and GPV and reticulated platelets in three female patients with AITP, before and after immunoadsorption treatment. None of the three patients' sera contained platelet antibodies prior to treatment. Thereafter, anti-GPIIb/IIIa, anti-GPIb/IX (n = 3), and anti-GPV (n = 1) were detectable in the patients' sera. These antibody specificities were also found in the eluates from the immunoadsorption columns. Only one patient had elevated levels of reticulated platelets. Immunoadsorption treatment did not induce a sustained increase of platelet counts in any patient. Immunoadsorption treatment in AITP can induce redistribution of antibodies into the circulation.     

Low incidence of specific anti-platelet antibodies detected by the MAIPA assay in the serum of thrombocytopenic MDS patients and lack of correlation between platelet autoantibodies, platelet lifespan and response to danazol therapy

We prospectively studied the mechanism of thrombocytopenia in 30 patients with myelodysplastic syndrome (MDS), who had a platelet count ≤70×10⁹/l , and marrow blasts ≤10% by analysis of platelet lifespan, platelet-bound IgG antibodies by platelet radioactive antiglobulin test (PRAT), and circulating and platelet-bound autoantibodies specifically directed against platelet glycoproteins by indirect and direct MAIPA test.   PRAT analysis was positive in 21/30 patients (70%), but antiplatelet antibodies were found in only eight by the MAIPA test. Platelet lifespan was shortened in five cases, only one of whom had a positive MAIPA test. Patients were treated by danazol (600 mg/d), and 9/26 evaluable patients (35%) responded, including three of the five patients with shortened platelet lifespan.   No correlation between platelet lifespan, results of PRAT and MAIPA tests, and response to danazol was found.   In conclusion, specific antiplatelet autoantibodies, reduced platelet lifespan, and efficacy of androgen therapy on platelets were seen in 20%, 17% and 35% of MDS with thrombocytopenia, respectively, but no correlation between these findings was found in this study.     

Antibodies against platelet glycoproteins and antiphospholipid antibodies in autoimmune thrombocytopenia

Abstract: Autoantibodies against platelet glycoproteins (anti-GP) are found in the majority of patients with autoimmune thrombocytopenia (AITP) as well as in thrombocytopenia associated with systemic lupus erythematosus (SLE). Some of these patients may have anti-phospholipid antibodies (anti-PL). To evaluate the pathogenetic significance of anti-PL and anti-GP antibodies in AITP and SLE patients, we investigated anti-cardiolipin (anti-CL), anti-phosphatidylserine (anti-PS) and anti-GP antibodies (anti-GPIIb-IIIa and anti-GPIb-IX) in 71 patients with AITP and 3 thrombocytopenic patients with SLE. Anti-GP antibodies were detected in 52 (70%) patients. Fifty-six (73%) patients showed anti-PL antibodies. Seven patients (6 AITP, 1 SLE) with both anti-GPIIb-IIIa and IgG anti-PL antibodies were followed during treatment with corticosteroids. Antibodies were measured before treatment and at the time of platelet-peak. Anti-GPIIb-IIIa antibodies decreased in all or became undetectable in five. In contrast, IgG anti-PS and IgG anti-CL antibodies decreased only moderately or remained positive. Adsorption experiments, using gelfiltered platelets, erythrocyte (Ec)-inside-out-vesicles and purified GPIIb-IIIa, showed that anti-GP and anti-PL antibodies have distinct specificities and do not crossreact. We conclude that anti-PL and anti-GP antibodies may be present simultaneously in some patients with immune mediated thrombocytopenia. Although anti-PS as well as anti-CL antibodies may be responsible for thrombocytopenia, we speculate that anti-GPIIb-IIIa antibodies are more related to the severity of thrombocytopenia.     

Provision of platelet support for fetuses and neonates affected by severe fetomaternal alloimmune thrombocytopenia

Severe fetomaternal alloimmune thrombocytopenia requires urgent treatment with compatible platelet concentrates. As prompt treatment is sometimes delayed owing to the unavailability of compatible platelets, we established an accredited platelet donor panel to provide effective and timely transfusion support for fetal and neonatal therapy. After a mass screening programme of over 60,000 blood donations, 45 HPA-1a-negative donors with no antibodies to HPA, HLA, red cell antigens and granulocytes/lymphocytes, and with low titre anti-A and/or -B were accredited. All accredited donors were fully genotyped for HPA-1, -2, -3 and -5 by PCR-SSP. Ninety-one per cent of the accredited donors were also negative for HPA-5b.