A female baby with a severe thrombocytopenia at 18 × 10⁹/l was born to a 29-year-old (gestation 2/partum 2) mother. Scattered petechiae were present on her legs, arms, chest and face, but there was no bleeding, infection, fever or hepatosplenomegaly. A platelet antibody screening immunocapture test was positive, which was performed on the mother’s serum 3, 12 and 38 days after delivery, but no platelet-specific antibodies were found by the monoclonal-antibody-specific immobilization of platelet antigen assay. The baby’s platelets and lymphocytes and the father’s platelets reacted strongly with the HLA antibodies present in the mother’s serum. The neonate was treated with intravenous human immunoglobulin (Tegeline^®, 1 g/kg per day) 1, 2 and 3 days after delivery. The platelet count rose from 18 × 10⁹/l on day 0 to 37 × 10⁹/l on day 3 and to 227 × 10⁹/l on day 12. No platelet transfusion was needed. Several factors which developed hereafter lead us to think that this neonatal alloimmune thrombocytopenia is due to the transplacental passage of maternal HLA antibodies to the baby.

Protein microarray technology provides a useful approach for the simultaneous serodetection of various antibodies in low sample volumes. To implement functional protein microarrays, appropriate surface chemistry must be designed so that both the protein structure and the biological activity can be retained. In the current study, two surface chemistries for protein microarrays and immunofluorescent assays were developed. Glass slides were functionalized with N-hydroxysuccinimide (NHS) ester via a monofunctional silane or maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer to allow covalent grafting of histone proteins. Analytical performance of these microarrays was then evaluated for the detection of anti-histone autoantibodies present in the sera of patients suffering from a systemic autoimmune disease, namely systemic lupus erythematosus (SLE), and the results were compared with those of the classical enzyme-linked immunosorbent assay (ELISA) and Western blot. The detection limit of our MAMVE copolymer microarrays was 50-fold lower than that of the classical ELISA. Furthermore, 100-fold less volume of biological samples was required with these miniaturized immunoassays.

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.

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DNA microarrays are a powerful experimental tool for the detection of specific genomic sequences and are invaluable to a broad array of applications: clinical diagnosis, personalized medicine, drug research and development, gene therapy, food control technologies, and environmental sciences. Alloimmunization to human platelet antigens (HPAs) is commonly responsible for neonatal alloimmune thrombocytopenia, post-transfusional purpura and platelet transfusion refractoriness. Using DNA microarrays, we developed a diagnosis to type the biallelic HPA-1 platelet group. The region for the human genomic DNA sequence that contains the polymorphism responsible for HPA-1 alleles was amplified by polymerase chain reaction (PCR). The expected DNA fragments were hybridized on DNA microarrays, and the data were analyzed using specially developed software. Our initial results show that the two HPA-1 antigens polymorphisms containing a single base difference were detected using DNA microarrays.

BackgroundNeonatal 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 MethodsPeptide 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-HPA1a human monoclonal antibody as a bait to isolate an aptamer that mimics the human platelet antigen HPA-1a. ResultsThis 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. ConclusionsThis 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.Key words: peptide aptamers, neonatal alloimmune thrombocytopenia, platelets, HPA-1a, diagnostic assay, MAIPA. Haematologica 2012; 97(5):696-704. doi:10.3324/haematol.2011 A novel assay for the detection of anti-human platelet antigen antibodies (HPA-1a) based on peptide aptamer technology Citation: Thibaut J, Mérieux Y, Rigal D, and Gillet G. A novel assay for the detection of anti-human platelet antigen antibodies (HPA-1a) based on peptide aptamer technology.

Four children with acute lymphoblastic leukaemia had autologous bone marrow (BM) or peripheral stem cell (PSC) transplantation with low dose of cyclosporine (CsA, 1 mg/kg/d i.v. during the first 28 d) to induce an autologous GVHD (auto‐GVHD). Two children did not have clinical auto‐GVHD and they relapsed 3 and 4 months after treatment. The 2 other children had clinical signs of auto‐GVHD (grade I and grade II): they both are in complete remission but after a first normal haematological recovery they had a prolonged period of aplasia until month 9 for 1 patient and still persistent at month 7 in the other case. We studied lymphocyte subsets reconstitution after transplantation in these patients. All patients had an important decrease in the CD4/CD8 ratio related both to a strong decrease in the CD4+ cells and a strong increase in the CD8+ cells. Most of the CD8+ cells were of the CD8bright+CD28‐ phenotype. These CD8bright+CD28‐ T‐cells represented from 33% to 68% of the total lymphocytes. We discuss the role of these cells after autologous transplantation with CsA, and wonder if these cells could mediate cytotoxicity. In conclusion, among 4 children who received autologous BM or PBC transplantation with low dose of CsA, we observed a complete remission after an auto‐GVHD and a prolonged period of aplasia in 2 patients and a relapse of leukaemia in 2 other patients. All these 4 patients had an increase in the CD8bright+CD28‐ T lymphocytes.

The rHPA-1 bead assay is a rapid 3-hour assay for the sensitive detection of HPA-1 antibodies. Its ease of use would enable prompt detection of maternal HPA-1a antibodies in suspected FMAIT cases, which is important supportive evidence for treatment by transfusion with HPA-1b1b PLTs.