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Background: The human platelet alloantigens (HPA) HPA-1a and HPA-5b are located on glycoproteins on the platelet surface and are the most relevant to cause neonatal alloimmune thrombocytopenia (NAIT). The antigens are defined by single nucleotide polymorphisms (SNPs) in the glycoprotein genes, and the antigen status can be determined by genotyping the SNPs. However, genotyping is time-consuming and costly depending on the method and sample throughput. Here, we tested the reliability of the evanescence wave based fluorescence (EVA) biosensor technology for the rapid phenotyping of the HPA-1a and HPA-5b antigens on blood donor samples in two laboratories. Methods: HPA-1a and HPA-5b phenotyping was performed on EDTA blood samples from 336 blood donors (Lyon: 216 donors; Mannheim: 120 donors) using EVA typing assays and the biosensor system (Davos Diagnostics, Davos, Switzerland). For genotyping, validated PCR-SSP and TaqMan-PCR methods were used. Results: HPA-1a phenotyping was positive for all samples with HPA-1aa (n = 244; EVA value 807 ± 167 U/s) and HPA-1ab (n = 82; 542 ± 110 U/s) genotypes. All samples (n = 10) with negative EVA values (Conclusion: EVA is a reliable method for rapid phenotyping of the clinically relevant HPA-1a and HPA-5b platelet antigens. All phenotyping results were 100% concordant with the HPA-1 or HPA-5 genotype. The test can be performed from only 10 µl of fresh or frozen blood samples within less than 15 min time-to-result.

BACKGROUND Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by human platelet (PLT) antigen (HPA) incompatibility. Beads coupled with recombinant β3 integrins, displaying the biallelic HPA-1 epitopes (rHPA-1), have been shown to detect HPA-1a alloantibodies implicated in FMAIT. This report describes a multicenter validation of the beads using the results of well-characterized samples to define the optimum parameters for analysis of a large cohort of 498 clinical samples. STUDY DESIGN AND METHODS Fifty-one blinded quality assurance (QA) samples were tested by six laboratories to standardize the rHPA-1 bead assay and to develop an algorithm for sample classification. Five laboratories retrieved samples from 498 independent FMAIT cases, previously tested by the monoclonal antibody-specific immobilization of PLT antigens (MAIPA) assay, from their local archives for testing with the rHPA-1 beads. The results were evaluated using a mathematical algorithm developed to classify the samples. RESULTS The QA samples gave a mean concordance of 94% between the bead and MAIPA assays, while 97% concordance was observed with the FMAIT samples. Of the 15 discrepant samples, seven were positive by the beads but negative by MAIPA, while the contrary was observed for eight samples. Overall, the bead assay achieved 98% sensitivity for HPA-1a antibody detection in FMAIT and 98.7% specificity compared to the local MAIPA. CONCLUSION 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.

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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 CD8 brigh+ CD28 - phenotype. These CD8 bright+ 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 CD8 bright+ CD28 - T lymphocytes.

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.

Resume Lors de la recherche des anticorps (Acs) anti-plaquettes (PS), la mise en evidence d'Acs anti-HLA de classe I est possible. Ces Acs ont ete recherches avec le test de « Monoclonal antibody-specific immobilization of platelet antigen (MAIPA) a l'aide d'un Acs monoclonal anti-β2 microglobuline. En cas de resultat positif, le depistage des Acs anti-HLA a ete pratique avec un test specifique (ELISA) et la confirmation effectuee par lymphocytotoxicite. Sur 263 patients positifs avec l'Acs anti-β2 microglobuline, 223 (84,8 %) sont positifs au depistage des Acs anti-HLA et 159 (71,3 %) confirmes en lymphocytotoxicite. Parmi ces 159 echantillons, 6 ont une densite optique faible, situee entre 0,100 et 0,149 pour 3 et, entre 0,150 et 0,199, pour les 3 autres. Les 60 echantillons avec une densite optique superieure ou egale a 1,500 au MAIPA sont tous positifs au depistage des Acs anti-HLA et 54 (90 %) sont confirmes en lymphocytotoxicite. Parmi 50 sujets controles negatifs en MAIPA, 4 (8,0 %) ont des Acs anti-HLA en lymphocytotoxicite. En clinique, dans certaines pathologies, la detection d'Acs anti-HLA de classe I represente un element d'information utile pour le diagnostic et le traitement. En presence d'une densite optique meme faible (

Resume La recherche d'une immunisation anti-plaquettes est un examen de plus en plus demande au laboratoire. Une etude sur 701 prelevements depistes positifs pour les anticorps anti-plaquettes a ete effectuee. Au cours du traitement des echantillons, plusieurs problemes pratiques sont observes. Les anticorps anti-plaquettes ont ete depistes a l'aide d'un test d'immunocapture et identifies par le test « monoclonal antibody-specific immobilization of platelet antigens a (MAIPA). La recherche des anticorps anti-plaquettes fixes et circulants a ete realisee. Au depistage, sur 701 prelevements positifs en anticorps anti-plaquettes, 95 etaient incomplets en qualite et en quantite (13,6 %). Sur ces 95 echantillons, la presence d'anticorps anti-plaquettes specifiques a pu etre montree dans 28 cas (29,5 %). Sur les 606 prelevements restants, l'identification n'a pas ete possible dans 21 cas (3,5 %). Les 585 prelevements complets soumis a identification se sont reveles porteurs d'anticorps anti-plaquettes specifiques dans 192 cas (32,8 %) repartis en 84 cas (43,8 %) avec allo-anticorps et 108 cas (56,8 %) avec auto-anticorps. Parmi les allo-anticorps anti-plaquettes, c'est la specificite anti-HPA 5b qui est la plus frequente. Au sein des auto-anticorps, c'est la specificite anti-GP IIb IIIa qui est predominante. La quasi-absence des renseignements cliniques transmis avec la demande d'examen n'a pas permis de preciser l'origine possible de l'immunisation anti-plaquettes chez les patients. Cette etude montre que des progres doivent etre accomplis en pratique sur les prelevements et les informations cliniques lors de la recherche des anticorps anti-plaquettes, que la specificite des tests de depistage doit etre estimee et amelioree et que de nouveaux systemes plaquettaires doivent etre introduits dans les tests d'identification.

Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by human platelet (PLT) antigen (HPA) incompatibility. Beads coupled with recombinant β3 integrins, displaying the biallelic HPA-1 epitopes (rHPA-1), have been shown to detect HPA-1a alloantibodies implicated in FMAIT. This report describes a multicenter validation of the beads using the results of well-characterized samples to define the optimum parameters for analysis of a large cohort of 498 clinical samples.Fifty-one blinded quality assurance (QA) samples were tested by six laboratories to standardize the rHPA-1 bead assay and to develop an algorithm for sample classification. Five laboratories retrieved samples from 498 independent FMAIT cases, previously tested by the monoclonal antibody-specific immobilization of PLT antigens (MAIPA) assay, from their local archives for testing with the rHPA-1 beads. The results were evaluated using a mathematical algorithm developed to classify the samples.The QA samples gave a mean concordance of 94% between the bead and MAIPA assays, while 97% concordance was observed with the FMAIT samples. Of the 15 discrepant samples, seven were positive by the beads but negative by MAIPA, while the contrary was observed for eight samples. Overall, the bead assay achieved 98% sensitivity for HPA-1a antibody detection in FMAIT and 98.7% specificity compared to the local MAIPA.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.

Resume De nombreuses techniques sont disponibles pour le depistage et l’identification des anticorps (Acs) anti-plaquettes (PLT) specifiques, mais l’efficacite de certaines demeure controversee. Pour l’apprecier, deux methodes ont ete evaluees simultanement en depistage, l’immunocapture (IC) et le « monoclonal antibody-specific immobilization of platelet antigen » (MAIPA). Parmi 737 echantillons, 95 (12,9 %) ont ete trouves positifs en IC directe mais negatifs en MAIPA direct et 21 (2,8 %) positifs uniquement en MAIPA direct. Dix huit echantillons (2,4 %) etaient positifs dans les deux methodes. Cent huit echantillons (14,7 %) etaient detectes positifs en IC indirecte seule. En MAIPA indirect seul, 23 echantillons (3,1 %) etaient positifs. Ils comprenaient 1 anti-HPA-1a, 9 anti-HPA-5b, 3 anti-GP Ia IIa, 3 anti-GP IIb III, 4 anti-GP Ib IX et 3 associations (2 anti-GP Ia IIa/IIb IIIa ; 1 anti-GP IIb IIIa/Ib IX). Vingt deux echantillons (3,0 %) etaient positifs dans les deux methodes. Enfin, 36 echantillons etaient positifs avec l’Acs monoclonal anti-β2 microglobuline en MAIPA indirect mais pas en IC. La detection des allo-Acs anti-PLT specifiques, particulierement la specificite anti-HPA-5b, ne semble pas performante en IC et l’introduction de deux tests de depistage devrait etre preconisee.

Decidual large granular lymphocytes (DLGL) are the most abundant lymphoid cell type found in the first trimester maternal decidua. The function of DLGL remains controversial, although freshly isolated DLGL have been shown to exert a weak NK activity. We report here the phenotypic characterization of two DLGL subpopulations by immunofluorescence, using mAb against CD56, PEN5 as well as adhesion molecules potentially involved in cell-cell contact between DLGL and trophoblasts. DLGL are CD56bri9ht and express the CD2, CD11 a, CD18, CD38 and CD50 molecules, dimly the CD54 molecule, and poorly the CD102 and CD69 molecules. A strong expression of the polysialylated form of N-CAM (or CD56) was also observed on the surface of DLGL. Finally, 40% of CD56bright DLGL cells express the PEN5 epitope, which is selectively expressed on CD56dim cells NK in the peripheral blood. No other phenotypic difference was detected between the CD56br|9htPEN5+ and the CD56brightPEN5~ DLGL populations. Our results show that DLGL are heterogeneous and suggest that the CD56br|9htPEN5+ DLGL subset belongs to the classical NK cell lineage.

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.

An evanescent wave fluorescence biosensor was combined with a DNA bio-barcode assay to resolve problems met in detection of poor biologic samples. Human platelet antigen (HPA) genotyping was used as a demonstrator. Our bio-barcode assay was based on magnetic carboxylatex particles and non-magnetic carboxylatex particles, both functionalized with oligonucleotides. It was assessed for detecting 84mer synthetic oligonucleotides as targets. The assay allows to specifically detect single nucleotide polymorphism with a detection limit of 2 pM of target nucleic acids. The fluorescence detection is achieved in 150 s.

Protein microarray technology has shown great advancements in the field of biomedical research and diagnosis, it allows to study and understand protein activities and protein - ligand interactions (e.g. detection of antigen-autoantibody interaction in autoimmune diseases. Autoantibodies are frequently targeted against antigens of the cell nucleus (double and single stranded DNA, histones, and nuclear antigens). The biological activities of proteins (e.g. enzymes, antibodies...) are controlled by peptides sequences of the active site. Consequently, we were interested in the investigation of peptide microarrays in order to further implement in situ peptide synthesis, in particular, deprotection reaction on glass supported peptides. In this work, a protected and biotinylated synthetic peptide was covalently immobilized onto amino functionalized glass surface by activation of its the C-terminus; this allows to orientate the peptide onto the surface. The peptide contains a fragment of the C-terminal end of the human histone H3 protein. The immobilized peptide was then deprotected by using concentrated trifluoroacetic acid solution. After the deprotection, surface stability and peptide grafting density were evaluated by indirect labelling of the immobilized peptide using Cy3 streptavidin conjugates. We also studied biological interaction of IgG polyclonal anti-histone H3 antibody with the immobilized peptide epitope to insure the efficiency of the acid deprotection. The specificity of the antibody interaction with the protected versus non protected peptides. This approach may be applied to in situ synthetic and prototected peptides, in order to elaborate a micro-immunoassay prototype for measurement of peptide-protein interactions on high density microarrays, and detection of antibodies in biological fluids such as serum.

In 1988, a new platelet-specific alloantigen, HPA-5b (Bra) was first described in four cases of neonatal alloimmune thrombocytopenia (NAIT) [1], and a new platelet system, HPA-5a/b (Brb/Bra) charac...

To acquire some biological markers associated with the occurrence of acute graft-versus-host disease (aGVHD) after allogeneic bone marrow transplant (BMT) in children, we have studied the lymphocyte subset reconstitution and the percentage of peripheral blood mononuclear cells bearing HLA-DR and HLA-DQ class II molecules. This study included 37 allogeneic BMT: either with ( n = 17) or without ( n = 20) aGVHD. Within 2 months after transplantation, we observed that patients with aGVHD had a unique mononuclear cell profile characterized by (i) a significant increase in the percentages of CD8 bright+ CD28 - T cells ( P = 0.05) and CD3 + T cells ( P = 0.001), (ii) an important decrease in the percentage of CD56 + cells ( P = 0.0001), and (iii) a decrease in the percentages of HLADQ + and HLA-DR + monocytes ( P = 0.001) and HLADQ + T lymphocytes ( P = 0.0001), in comparison with patients without aGVHD. Moreover, statistical studies indicate that there was a positive correlation between CD 8bright+ CD28 - and CD3 + T cells, whereas CD3 + T cells were negatively correlated to CD56 + cells. We did not find any statistical correlation between the percentages of HLA-DQ + or HLA-DR + cells and the percentages of these lymphocyte subsets. Therefore, in this study done in children, we suggest that patients with (i) less than 20% of DQ + monocytes, (ii) less than 25% of CD56 + lymphocytes, and (iii) an enhanced percentage of CD 8bright+ CD28 - T cells are strongly associated with aGVHD. Unfortunately, these biological markers of aGVHD may not precede the clinical manifestations of the disease.