Platelet diagnostics

Our research & development group of 4 European laboratories was set up in 2001 with the objective to develop simple and affordable assays for HPA platelet typing and antibody detection. One of the first steps of the project consisted in defining a standard protocol for the MAIPA (Monoclonal Antibody Immobilization of Platelet Antigens) assay as a reference protocol. Results - The plate-based technique produced very similar results between participants. Good signal/noise ratios were obtained with all HPA specificities investigated. No erroneous results were obtained. - With the consensus tube method a false-negative reaction was obtained with clinically significant HPA-1 antibodies, anti-GpIbIX antibodies and reactions with HPA-5 system antibodies were less clear. In addition greater variation of results was observed between workshop participants. Conclusion This study performed with the same reagents and under the same conditions, showed a lower sensitivity with the tube-based MAIPA when compared with the microplate one. A complementary study comparing three methods (routine tube, consensus tube and microplate method) was performed after the workshop and confirmed the results. Consequently, the group decide to adopt the microplate MAIPA technique as reference technique for the future. Lyophilized platelets One of the major drawbacks in the platelet immunology field is the lack of stable reagents to detect allo and autoantibodies. The French team has had the opportunity of getting freezed dried platelets for potential clinical use. We have decided to use them, instead of fresh platelets as is usually done, to check their capacity in the MAIPA test. Our first step consisted in controlling the surface glycoprotein expression on glycoproteins such as the GPIb, GPIIb, GPIV, CD109 etc. The level of expression with lyophilized platelets after rehydration turned out to be similar to the one with fresh platelets. The same in the MAIPA tests, lyophilized platelets gave results similar to the results obtained with fresh platelets. This new reagent is being validated by other laboratories (CR2, British team /CR4 German team and CR5 Swiss team) to check that all types of allo and autoantibodies can be detected.

DiaMed / ADS in collaboration with Leuze electronic has developed a new technology best described as real-time Fluorescence Immuno-Assay also known as TIRF. This basic technology has been exploited to transform our 40 minute HPA-1a ELISA to a 5 min real-time FIA. The TIRF Assay is completed in 5 minutes with just two pipetting steps and no washing step. A field trial indicated good performance of the real-time FIA with ELISA and genotyping. For antibody assays we developed a MAIPA / real-time FIA procedure to be performed in 1 h. Basically we incubate platelets and human plasma to be examined in the same way as is done for a conventional MAIPA and then follow the MAIPA wash procedures. The major difference is the detection of the complex monoclonal antibody / platelet glycoprotein / human immune globulin where our real-time FIA uses a one-step binding / detection step and gives a result in ten minutes. We have proven the feasibility of the new fast MAIPA with gpIIbIIIa, an optimisation procedure and the application to the other immunologically relevant platelet glycoproteins gpIaIIa, gpIbIX, CD109 and HLA will be the necessary. A development in 2005 for a one-hour MAIPA test is foreseen using the real-time fluorescence immuno assay. To expand the HPA typing range further than the HPA-1a typing an approach to HPA genotyping has been evaluated in a feasibility study. Using the real-time fluorescence immuno assay platform and HPA-1a oligonucleotides as analyte we can in 15minute hybridisation and detection determine the oligonucleotide concentration with an analytical sensitivity of 5 pmol/L

Gov & CD109 The Cambridge team established the molecular basis of the Gov alloantigens and typing assays based on PCR amplification. The relevant fragment of the CD109 gene have been developed. Appropriate monoclonal antibodies against CD109 have been identified for the use in MAIPA. The molecular basis of the Gov antigen system has been protected by a patent application between the Universities of Toronto and Cambridge. Recombinant CD109 fragments for Gov antibody detection are currently being expressed in C41 E. Coli. GPVI The Cambridge team determined the single nucleotide polymorphism (SNP) map of GPVI. Over 1100 donors have been genotyped for the GPVI SNPs. GPVIaa and GPVIbb donors have been identified. Platelets from these homozygous donors and recombinant GPVI antibodies can now be used in the MAIPA to determine whether GPVI is an alloantigen.

The German team developed and evaluated an improved MAIPA method based on simultaneous detection of various platelet specific immunoglobulin G (IgG) and IgM antibodies immobilized on beads and detected by flow cytometry. 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 various 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 platelet than other methods.

The Consortium decided to collaborate with an experienced computing firm, the Dendrite company, to have a software for the collection and analysis of the clinical and biological cases of Neonatal Alloimmune thrombocytopenia (NAIT) developed. Dendrite Clinical Systems provide a sophisticated clinical outcomes database management system that creates an environment in which the analysis and reporting of data becomes biologically and clinically meaningful. The Dendrite software is a unique clinical outcomes software, providing clinical users with the ability to track time related clinical data for analysis of any medical or surgical procedure, all within a single software environment. The software allows an extensive array of in built analytic and risk modelling functionalities: - Simple distribution analyses (single group, multi group descriptive statistics squared with Chiand Yates tests, t-tests - Longitudinal Analysis (Kaplan Meier, Cox Regression, and Hazard curves for both survival and event free analysis) - Sequential Outcome Analysis (CUSUM, CRAM, CRAO, VLAD, SPRTS) - Volume/Outcome Analysis (Funnel Plots) - Risk Scoring Evaluation (ROC curves and calibration plotting) - Risk Scoring Algorithms (POSSUM, Parsonnnet, Cleveland Clinic Scoring, EuroSCORE, Bayes and Neural Networks) - Advanced Data Analysis and links to SPSS/S-Plus/Crystal Reports

The German partner performed complete sequence analysis of P-selectin and its ligand PSGL-1 in healthy blood donors and patients with coronary heart disease (CHD). By using PCR-SSP they genotyped 261 CHD patients and 214 controls for 5 SNPs in P-selectin and 2 SNPs in PSGL-1. The P-selectin 715P allele was more frequent among CHD patients with hypercholesterolemia compared to patients with normal cholesterol levels. A SNP (M62I) in the PSGL-1 gene was found close to the P-selectin binding site and the 62I allele revealed a higher prevalence in the control group indicating a protective effect of the mutation. In addition to the SNPs the PSGL-1 gene also revealed a variable number of tandem repeats (VNTR) polymorphism. The results of case/control studies showed that the PSGL-1 VNTR polymorphism is not a genetic risk factor for CHD. The P-selectin and PSGL-1 variants may represent the molecular basis of alloantigens. More than 28,683 human genes were investigated by microarray analysis and in the receptor category 221 of 1,056 (20.9 %) analyzed genes were detectable. Among these, candidates were selected for further investigation. The protein expression of IL-4R, IL-10R, IL12-R, IL-18R and CCR5 could be confirmed by flow cytometrical analysis of platelets. The interleukin, chemokine and cell adhesion receptors may be important for the role of platelets in inflammation. These membrane proteins may also carry novel alloantigens.

Most blood group alloantigens specific for red cells and platelets are based on single nucleotide polymorphisms (SNPs) in genes encoding relevant membrane proteins. Using five Human Platelet Antigen (HPA) systems as a model a 4th generation microarray technique for SNP typing was established by CR4. In parallel, a 3rd generation technique, the TaqMana assay, was developed by CR2. Both techniques were validated by using a unique panel of 71 blinded DNA samples containing at least 15 aa, bb, and ab genotypes for the HPA-1, -2, -3, -5, and -15 systems. Unambiguous and concordant results were obtained with both techniques for all samples. The data validate the use of microarray for large scale SNP typing for clinically relevant blood group alloantigens.

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