Hagop Bessos

Application of a Time–Resolved Fluoroimmunoassay for the Analysis of Normal Prion Protein in Human Blood and Its Components

Background and Objectives: To quantify the cellular isoform of prion protein (PrPc) in human blood using a new time‐resolved dissociation‐enhanced fluoroimmunoas‐say (DELFIA®). Materials and Methods: The DELFIA was optimised for human blood samples and applied to isolated cell and plasma fractions from blood donations. The physicochemical properties of PrPc were analysed. Results: 26.5% of blood PrPc was associated with the platelet fraction, 0.8% with polymorphonuclear leucocytes, 2.4% with mononuclear leucocytes, 1.8% with red cells and 68.5% with plasma (mean values from 4 processed donations). Conclusion: The majority of blood PrPc is found in the platelet and plasma compartments.

Prospective epidemiologic study of the outcome and cost‐effectiveness of antenatal screening to detect neonatal alloimmune thrombocytopenia due to anti‐HPA‐1a

BACKGROUND: To assess the value of antenatal screening to detect neonatal alloimmune thrombocytopenia (NAIT) due to anti‐HPA‐1a, a prospective study was carried out to quantify the potential clinical benefits and determine whether screening would be cost‐effective.

The application of a new quantitative assay for the monitoring of integrin-associated protein CD47 on red blood cells during storage and comparison with the expression of CD47 and phosphatidylserine with flow cytometry.

BACKGROUND: After the introduction of universal leukoreduction, the role of factors other than white blood cells in red cell (RBC) storage lesion is attracting increasing attention. These include changes in the levels of CD47 and phosphatidylserine (PS) markers on RBCs during storage. The aim of this study was to monitor these changes with both flow cytometry (FACS) and a newly developed quantitative enzyme‐linked immunosorbent assay (ELISA).

Characterization of the alloreactive helper T-cell response to the platelet membrane glycoprotein IIIa (integrin-β3) in human platelet antigen-1a alloimmunized human platelet antigen-1b1b women

BACKGROUND: The aims were to characterize the helper T‐cell response to platelet (PLT) glycoprotein (GP) IIIa, which stimulates the alloimmune antibody response to human PLT antigen (HPA)‐1a, to identify immunodominant epitopes and to examine the HLA Class II associations.

The release of prion protein from platelets during storage of apheresis platelets

BACKGROUND: Recent studies using a time‐resolved fluoroimmunoassay method (dissociation‐enhanced lanthanide fluoroimmunoassay) showed that platelets and plasma are the main reservoir of the normal isoform of cell‐associated prion protein (PrPc) in human blood. The aims of the present study were to monitor PrPc levels in various fractions of apheresis platelets during storage by using the DELFIA method and to assess the association of this release with alpha‐granule protein β‐thrombo‐globulin and cytoplasmic LDH.

A whole blood assay for platelet HPA1 (PLA1) phenotyping applicable to large-scale screening.

Any future programme of antenatal screening of pregnancies for risk of neonatal alloimmune thrombocytopenia will have as a major requirement the availability of cost‐effective assays which can be applied to large numbers of samples. To address this, we developed a competitive ELISA to type whole blood samples for the platelet alloantigen HPA1a, based on the use of purified glycoprotein (GP) IIb/IIIa from donors of known HPA1 genotype along with well characterized anti‐HPA1a antiserum. Microtitre plates were coated with purified GPIIb/IIIa from donors of genotype HPA1a1a/3a3a. Anticoagulated whole blood of unknown HPA1 type was added to each well followed by anti‐HPA1a. Residual antiHPA1a antibody not bound to the platelets in the test blood sample, bound to the immobilized HPA1a on the plate and was quantitated by standard ELISA. 475 blood donors were typed by the assay and the results compared in a blinded comparison with typing in the Capture‐Ptm assay. Concordance was 100% (468 HPA1a positive and seven HPA1a negative). The HPA1 type of control samples stored as whole blood could be discriminated by this assay for up to 23 d of storage at 4°C. This assay should be suitable for use in large‐scale population screening programmes.

A new competitive binding enzyme-linked immunosorbent assay for glycocalicin in plasma and platelet concentrate supernatants

A new competitive binding enzyme-linked immunosorbent assay (CB ELISA) for glycocalicin (GC) was developed using GC coated wells and a monoclonal antibody (MAb) to glycoprotein Ib (AN51). The principal stages of the CB ELISA consisted of coating the plate with GC extract overnight, blocking with 3% BSA, incubating the wells with test or standard sample dilution and AN51, and a final incubation with horseradish peroxidase-conjugated goat anti-mouse IgG. Serial dilutions of purified GC, starting in 2% BSA, yielded standard curves which were linear between 10 and 0.4 micrograms/ml. Parallel curves were obtained for platelet concentrate supernatants and for citrated plasma. We used the ELISA to measure GC levels in platelet concentrates during storage. The results indicated that soluble GC increased progressively during storage from 3.3 to 6.7 micrograms/ml, while GC levels in platelet-poor plasma remained at 1.9-2.2 micrograms/ml. These results show that the new CB ELISA is a simple and short assay for the direct measurement of GC in plasma solutions, and may be of use in clinical studies.

An International Trial Demonstrates Suitability of a Newly Developed Whole‐Blood ELISA Kit for Multicentre Platelet HPA‐1 Phenotyping

Background: Neonatal alloimmune thrombocytopenia (NAIT) is in most cases due to pregnant women with HPA–1b platelet phenotype producing antibodies to HPA–1a platelets of the fetus, which may lead to intracranial haemorrhage with subsequent death or life–long morbidity. The availability of sensitive, reliable, straightforward, and inexpensive assays would enable large–scale screening in pregnancy and may help avoid NAIT. Methods: A recently developed enzyme–linked immunosorbent assay (ELISA) was produced in kit form incorporating modified reagents and enabling distribution to 21 international Platelet Immunology and Blood Centres (see Acknowledgments). The kits were assessed using anticoagulated whole–blood samples stored up to 10 weeks at 4°C. Each centre tested its own blood samples most of which had been previously typed by established assays. Results: Of the 152 samples that were tested, all 31 HPA–1b phenotypes were correctly identified by the kit. The respective mean ± standard deviation of the specific absorbances of HPA–1b and HPA–1a samples were: 0.52±0.15 and 0.12±0.06 (p<0.0005). In addition, the modified ELISA showed 100% concordance with PCR–SSP in the phenotyping of 93 donors. Finally, a comparison between freshly prepared and stored kits showed that the kit was stable for at least 2 years at 4°C. Conclusions: The international trial showed that the modified whole–blood ELISA kit is very well suited for wide–scale screening in pregnancy. Moreover, the ELISA kit could be used for large–scale phenotyping of blood donors, with HPA–1b–typed individuals getting invited to become apheresis platelet donors for patients with NAIT.

Direct comparison between two quantitative assays in the measurement of maternal anti-HPA-1a antibody in neonatal alloimmune thrombocytopenia (NAIT)

BACKGROUND Around 80% of severe neonatal alloimmune thrombocytopenia (NAIT) cases in the Caucasian population are due to anti-HPA-1a antibodies. However, the relationship between anti-HPA-1a antibody quantity and severity of NAIT has been subject to debate, possibly due to discrepant results between studies incorporating different assays (such as ELISA and MAIPA) and the number of samples. The aim of this study was to compare the level of maternal anti-HPA-1a antibodies in the same samples, using two different methods; quantitative ELISA and quantitative MAIPA. At the same time, the relationship between the maternal anti-HPA-1a antibody quantity and the newborn platelet count was examined. MATERIALS AND METHODS Plasma samples were obtained from HPA-1a negative mothers giving birth to children with different platelet counts (i.e. NAIT vs normal platelet count). The antibody levels were quantified blindly in the respective assays using standards calibrated against the international standard NIBSC 03/152. RESULTS A linear correlation was observed between quantitative MAIPA and quantitative ELISA results. However, there were many individual variations giving a mean ratio of disagreement between the quantitative ELISA and quantitative MAIPA of 2.84. Furthermore, regression analysis revealed a significant relation between anti-HPA-1a antibody quantity measured by MAIPA, but not ELISA, and the newborn platelet count. CONCLUSION The results indicate that differences observed between various studies with regard to NAIT severity and anti-HPA-1a quantity could partly be due to different assays used.

The cellular immunobiology associated with fetal and neonatal alloimmune thrombocytopenia

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal antibodies that cross the placenta in connection with pregnancy and destroy fetal platelets. Recently, maternal T cell responses associated with FNAIT have been studied at the clonal level. These T cell clones recognize an integrin β3 epitope, which is anchored to the HLA-DRB3∗0101-encoded MHC molecule DR52a. The same MHC allele is strongly associated with FNAIT. As the production of pathological antibodies reactive with fetal platelets is likely dependent on these T cell responses, there exists a potential for preventing FNAIT by targeting these T cells.