A multicenter validation of recombinant ß3 integrin-coupled beads to detect human platelet antigen-1 alloantibodies in 498 cases of fetomaternal alloimmune thrombocytopenia.

BACKGROUND: Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by human platelet (PLT) antigen (HPA) incompatibility. Beads coupled with recombinant ß3 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.

Accurate quantitation of D+ fetomaternal hemorrhage by flow cytometry using a novel reagent to eliminate granulocytes from analysis.

BACKGROUND: Quantitation of fetomaternal hemorrhage (FMH) is performed to determine the dose of prophylactic anti-D (RhIG) required to prevent D immunization of D- women. Flow cytometry (FC) is the most accurate method. However, maternal white blood cells (WBCs) can give high background by binding anti-D nonspecifically, compromising accuracy. STUDY DESIGN AND METHODS: Maternal blood samples (69) were sent for FC quantitation of FMH after positive Kleihauer-Betke test (KBT) analysis and RhIG administration. Reagents used were BRAD-3-fluorescein isothiocyanate (FITC; anti-D), AEVZ5.3-FITC (anti-varicella zoster [anti-VZ], negative control), anti-fetal hemoglobin (HbF)-FITC, blended two-color reagents, BRAD-3-FITC/anti-CD45-phycoerythrin (PE; anti-D/L), and BRAD-3-FITC/anti-CD66b-PE (anti-D/G). PE-positive WBCs were eliminated from analysis by gating. Full blood counts were performed on maternal samples and female donors. RESULTS: Elevated numbers of neutrophils were present in 80% of patients. Red blood cell (RBC) indices varied widely in maternal blood. D+ FMH values obtained with anti-D/L, anti-D/G, and anti-HbF-FITC were very similar (r = 0.99, p < 0.001). Correlation between KBT and anti-HbF-FITC FMH results was low (r = 0.716). Inaccurate FMH quantitation using the current method (anti-D minus anti-VZ) occurred with 71% samples having less than 15 mL of D+ FMH (RBCs) and insufficient RhIG calculated for 9%. Using two-color reagents and anti-HbF-FITC, approximately 30% patients had elevated F cells, 26% had no fetal cells, 6% had D- FMH, 26% had 4 to 15 mL of D+ FMH, and 12% patients had more than 15 mL of D+ FMH (RBCs) requiring more than 300 µg of RhIG. CONCLUSION: Without accurate quantitation of D+ FMH by FC, some women would receive inappropriate or inadequate anti-D prophylaxis. The latter may be at risk of immunization leading to hemolytic disease of the newborn.

Detection of human platelet antigen-1a alloantibodies in cases of fetomaternal alloimmune thrombocytopenia using recombinant ß3 integrin fragments coupled to fluorescently labeled beads.

BACKGROUND: Testing for alloantibodies against human platelet antigens (HPAs) is essential for the clinical diagnosis of fetomaternal alloimmune thrombocytopenia (FMAIT), posttransfusion purpura, and platelet (PLT) refractoriness. Most of the methods currently used for HPA alloantibody detection rely on the availability of panels of HPA-typed PLTs and some rely on validated monoclonal antibodies (MoAbs) against the PLT glycoproteins. Recombinant ß3 integrins displaying the HPA-1a (rHPA-1a) or HPA-1b (rHPA-1b) epitopes have been produced as an alternative source of antigen. The suitability of these integrin fragments was evaluated for the development of an HPA-1a alloantibody screening assay, using Luminex xMAP technology. STUDY DESIGN AND METHODS: A 3-plex bead assay was developed by coupling biotinylated rHPA-1a, rHPA-1b, and recombinant glycoprotein VI to LumAvidin microspheres. Forty patient samples referred for FMAIT diagnostic testing, which were previously screened by the MoAb-specific immobilization of PLT antigens (MAIPA) assay, were used to assess the assay. RESULTS: The rHPA-1a- and rHPA-1b-coupled beads were able to detect HPA-1a and HPA-1b alloantibodies in all patient samples tested that were previously confirmed to contain HPA-1-specific antibodies. Furthermore, HLA Class I antibodies did not cross-react with the coupled beads. CONCLUSION: The 3-plex bead assay can be used to detect HPA-1a antibodies with sufficient specificity and sensitivity for use in the clinical setting of FMAIT. The development of other recombinant integrin fragments with the use of Luminex xMAP technology may assist in providing more rapid HPA antibody detection, enabling prompt diagnosis of alloimmune PLT disorders.

VEGF165b, an inhibitory vascular endothelial growth factor splice variant: mechanism of action, in vivo effect on angiogenesis and endogenous protein expression.

Growth of new blood vessels (angiogenesis), required for all tumor growth, is stimulated by the expression of vascular endothelial growth factor (VEGF). VEGF is up-regulated in all known solid tumors but also in atherosclerosis, diabetic retinopathy, arthritis, and many other conditions. Conventional VEGF isoforms have been universally described as proangiogenic cytokines. Here, we show that an endogenous splice variant, VEGF(165)b, is expressed as protein in normal cells and tissues and is circulating in human plasma. We also present evidence for a sister family of presumably inhibitory splice variants. Moreover, these isoforms are down-regulated in prostate cancer. We also show that VEGF(165)b binds VEGF receptor 2 with the same affinity as VEGF(165) but does not activate it or stimulate downstream signaling pathways. Moreover, it prevents VEGF(165)-mediated VEGF receptor 2 phosphorylation and signaling in cultured cells. Furthermore, we show, with two different in vivo angiogenesis models, that VEGF(165)b is not angiogenic and that it inhibits VEGF(165)-mediated angiogenesis in rabbit cornea and rat mesentery. Finally, we show that VEGF(165)b expressing tumors grow significantly more slowly than VEGF(165)-expressing tumors, indicating that a switch in splicing from VEGF(165) to VEGF(165)b can inhibit tumor growth. These results suggest that regulation of VEGF splicing may be a critical switch from an antiangiogenic to a proangiogenic phenotype.

Protein distribution during human erythroblast enucleation in vitro.

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes.

Intravascular survival of red cells coated with a mutated human anti-D antibody engineered to lack destructive activity.

Alloimmune feto-maternal destruction of blood cells is thought to be mediated by binding of alloantibodies to Fc receptors on effector cells. Blocking the antigen using inert antibodies might prolong cell survival. We have performed a "proof of principle" study in volunteers to measure the intravascular survival of autologous red cells coated with human recombinant IgG antibody containing a novel constant region, G1Deltanab, devoid of in vitro cytotoxic activity. RhD-positive red blood cells (RBCs), labeled with chromium-51 or technetium-99m, were separately coated to equal levels with wild-type IgG1 or G1Deltanab anti-D antibody (Fog-1). After re-injection, there was complete, irreversible clearance of IgG1-coated RBCs by 200 minutes, concomitant with appearance of radiolabel in plasma. Gamma camera imaging revealed accumulation in spleen and, at higher coating levels, in liver. In contrast, clearance of G1Deltanab-coated cells was slower, incomplete, and transient, with whole blood counts falling to 7% to 38% injected dose by about 200 minutes before increasing to 12% to 67% thereafter. There was no appearance of plasma radiolabel and no hepatic accumulation. These findings suggest that G1Deltanab-coated RBCs were not hemolysed but temporarily sequestered in the spleen and that our approach merits investigation in larger studies.

Eosin-5-maleimide binding to band 3 and Rh-related proteins forms the basis of a screening test for hereditary spherocytosis.

Flow cytometric analysis of eosin-5-maleimide (EMA) binding to red cells is a screening test for the diagnosis of hereditary spherocytosis (HS). The present study used chemical modifications to determine the integral membrane proteins that react with EMA. The predominant interaction of EMA, contributing c. 80% of fluorescence, was with the epsilon-NH2 group of lysine in band 3 protein, as previously reported. The remainder of the EMA fluorescence was attributable to labelling of accessible sulfhydryl groups on intact red cells. This reaction was heat labile. Three molecules containing sulfhydryl groups were shown to be associated with the Rh blood group protein complex by sodium dodecyl sulphate polyacrylamide gel electrophoresis. These were CD47 and the Rh-associated glycoprotein, both in the Mr 40-60 kD region, and the Rh blood group proteins in the 30-32 kD region. Immunoprecipitation, using specific monoclonal antibodies and antibody binding studies by flow cytometry, showed that the relative content of these three membrane proteins was lower in HS than normal red cells. Thus, the high predictive value of the EMA binding test for HS reflects changes in the relative amounts of the Rh-related integral membrane proteins as well as band 3 in HS red cells.

Monoclonal antibodies inhibit prion replication and delay the development of prion disease.

Prion diseases such as Creutzfeldt-Jakob disease (CJD) are fatal, neuro-degenerative disorders with no known therapy. A proportion of the UK population has been exposed to a bovine spongiform encephalopathy-like prion strain and are at risk of developing variant CJD. A hallmark of prion disease is the transformation of normal cellular prion protein (PrP(C)) into an infectious disease-associated isoform, PrP(Sc). Recent in vitro studies indicate that anti-PrP monoclonal antibodies with little or no affinity for PrP(Sc) can prevent the incorporation of PrP(C) into propagating prions. We therefore investigated in a murine scrapie model whether anti-PrP monoclonal antibodies show similar inhibitory effects on prion replication in vivo. We found that peripheral PrP(Sc) levels and prion infectivity were markedly reduced, even when the antibodies were first administered at the point of near maximal accumulation of PrP(Sc) in the spleen. Furthermore, animals in which the treatment was continued remained healthy for over 300 days after equivalent untreated animals had succumbed to the disease. These findings indicate that immunotherapeutic strategies for human prion diseases are worth pursuing.

Micromolar A and B blood group active trisaccharides abolish human complement haemolytic activity assayed by erythrocytes.

BACKGROUND: At present, there is really no satisfactory treatment of severe haemolytic transfusion reactions involving the ABO system other than the use of steroids that at best are palliative in their effects. In contrast, the use of micromolar concentrations of A or B blood group active trisaccharides that are inexpensive and readily available may prevent lysis by generating soluble immune complexes (ICs) that consume complement. The purpose of this study was to determine the total lytic activity of human serum and to estimate the extent to which trisaccharides can exhaust this capacity. METHODS: We measured complement consumption by (ICs) formed between anti-blood group antibodies and A or B blood group active sugars on erythrocytes (solid phase) or soluble components carrying trisaccharides (fluid phase) in AB serum. A direct complement-mediated lysis (DCL) assay measured the solid-phase reaction and an indirect complement consumption assay (CCA) allowed determination of the fluid-phase reaction. In CCA, the residual lytic activity of AB serum was measured following preincubation with various ICs. RESULTS: Based on over 4000 data points a new mathematical model of complement consumption was formulated. Its predictions deviated by less than 6% in DCL and 9% in CCA when compared with the experimentally accessible data. The new model describes the dynamics of complement consumption including the soluble phase of the reaction. CONCLUSIONS: The mathematical model extrapolation predicts that: (1) in undiluted human serum up to 40% of the physiological erythrocyte concentration could be lysed (when antibodies did not limit lysis); and (2) a 40 microM (or less) concentration of blood group active trisaccharides per patient is sufficient to block the haemolytic complement activity.