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.

The Influence of the HLA-DRB1 rheumatoid arthritis shared epitope on the clinical characteristics and radiological outcome of psoriatic arthritis.

OBJECTIVE: To investigate the associations of the HLA-DRB1 rheumatoid arthritis shared epitope (SE) with clinical characteristics and radiological outcome in patients with psoriatic arthritis (PsA). METHODS: One hundred fifty-eight patients with well documented PsA and 250 controls were typed for HLA-DRB1 alleles including the SE by polymerase chain reaction. Clinical data collected on the patient group included disease subset, swollen and tender joint counts, the psoriasis area severity index (PASI), and the presence of radiological erosions. Clinical and radiological associations with HLA-DRB1 and SE alleles were determined. RESULTS: There was an increased frequency of HLA-DR7 (41 vs 25%; puncorr = 0.001, OR 2.02, pcorr = 0.01) and a decreased frequency of HLA-DR2 (19 vs 28%; puncorr = 0.03, OR 0.59, pcorr = 0.3) in the patient population compared with controls. There was no significant difference in the frequency of HLA-DR1 and HLA-DR4 between patient and control populations. There was no significant difference in the prevalence of SE alleles between the patient and control populations (48 vs 54%). There was no increase in the prevalence of the SE in the polyarthritis subgroup, but there was a marginal decrease in those who remained in the oligoarthritis subgroup. There were no differences with respect to sex, age of onset of disease, family history, Health Assessment Questionnaire score, joint score, skin score, or nail score between those patients who were SE positive and those who were SE negative. However, significantly more patients who were SE positive developed radiological erosions (60 vs 43%; p = 0.03, OR 2.11). CONCLUSION: Overall, the prevalence of the SE in patients with PsA did not differ from our control population. However, it was overrepresented in those who developed radiological erosions. It is possible that the SE does have a role in the clinical severity of PsA.