Comparison of Next-Generation Sequencing-Based Human Leukocyte Antigen Typing with Clinical Flow Cytometry and Allele-Specific PCR Melting Assays for HLA-B27 Genotyping.

BACKGROUND: Due to the strong association between ankylosing spondylitis and Human Leukocyte Antigen (HLA)-B27, accurate identification of HLA-B27 is important in the diagnosis of patients with suspected spondyloarthritides. For this study, we compared a high-resolution HLA-B typing method to the clinical flow cytometry and allele-specific PCR melting assays to determine clinical benefits of high-resolution testing. METHODS: Residual clinical samples submitted for HLA-B27 testing by flow cytometry were tested by single-locus HLA-B genotyping using next-generation sequencing (NGS), and PCR with melting curve analysis, currently used as a reflex test for indeterminate flow cytometry results. RESULTS: Fifty out of the 51 samples (98%) positive by flow cytometry confirmed as HLA-B27 positive by PCR melting assay and by NGS. The sample that did not confirm was genotyped as HLA-B*07:02. All the samples negative by flow cytometry were confirmed as HLA-B27 negative by both PCR melting assay and NGS. For the group that was indeterminate by flow cytometry, 84.5% (n = 49) typed as positive for HLA-B27, while 15.5% (n = 9) were negative for HLA-B27 but positive for HLA-B*07:02. NGS was the only method able to distinguish between pathogenic and nonpathogenic HLA-B27 variants, in contrast to the flow cytometry or the PCR melting assays. CONCLUSIONS: Single-locus NGS is superior to flow cytometry and PCR melting assay for the unambiguous identification of HLA-B27 variants, and uniquely able to distinguish between pathogenic and nonpathogenic B27 alleles. Due to its high accuracy, it may be a feasible superior alternative to flow cytometry and traditional molecular methods for clinical HLA-B27 testing.

ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 2 - reagent selection and assay optimization for high-sensitivity testing.

Since publication in 2010 of the International Clinical Cytometry Society (ICCS) Consensus Guidelines for detection of Paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometery, a great deal of work has been performed to develop, optimize, and validate a number of high-sensitivity assays to detect PNH phenotypes in both red blood cells (RBCs) and white blood cells (WBCs, neutrophils, and monocytes). This section (Part 2) of the updated ICCS PNH Consensus Guidelines will focus on specific instrument setup for these PNH assays, the identification and proper testing of appropriate antibody conjugates and combinations therof, and basic assay design. © 2017 International Clinical Cytometry Society.

Rare event counting of CD59- red cells in human blood: A 47-month experience using PNH consensus guidelines for WBC and RBC testing in a reference lab.

INTRODUCTION: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disorder characterized by increased complement-mediated lysis of erythrocytes (RBCs) because of low/absent glycophosphatidylinositol (GPI) anchors of numerous cell surface proteins. METHODS: Rare event analysis was applied to 120 million RBCs (12 normal individuals) and 102 million RBCs (102 normal individuals) to establish a reference range and verify a methodology for rare event analysis. Patient PNH testing (n = 10,984) was performed over 47 months using the 2010 consensus guidelines with CD59-PE and glycophorin A-FITC for RBCs and FLAER-Alexa 488, CD33-PerCP-Cy5.5, CD15-APC, CD14-APC-Cy7, and CD24-PE for WBCs. RESULTS: The distribution of CD59- RBCs in the normal population was asymmetric with a mean of 5.9, median between 3 and 4, and mode of 2 per million RBCs. The normal range of CD59- RBCs was 0-17/million RBCs. A natural cutoff of 2.5% of the peak expression of CD59 delineates CD59+ from CD59- populations. The incidence of GPI- samples received by the laboratory was 6-7% without correlation to age (P = 0.35) or sex (P = 0.45). The percentage of GPI- neutrophils and monocytes were strongly correlated (R(2) = 0.96) and usually greater than the percentage of GPI- RBCs. CONCLUSION: PNH RBC testing is a good example of rare event analysis applied to clinical cytometry­only 2.5 min are required to collect 1 million RBCs. With an established normal range of CD59- RBCs, the correlation between total cell count and sensitivity for detecting an abnormal population can be calculated using Poisson statistics.