FLAER-negative CD15+ neutrophils can be used for the simplified screening of suspected PNH cases.

BACKGROUND: The flow cytometry analysis of GPI-linked proteins on red blood cells and leukocytes is crucial for paroxysmal nocturnal hemoglobinuria (PNH) diagnostics. However, the commonly used multicolor panels cannot be implemented in low-resourced hematology laboratories. In order to develop a simple prediagnostic test for PNH screening, we analyzed the diagnostic accuracy of the two-color (FLAER/CD15) detection of GPI-deficient neutrophils. METHODS: We reanalyzed multicolor data set of 1594 peripheral blood samples of patients screened for PNH applying only two markers (FLAER/CD15). The quantitative positivity/negativity was reported. Then, these results were compared in a blinded manner with previously obtained multicolor data from the same samples. RESULTS: Among the 1594 samples included in the study, 507 samples were PNH-positive by the multicolor assay. The two-color method revealed 510 PNH-positive samples. The detailed examination of this discrepancy revealed 12 false-positives and 9 false-negatives. Therefore, FLAER/CD15 screening method displayed 98.90% of the diagnostic specificity and 98.22% of the sensitivity. CONCLUSION: This simple two-color evaluation of FLAER-negative neutrophils is a highly effective screening test for PNH. Although this approach is not intended to replace the multicolor diagnostic procedure, it could minimize the number of patients requiring a conventional multicolor flow cytometric assay.

An inter-laboratory comparison of PNH clone detection by high-sensitivity flow cytometry in a Russian cohort.

OBJECTIVES: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by partial or absolute deficiency of glycophosphatidyl-inositol (GPI) anchor-linked surface proteins on blood cells. A lack of precise diagnostic standards for flow cytometry has hampered useful comparisons of data between laboratories. We report data from the first study evaluating the reproducibility of high-sensitivity flow cytometry for PNH in Russia. METHODS: PNH clone sizes were determined at diagnosis in PNH patients at a central laboratory and compared with follow-up measurements in six laboratories across the country. Analyses in each laboratory were performed according to recommendations from the International Clinical Cytometry Society (ICCS) and the more recent 'practical guidelines'. Follow-up measurements were compared with each other and with the values determined at diagnosis. RESULTS: PNH clone size measurements were determined in seven diagnosed PNH patients (five females, two males: mean age 37 years); five had a history of aplastic anemia and three (one with and two without aplastic anemia) had severe hemolytic PNH and elevated plasma lactate dehydrogenase. PNH clone sizes at diagnosis were low in patients with less severe clinical symptoms (0.41-9.7% of granulocytes) and high in patients with severe symptoms (58-99%). There were only minimal differences in the follow-up clone size measurement for each patient between the six laboratories, particularly in those with high values at diagnosis. CONCLUSIONS: The ICCS-recommended high-sensitivity flow cytometry protocol was effective for detecting major and minor PNH clones in Russian PNH patients, and showed high reproducibility between laboratories.

Application of flow-FISH for dynamic measurement of telomere length in cell division.

This method makes it possible to measure the fluorescence of a DNA probe in cells with known division number and targeted surface antigen. In fact, this method is a combination or consistent application of three other methods: cell tracking by vital dye, surface immunophenotyping, and flow-FISH. The idea in developing this method was to study telomere length changes in cells with known surface antigen after every new cell division. First, the in vitro cell culturing and staining with CFSE vital dye are performed. Then, cells are stained with surface MAbs labeled with biotin, followed by incubation with streptavidin-labeled fluorochrome. After that, cells are fixed with BS(3) reagent followed by the flow-FISH procedure with PNA-probe complementary to telomere DNA repeats. Finally, in one tube, it is possible to determine telomere length in surface antigen-labeled cells that have made the exact same number of divisions after incubation.

Distribution function approach to the study of the kinetics of IgM antibody binding to FcγRIIIb (CD16b) receptors on neutrophils by flow cytometry.

Though flow cytometry provides the entire distribution of cellular fluorescence (i.e., "fluorescence profile"), only mean fluorescence data are usually considered in studies of ligand-receptor binding. In this study, we presented a method of the treatment of the temporal evolution of the whole fluorescence profile with a comprehensive statistical approach extended to the reversible binding case. The method was demonstrated in the study of the 1D3 IgM monoclonal antibodies binding to FcγRIIIb receptors (CD16b) on neutrophils. Kinetic experiments were carried out using a FACSCalibur (Becton Dickinson, USA) flow cytometer. For each of four donors, we obtained the distribution of the number of FcγRIIIb surface receptors for neutrophils and the rate constants per receptor: the association rate constant of (2.7±0.4)×10(7) M(-1) min(-1), and the dissociation rate constant of (1.3±0.4)×10(-1) min(-1). Based on the obtained values, the size of the receptor reaction site was estimated at approximately 1 nm. It was found, that cell receptors distributions differed sufficiently between donors in mean and the skewness values, whereas the coefficient of variation (i.e., the ratio of the standard deviation to the mean) did not vary significantly.