Flow cytometric detection of erythrocyte osmotic fragility

Abstract

Background: Hereditary spherocytosis (HS) is the most common cause of inherited chronic hemolysis.The various tests developed for HS screening have many shortcomings. The purpose of this study was to develop a new, simple, reliable test using flow cytometry.Methods: In this new test, deionized water, a hemolysis-inducing agent, is spiked to a red cell suspension during acquisition, and the count of red cells is measured sequentially in real-time using flow cytometry. In this study, the optimal parameters for this test were established, and the discriminatory power of the adopted protocol was verified among several groups with various degrees of osmotic fragility (OF).Results: The established protocol successfully discriminated between blood analyzed immediately and blood stored for 24 h (P < 0.00001). With healthy individuals as a reference, the OF measured by this protocol was significantly decreased in patients with iron deficiency anemia (P = 0.030) and was significantly increased in HS patients (P = 1.1 × 10 29 ). This new test found all 11 HS patients to have increased OF.Conclusions: This new test was verified to be simple, quantitative, objective, and cost-effective for the measurement of OF. We suggest this test as another effective approach for HS screening, although its assay performance will require further verification through more clinical trials. q 2008 Clinical Cytometry

Flow cytometric osmotic fragility—An effective screening approach for red cell membranopathies

“…As found earlier, a delay between the preparation of the final red cell suspension and the installation at the port (acquisition), led to a significant reduction in the percentage of residual red cells (9). Hence it is very important to proceed to the next step immediately after the blood is diluted.…”

“…The method described by Won and Suh was followed (9). The red cell suspension was prepared by a two-step dilution with normal saline.…”

“…Recently a flow cytometric method based on osmotic fragility has been described for measuring the residual red cell counts after the cells are exposed to deionized water (9). We evaluated the usefulness of this approach for screening for red cell membrane protein disorders and compared the results of the flow cytometric osmotic fragility test with the E5 0 M dye binding test.…”

Esferocitosis hereditaria. Revisión. Parte I. Historia, demografía, etiopatogenia y diagnóstico

“…59,60 Actualmente, se dispone de nuevas pruebas diagnósticas, más específicas y sensibles, que presentan, además, la ventaja de requerir menor cantidad de sangre para el procesamiento. 8,[61][62][63][64][65] En nuestro país, algunas de estas -criohemólisis hipertónica (CH), citometría de flujo con 5'eosina maleimida (5'EMA-CF), FOE por citometría de flujo (FOE-CF)-se comenzaron a implementar a partir del año 2007.…”

“…Es decir que se evalúa el porcentaje de eritrocitos remanentes, en lugar de evaluar la hemólisis producida en el medio hipotónico. La prueba fue descrita hace muy pocos años, 64 de manera que aún no se ha difundido ampliamente. Los resultados han sido reproducidos por diferentes grupos.…”