Flow cytometric osmotic fragility test: Increased assay sensitivity for clinical application in pediatric hematology

Abstract

Background: Osmotic fragility test (OFT) is widely considered as a sensitive indicator of red blood cells' sensitivity to the hypotonic solution. It is often used as a screening test for the diagnosis of hereditary spherocytosis (HS). Nowadays, the osmotic fragility test based on flow cytometric analysis (FCM OF) is widely used in laboratory practice. The purpose of this study was to optimize the assay sensitivity and to validate its clinical application in the diagnostic screening of childhood anemias.Methods: The study was conducted on 175 children suffering from various types of anemia (including 30 children with proven hereditary spherocytosis, HS) and 16 healthy subjects. All children were aged between 3 months and 17 years, including 94 boys and 97 girls. FCM OF was performed on every subject according to two different analysis time patterns (hemolysis was analyzed for 214 or 300 s) using Cytomics FC500 flow cytometer.Results: Significant higher sensitivity was demonstrated by the tests carried out according to the longer analysis time pattern (90.0 vs. 83.33%). The level of specificity of both the analysis patterns was similar. When an extended analysis time was used, the percentage of red cell survival levels in HS patients were significantly lowered compared to the same cases analyzed with shorter incubation times and all other non-HS anemic cases (9.31 6 4.69 vs. 35.59 6 15.30%, P < 0.05). During the shorter analysis time, the values obtained were 13.76 6 7.92% for HS and 48.18 6 19.04% for non-HS, P < 0.0001. The 300-s test is very useful in distinguishing thalassemia patients from patients with other types of anemias (94.74% sensitivity and 90.12% specificity) and provided the values of remaining red blood cells as 70.46 6 12.29% for thalassemia and 27.16 6 13.01% for nonthalassemia subjects, P < 0.0001. Conclusion: Flow cytometric osmotic fragility test with a longer (300-s) analysis time demonstrated an increased sensitivity in detecting HS in anemic children. V C 2017 International Clinical Cytometry Society

Screening of hereditary spherocytosis and pyruvate kinase deficiency by automated blood count using erythrocytic and reticulocytic parameters

“…This assay can be completed with osmotic fragility tests, which are challenging to implement, and lack of sensitivity and specificity . Furthermore, osmotic fragility test by flow cytometry is emerging and could become a promising alternative test . Additionally, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) has also been implemented in diagnosis of atypical cases and discriminates HS from congenital dyserythropoietic anemias type II (CDA II) .…”

Flow Cytometric Analysis of Erythrocytes Osmotic Fragility in Hereditary Spherocytosis: A Case‐Controlled Study Evaluating the Best Anticoagulant, Sample Pre‐Treatment and NaCl Concentration for Reliable Screening of this Red Blood Cell Membrane Disorder

“…In this study the events acquisition was performed for 100 s mimicking the events acquisition time after the distilled water spiking of Warang et al protocol, which has a total assay time of 140 s . Recently, Ciepiela et al described that increasing the events acquisition time after the distilled water spiking increased the assay sensitivity—total assay time of 300 s . The application of this increased events acquisition time after the distilled water spiking was not tested in our protocol and should be examined in future studies.…”

Overhydrated stomatocytosis associated with a complexRHAGgenotype including a novelde novomutation

“…Readings were taken at saline concentrations ranging from 9.0 g/L (308 mOsmol/L) to 1.0 g/L (34.2 mOsmol/L) NaCl. However, flow cytometric OFT revealed 12.36% residual red cells (reference range 35.59%±15.30%3) (figure 1C). Flow cytometric eosin-5′-maleimide dye’s mean channel fluorescence ratio of patient versus normal controls was 0.94, thus excluding hereditary spherocytosis.…”