Comparison of a modified flow cytometry osmotic fragility test with the classical method for the diagnosis of hereditary spherocytosis.

BACKGROUND: Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia. The flow cytometric test using eosin-5'maleimide (EMA) is a well-established diagnostic method. However, in order to improve HS detection, it is recommended that EMA and an osmotic fragility test (OFT) both be performed. OFT is time consuming and labor intensive. We used a flow cytometric (FOFT) adaptation of the classical OFT reported by Yamamoto. We compare the FOFT to the classical OFT including practical data and propose options for simplifying this method. METHODS: Suspected and known HS patients and controls were tested by the following methods: EMA, OFT, and FOFT including some modifications. RESULTS: The FOFT method is robust and correlates to loss of red blood cells. OFT and FOFT gave similar results in healthy controls and four HS patients. Normal range for FOFT in 70 adults is shown and can be used as a reference value. Neonates should have their own normal range defined. Overnight sample incubation at 37°C did not add information to the FOFT results. CONCLUSION: Our modified Yamomoto FOFT can replace the classic OFT as the addition to EMA for the diagnosis of HS. The use of flow cytometry in both these methods requires small sample volume, is reproducible, simpler, and produces results more rapidly.

Adult pre B-cell acute lymphoblastic leukemia with unusually large proportion of bone marrow CD45 bright/high SSc blasts.

BACKGROUND: We present a pre B-ALL patient with the rare clinical manifestation of extramedullary disease, and a normal hemogram. This patient's blasts expressed bright CD45 and high side scatter (SSc) placing the cells in the monocyte gate. METHODS: Samples from peripheral blood and bone marrow (BM) aspirate from a 50-year-old female patient were immunophenotyped by multiparametric flow cytometry. RESULTS: Flow cytometry studies of the BM aspirate showed a large monocyte gate with 90-95% of the cells expressing an abnormal B cell phenotype. Peripheral white blood cells count was normal and cytogenetic analysis of the BM revealed a normal karyotype. CONCLUSION: It was not possible, based on CD45/SSc to identify a lymphoblast population in this pre B-ALL patient. Although bright expression of CD45 B-ALL blasts has been associated with poor prognosis to the best of our knowledge, the combination of bright CD45 blasts with high SSc has not been reported. As CD45 expression vs. SSc is routinely measured in the diagnostics of acute leukemias, a possible association between CD45 bright positivity and extramedullary disease or prognosis warrants further exploration. © 2015 International Clinical Cytometry Society.

Lymphocyte Subset Reference Ranges in Healthy Israeli Adults.

BACKGROUND: Reference ranges for adult peripheral blood lymphocyte subsets have been established in a few countries. To the best of our knowledge no broad lymphocyte subset analysis of the Israeli population has been reported.  Objectives: To establish reference ranges for healthy adults in Israel and to describe age- and gender-specific differences, if present. OBJECTIVES: To establish reference ranges for healthy adults in Israel and to describe age- and gender-specific differences, if present. METHODS: Lymphocyte subsets CD3, CD3/CD4, CD3/CD8, CD3-/CD16+/CD56+, CD3/TCRαß, CD3/TCRγδ, and CD19 were examined by flow cytometry in 326 subjects. Samples were subdivided according to age and gender. RESULTS: Women of all ages had a significantly higher percentage and absolute counts of CD3/CD4 cells than their male counterparts. Higher CD3/CD4 cells were observed also in the older population (> 50 years). CD3/CD8 and CD3-/CD16+/CD56+ were higher in males. Older males had a lower total lymphocyte percentage and CD19 cells compared to younger men. No significant gender-related differences were observed in percent and number of CD19, CD3/TCRαß or CD3/TCRγδ at all ages. CONCLUSIONS: These reference values could be useful in further studies for assessing changes that occur in different populations in human pathology.