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.

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.

Aggregation of polyQ proteins is increased upon yeast aging and affected by Sir2 and Hsf1: novel quantitative biochemical and microscopic assays.

Aging-related neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's diseases, are characterized by accumulation of protein aggregates in distinct neuronal cells that eventually die. In Huntington's disease, the protein huntingtin forms aggregates, and the age of disease onset is inversely correlated to the length of the protein's poly-glutamine tract. Using quantitative assays to estimate microscopically and capture biochemically protein aggregates, here we study in Saccharomyces cerevisiae aging-related aggregation of GFP-tagged, huntingtin-derived proteins with different polyQ lengths. We find that the short 25Q protein never aggregates whereas the long 103Q version always aggregates. However, the mid-size 47Q protein is soluble in young logarithmically growing yeast but aggregates as the yeast cells enter the stationary phase and age, allowing us to plot an "aggregation timeline". This aging-dependent aggregation was associated with increased cytotoxicity. We also show that two aging-related genes, SIR2 and HSF1, affect aggregation of the polyQ proteins. In Δsir2 strain the aging-dependent aggregation of the 47Q protein is aggravated, while overexpression of the transcription factor Hsf1 attenuates aggregation. Thus, the mid-size 47Q protein and our quantitative aggregation assays provide valuable tools to unravel the roles of genes and environmental conditions that affect aging-related aggregation.