Establishment Of A Multiparametric Flow Cytometry Assay For Measuring The Absolute And Relative Numbers Of Human Lymphocyte Subsets And TS Application In I Wiskott-Aldrich Syndrome

Objective:To establish a multiparametric flow cytemetry (FCM) assay for measuring the absolute and relative numbers of human lymphocyte subsets using the lysed whole blood method and the application of this method in Wiskott-Aldrich syndrome(WAS).Method:500μl peripheral blood (PB) was collected in a vacutainer tube containing liquid ethylenediaminetetraacetic acid (EDTA) as an anticoagulant and processed within 2 hour from collection. The percentage values of studied subsets obtained by cytometric reading were converted into absolute values (cells/μl) according to absolute number of lymphocyte count provided by the haematological analyser. To prove whole blood assay is reliable, whole blood and PBMC were detected by this method from the same samples. PB was collected from 20 children with WAS,who came to Children’s Hospital of Chongqing Medical University for medical treatment from June 2014 to March 2015.These patients had diagnosed with WAS by detection of WAS gene and WASp.The lymphocyte subsets of these patients were detected by the established method.Results:The relative and absolute numbers of 14 lymphocyte subsets are analyzed. The absolute numbers of lymphocyte subsets are determined by a certain formula. There was no statistical difference between whole blood and PBMC samples. The differences in relative and absolute numbers of lymphocyte subsets, which are normally distributed, between the two groups detected by the WAS patients and heath control(HC) respectively were determined by paired t-test. On the contrary, if the data are not normally distributed, Wilcoxon’s signed rank tests were performed. The percentages and absolute numbers of Th cells in WAS were lower than HC; the percentages and relative number of NK cells in WAS was significantly higher than that in HC; the percentage and absolute number of B cells was significantly decreased in WAS patients; CD8 naive subset’s percentages and absolute numbers were significantly reduced in children with WAS,on the contrary, CD8 EM subset’s percentage and absolute number were increased; CD8 TEMRA and CD8 CM subsets were increased in WAS,which was a significant difference compared with HC. B cell and it’s 4 subsets(Memory B cell,Naive B cell,Transitional B cell and plasmablasts) were significantly lower than that of HC.Conclusion:We raised a method to quantitatively assess 20 lymphocyte subsets using lysed whole blood method, which appeared to be an effective and rapid test for comprehensive assessment of immune function, provided clues and evidence for the diagnosis of immune diseases, and played an important role in clinical treatment. In this study,we examined quantity changes of lymphocyte subsets in 20 WAS patients. We found that subsets of peripheral blood B cells significantly decreased which is an abnormal distribution of peripheral blood B cells because of the defection of WASp and resulting in perturbing the homeostasis of B cell; Naive T cells were reduced and effector memory T cells increased with no infection, which may be related to abnormal T cell immune response. In addition,the study found that the number of DNT cells, B cells, and transitional B cells were nearly 0 in some WAS patients; In some cases, CD8 TEMRA significantly increased, reaching 50% of T cells,which need further medication analysis.