[Application of measuring human peripheral NK cell activity with flow cytometry in diagnosis for hemophagocytic lymphohistiocytosis]

Abstract

The aim of study was to establish an accurate and stable technique for the detection of NK cell activity in the diagnosis of hemophagocytic lymphohistiocytosis (HLH). 21 suspected acquired HLH patients and 20 healthy subjects as controls were enrolled in the study, and the suspected HLH patients were divided into confirmedly diagnosed group and excluded group according to HLH-2004 diagnostic criteria. The plasmid pEGFP-N1 was transfected into K562 cells. After scanned with G4l8 and monoclone, the EGFP-K562 cell line stably expressing enhanced green fluorescent protein was obtained. PBMNC and EGFP-K562 were mixed at the effector to target ratio of 10:1. After incubation for of 2 hours, propidium iodine (PI) was added to stain dead cells, and then cytotoxic activity was analyzed by using flow cytometry. Meanwhile, the cytotoxic activity of NK cells in peripheral blood on K562 cells was detected by LDH release assay, and was compared with results detected by flow cytometry. The results showed that a K562 cell line stably expressing EGFP was constructed and were used to measure NK cytotoxicity against the target cells by flow cytometry without pre-staining or pre-labeling target cells. There was significant difference in killing rate of NK cells between the diagnostic group and the control group. This new technique correlated strongly with the results by LDH release assay. In conclusion, this study provides a novel, simple, rapid, repeatable and reproducible method to measure NK activity by flow cytometry using EGFP-K562 without pre-staining or pre-labeling target cells. It may be widely used in the diagnosis of hemophagocytic lymphohistiocytosis.