Flow Cytometry Assay Measures NK Cell Function

The TINKL assay is a sensitive flow cytometry-based assay for measuring NK cell function; its clinical application has been evaluated and the activity measured, which is independent of the number of NK cells in the assay.

Immunologists at The Canberra Hospital (Garran, ACT, Australia) collected blood from 70 healthy adult male, and 53 healthy female volunteers. Their mean age (± standard deviation) was 38 ± 11 years, ranging from 19 to 65 years. Venous blood was collected in vacutainers containing ethylenediaminetetraacetic acid (EDTA), and samples were processed within 24 hours. Peripheral blood mononuclear cells (PBMC) were isolated, cryopreserved, and stored in liquid nitrogen. On the day prior to assay, PBMC were thawed and maintained in culture overnight.

The TINKL assay was conducted using NK-sensitive erythroleukaemic cell K562 was used to measure natural killing, and the NK-resistant Raji B lymphoblastoid cell in the presence of the CD20 monoclonal antibody Rituximab was used to measure antibody-dependent killing. Peripheral blood NK cell function measured by the TINKL assay was assessed in repeated samples of 123 healthy adults.

The PBMC were assayed in 21 trials over a three-month period. NK loss was 31% ± 10% (range 7% to 55%) as a consequence of natural killing, and 39% ± 10% (range 14% to 64%) as a consequence of antibody-dependent killing. The variation in NK cell function in the population measured by the TINKL assay is greater than can be accounted for by interexperimental variability. The intraexperimental coefficient of variation (CV) was on average 11% for natural killing and 3% for antibody-dependent killing, compared to 14% and 9% respectively for the interexperimental variation.

The authors concluded that their data provides additional information on the TINKL assay relevant to testing NK cell function in a clinical setting. The intra-experimental variability and the inter-experimental variability of the assay have been quantified, and the large range in NK cell function in a population of healthy donors is now chronicled. The study was published on April 6, 2013, in the Journal of Immunological Methods.

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