Analytical And Clinical Validation Of A Diagnostic Test For Evaluation Of NK Cell Subsets And NKT Cells

Abstract

RATIONALE: Natural killer (NK) cells have both cytotoxic and cytokine-producing effector functions. NK cells are not a homogeneous subset and the ability to quantitatively evaluate various NK cell subsets, and functional proteins associated with these cell populations is clinically relevant.METHODS: A multiparametric flow cytometric assay was developed and analytically validated. CD45, CD3, CD16 and CD56 were used for total NK/NKT cell and NK subset quantitation. Additional markers (CD27, CD69, CD107a/b, NKp46, NKG2D, Granzyme A, Granzyme B, Perforin and IFNγ) characterizing NK cell functions were also used.RESULTS: Most NK-specific markers are only stable for up to 24h at ambient temperature, necessitating prompt analysis of whole blood samples. The inter- and intra- assay precision for the various subsets were typically within a coefficient of variation (CV) of 20%, for well-represented populations, and less than 10 cells absolute difference for rare events. Reference values (mid 95 percentile) for all subsets were established using 127 healthy adult donors. The precision at the limits of detection (1 - 201103cells/μL) were established for total NK cells as 10.31% at lower limits and 5.48% at upper limits. The assay was clinically validated with 21 patients representing a spectrum of primary or secondary immunological disorders, with various NK cell defects.CONCLUSIONS: We have a developed a clinical diagnostic assay for the quantitation of various NK cell/NKT subsets, including expression of functionally relevant markers, whose analytical parameters have been established, and can be used to evaluate NK cell populations in a variety of specific clinical and immunological contexts. RATIONALE: Natural killer (NK) cells have both cytotoxic and cytokine-producing effector functions. NK cells are not a homogeneous subset and the ability to quantitatively evaluate various NK cell subsets, and functional proteins associated with these cell populations is clinically relevant. METHODS: A multiparametric flow cytometric assay was developed and analytically validated. CD45, CD3, CD16 and CD56 were used for total NK/NKT cell and NK subset quantitation. Additional markers (CD27, CD69, CD107a/b, NKp46, NKG2D, Granzyme A, Granzyme B, Perforin and IFNγ) characterizing NK cell functions were also used. RESULTS: Most NK-specific markers are only stable for up to 24h at ambient temperature, necessitating prompt analysis of whole blood samples. The inter- and intra- assay precision for the various subsets were typically within a coefficient of variation (CV) of 20%, for well-represented populations, and less than 10 cells absolute difference for rare events. Reference values (mid 95 percentile) for all subsets were established using 127 healthy adult donors. The precision at the limits of detection (1 - 201103cells/μL) were established for total NK cells as 10.31% at lower limits and 5.48% at upper limits. The assay was clinically validated with 21 patients representing a spectrum of primary or secondary immunological disorders, with various NK cell defects. CONCLUSIONS: We have a developed a clinical diagnostic assay for the quantitation of various NK cell/NKT subsets, including expression of functionally relevant markers, whose analytical parameters have been established, and can be used to evaluate NK cell populations in a variety of specific clinical and immunological contexts.