Lab Med Online.  2023 Oct;13(4):349-355. 10.47429/lmo.2023.13.4.349.

Establishing a Reference Interval for Flow Cytometry-Based NK Activity (FC-NKA) Assay in Healthy Korean Population

• Kang H
• Choi AR
• Ryu JH
• Oh EJ

• KMID: 2552762
• DOI: http://doi.org/10.47429/lmo.2023.13.4.349

Abstract

Background
Flow cytometry (FC)-based natural killer cell activity (NKA) assays are commonly used in clinical laboratories. As low or no NKA is indicative of hemophagocytic lymphohistiocytosis (HLH), the precise determination of NKA levels is essential. The study aims to establish a reference interval for FC-NKA in healthy Korean subjects and determine the optimal cutoff for diagnosing HLH.
Methods
In a single-center retrospective study, internal quality control data from a total of 143 healthy individuals were analyzed. The FC-NKA assessed NK cytotoxicity by culturing effector cells with carboxyfluorescein diacetate succinimidyl ester-labeled K562 cells at an effector-to-target ratio of 32:1 for 4 hours. We compared the assay results among subgroups stratified by age and sex, as well as between healthy controls and HLH patients.
Results
The FC-NKA results did not vary significantly depending on age and sex. The reference interval drawn from the 2.5–97.5 percentile of the population was 13.8–82.9%. The lower limits of FC-NKA for the 5th and 10th percentiles were found to be 17.9% and 26.8%, respectively. Using receiver operating characteristic analysis, an optimal cutoff value of 20.0% was identified for HLH diagnosis with a sensitivity and specificity of 93.8% and 93.0%, respectively. The intra-individual coefficient of variation was determined to be 13.4% for the analysis of sequential samples from a single blood donor (N = 8).
Conclusions
The reference interval of the FC-NKA was determined for healthy Korean subjects, and the suggested cutoff level has the potential to be utilized for the diagnosis of HLH.

Keyword

Natural killer (NK) cell; Flow cytometry (FC); Cytotoxicity; Reference interval

Figure

• 

  Fig. 1 Percent of lysed K562 cells in separate subgroups according to age and sex. Age and gender subgroups are compared for the FC-based NK activity (FC-NKA) results. Data are presented in terms of the median with the interquartile range (IQR) and the tick marks indicate minimum and maximum values. Two-way analysis of variance (ANOVA) was performed, and the statistical significance between males and females in the same age group is shown in black. The statistical significance between different age groups in males is shown in blue, and in females, in red. Abbreviation: ns, not significant.

• 

  Fig. 2 Comparison of %K562 cytolysis in healthy controls and HLH patients and ROC curve for FC-NKA assay. (A) Comparison of %K562 cytolysis in healthy controls and in patients diagnosed with hemophagocytic lymphohistiocytosis (HLH). Data are presented as medians with interquartile range (IQR), and the tick marks indicate minimum and maximum values. (B) Receiver operating characteristic (ROC) curve for flow cytometry-based NK activity (FC-NKA) assay. ****P<0.0001 by Mann-Whitney test.

• 

  Fig. 3 Percentage of lysed K562 cells across lots and interpreters. Each dot represents a single individual result. The horizontal line indicates median with interquartile range (IQR). Abbreviation: ns, not significant.

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