유세포검사에 의한 림프구아형검사에서 Gating 시 CD45의 필요성 및 NK세포 측정 시 CD16의 중요성
Flow Cytometric Assays for Lymphocyte Subset Enumeration: CD45 is Inevitable for Lymphocyte Gating and CD16 is Essential for NK Cells
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배경: Clinical and Laboratory Standards Institute 가이드라인(H42-A2)에서는 림프구아형분석을 위하여 ‘CD45/SSC’ gating법을 사용하고 NK세포아형 측정 시 CD16를 사용하지 않고 CD56만을 사용할 것을 권장하고 있다. 또한 CAP의 유세포검사 체크리스트에서는 림프구아형검사 시 gating을 위하여 특정한 gating방법의 사용을 권장하지 않고, 단지 gate 순도에 따라 림프구아형검사의 결과를 교정할 것을 권장하고 있다. 방법: 102명의 환자군과 12명의 정상 대조군으로부터 EDTA 혈액을 채취하여 세 가지의 gating법(CD45/SSC, FSC/SSC, FSC/SSCgating 후 gate 순도를 이용한 교정)으로 림프구아형검사를 실시하여 그 결과를 각각 비교하였고, NK세포 가운데 CD56-/CD16+ NK세포아형의 백분율을 평가하였다. 결과: 림프구백분율이 증가할수록 림프구 gate의 순도가 증가하는 것을 알 수 있었으나 높은 림프구 순도를 나타내는 특정한 림프구백분율은 알 수 없었다. T세포의 경우 gating법에 따라 결과가달라졌는데(CD45/SSC와 FSC/SSC 비교 시 결과값의 차이P = 0.027, CD45/SSC와 FSC/SSC gating 후 림프구순도로 교정 비교 시 결과값의 차이 P = 0.002), FSC/SSC gating 후 림프구순도로교정하는 방법이 FSC/SSC gating법에 비해 정확도가 증가되지 않았다. CD56-CD16+ NK세포아형은 총 NK세포의 17.1% (평균)를차지하였으며 환자군(13.1-25.5%)에서 대조군(9.5%) 보다 더 높은비율을 차지하였다. 결론: 유세포검사에 의한 림프구아형 측정에서 CD45는 gating 시반드시 사용되어야 하고, NK세포 측정 시 CD16 사용이 필수적이다.
Abstract
Background: Clinical and Laboratory Standards Institute (CLSI) guidelines (H42-A2) recommend the “CD45/SSC” gating method for assays on lymphocyte subset enumeration and CD16 exclusion for assays enumerating NK cells. In contrast, the Flow Cytometry Checklist (06/17/2010) of the College of American Pathology does not recommend a specific lymphocyte gating method, but recommends the correction of lymphocyte subset results for lymphocyte gate purity. Methods: We compared lymphocyte subset results of EDTA-treated blood from 102 patients with various diseases and 12 normal controls, using 3 lymphocyte gating methods (CD45/SSC, FSC/SSC, and lymphocyte gate purity correction after FSC/SSC gating), and assessed the proportion of CD56-/CD16+ NK cells within the total NK cell population. Results: Lymphocyte gate purity increased as the percentage of lymphocytes increased. However, lymphocyte subsets that consistently showed high lymphocyte gate purity could not be identified. The purity of the T cell population differed significantly depending on the gating method used:CD45/SSC vs. FSC/SSC, P =0.027; CD45/SSC vs. gate purity correction after FSC/SSC, P =0.002. However, the lymphocyte gate purity correction after FSC/SSC gating did not significantly improve the accuracy of the lymphocyte subset enumeration assay using FSC/SSC gating. The subset of CD56-CD16+ NK cells, constituted an average of 17.1% of total NK cells. Patients had higher proportions of CD56-CD16+ NK cells (13.1-25.5%)than did the normal controls (9.52%). Conclusions: In flow cytometric assays to evaluate lymphocytic subsets, the CD45 is inevitable for lymphocyte gating, whereas the measurement of CD16 is essential for the evaluation of NK cell proportions.
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