Abstract:

Objective To establish the classification reference method of peripheral blood leukocytes by flow cytometry，and to verify the feasibility of its classification. Methods An 8-colour flow cytometry leukocyte counting protocol was established using a combination of CD16-Pacific Blue，CD45-V500，SYTO16，CD7-PE，CD123-PerCP-Cy5.5，CD14-PE-Cy7，CD3+CD19-APC and CD11b-APC-Cy7 antibodies. Peripheral blood samples were collected from 50 healthy subjects with ethylenediaminetetraacetic acid dipotassium anticoagulation，and leukocyte classification was performed by full-spectrum flow cytometry（flow method），fully automated hematology analyzer（instrument method） and microscopy（manual method）. Passing & Bablok regression was used to compare the determination results of the 3 methods for paired comparison. The manual method was used as a reference method to evaluate the consistency of the 3 methods of leukocyte classification methods. Results An 8-colour flow cytometry leukocyte counting protocol was established successfully. The linear probability values（P） of regression equations for the classification results of instrument method and flow method were >0.05. The slopes of the regression equations for neutrophil（NEUT），lymphocyte（LYMPH），monocyte（MONO），eosinophil （EOS）and basophil （BASO）of the 2 methods were 1.01，0.98，0.97，1.00 and 2.33，respectively，and the intercepts were -1.53，0.90，-0.11，0.01 and -0.23，respectively. The linear probability values（P） of regression equations for the classification results of manual method and instrument method were >0.05. The slopes of the regression equations for NEUT，LYMPH，MONO，EOS，and BASO of the manual method and flow method were 0.98，0.94，0.68，0.86 and 0.50，respectively，and the intercepts were -0.96，2.64，2.74，0.29 and 0.30，respectively. The slopes of the regression equations for NEUT，LYMPH，MONO，EOS，and BASO by the manual method and instrument method were 0.99，0.94，0.76，0.93 and 0.12，with the intercepts of -1.13，1.79，2.69，0.25 and 0.26，respectively. The slopes of the regression equations for NEUT，LYMPH，MONO，EOS，and BASO by the leukocyte classification was evaluated by the manual method of leukocyte classification，and the classification results of flow method and instrument method were within the confidence interval，but the flow method and the instrument method were evaluated as superior to the flow method and the manual method from the methodological comparison for correlation. Conclusions The 8-colour flow cytometry leukocyte classification method has been established. The flow method is correlated with the reference method，and the results are within the confidence interval of the reference method. The methodological agreement between the flow method and the instrument method is better than that of the reference method，and thus can further improve the accuracy of leukocyte classification in clinical routine blood tests.

Key words: Leukocyte classification, Flow cytometry, Reference method