Abstract: ObjectiveTo investigate the detection results of lymphocyte subsets in patients with high direct bilirubin（D-Bil) after processing their samples using two methods,and compare the diferences between the two methods.MethodsForty hyperbilirubin patients detected lymphocyte subsets at Xianyang Hospital of Yan'an University from October 2023 to September 2024, including 25 high D-Bil（D-Bil>130 pmol/L)and 15 high indirect bilirubin(I-Bil)(I-Bil>130 pmol/L) patients,were selected. The samples with high D-Bil were processed using two methods: the dilution method（adjusting 50 pL of whole blood to 5 pL) and the staining-lysis-washing method. The absolute counts of lymphocyte subsets were detected by flow cytometry,and the results of the two methods were analyzed. ResultsThe division of CD3 and CD3* cell populations in high I-Bil samples was not affected by the concentration of bili-rubin,while that in high D-Bil samples was influenced by the concentration of bilirubin.With the increase of D-Bil concentration, the CD3 cell population gradually approached the CD3* cell population. When the concentration of D-Bil> 130pmol/L,the gating of CD3* cell population was affected. Both the dilution method and the staining-lysis-washing method could eliminate the interference of D-Bil on the fluorescein isothiocyanate （FITC) fluorescence signal in high D-Bil samples，and there was no statistically significant difference in the absolute counts of CD45*，CD3*，CD3*CD4*，CD3*CD8*，CD3-CD19*，and CD3-CD16' CD56* cells after treat-ment with the two methods（P>0.05). ConclusionWhen the concentration of D-Bil>130 pmol/L, the gating of the CD3* cell population can be affected. Both the dilution method and the staining-lysis-washing method can eliminate the interference of D-Bil on the FITC fluorescence signal without affecting the accuracy of the results.