Objective To develop a novel drug lymphocyte stimulation test( DLST) using cell counting kit- 8( CCK- 8) assay,and to investigate its application in the diagnosis of acute drug- induced liver injury( DILI). Methods The patients with acute DILI who were admitted to Beijing You An Hospital from January 2011 to December 2014 were screened,and the suspected drugs for liver injury were collected. The patients' peripheral blood mononuclear cells( PBMCs) were isolated,cultured in vitro,and then incubated with the suspected drugs. The CCK- 8 assay was used to measure the proliferation of lymphocytes. A positive control group was established for each experiment and was stimulated by phytohemagglutinin. Meanwhile,PBMCs from healthy subjects were enrolled as the negative control group,and were exposed to the same drugs and measured simultaneously. The results of DLST were introduced into the Roussel Uclaf Causality Assessment Method( RUCAM) scoring system to assess their effects on the sensitivity of the diagnosis of acute DILI. The chi- square test was used for comparison of categorical data between groups. Results Preliminary experiments were performed to determine the experimental conditions of DLST based on CCK- 8 assay. A total of 61 patients with acute DILI were enrolled. DLST based on CCK- 8 assay was completed in 52 patients who received a total of 75 drugs,and the results were identified to be reliable in 48 patients with 70 drugs. A total of 19 patients with22 drugs achieved positive results of DLST,with positive rates of patients and drugs being 39. 6% and 31. 4%,respectively,and a specificity of 93. 8%. Thirteen( 38. 2%) out of 34 traditional Chinese medicines and 9( 25%) out of 36 Western medicines achieved positive results of DLST,and the median stimulation index was 2. 29( 1. 81- 14. 20). After the results of DLST were introduced into the RUCAM scoring system,the proportion of drugs assessed as“highly probable”or “probable”were increased from 74. 7% to 92. 0%( χ2= 8. 112,P =0. 004). Conclusion CCK- 8 assay can be used to perform DLST,and the introduction of DLST results into the RUCAM scoring system can significantly improve the sensitivity of RUCAM in the diagnosis of DILI. Compared with the conventional DLST,DLST based on CCK-8 has the advantages of few requirements for laboratories,a simple and timesaving procedure,and low costs,and can be further used and verified in clinical practice.
DownLoad: