Optimal gating strategies for determining bronchoalveolar lavage CD4/CD8 lymphocyte ratios by flow cytometry

Alterations in bronchoalveolar lavage (BAL) CD4/CD8 T cell subset ratios have been demonstrated in a variety of different respiratory disorders and the measurement of these changes may be diagnostically helpful. Flow cytometry (FCM) is a precise technology that offers many advantages over conventional cytospin techniques to determine T cell subset ratios in tissue fluids such as BAL. However, the optimum gating strategies for evaluating these parameters by FCM have not been evaluated. Here, the CD4/CD8 ratios in 33 BAL samples were compared using three different methods by FCM with two different flow cytometers. Bland Altman analysis demonstrated clinically insignificant differences between two simplified staining and gating strategies and a more complex "gold standard" method. These findings confirm the precision of FCM for BAL T cell subset ratio analysis and suggest that the optimal gating strategy may be a simple panel using only CD45, CD4 and CD8.