Improved immnunophenotyping of lymphocytes in bronchoalveolar lavage fluid(BALF) by flow cytometry

Background: The immnunophenotyping of lymphocytes in bronchoalveolar lavage fluid (BALF) is of particular importance in the differential diagnosis of interstitial lung disorders. The standard method of lymphocyte phenotyping is peroxidase-anti-peroxidase technique (PAP). However, it was time-consumi ng and experience-dependent. Flow cytometric (FCM) analysis of BALF lymphoc ytes was introduced to overcome these disadvantages. Unfortunately, when th e number of cells counted was small, FCM could not distinguish lymphocytes from other cells and particles in BALF by light scattering. Methods: We est ablished a tri-color flow cytometric approach to phenotyping of lymphocytes in BALF. FITC-CD45/PE-CD14 antibodies were used to gate lymphocytes and ex clude other contamination. Propidium iodide (PI) was introduced to distingu ish lymphocytes from debris. Forty-three BALF species were tested by flow c ytometer as well as peripheral blood as a control group by conventional PAP method. Results: The variation of FCM (CV < 1.0%) was much lower that that of PAP method (CV > 9.8%). Meanwhile, we found that BALF had more clinical significance than peripheral blood in T subset analysis (p < 0.01). There were characteristic changes in some lung diseases. Both CD3 and CD4 were si gnificantly increased with decreasing CD8 in sarcoidosis (n = 14). Idiopath ic pulmonary fibrosis (n = 16) demonstrated the reverse tendency: CD8 rose but both CD3 and CD4 dropped. As for lung cancer (n = 7), CD3 was normal bu t the CD4/CD8 ratio declined. Tuberculosis of the lungs (n = 6) showed a no rmal CD3, CD4 and CD8. Conclusions: The high precision and reliability of t ri-color flow cytometric approach to phenotyping of lymphocytes in BALF sug gested that it should be used as a routine test, especially of BALE, which was often contaminated by inorganic particles. (C) 2001 Elsevier Science B. V. All rights reserved.