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

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-consuming and experience-dependent. Flow cytometric (FCM) analysis of BALF lymphocytes was introduced to overcome these disadvantages. Unfortunately, when the number of cells counted was small, FCM could not distinguish lymphocytes from other cells and particles in BALF by light scattering.We established a tri-color flow cytometric approach to phenotyping of lymphocytes in BALF. FITC-CD45/PE-CD14 antibodies were used to gate lymphocytes and exclude other contamination. Propidium iodide (PI) was introduced to distinguish lymphocytes from debris. Forty-three BALF species were tested by flow cytometer as well as peripheral blood as a control group by conventional PAP method.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 significantly increased with decreasing CD8 in sarcoidosis (n=14). Idiopathic 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 but the CD4/CD8 ratio declined. Tuberculosis of the lungs (n=6) showed a normal CD3, CD4 and CD8.The high precision and reliability of tri-color flow cytometric approach to phenotyping of lymphocytes in BALF suggested that it should be used as a routine test, especially of BALF, which was often contaminated by inorganic particles.