External quality assessment of flow cytometric bronchoalveolar lavage cellular analysis: 20 years' experience in The Netherlands.

BACKGROUND: Bronchoalveolar (BAL) cellular analysis can be supportive in the diagnosis of interstitial lung disease. The flow cytometric analysis of BAL fluid cells is complicated by cell fragility and adherence and autofluorescence of macrophages, making conventional analysis of BAL fluid cells as done in external quality schemes (EQA) for blood lymphocyte subsets, not representative. Following a procedure for stabilized BAL cells, a separate EQA was set up. The results of 20 years' experience are presented. METHODS: From each round between 2000 and 2020 the following flow cytometric parameters were recorded from each participant: total lymphocyte population (TLY), CD3+ lymphocytes, CD3+ CD4+ lymphocytes, CD3+ CD8+ lymphocytes, CD3- CD16+/56+ lymphocytes, CD19+ lymphocytes and CD103 + CD3+ lymphocytes. In addition, the eosinophils and neutrophils were recorded. The mean and standard deviation of each parameter per round were calculated. The 40 rounds were divided in four respective groups of 10 in order to compare the results as function of time. In addition the interpretation of the results of participants was scored. RESULTS: The median SD in the four groups was below 10% for all parameters except for TLY and the CD103+ CD3+ lymphocytes. No improvement in time was observed for any (sub)population except for the CD3+ CD4+ subset. Interpretation of the results varied based on disease, with greatest consensus for sarcoidosis cases and lowest for nonspecific interstitial lung disease cases. CONCLUSIONS: A dedicated EQA for BAL fluid cellular analysis appears to be justified as the test material is substantially different from that of peripheral blood. We show that adequate analytical and post-analytical quality control can be achieved.

Comparison of three methods to stabilize bronchoalveolar lavage cells for flowcytometric analysis.

BACKGROUND: Flowcytometric analysis of lymphocytes and their subpopulations in bronchoalveolar lavages (BAL) can support the diagnosis of interstitial lung diseases. This analysis should be done within 4 hr after lavage due to rapid cell deterioration. We tested three methods in order to stabilize for at least 28 days the BAL cell populations to allow delayed flowcytometric analysis in order to facilitate external quality assurance (EQA). METHODS: We compared an in-house, dual-step stabilization method for BAL cells with results of two different commercial available stabilization reagents: TransFix® and Streck Cell Preservative™. All three methods were compared with native BAL cells as reference. BAL samples from six patients were tested on six occasions following stabilization from 1 to 28 days by flow cytometry. RESULTS: Following stabilization and storage at 4°C, BAL cell suspensions had stable light scatter patterns and lymphocyte subsets. As expected, rapid deterioration of cells was seen with native BAL cells. The stabilized lavages showed more stable counts of WBC and lymphocyte populations with only minor differences found between the three methods. CONCLUSIONS: If analysis of the BAL cells is performed more than 24 hr after the lavage, stabilized BAL cells are superior to native cells. The in-house method can be used for EQA purposes with stability for at least 28 days. The TransFix and Streck methods might be useful for postponed diagnostic analysis of lavage cells but did not meet our 28 days criterion defined needed for EQA purposes.