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.

Applicability and reproducibility of acute myeloid leukaemia stem cell assessment in a multi-centre setting.

Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection. Here we present the validation experiments of the assay in several large AML research centres, both in Europe and the United States. Variability within instruments and sample processing showed high correlations between different instruments (Rpearson  > 0·91, P < 0·001). Multi-centre testing introduced variation in reported LSC percentages but was found to be below the clinical relevant threshold. Clear gating protocols resulted in all laboratories being able to perform LSC assessment of the validation set. Participating centres were nearly unanimously able to distinguish LSChigh (>0·03% LSC) from LSClow (<0·03% LSC) despite inter-laboratory variation in reported LSC percentages. This study proves that the LSC assay is highly reproducible. These results together with the high prognostic impact of LSC load at diagnosis in AML patients render the one-tube LSC assessment a good marker for future risk classification.