QUANTIFICATION OF CELL LOSS DURING BRONCHOALVEOLAR LAVAGE FLUID PROCESSING - EFFECTS OF FIXATION AND STAINING METHODS

Discrepancies have been reported in differential cell counts according to the diverse processing methods used in bronchoalveolar lavage (BAL ) fluid management. The differences have proved to be mainly the resul t of selective lymphocyte loss, while the exact mechanisms of the phen omenon remain controversial. Observing a similar variation in differen tials from differently stained identical smears, we quantified the cel l loss due to staining procedures from 45 consecutive satisfactory BAL procedures. To do this, we compared relative lymphocyte recovery on n eat pooled lavage in a hemocytometer with that from smears and cytopre ps fixed and stained in different ways. We found (1) A significant lym phocyte loss (p < 0.05) whatever the staining method. (2) Different me thods of fixation and staining lead to considerable variation in diffe rentials from slides otherwise identically managed. The loss is higher during air-drying fixation followed by staining with an aqueous mediu m such as Diff-Quik than on spray-fixed slides stained in an alcohol m edium such as Papanicolaou stain. (3) The effect of lymphocyte loss on differentials is more important when the initial lymphocytosis is les s than 35%, and decreases to nonsignificance when it exceeds 70%. The role of cytocentrifugation or other manipulations in cell loss probabl y has been overestimated because unknown effects of staining methods w ere also attributed to these manipulations. We suggest that lymphocyte loss could arise from poor adherence on slides, which is exacerbated during aqueous staining if no artifice (e.g., spray fixation), is used to hold them. Thus, the definition of the long-awaited standard proce dure for an accurate differential count of BAL fluid must take into ac count fixation and staining methods.