Am. J. Respir. Crit. Care Med., Vol 149, No. 3, 03 1994, 636-640.
Quantification of cell loss during bronchoalveolar lavage fluid processing. Effects of fixation and staining methods
H Moumouni, P Garaud, P Diot, E Lemarie and P Anthonioz
Department des Maladies Respiratoires, Centre Hospitalier Universitaire de Tours, France.
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 result of
selective lymphocyte loss, while the exact mechanisms of the phenomenon
remain controversial. Observing a similar variation in differentials from
differently stained identical smears, we quantified the cell loss due to
staining procedures from 45 consecutive satisfactory BAL procedures. To do
this, we compared relative lymphocyte recovery on neat pooled lavage in a
hemocytometer with that from smears and cytopreps fixed and stained in
different ways. We found (1) A significant lymphocyte loss (p < 0.05)
whatever the staining method. (2) Different methods of fixation and
staining lead to considerable variation in differentials from slides
otherwise identically managed. The loss is higher during air-drying
fixation followed by staining with an aqueous medium such as Diff-Quik than
on spray-fixed slides stained in an alcohol medium such as Papanicolaou
stain. (3) The effect of lymphocyte loss on differentials is more important
when the initial lymphocytosis is less than 35%, and decreases to
nonsignificance when it exceeds 70%. The role of cytocentrifugation or
other manipulations in cell loss probably has been overestimated because
unknown effects of staining methods were 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 procedure for an accurate differential count
of BAL fluid must take into account fixation and staining methods.
