Quantification of cell loss during bronchoalveolar lavage fluid processing. Effects of fixation and staining methods.

Abstract

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.

Lymphocyte Subsets in Bronchoalveolar Lavage Fluid of Children with Lung Infiltrates

“…The most representative age group was up to 5 years. 71% of the patients with lung infiltrates in our study were below the age of 5, as other authors have reported (9,10). In our study, the presentation of CD4+and CD8+ lymphocytes in BAL in the studied group shows a small percentage of CD8+ lymphocytes as an immune response in 8-10% of patients, while the cellular response of CD4 +lymphocytes in the sample itself was present up to 14% in the entire group of the diseased children.…”

Effects of on‐slide fixation on the cell quality of cytocentrifuged equine bronchioalveolar lavage fluid

“…To date, the effects of on‐slide fixation on BALF samples have only been determined concerning lymphocyte loss and identification of mast cells in human BALF and included a wet‐spray fixation method, and formaldehyde and methanol fixation methods . In equine clinical diagnostics, fixatives such as a formalin saline solution and Saccamanno fixative (an alcohol‐based solution) have been used exclusively as prefixatives added to BALF prior to slide preparation but on‐slide fixation methods on the prepared slide as performed in the present study have not been published.…”

Hope-Bal

“…BAMBI, BMP and activin membrane-bound inhibitor. (Xaubet et al 1991;Moumouni et al 1994), there is still no notable method for long-term storage of BAL. Therefore, this study presents a novel and simple method for fixation of BAL cells that allows a plethora of applications besides long-term storage.…”