Quantification of Cells Recovered by Bronchoalveolar Lavage: Comparison of Cytocentrifuge Preparations with the Filter Method

Abstract

Controversy exists as to the appropriate methods to use in the processing of bronchoalveolar lavage (BAL) fluid for total cell numbers and cellular differential analysis. It has been shown that cell losses (primarily lymphocytes) occur by the most commonly employed methods. Therefore, we examined the total cell and differential counts obtained by several methods of cytocentrifuge preparation and by the filter preparation in 46 consecutive patients with interstitial lung disease and 29 healthy volunteers undergoing bronchoalveolar lavage. The retrieved lavage fluid was pooled, and an aliquot was used to determine the total cell count, cell viability, and the differential cell count by the filter and cytocentrifuge techniques. The remaining fluid was centrifuged (800 g for 10 min), and the cell pellet was resuspended in Hank's balanced salt solution without Ca²⁺ and Mg²⁺. An aliquot of these centrifuged and resuspended cells was used for repeat determination of the cell viability, total cell count, and cellular differential by cytocentrifuge technique. Autologous serum was added to another aliquot of these centrifuged and resuspended cells to arrive at a 10% protein solution, and the cellular differential obtained by cytocentrifuged preparation. We found that (1) measurement of the total cells recovered by lavage is most accurate if determined on the original uncentrifuged, pooled lavage fluid, (2) the centrifugation and resuspension of lavage cells causes a generalized loss of total cells with a decrease in cell viability, (3) the addition of serum to the cell suspension prior to preparation of the cytoprep slides results in a selective loss of lymphocytes, and (4) the use of uncentrifuged, pooled lavage fluid for the cytopreparation yields identical cell differential counts compared with those of the filter technique. Thus, quantification of the total cells recovered by BAL is most accurate when counted on an aliquot of the original cell suspension. Further, cytopreparations yield accurate quantification of lavage cellular constituents if serum is not added to the solution.