INTERLABORATORY EVALUATION OF THE QUANTIFICATION OF RAT SPLENIC LYMPHOCYTE SUBTYPES USING IMMUNOFLUORESCENT STAINING AND FLOW CYTOMETRY

Abstract

The quantification of rat splenic lymphocyte populations using immunofluorescent staining and flow cytometry was evaluated in an interlaboratory study involving 6 independent facilities employing a common protocol. The study had three specific aims: (1) to establish baseline values for rat splenic lymphocyte populations, (2) to examine variability in flow cytometry data both within and am ong laboratories, and (3) to evaluate single versus dual cell labeling of T-cell subpopulations, as well as quadrant and histogram analysis procedures. Splenic lymphocytes were enumerated following the lysis of red blood cells (RBC) with ammonium chloride. B-cells (anti-rat kappa light chain; clone OX12) were exam ined using a single im munofluorescent label, whereas T-cells (anti-CD5; OX19), T (T) (anti-CD4; W3/ 25), and T (T) helper h cytotoxic/ su ppressor cyt/ su p (anti-CD8; OX8) cells were examined using both single and dual labeling. Cell recoveries and viabilities reported by each laboratory following the lysis of RBC ranged from 27 to 87% and 82 to 95%, respectively. Splenic differential counts indicated that approximately 20% of rat splenocytes are not lymphocytes. For quantitating relative and absolute num bers of lymphocytes, intralaboratory variability was sim ilar across analysis and labeling procedures. Intralaboratory variability was higher and interlaboratory reference ranges larger when absolute versus relative numbers of lymphocytes were compared. Statistical performance indices indicated that no analysis or labeling method was consistently performed among the laboratories. Since monoclonal antibodies for labeling rat T and T cells recognize cross-reacting epitopes on other h cyt/ su p cell populations, the authors recomm end dual labeling of T-cell subpopulations and quadrant analysis procedures. The following are the overall mean relative (%) and absolute numbers, respectively, for each of the various splenic lymphocyte populations obtained in this study using dual labeling of T and T cells h cyt/ sup and quadrant analysis: 20 - 1 (2.2- 0.2 2 108) W3/ 25+OX19+ cells; 17 - 2 (1.8 - 0.3 2 108) OX8+OX19+ cells; 43 - 2 (4.7- 0.5 2 108) OX19+ cells; and 39 - 2 (4.3- 0.5 2 108) OX12+ cells. This study indicates that additional standardization of sample preparation and analysis procedures (i.e., RBC lysis and cell gating) may be needed in the use of flow cytometry in imm unotoxicology studies.

Keywords: :

• Keywords Flow Cytometry, Immunofluorescent Staining, Lymphocytes, Rat, Spleen