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

The quantification of rat splenic lymphocyte populations using immunof luorescent staining and flow cytometry was evaluated in an interlabora tory study involving 6 independent facilities employing a common proto col. The study had three specific aims: (1) to establish baseline valu es for rat splenic lymphocyte populations, (2) to examine variability in flow cytometry data both within and among Laboratories, and (3) to evaluate single versus dual cell labeling of T-cell subpopulations, as well as quadrant and histogram analysis procedures. Splenic Lymphocyt es were enumerated following the lysis of red blood cells (RBC) with a mmonium chloride. B-cells (anti-rat kappa light chain; clone OX12) wer e examined using a single immunofluorescent label, whereas T-cells (an ti-CD5; OX19), T-helper (T-h) (anti-CD4; W3/25), and T-cylotoxic/suppr essor (T-cyt/sup) (anti-CD8; OX8) cells were examined using both singl e 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 approxim ately 20% of rat splenocytes are not lymphocytes. For quantitating rel ative and absolute numbers of lymphocytes, intralaboratory variability was similar across analysis and labeling procedures. Intralaboratory, variability was higher and interlaboratory reference ranges larger wh en absolute versus relative numbers of lymphocytes were compared. Stat istical performance indices indicated that no analysis or labeling met hod was consistently performed among the laboratories. Since monoclona l antibodies for labeling rat T-h and T-cyt/sup cells recognize cross- reacting epitopes on other cell populations, the authors recommend dua l 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 o btained in this study using dual labeling of T-h and T-cyt/sup cells a nd quadrant analysis: 20 +/- 1 (2.2 +/- 0.2 X 10(8)) W3/25OX19(+) cel ls; 17 +/- 2 (1.8 +/- 0.3 x 10(8)) OX8(+)OX19(+) cells; 43 +/- 2 (4.7 +/- 0.5 x 10(8)) OX19(+) cells; and 39 +/- 2 (4.3 +/- 0.5 x 10(8)) OX1 2(+) cells. This study indicates that additional standardization of sa mple preparation and analysis procedures (i.e., RBC lysis and cell gat ing) may be needed in the use of flow cytometry in immunotoxicology st udies.