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

In phase one of an interlaboratory study, baseline values for rat splenic lymphocyte populations were established. In phase two, rat splenic lymphocyte populations were evaluated using immunofluorescent staining and flow cytometry following exposure to the immunosuppressive agent cyclophosphamide (CY). The study involved four independent facilities employing a common protocol. All laboratories purchased animals and reagents from the same sources. The objective of phase two was to determine whether each laboratory could detect a significant change in the same splenic lymphocyte population(s) at the same or similar CY dose levels. Crl:CD R BR male rats were dosed by the intraperitoneal route with 1, 3, or 10 mg / kg CY for 4 days. On day 5, spleen cell number and weights were obtained and splenic lymphocytes were evaluated following the lysis of red blood cells with ammonium chloride. Splenic lymphocyte populations were enumerated with monoclonal antibodies using the dual labeling of T-cell subpopulations and quadrant analysis procedures. The no observable adverse effect level (NOAEL) for spleen weight was 1 mg / kg for two laboratories and 3 mg / kg for the remaining two laboratories. For spleen cell number, the NOAEL was 1 mg / kg for three of the laboratories and between 3 and 10 mg / kg for the fourth. For the relative percentages of each splenic lymphocyte population, three of the four laboratories were within one dose level of each other for the NOAEL. The NOAEL for the W3/25+OX19+/OX8+OX19+ ratio was 3 mg/kg for three of the laboratories and < 1 mg / kg for the remaining laboratory. With the exception of the absolute numbers of OX8+OX19+ cells, the NOAELs for each absolute lymphocyte population were within one dose level for three of the laboratories. The fourth laboratory had NOAELs for each absolute population of > 10 mg/kg. Using flow cytometry, each of the laboratories identified CY as an immunotoxic agent.