PHASE-2 OF AN INTERLABORATORY EVALUATION OF THE QUANTIFICATION OF RATSPLENIC LYMPHOCYTE SUBTYPES USING IMMUNOFLUORESCENT STAINING AND FLOW-CYTOMETRY

In phase one of art intel-laboratory study, baseline values for rat sp lenic lymphocyte populations were established. In phase two rat spleni c lymphocyte populations were evaluated using immunofluorescent staini ng and flow cytometry following exposure to the immunosuppressive agen t cyclophosphamide (CY). The study involved four independent facilitie s employing a common, protocol. All laboratories purchased animals and reagents from the same sources. The objective of phase two was to det ermine whether each Laboratory could detect a significant change in th e same splenic lymphocyte population(s) at the same or similar CY dose levels. Crl:CD(R) BR male rats were dosed by the intraperitoneal rout e with 1, 3, or 10 mg/kg CY for 4 days. On day 5, spleen cell number a nd weights were obtained and splenic lymphocytes were evaluated follow ing the lysis of red blood cells with ammonium chloride. Splenic lymph ocyte populations were enumerated with monoclonal antibodies using the dual labeling of T-cell subpopulations and quadrant analysis procedur es. The no observable adverse effect level (NOAEL) for spleen. weight was 1 mg/kg for true laboratories and 3 mg/kg for the remaining two la boratories. For spleen cell number the NOAEL was I mg/kg for three of the laboratories and between 3 and 10 mg/kg for the fourth. For the re lative percentages of each splenic lymphocyte population, three of the four laboratories were within one dose level of each other for the NO AEL.