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  • Author or Editor: Robert M. Gogal Jr x
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Objective—To compare results of a nonradioactive colorimetric microplate assay with results of a traditional radioactive proliferation assay for determination of its use as a reliable and accurate alternative method for determination of proliferative activity of feline lymphocytes.

Sample Population—Blood samples from 10 clinically normal domestic shorthair cats.

Procedure—Double-density gradient separation was used to isolate mononuclear cells. Isolated cells were stimulated with various concentrations of concanavalin A (Con-A) and cultured for 72 hours. Lymphocyte proliferation was measured by radioactive ([3H]thymidine) and nonradioactive (colorimetric) techniques. Immunophenotypic analysis with felinespecific CD4+ and CD8+ monoclonal antibody was performed, using flow cytometry.

Results—Mononuclear cells were successfully isolated (97 to 99% purity and viability) from blood samples. A similar dose-dependent proliferative response to Con-A stimulation was measured with [3H]thymidine incorporation and the colorimetric assay. For both techniques, concentrations of 0.1 and 1.0 µg of Con-A/ml were submitogenic, and 100 µg/ml was toxic to cultured cells. For both techniques, maximal proliferation was observed with 5 µg of Con-A/ml.

Conclusion and Clinical Relevance—These results indicate that the nonradioactive colorimetric technique is a reliable and accurate method for measuring proliferative activity of feline lymphocytes. Clinically, this assay can be used as part of a screening process to determine immunocompetence of at-risk cats and to evaluate treatments for cats with immune-mediated or T-cell-dependent diseases. (Am J Vet Res 2001; 62:567–571)

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in American Journal of Veterinary Research


Objective—To determine whether immune function can be accurately assessed in blood samples obtained from horses and refrigerated overnight and whether a nonradioactive lymphocyte proliferation assay can be used to evaluate samples obtained from horses.

Sample Population—224 blood samples from 28 clinically normal adult horses.

Procedure—Heparinized blood samples were collected. Each sample was divided into 2 equal aliquots. One aliquot was refrigerated overnight to simulate overnight shipping of blood samples, and the other aliquot was evaluated on the day of blood collection. Lymphocytes were isolated and enumerated by use of a modified single-gradient procedure. Cell viability and function were assessed by use of cytologic examination, flow cytometry, and mitogen-induced proliferation assays. Lymphocyte proliferation in response to T- and B-cell mitogens was measured by use of [3H]-thymidine incorporation and a nonradioactive lymphocyte proliferation assay.

Results—Lymphocytes refrigerated for up to 24 hours continued to be acceptable for use in immunologic analysis on the basis that they maintained viability and did not have significant alterations in lymphocyte subsets, except for CD8, when compared with freshly isolated lymphocytes. Furthermore, results for mitogeninduced lymphocyte proliferation assays were also comparable between fresh and refrigerated aliquots.

Conclusions and Clinical Relevance—The nonradioactive lymphocyte proliferation assay is a reliable alternative to [3H]-thymidine assay for assessing proliferation of equine lymphocytes. Collectively, our results imply that blood samples refrigerated and shipped overnight to a laboratory can be used to perform cellular-immune assays; results of those assays would enhance a clinician's diagnostic abilities to monitor the efficacy of treatment. (Am J Vet Res 2003;64:1003–1009)

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in American Journal of Veterinary Research