Immunologic analysis of blood samples obtained from horses and stored for twenty-four hours

Sharon Witonsky Department of Large Animal Clinical Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0442.

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Robert M. Gogal Jr Center for Molecular Medicine and Infectious Disease, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0442.
Department of Biomedical Sciences, College of Osteopathic Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0442.

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Virginia Buechner-Maxwell Department of Large Animal Clinical Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0442.

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S. Ansar Ahmed Center for Molecular Medicine and Infectious Disease, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0442.

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Abstract

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)

Abstract

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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