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)
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
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;