Stability of immunophenotypic lymphoid markers in fixed canine peripheral blood for flow cytometric analysis.
BACKGROUND
Flow cytometric analysis of blood samples for immunophenotyping lymphoproliferative diseases has become popular in veterinary medicine. Unfortunately, the use of this technique has been limited by the necessity to test samples within a short time frame after blood collection. A possible solution to this problem is the use of fixative products to preserve the stability of lymphoid antigens.


OBJECTIVE
The aim of this study was to evaluate the expression of 5 lymphoid surface markers (CD3, CD4, CD8, CD21, and CD45) in blood samples collected in K3-EDTA and Cyto-Chex BCT tubes from healthy dogs.


METHODS
Blood from 8 dogs was collected in K3-EDTA and Cyto-Chex BCT tubes and analyzed by flow cytometry at 6 hours, one day, 3 days, and 7 days after collection. Lymphocyte percentage, lymphocyte mor-phology, expression of lymphoid surface markers and mean fluorescence intensity (MFI) were recorded at each time point and compared.


RESULTS
Lymphocyte percentage and morphology were preserved up to 3 days in samples collected in Cyto-Chex BCT, and lymphocyte percentage was mildly decreased on day 7. CD4, CD8, and CD21 were stable in Cyto-Chex BCT up to 7 days, whereas CD3 and CD45 showed a significant decrease in expression from day 3, with a decrease on average of 21% and 2.4%, respectively, on day 7. MFI was significantly decreased on day 7 for all markers except CD21.


CONCLUSIONS
These findings indicate that storage of samples in Cyto-Chex BCT affects lymphoid marker expression and caution should be exercised when interpreting data produced on such samples.
Department of Veterinary Medicine, University of Cambridge, Cambridge, UK.
Francesco
F
Cian
Maciej
M
Guzera
Simon
S
Frost
Stefaan
S
Van Poucke
Stefano
S
Comazzi
Joy
J
Archer
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D015415:Biomarkers / Q000097:blood; D001773:Blood Cells / Q000166:cytology / Q000276:immunology; D001793:Blood Preservation / Q000662:veterinary*; D004285:Dogs; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D016130:Immunophenotyping / Q000662:veterinary; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology*; D008297:Male; D013048:Specimen Handling / Q000295:instrumentation / Q000662:veterinary*; D013997:Time Factors
D000954:Antigens, Surface; D015415:Biomarkers
Blood cell storage; Cyto-Chex BCT; surface markers