Stability of immunophenotypic lymphoid markers in fixed canine peripheral blood for flow cytometric analysis

Abstract

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. Objectives: 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 morphology, 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 CytoChex 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.

The use of flow cytometry for immunophenotyping lymphoproliferative disorders in cats: a retrospective study of 19 cases

“…Although Streck Cell Preservative has not yet been validated for use in cats, it is routinely used by the authors for collection and shipment of feline and canine specimens for flow cytometric immunophenotyping. The authors' own experience shows that Streck Cell Preservative does not alter the expression of feline antigens or canine antigens …”

“…The authors' own experience shows that Streck Cell Preservative does not alter the expression of feline antigens or canine antigens. 37 Authors' preliminary studies (data not shown) indicate that flow cytometric immunophenotyping is also useful in the diagnosis and characterization of non-FeLV-associated feline leukaemia, although further studies are warranted.…”

Gamma delta T‐cell large granular lymphocyte lymphoma in a dog

“…All antibodies were from Serotec (Oxford, UK) except for anti‐TCRγδ provided by Leukocyte Antigen Biology Laboratory (UC Davis, CA, USA). Additional single color analysis for CD11d (clone CA11.8H2; AbD Serotec, Raleigh, North Carolina, USA) was performed at the Department of Veterinary Medicine, University of Cambridge with a FACSCalibur flow cytometer and CellQuest software (Becton‐Dickinson) . Overall, gated lymphoid cells were positive for CD3, CD5, CD45, and γδ‐TCR, and negative for the other antibodies tested (CD4, CD8, CD11d, CD21), confirming a γδ T‐cell origin.…”

A case of canine high‐grade T‐cell lymphoma immunophenotypically consistent with T‐zone lymphoma

“…Additionally, the loss of CD45 on T cells may have been secondary to delayed processing of the lymph node samples. A recent article showed that the expression of CD3 and CD45 decreased significantly starting 3 days after collection . Finally, it is possible that there was a continuum between the two disease processes.…”