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Updated: May 3, 2026
Flow Cytometric Characterization of Murine B Cell Development
Published on: January 22, 2021
Stability of immunophenotypic lymphoid markers in fixed canine peripheral blood for flow cytometric analysis
Francesco Cian1, Maciej Guzera, Simon Frost
1Department of Veterinary Medicine, University of Cambridge, Cambridge, UK.
Insights
Blood sample preservation for canine lymphoproliferative disease diagnosis using Cyto-Chex BCT tubes shows variable stability for lymphoid markers. CD3 and CD45 expression decreased significantly by day 7, impacting flow cytometry results.
Area of Science:
- Veterinary immunology
- Flow cytometry applications
- Diagnostic hematology
Background:
- Flow cytometry is crucial for immunophenotyping canine lymphoproliferative diseases.
- Limited sample stability necessitates rapid analysis, posing logistical challenges.
- Preservative tube technology offers a potential solution for extended sample viability.
Purpose of the Study:
- To assess the stability of five key lymphoid surface markers (CD3, CD4, CD8, CD21, CD45) in canine blood.
- To compare antigen expression in samples collected in K3-EDTA versus Cyto-Chex BCT tubes.
- To evaluate the impact of storage duration on flow cytometric analysis.
Main Methods:
- Blood from 8 healthy dogs was collected in K3-EDTA and Cyto-Chex BCT tubes.
- Samples were analyzed via flow cytometry at 6 hours, 1, 3, and 7 days post-collection.
- Lymphocyte characteristics and surface marker expression (CD3, CD4, CD8, CD21, CD45) were quantified.
Main Results:
- Lymphocyte percentage and morphology remained stable in Cyto-Chex BCT for up to 3 days.
- CD4, CD8, and CD21 expression were stable for 7 days in Cyto-Chex BCT.
- CD3 and CD45 expression significantly decreased from day 3, with reduced mean fluorescence intensity (MFI) by day 7 for most markers.
Conclusions:
- Cyto-Chex BCT tubes impact lymphoid marker expression, particularly for CD3 and CD45.
- Interpretation of flow cytometry data from Cyto-Chex BCT samples requires caution.
- Further studies are needed to optimize protocols for extended sample preservation in veterinary diagnostics.
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 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.
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