Related Experiment Video
Updated: Mar 23, 2026
A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release
Published on: November 21, 2016
Identification of a suitable internal control for fluorescence analysis on canine peripheral blood samples
F Riondato1, V Martini2, A Poggi1
1Department of Veterinary Sciences, University of Turin, Largo P. Braccini 2, 10095 Grugliasco, Turin, Italy.
Insights
CD4 is the best internal control for canine flow cytometry analysis, showing stable fluorescence intensity across subjects and storage. This finding improves diagnostic and research comparisons in veterinary medicine.
Area of Science:
- Immunology
- Veterinary Medicine
- Flow Cytometry
Background:
- Reliable fluorescence intensity (FI) detection via flow cytometry (FC) is crucial for accurate measurements.
- Internal controls are essential for standardizing FC measurements due to instrument and sample variability.
- Commercially available bead standards are costly, limiting their use in veterinary diagnostics.
Purpose of the Study:
- To evaluate the stability of main lymphocyte antigen expression in canine peripheral blood.
- To identify a suitable antigen for use as a stable internal control in flow cytometry analyses.
- To assess antigen MFI variability within subjects, between subjects, and after sample storage.
Main Methods:
- Peripheral blood from 18 healthy dogs was analyzed using flow cytometry for CD3, CD5, CD4, CD8, CD21, and cyCD79b expression.
- Mean Fluorescence Intensity (MFI) was calculated as Relative Fluorescence Intensity (RFI) using fluorescent microbeads.
- Variability was assessed using coefficient of variation (CV) for cell-to-cell and inter-subject comparisons, and after 24-hour storage.
Main Results:
- CD4 demonstrated the lowest cell-to-cell and dog-to-dog variability among the tested lymphocyte antigens.
- CD4 expression showed optimal stability after 24-hour storage at 4°C.
- Breed, sex, and age did not significantly influence CD4 RFI in a larger cohort of 21 dogs.
Conclusions:
- CD4 is identified as the most suitable antigen for use as an internal control in canine flow cytometry MFI analyses.
- Normalizing fluorescence data using CD4 MFI can enhance the comparability of results across different laboratories and studies.
- Further research is recommended to validate these findings with diverse flow cytometry methodologies.
Abstract:
Reliable detection of fluorescence intensity (FI) by flow cytometry (FC) is fundamental. FI depends on instrument settings and sample processing procedures: thus, measurements should be done using internal controls with known FI. Commercially available beads-based standards are expensive, thus reducing their usability in the veterinary practice. Cell subsets with stable mean FI (MFI) within the population have been proposed as acceptable surrogates in human medicine. In veterinary medicine, no data exist about stability of antigen expression among different subjects or upon sample storage. The aim of the present study was to evaluate MFI variability of main lymphocytes antigens among the lymphoid cells within each subject, among different subjects, and upon 24-h storage, in order to identify the antigen most suitable as stable internal control in MFI analyses. Peripheral blood samples from 18 healthy dogs were analysed by FC within 3h from sampling to assess the expression of CD3, CD5, CD4, CD8, CD21 and cyCD79b using conjugated monoclonal antibodies. Analyses were restricted to the lymphoid population. Fluorescent microbeads were added to each tube, and antigen MFI was calculated as Relative Fluorescence Intensity RFI (CD/beads). Fluorescence histogram CV (fhCV) for each CD was regarded as an index of the variability of expression among lymphocytes within each subject (cell-to-cell variability); whereas the CV of RFI was regarded as an index of inter-subjects variability (dog-to-dog variability). In 11 cases, FC analyses were repeated after 24h storage at 4°C and RFI and CVs of fresh and stored samples were compared to assess variability linked to storage. CD4 was identified as the best antigen to be used as an internal control for MFI analyses in canine peripheral blood samples because of low cell-to-cell and dog-to-dog variability, and optimal stability upon 24-h storage. Blood samples from a second group of 21 healthy dogs were labelled only with CD4, in order to assess the influence of breed, sex and age on the expression of CD4 in a larger case series. Based on univariate GLMs, none of these variables influenced CD4 RFI. Normalizing fluorescence data using lymphoid CD4 MFI as a reference would improve the comparison of results obtained by different laboratories, patients or times in diagnostic and research analyses of FI. Further studies are needed to confirm our results with different FC approaches.
