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Published on: February 8, 2016
Rapid, effective and user-friendly immunophenotyping of canine lymphoma using a personal flow cytometer
Stratos Papakonstantinou1, Inese Berzina1, Amanda Lawlor1
1School of Veterinary Medicine, University College Dublin (UCD), Dublin, Ireland.
Insights
A new personal flow cytometer offers rapid, user-friendly canine lymphoma immunophenotyping. This accessible technology provides results comparable to conventional methods, aiding diagnosis and prognosis in veterinary medicine.
Area of Science:
- Veterinary Medicine
- Immunology
- Oncology
Background:
- Conventional flow cytometry for canine lymphoma diagnosis is limited by cost, space, and expertise.
- The Guava EasyCyte Plus personal flow cytometer addresses these limitations.
Purpose of the Study:
- Adapt a canine lymphoma immunophenotyping protocol for a personal flow cytometer.
- Demonstrate its practicality for widespread veterinary application in diagnosing and prognosing canine lymphoma.
- Assess performance using two monoclonal antibodies (CD3 and CD21) compared to conventional cytometers.
Main Methods:
- Adapted immunohistochemical protocol for personal flow cytometry.
- Used monoclonal antibodies against CD3 and CD21 for T and B lymphocyte immunophenotyping.
- Compared results with conventional flow cytometers and automated hematology analyzers.
Main Results:
- Protocol completion in 2-3 hours.
- Personal flow cytometer lymphocyte counts highly correlated with automated analyzers (r=0.97).
- Immunophenotype counts were indistinguishable from conventional flow cytometers (blood: r=0.95, lymph node: r=0.98).
Conclusions:
- Two monoclonal antibodies are sufficient for rapid canine lymphoma immunophenotyping.
- Personal flow cytometer is as effective as conventional methods.
- Personal flow cytometer offers greater accessibility, user-friendliness, and requires lower sample volumes.
Background:
Widespread use of flow cytometry for immunophenotyping in clinical veterinary medicine is limited by cost and requirement for considerable laboratory space, staff time, and expertise. The Guava EasyCyte Plus (Guava Technologies, Hayward, CA, US) is the first, personal, bench-top flow cytometer designed to address these limitations.
Objective:
The aim of this study was to adapt the immunohistochemical protocol used for immunophenotyping of canine lymphoma to the personal flow cytometer for rapid, effective and user-friendly application to the diagnosis and prognosis of canine lymphoma and to demonstrate its practicality for widespread veterinary application. Performance of the personal flow cytometer for immunophenotyping T and B lymphocytes in blood and lymph nodes from normal dogs and dogs with lymphoproliferative disease, was assessed using only two monoclonal antibodies (against CD3 and CD21), and by comparison with analysis using two conventional flow cytometers.
Methods:
26 dogs with lymphoproliferative disease (23 with lymphoma, 3 with lymphocytic leukaemia) were studied along with 15 controls (2 non-lymphoma lymph nodes and 13 non-leukemic bloods. Lymphocytes were immunostained with fluorescent-labeled, monoclonal antibodies against CD3 and CD21. To assess the effectiveness of the personal flow cytometer in discrimination between T and B cell immunophenotypes, T and B cell counts for half the samples (14 blood and 11 lymph node) were also determined using the same method and conventional flow cytometers (FACSCalibur, Cyan Dako). To assess the effectiveness of the personal flow cytometer in discriminating between leukocyte types, lymphocyte differential counts were determined for 21 blood samples and compared with those from automated hematology analyzers (CELL-DYN 3500, n=11 and ADVIA 2120, n=10). Quality and sub-cellular distribution of immunostaining was assessed using fluorescence microscopy.
Results:
The protocol for immunophenotyping took 2 to 3 hours to complete from the point of receipt of sample to reporting of immunophenotype. The personal flow cytometer differential lymphocyte counts correlated highly (n=20; r=0.97, p<0.0001) with those of automated haematology analyzers. The personal flow cytometer counts consistently, but mildly, underestimated the percentages of lymphocytes in the samples (mean bias of -5.3%.). The personal flow cytometer immunophenotype counts were indistinguishable from those of conventional flow cytometers for both peripheral blood samples (n=13; r=0.95; p<0.0001; bias of -1.1%) and lymph node aspirates (n=11,r=0.98; p<0.001; bias of 1%). All but one leukemic and one lymphomatous lymph node sample, out of 26 samples of dogs with lymphoproliferative disease analyzed, could be immunophenotyped as either B or T cells.
Conclusions:
We conclude that use of only 2 monoclonal antibodies is sufficient for immunophenotyping most cases of canine lymphoma by flow cytometry and enables rapid immunophenotyping. The personal flow cytometer may be as effectively used for immunophenotyping canine lymphoma as conventional flow cytometers. However, the personal flow cytometer is more accessible and user-friendly, and requires lower sample volumes.
