Poster Presentation Australasian Cytometry Society 44th Annual Conference and Workshop

Comparison Of A Dye Based Flow Cytometry And Tritiated Thymidine Proliferation Method For The Assessment Of Lymphocyte Proliferation In Clinical Samples Submitted For Routine Testing (24550)

Sarah Brown 1 , Stephanie Phung 1 , Samuel Neeling 2 , Grace Gong 2 3 , Christine Bundell 1 2
  1. University of Western Australia, School of Biomedical Sciences, Nedlands, Western Australia, Australia
  2. PathWest Laboratory Medicine, QEII Medical Centre, Nedlands, Western Australia, Australia
  3. Clinical Immunology, Perth Children's Hospital, Nedlands, Western Australia, Australia

Background: Lymphocyte responses to mitogens are used to investigate suspected immune deficiencies in patients.  The current diagnostic laboratory approach uses tritiated thymidine incorporation following ex vivo culturing with mitogens.  This radioactive assay has complex logistical requirements requiring long term storage of waste material.

We investigated a flow-based method for assessing lymphocyte proliferation and specifically T-cell proliferation in peripheral blood mononuclear cells (PBMC) by measuring Violet Proliferation Dye 450 (VPD450) incorporation over 120 hours of cell culture.  New generations of progeny cells show a reduction in VPD450 fluorescence intensity which correlates with cycles of lymphocyte proliferation. This flow-based method is proposed as an alternative method which eliminates the use of radioactive material and allows proliferation of subsets of T-cells to be investigated.  

Method: Fresh peripheral blood was obtained from healthy volunteers and patients under investigation for immune deficiency.  PBMCs were cultured with mitogens concanavalin-A and phytohemagglutinin at increasing concentrations and either pulsed with tritiated thymidine 18 hours prior to harvesting at 72 hours or preincubated with 10mM VPD450 and then incubated with the mitogens for 120 hours. VPD450 cultures were prepared for flow cytometry and stained with fluorescent markers conjugated to anti CD3, CD4 and CD8.

Results: Proliferation measured by flow cytometry was evident for both mitogens at all concentrations after 120 hours (Phytohemagglutinin: Figure 1). Parental populations were diminished after 120hours as proliferation occurred.   Lymphocyte proliferation was evident in all samples tested.  Consistent with the cell population analysis, CD8 T-cell proliferation was not detected in a patient shown to have absent CD8 T-cells.

Conclusion: A flow-based method is adequate for assessing lymphocyte proliferation in freshly prepared patient PBMCs incubated with mitogens for 120 hours. The results obtained from this assay are comparable to the existing complex tritiated thymidine method and eliminate the need for radiation protocols and safety management. 

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