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Low cost CD4 enumeration using generic monoclonal antibody reagents and a two-color user-defined MultiSET protocol
Kovit Pattanapanyasat1, Hla Shain, Varipin Prasertsilpa
1Center of Excellence for Flow Cytometry, Office for Research and Development, Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand. grkpy@mahidol.ac.th
Background:
The standard three-tube, three-color flow cytometric method utilizing the TriTEST reagents in conjunction with the MultiSET software commonly used in most laboratories in Thailand for CD4 enumeration is expensive and thus unavailable to most HIV-infected patients. A more affordable method, i.e., the PanLeucogating protocol using only two monoclonal antibody reagents, has been described but requires the use of the CellQUEST software that does not have automatic gating and reporting facilities. We describe a simple protocol that utilizes a two-color user-defined protocol with the automated MultiSET software for the acquisition, analysis, and reporting of CD4 results.
Methods:
A two-color user-defined protocol was set up following instructions in the Becton Dickinson Biosciences MultiSET manual, adhering strictly to the information regarding the Gate and Attractor Hierarchy for analyzing various reagent combinations. This simple two-color user-defined MultiSET software was evaluated using generic monoclonal reagents in comparison with the standard TriTEST/MultiSET protocol.
Results:
The two-color user-defined MultiSET software is easy to use. It requires only modification of the original MultiSET program and the results obtained are comparable with those derived from the standard TriTEST/MultiSET protocol.
Conclusion:
The use of this easy and reliable two-color user-defined MultiSET protocol represents an affordable alternative to CD4 testing in resource-poor settings.
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