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(4) CD4 Double Platform. |
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Any haematological analyser plus any flow cytometer can be used in tandem as a "double platform" to obtain an absolute CD4 count [8]. |
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A 93% saving on reagent costs has been documented when compared to the 6 tube standard CDC protocol [8]. This saving has been achieved by replacing the CDC protocol of 6 tubes, each containing 2 MAbs (6x2), with a single tube containing CD4 MAb. A mean difference in the absolute CD4 counts measured by the two methods remained as low as <10/ul. Although potential savings can be achieved using CD4 "only", quality control on the testing is poor or non-existant. Inter-laboratory variations are high between laboratories using the double-platform option, indicating that there are pitfalls in the system in terms of matching the respective lymphoid populations on the two component instruments. Lymphocytes used as the common denominator introduce error. More accurate points of reference to ensure better quality control viz. PanLeucogating. |
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Legend to 'a': These two machines need to work in close co-ordination to identify the same populations of common denominator. These are either White Blood Cells (WBC) or Lymphocytes (Lymphs). CD4 T cell absolute counts can be calculated using the CD4% among lymphocytes (defined by Flow Cytometry) and multiplying this % value by the Absolute Lymphocyte Count (ALC; generated on a haematology analyser). This procedure is however not optimal because lymphocyte counts are imprecise (shown above) and not reliably matched to lymphocytes defined by various flow cytometric methods. A more reliable parameter is to use the Total White blood cell count as the common denominator for both instruments, referred to as "PanLeucogating". |
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CD4+/CD45 double staining and PanLeucogating on a double platform provides precise, affordable CD4 counts. |
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On haematological analysers the WBCC (leucocyte counts) are more reliable than Absolute Lymphocyte counts. On flow cytometers total WBCC are easily gated after staining with CD45 antibody. We show in [12] that on this 'double-platform' system the most reliable CD4 counts can be achieved using the concept of 'Pan-Leucogating'. This gating strategy facilitates the calculation of an absolute CD4 count using a WBCC (obtained on the haematological analyser) and a CD4% value (of CD4+ cells)of the CD45+ gated WBCC population (obtained on a flow cytometer). |
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It is therefore recommended that double platform CD4 enumeration should instead use the more reliable, quality controlled parameter WBC as the basis from which to measure the CD4 T cells, instead of using the lymphocyte population. This method produces a more accurate and reproducible CD4 absolute count. |
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Legend: The PanLeucogated CD4 counts obtained on 'double-platform' (DP) show a very high agreement with the absolute CD4 counts observed on the volumetric 'single platform' (SP) flow cytometer. This is documented using Bland-Altman statistical analysis (figure above). This correlation is excellent despite the fact that these samples were fixed using TransfixTM and investigated using DP PanLeucogating in Johannesburg and SP Volumetric FCM in London. The outliers numbered are fixation artefacts (see [12] for details) . |
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PanLeucogated Absolute CD4+ T cell enumeration using white blood cells (defined by means of antibody, nuclear dyes or other means) as the basis from which to perform an absolute CD4+ T cell count directly is registered under a provisional patent (Application No: 20015700) at the Registrar of Patents, Designs, Trade Marks and Copyright, RSA. South Africa is a signatory of the Paris Convention for the Protection of Industrial Property. |
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