Flow Cytometry
Manual methods using cell selection and microscope counting techniques can be used, however, automated counting of CD4+ Helper T lymphocytes in blood is usually performed on a flow cytometer. Although more expensive, this method is more accurate and less labour intensive.
The flow cytometer uses fluorescent cell surface markers (usually these are labelled antibodies) to identify the subset of CD4+ Helper T lymphocytes present in blood samples.
There are two approaches regarding the type of cell surface markers used.
1) Conventional CD4 counting (CD3 and CD4 labelling)
The conventional method makes use of fluorescent antibodies to CD3 and CD4 cell surface receptors. CD3 is part of the T cell receptor (TCR) and is expressed on CD8+ Cytotoxic T lymphocytes and CD4+ Helper T lymphocytes. CD4 is expressed on Helper T lymphocytes and monocytes. Using fluorescence detection on a flow cytometer the fraction of total lymphocytes double-positive for CD3 and CD4 (i.e. CD4+ Helper lymphocytes) can be measured. For single platform assays the absolute number of the double-positive cells can be inferred from calibrated bead standards or by determining the volume of blood used with a volumetric flow cytometer. The dual-platform method relies on a full blood count by a haematology analyser to provide a value for the total lymphocyte count, while the flow cytometer gives a percentage of lymphocytes that are CD4+ Helper T lymphocytes. Together these readings can be used to calculate absolute CD4 counts. The most reliable and robust counting procedure is the single platform assay.
2) CD4 counting by the PanLeucogate method (CD45 and CD4 labelling)
An alternative labelling method uses fluorescent antibodies to CD45 and CD4 cell surface receptors. CD45 is expressed on all leucocytes and is not expressed on mature red blood cells (erythrocytes) or platelets (thrombocytes). It therefore serves as a marker of total white blood cells. CD4 is expressed on Helper T lymphocytes and monocytes. The dual platform method requires a haematology analyzer to provide only the total leukocyte count. This is the reason PanLeucogating is preferred when using the dual platform method, only a total white cell count is needed from the haematology analyzer and the need for a differential count (the lymphocyte count) from the haematology analyser is no longer necessary. The leucocytes serve as a common counting parameter when using the flow cytometer and haematology analyzer. Since CD4 is expressed on both Helper T lymphocytes and monocytes the light scatter parameters available on the flow cytometer are used to differentiate between them because of differences in complexity between the populations. This method can also be used on a single platform basis using calibrated bead standards or a volumetric flow cytometer to calculate the absolute CD4 count.
