Primary CD4 gating

(3) Primary CD4 gating.

"Primary immunological gating" is a simple concept based on the fact that various cell types can be more readily distinguished by immunological reagents (i.e. specific monoclonal antibodies; MAbs) then by their morphology (i.e. scatter features).

Beverley et al. & Loken et al. [9] suggested using CD45 MAbs to identify all leucocytes, and within the leucocytes, to discriminate between lymphocytes, monocytes and granulocytes on the basis of their different CD45 staining intensity. At that time this process was referred to as "back-gating"; this term means the same as: "primary immunological gating using CD45 MAb".

Mandy et al. [10] then introduced primary CD3 gating (or T-cell gating) as this MAb is fully specific for T lymphocytes.

Haemopoietic precursor cells can be counted using primary CD34 gating.

Mercolino et al. [13] introduced primary immunological gating to identify, using a volumetric machine, the absolute numbers of CD3+(T), CD19+ (B) and CD16+(NK) cells. Their sum, when added together, is the value of ImmunoSum or LymphoSum, i.e. a total lymphocyte count precisely determined by primary immunological gating.

These methods tend to be superior to morphology (or light scatter of cells) in identifying the relevant leucocytes or their subsets. The frugality of scatter features, as opposed to the robustness of immunological staining, is also illustrated by the fact that a variable proportion of cells can decrease their viability during travelling and show apoptotic scatter outside the proper scatter-gate (that has been drawn based on viable cells) but are still recognized by antibodies and therefore properly counted by immunological gating.

Legend to a:: CD4 staining intensity (y axis) and side scatter (x axis) are displayed, and CD4++ small T lymphocytes are gated (R1'). This is referred to as "primary CD4 gating" [11].

Primary CD4 gating" has also been successfully introduced (a) to save costs in monitoring HIV disease.

In routine clinical service >600 samples were tested using volumetric absolute counting with the full ORTHO reagent panel, identifying lymphocyte counts (by ImmunoSum) and absolute CD3 CD4, CD8, CD19(B). CD16(NK) cell numbers [11] . The same samples were run to use only the CD4 staining results for CD4 absolute counts (see 'b'). Only one sample (red arrow) showed a discrepancy. This however had abnormally low CD4 staining intensity.

When volumetric FCM is used in adults, a single CD4 antibody is sufficient to get reliable CD4 counts.

If the resources are poor and the clinicians request only CD4 counts, then it is not justifiable to perform a full panel of investigation with inherently high costs [7].

If clinicians are also interested in T lymphocyte and CD8 counts and resources are available for more than a single CD4 antibody an additional tube can also be set up for CD8 counts. These issues are discussed in [11].

In paediatric samples, however, staining with a single CD4 MAb may not be fully adequate.

In children CD4% values are more informative because absolute lymphocyte counts are age-dependent and therefore absolute CD4 counts are also highly variable. To obtain reliable CD4% values CD4+CD45 double staining is the optimal cost-saver since accurate manual gating for lymphocytes is time-consuming [12].

If clinicians are also interested in T lymphocyte and CD8 counts and resources are available for more than a single CD4 antibody, an additional tube can also be set up for CD8 counts. These issues are discussed in [11].

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