Barbara J. Bain - Immunophenotyping for Haematologists

Figure 1.3 Delineation of peripheral blood or bone marrow leucocyte populations using CD45 expression and SSC.

A plot of CD45 expression and SSC is not only useful for separating normal cell populations but also helps identify precursor cell populations, which frequently show only weak CD45 expression ( Figure 1.3).

CD19 versus SSC ( Figure 1.4) and CD34 versus SSC ( Figure 1.5) plots are useful for isolating B cells and blast cells, respectively.

Back gating is a process whereby a target population identified in one approach can be tracked in another. For example, CD34+ myeloblasts can be isolated using CD34 versus SSC, then colour tracked into the FSC versus SSC plot to show cell size and granularity. With modern multichannel instruments it is possible to study 6–8 or more antigens in a single tube. If multiple tubes are studied, several core antibody‐fluorochrome conjugates can be included in each tube analysed so that cross‐comparison between the same cells stained with different antibody panels in different tubes is possible.

Figure 1.4 Delineation of peripheral blood or bone marrow B‐cell populations using CD19 expression and SSC.

Figure 1.5 Delineation of peripheral blood or bone marrow CD34+ blast populations using CD34 expression and SSC.

Flow cytometric immunophenotyping is used particularly in the investigation of haematological neoplasms, but there are other roles ( Table 1.2).

Following analysis, the immunophenotyping laboratory will issue a report detailing the characteristics of any abnormal population identified and offering an interpretation. The strength of expression of any antigen is also of relevance. This may be expressed as

Table 1.2 Role of flow cytometric immunophenotyping.

AML, acute myeloid leukaemia; B‐ALL, B‐lineage acute lymphoblastic leukaemia; CD, cluster of differentiation; FLAER, fluorescent aerolysin; HIV, human immunodeficiency virus; HLA‐DR, human leucocyte antigen‐DR; PNH, paroxysmal nocturnal haemoglobinuria; RNA, ribonucleic acid; SAP, SLAM‐associated protein; T‐ALL, T‐lineage acute lymphoblastic leukaemia; XIAP, X‐linked inhibitor of apoptosis

*This is not part of conventional immunophenotyping but represents a flow cytometric immunophenotyping technique incorporated into an automated instrument for performing blood counts.

1 –, ±, +, ++;

2 negative, weak, moderate, strong;

3 negative, dim, moderate, bright;

4 hi, lo.

It should be noted that ± indicates weak expression whereas +/– indicates that expression may be positive or negative.