Methods Of Describing Antigen Expression

Antigen expression can be characterized according to antigen density, which is described by terms such as bright versus dim staining of antibodies or low versus high antigen expression. These descriptions relate to the intensity with which a fluorescence-conjugated antibody stains a given cell. Fluorescence intensity can be helpful diagnostically when the same antigen is expressed on contaminating normal and leukemic cells with differential density and gating procedures are inefficient in selecting the leukemic cell population. For instance, CD45 fluorescence intensity of staining is characteristically low on myeloid precursor cells, lower than on normal lymphocytes or more mature myeloid cells, which allows their separation in a flow differential.83 Alternatively, fluorescence intensity may vary among various leukemia subtypes. For instance, staining with antibody to the uncommitted cell marker, terminal transferase (TdT), in leukemic myeloblasts is significantly weaker than TdT staining in leukemic lymphoblasts.84 Furthermore, abnormally high or low expression of an antigen by a leukemic cell when compared with its normal counterpart can provide a leukemia-specific immunologic fingerprint exploitable for the monitoring of minimal residual disease.85 If fluorescence intensity is the interpretation of choice, antibody reactivity will be interpreted as negative, dimly positive, moderately positive, brightly positive, or indeterminate.

Particularly for multicenter studies that require comparison of fluorescence intensity data from several laboratories, the use of microbead calibration standards, which provide a reliable quantitative assessment of antigen density, is currently recom mended. Flow cytometry data are expressed in the form of molecular equivalents of soluble fluorochrome (MESF), which are calculated from the peak fluorescence of cells with given antibodies relative to that of a set of four or five microbead standards with different titers of MESF units.86

Other clinical laboratories describe the percentages of blast cells positive for the various antigens, which are calculated from the proportion of cells that stain with a given antibody with greater intensity than the brightest cells of the negative control or of nonstaining cells present in the cell population. To date, it has been accepted strategy to define antigen positivity of a leukemia population based on arbitrary cutoff levels for antibody staining cells, which range from 1 to 50 percent. Within a given study, the same cutoff level applies to all antigens tested. Recently, a biologically and clinically more meaningful approach has been recommended, which involves the retrospective determination of cutoff points defining the level of antibody staining blast cells that distinguishes between responding and nonresponding patients. Such response-driven cutoff points can then be tested for their validity in a prospective fashion.40,80,87,88

Thus, for both methods of antibody binding analysis, strategies to interpret the findings, that is, to define the level of brightness of staining or the cutoff level of percentage of staining cells that defines positivity, are variable and open to ongoing discussion. Either method has apparent flaws, which can affect the results of statistical analyses and the overall usefulness of immunophenotyping for the biologic characterization of leukemia subsets.

Whatever the method of data interpretation, it is absolutely essential that antibody binding be reported exclusively for the leukemic cell population, with exclusion of contaminating normal cells.89 The physician faced with interpreting an immunophenotype should pay particular attention to any indication that the data refer to either total white cells, total mononuclear cells, or blast cells. Provided that the abnormal cell population was identified and antigen characteristics of that gated population are exclusively reported, the choice between using qualitative (positive or negative based on fluorescence intensity) or quantitative descriptors of antibody binding (percentage of staining cells) becomes less significant. Gating on blast cells can be accomplished by clustering cell components based on CD45 fluorescence intensity combined with scatter characteristics, provided the leukemic blasts express CD45 (a considerable fraction of ALL are partially or completely CD45 negative). Alternatively, blasts cells are selected from a heterogeneous population of normal and abnormal cells with the help of an antibody that recognizes an antigen expressed by the leukemic cells in multicolor flow cytometry.89 Care must be taken to use an antibody that will distinguish between normal and abnormal cells of the same lineage. For instance, in the case of a T-cell ALL, CD7 would equally recognize both contaminating normal lymphocytes and the blast cells. Figure 15-2 demonstrates that both methods yield equivalent results, although occasionally gating based on a leukemic cell-associated antigen produces a clearer representation of the blast cell population. We recommend pretesting leukemic specimens with limited antibody panels to determine the ideal antibody for the separation of blast cells (e.g., CD34, CD117, HLA-DR), which will subsequently be added to every antibody tube in the full testing panel (Paietta E, unpublished).

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