Clinical Applications Of Flow Cytometry

Routine applications of flow cytometry in the clinical laboratory include enumeration of peripheral blood CD4+ T-cells in patients with human immunodeficiency virus (HIV) infection, enumeration of CD34+ stem cells in peripheral blood and bone marrow for use in stem cell transplantation, and immunopheno-typic characterization of acute leukemias, non-Hodgkin's lymphomas, and other lymphoproliferative disorders. In certain instances, the DNA content, or ploidy status of tumor cells, which is readily measured by flow cytometry using a DNA-binding dye, might have prognostic value. In recent years, flow cytometry has also been used to monitor patients with leukemia or lymphoma who have already been treated and have attained a clinical remission. The detection of so-called "minimal residual disease" by flow cytometry in these patients predicts a higher likelihood of relapse and might permit more precise tailoring of additional therapy.

2.1. CD4+ T-CELL ENUMERATION AND OTHER ASSAYS OF IMMUNE DEFICIENCY A principal mechanism whereby HIV abolishes normal immune function is infection and depletion of a subset of T-cells bearing the CD4

Fig. 2. Fluorescence signals obtained using two fluorochromes (FITC, FL1; PE, FL2) with overlapping emission spectra before (A) and after (B) compensation.

molecule (8-10). The extent of depletion of CD4+ T-cells in flow cytometry has been used to calculate the percentage of

HIV-infected patients correlates with prognosis. Moreover, in white blood cells that are T-cells (CD3+) that also express the conjunction with HIV viral load measurement, CD4+ T-cell T-cell subset antigen CD4 (Fig. 4). In this methodology, the enumeration provides a quantitative assay for the efficacy of absolute number of CD4+ T-cells is calculated by multiplying therapy in patients treated with antiretrovirals. Traditionally, the absolute lymphocyte count (measured using a routine

Fig. 3. Example of electronic gating. (A) All B-cells are displayed and all are positive for the B-cell antigen CD20. Selective gating on B-cells whose CD20 expression is comparatively dim (R3) or bright (R4), however, discloses that the predominant population of abnormal CD20(dim)+ B-cells expresses monotypic kappa light chain (B) whereas the few remaining normal CD20(bright)+ B-cells comprise a mixture of kappa- and lambda-bearing cells (C).

Fig. 3. Example of electronic gating. (A) All B-cells are displayed and all are positive for the B-cell antigen CD20. Selective gating on B-cells whose CD20 expression is comparatively dim (R3) or bright (R4), however, discloses that the predominant population of abnormal CD20(dim)+ B-cells expresses monotypic kappa light chain (B) whereas the few remaining normal CD20(bright)+ B-cells comprise a mixture of kappa- and lambda-bearing cells (C).

hematology analyzer) by the percentage of CD4+ T-cells determined flow cytometrically.

Interlaboratory reproducibility of CD4+ T-cell enumeration can be enhanced by eliminating the requirement for separate measurement of the white blood cell count. In so-called single-platform methods, a precise volume of blood is added to a commercially prepared tube containing a known number of microfluorospheres as well as an appropriate combination of fluorochrome-conjugated antibodies. Because the total number of microfluorospheres is known, the absolute number of lymphocytes (which are characteristically brightly positive for CD45) can be calculated by comparison with the number of microfluorospheres detected during the acquisition period. This value is then multiplied by the percentage of CD4+ T-cells to yield the absolute CD4+ T-cell count.

It should be noted that flow cytometric evaluation of immune deficiency is not limited to acquired immunodeficiency. There are many forms of primary (congenital) immunodeficiency,

Fig. 4. Peripheral blood specimen, gated on CD3+ T-cells. In this manner, the percentage of CD4+ T-cells (lower right quadrant) and CD8+ T-cells (upper left quadrant) can be quantified.

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