Flow Cytometry

Flow cytometry (FC) is an analytical technique that again utilizes specific antibodies.59,60 FC differs from IHC in that whereas IHC utilizes formalin-fixed, paraffin-embedded soft tissue, FC makes use of suspensions of live cells. The cells may be leukocytes isolated from

FIGURE 12.9. Combination of routine and ancillary testing. For the superior lateral orbital lesion shown, the clinical differential diagnosis included lacrimal gland tumor, inflammatory infiltrate, and lymphoma. (A) Intraoperative frozen section analysis showed that the lesion is not an epithelial tumor. (B) The paraffin-embedded sections were more compatible with one of the latter two diagnoses. (C) The combination of flow cytometry results [CD19 ( + ), CD20 ( + ), with K-chain restriction-B-cell lymphoma], morphologic study of the routine H&E sections (mixture of small and large lymphocytes), along with IHC staining of the paraffin tissue (CD3 ( + ), CD10 ( — ), and bcl-2 ( —) generated a final diagnosis of an intermediate-grade large B-cell lymphoma of the orbit.

the peripheral blood, a cell suspension derived from a solid lymph node, or cells obtained from a fine-needle aspiration. Regardless of the source, the cells are ultimately incubated with labeled/tagged antibodies. Once prepared, the cells are analyzed on an instrument called a cell sorter.

The data produced from this analysis allow for the differentiation and relative enumeration of the cells. For example, analysis of leukocytes from the peripheral blood would produce a graphic distribution as shown in Figure 12.15. The distribution of the data signals generated is based on two concepts: forward scatter and side scatter. Forward scatter varies depending on the size of the cell, mature lymphocytes being the smallest leukocyte and the monocyte/ granulocyte lineage being larger. The second concept, that of side scatter, is a reflection of the cytoplasmic complexity of the cells.

Just as in IHC, the concept of analytical panels is utilized in FC. Because it uses antibodies that are reactive to specific cellular surface antigens, the FC profile, again, when coupled with a surgical biopsy specimen (e.g., a bone marrow core biopsy of bone), will allow for a much more specific diagnosis. Some of the more routinely used antibody panels in FC are listed in Table 12.2.52,53,61,62

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