Phenotyping of MCs in Extramedullary Organs

3.2.1. Other Hematopoietic Organs Analyzed in Patients With Mastocytosis

In almost all patients with suspected SM, the bone marrow is affected and is the primary organ to be analyzed (22,23). Other "hematopoietic" organs

10° 101 102 103 104 10° 101 102 103 104 Mean fluorescence intensity Mean fluorescence intensity

Fig. 3. Expression of CD203c on bone marrow mast cells (MCs). (A) Normal bone marrow MCs express low but detectable amounts of CD203c. The graph shows a histogram of MCs stained with the PE-labeled CD203c monoclonal antibody (MAb) 97A6. (B) In patients with systemic mastocytosis (SM), the levels of CD203c expressed on bone marrow MCs usually are higher as compared with those found on MCs in the normal bone marrow. A comparison of the two samples analyzed (A vs B) suggests an approximately fivefold higher expression of CD203c on MCs in a patient with SM (B) compared with normal MCs (A). Note logarithmic scale of immunofluorescence intensity.

10° 101 102 103 104 10° 101 102 103 104 Mean fluorescence intensity Mean fluorescence intensity

Fig. 3. Expression of CD203c on bone marrow mast cells (MCs). (A) Normal bone marrow MCs express low but detectable amounts of CD203c. The graph shows a histogram of MCs stained with the PE-labeled CD203c monoclonal antibody (MAb) 97A6. (B) In patients with systemic mastocytosis (SM), the levels of CD203c expressed on bone marrow MCs usually are higher as compared with those found on MCs in the normal bone marrow. A comparison of the two samples analyzed (A vs B) suggests an approximately fivefold higher expression of CD203c on MCs in a patient with SM (B) compared with normal MCs (A). Note logarithmic scale of immunofluorescence intensity.

(spleen, liver, lymph nodes, peripheral blood) may also be affected in these patients, and sometimes it may be of importance to know whether these organs contain MC infiltrates (25,26). Phenotyping of MCs in these organs usually is performed by immunohistochemistry but not by surface marker studies (see Notes 4 and 5 [25]). An exception is blood. Thus, in patients with MC leukemia, circulating MCs can be detected by flow cytometry (27-29). In typical MC leukemia, the percentage of MCs in the peripheral blood exceeds 10% of all nucleated white blood cells (22,30). However, in some cases (e.g., aleukemic MC leukemia), the percentage of MCs is less than 10% (22,28). In these patients, cell surface phenotyping of MCs in the peripheral blood is sometimes helpful to confirm the presence of MCs and to determine their phenotype (28).

3.2.2. MC Phenotypingin Extrahematopoietic Tissues

MCs can also be examined phenotypically in nonhematopoietic organs, such as the lung, skin, or gastrointestinal tract (31-35). However, cell surface marker analysis on these MC subsets usually cannot be performed in patients with SM but instead has to be performed in surgical specimens obtained from patients who suffer from other neoplastic disorders (31-35). Likewise, lung tissue can be obtained at surgery (lobectomy) from patients with bronchiogenic carcinoma. Tissue MCs are isolated as follows:

1. Tissue is chopped into small fragments and washed extensively in Mg2+/Ca2+-free Tyrode's buffer (31-35).

2. Tissue fragments are incubated in collagenase type II (2 mg/mL) at 37°C for 1-3 h (31-35). The additional use of other enzymes may increase the percentage of MC in the final suspension samples. However, many of these enzymes (but not collagenase) are known to degrade various cell surface antigens and, therefore, their use is not recommended for MC isolation.

3. Dispersed cells are separated from the tissue by filtration through Nytex cloth, washed, and recovered in RPMI 1640 medium plus 10% fetal calf serum.

Immunophenotyping of MCs in lung cell suspensions can be performed in the same way as described for cell marker analysis of bone marrow MCs. Again, MCs are detected by CD117 and are delineated from other (CD117+) tissue cells by multicolor flow cytometry (17,35). The MAbs applied are the same as those used to phenotype bone marrow MCs (Table 2 [17,35]). Apart from flow cytometry, however, suspended MCs also can be analyzed by combined toluidine blue/immunofluoresence staining technique (see Subheading 3.3.).

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