Cytochemistry

Case Study

After studying the material in this chapter, the reader should be able to respond to the following case study:

A 38-year-old woman came to the physician with a 2-month history of gingival bleeding and gingival hypertrophy. The CBC revealed the following: WBCs, 64 × 10⁹/L; Hb, 7.5 g/dL; and platelets, 36 × 10⁹/L. The peripheral blood film and the bone marrow specimen demonstrated greater than 80% abnormal mononuclear cells. The results of cytochemical testing of these cells were as follows:

Myeloperoxidase: negative

Sudan black B: negative

α-Naphthyl butyrate esterase: positive

Periodic acid–Schiff: negative

1. What cell lines stain positive for esterases?

2. What general diagnosis is suggested by the CBC values?

3. Based on morphology and cytochemical staining, what is the classification of this disorder?

Introduction and Principle

Cytochemistry is the study of the chemical constituents of cells. These elements may be enzymatic (e.g., peroxidase) or nonenzymatic (e.g., lipids and glycogen). The cellular morphology of peripheral blood and bone marrow often provides a provisional diagnosis, and since the early twentieth century, cytochemical staining of cells has provided a useful adjunct for differentiation of hematopoietic diseases, especially acute leukemias. Immunophenotyping, high-resolution cytogenetic studies, and molecular genetic analysis have reduced the importance of cytochemical testing. Microarray technology can quantify the expression of thousands of genes in a single analysis.1,2 However, cytochemical testing still has limited use in many laboratories, especially in equivocal cases or where more sophisticated technology is not available.^3,4 (Leukemias are discussed in Chapters 29, 36, and 37; cytogenetics, in Chapter 31; molecular diagnostics, in Chapter 32; and flow cytometry, in Chapter 33.)

Acceptable Specimens and Fixatives

Many specimen types are adequate for cytochemical studies. Smears and imprints made from bone marrow, lymph nodes, spleen, or peripheral blood are preferred. In enzymatic techniques, fresh smears are used whenever possible to ensure optimal enzyme activity. Smears for nonenzymatic stains, such as periodic acid–Schiff (PAS) or Sudan black B (SBB), may remain stable for months if stored at room temperature.

Certain elements may be inhibited during the fixation of smears and imprints; it is important to use the proper fixative for the desired cytochemical stains. Fixatives containing alcohol (methanol or ethanol), acetone, formaldehyde, or a combination of these are commonly used for most cytochemical studies.

Myeloperoxidase (MPO) (Figures 30-1 and 30-2) is an enzyme found in the primary granules of neutrophils, eosinophils, and, to a certain extent, monocytes. Lymphocytes do not exhibit MPO activity. This stain is useful for differentiating the blasts of acute myeloid leukemia (AML) from those of acute lymphoblastic leukemia (ALL).

Figure 30-1 Positive reaction to myeloperoxidase stain in early myeloid cells. Note Auer rod at arrow (bone marrow, ×1000).

Principle

When hydrogen peroxide is present, MPO oxidizes dye substrates, creating black to red-brown staining (depending on the substrate) at the site of the activity. Benzidine had been the substrate most often used, but because of its carcinogenic properties, other substrates such as 3,3′-diaminobenzidine or p-phenylenediamine dihydrochloride and catechol are currently used.5–7

Interpretation

MPO is present in the primary granules of granulocytic cells, beginning at the promyelocyte stage and continuing throughout maturation. Leukemic myeloblasts also are usually positive for MPO. In many cases of the AMLs (without maturation, with maturation, and promyelocytic leukemia), it has been found that more than 80% of the blasts show MPO activity. Auer rods found in leukemic blasts and promyelocytes test strongly MPO positive. Because of their strong MPO positivity, many Auer rods that could not be seen with a Wright-Giemsa stain can be seen with the MPO stain.

Monocytes are MPO negative to weakly or diffusely positive. In contrast, lymphoblasts and lymphoid cells are MPO negative; in patients with ALL, fewer than 3% of the blasts show peroxidase positivity.8–10

It is important that the reaction only in the blast cells be used as the determining factor for the differentiation of acute leukemias. This is true for MPO and for the other cytochemical stains used in determining cell lineage that are mentioned in this chapter. The fact that maturing granulocytes are MPO positive is normal and has little or no diagnostic significance.

Sudan Black B

SBB staining (Figure 30-3) is another useful technique for the differentiation of AML from ALL. The staining pattern is quite similar to that of MPO; SBB staining is possibly a little more sensitive for the early myeloid cells.

A positive α-naphthyl butyrate esterase reaction is also seen in monocytes. α-Naphthyl butyrate is less sensitive than α-naphthyl acetate, but is more specific. Granulocytes and lymphoid cells generally show a negative reaction (Figure 30-6). In myelomonocytic leukemia, positive AS-D chloroacetate activity and positive α-naphthyl butyrate or α-naphthyl acetate activity should be seen because myeloid and monocytic cells are present. In myelomonocytic leukemia, at least 20% of the cells must show monocytic differentiation that is nonspecific esterase positive and is inhibited by sodium fluoride. In the pure monocytic leukemias, 80% or more of the blasts are nonspecific esterase positive and specific esterase negative.