Figure 11.1 Comparison of myeloblast and lymphoblast morphology.
Figure 11.1 Comparison of myeloblast and lymphoblast morphology.
Table 11.4 O Cytochemical Reactions in Acute Leukemia
Cytochemical Reaction Cellular Element Stained Blasts Identified
Myeloperoxidase (MPO) Sudan Black B (SBB) Specific esterase Nonspecific esterase (NSE) Terminal deoxynucleotidyl transferase (TdT)
Neutrophil primary granules Phospholipids Cellular enzyme Cellular enzyme Intranuclear enzyme
Myeloblasts strong positive; monoblasts faint positive Myeloblasts strong positive; monoblasts faint positive Myeloblasts strong positive Monoblasts strong positive Lymphoblasts positive
Variable, coarse or block-like positivity often seen in lymphoblasts and pronormoblasts, myeloblasts usually negative although faint diffuse reaction may occasionally be seen
Primary granules of myeloid cells contain peroxidase. The granules are found in the late myeloblast and exist throughout all the myeloid maturation stages. Because primary granules are absent in myeloblasts, there is limited MPO activity in early myeloblasts; however, those blasts that are closer to maturing to the promyelocyte stage will stain positive. Promyelocytes, myelocytes, metamyelocytes, and band and segmented neutrophils will stain strongly positive, indicated by the presence of blue-black granules. Monocytic granules stain faintly positive. Because lymphoid cells, nucleated red cells, and megakaryoblasts lack this enzyme, they will stain negative. This negative reaction is useful in initially differentiating ALL from AML. Eosinophils will also stain positive for MPO. Auer rods are strongly positive for peroxidase. The enzymatic activity in blood smears will fade over time, so slides should not be held for staining for longer than 3 weeks. The MPO stain will be positive in acute myeloid leukemia (>3% positive), acute promyelocytic leukemia (90% to 100% positive), acute myelomonocytic leukemia (including AMML with abnormal eosinophils, AMML Eo [variable, >3% positive]), acute monoblastic and monocytic leukemia (variable), and in the myeloblasts present in acute ery-throid leukemia (myeloblasts, >3% positive).
Phospholipids and other intracellular lipids are stained by SBB. Phospholipids are found in the primary (nonspecific) and secondary (specific) granules of neu-trophilic cells and eosinophils, and in smaller quantities in monocytes and macrophages. The stain will be negative in lymphocytes, although rarely the azurophilic granules of lymphoblasts may demonstrate positivity. The SBB pattern of staining mimics the MPO stain in that it is very sensitive for granulocyte precursors, increases in staining intensity with the later stages of granulocytic maturation, and stains weakly positive with monocytic cells. Thus, this stain can also be used to differentiate AML from ALL. A distinct advantage of the SBB over the MPO stain is that the SBB-stained slides are stable for a longer period of time.
Specific Esterase (Naphthol AS-D Chloracetate Esterase)
The specific esterase enzyme is present in the primary granules of myeloid cells. Accordingly, myeloblasts and other neutrophilic cells in AML will stain positive. This stain will also be positive in basophils and mast cells but is negative in eosinophils, monocytes, and lymphocytes. The specific esterase enzymatic reaction is stable in paraffin-embedded tissue sections, making this an extremely useful stain for identifying cells of myeloid lineage in extramedullary myeloid tumors.20
Nonspecific Esterase (Alpha-Naphthyl Butyrate or Alpha-Naphthyl Acetate Esterase)
These stains are used to identify monocytic cells and will stain negative with granulocytes. Different substrates are available, with alpha-naphthyl butyrate stain considered more specific and alpha-naphthyl acetate stain being more sensitive. Many cells in addition to monocytes will stain positive (macrophages, histio-cytes, megakaryoblasts, and some carcinomas), so the sodium fluoride inhibition step is used to differentiate the positivity. With this step, following initial staining, the NSE activity of monocytes and macrophages is
166 Part III • White Cell Disorders inhibited (reaction was positive, then reaction is negative), whereas the activity of the other cells remains positive. Mature T lymphocytes will stain a coarse dot-like pattern. The NSE stain is used to identify the monoblast and promonocyte populations in acute monoblastic leukemia and acute myelomonocytic leukemia. However, in 10% to 20% of cases of acute monoblastic leukemia, the NSE stain is weakly positive or negative. In these cases, immunophenotyping may be used to confirm monocytic differentiation.19
Terminal deoxynucleotidyl transferase (TdT) is an intranuclear enzyme found in stem cells and immature lymphoid cells within the bone marrow, but not in mature B lymphocytes. It is present in 90% of acute lymphoblastic leukemias and in only 5% to 10% of acute myeloblastic leukemias.21 It has also been demonstrated in one-third of cases of the blast crisis stage of chronic myelogenous leukemia and is a good prognostic indicator in these patients.22,23
Immunophenotyping can help to classify the clone of leukemic blasts by using monoclonal antibodies directed against cell surface markers. The specific lineage and stage of maturation can be tagged, and this information then is used to indicate appropriate therapy and can be correlated to prognosis. Immunophenotyp-ing can be done by flow cytometry or by immuno-histochemistry methods. Multiple antigens can be detected simultaneously on a single cell using flow cytometry. A selected panel of monoclonal antibodies is presented in Table 11.5.
The assignment of the particular nomenclature for the type of leukemia is based on the combined morphologic, immunophenotypic, and cytochemical information, as well as any unique clinical presentation. The information is used to suggest the best approximation of the subtype of leukemia, recognizing that our knowledge is sometimes imperfect and that changes in these classifications may again take place in the future as our understanding of the science of leukemia improves.
The morphological variants of acute myeloid leukemia may occur as a primary presentation or may be the result of a clonal evolution from other disorders such as the myeloproliferative disorders of chronic myeloge-nous leukemia, idiopathic myelofibrosis, or essential
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