Isj

Table 3.1

continued

Diagnostic study

Specimen requirements

Tests performed

Diagnostic utility

Comments

Flow cytometry

4 mL bone marrow in 4 mL (EDTA) lavender top tube; 4 mL whole blood in 4 mL (EDTA) lavender top tube, 7 mL (ACD) yellow top tube or 4 mL heparinized (sodium or lithium) green top tube; or 4 mL bone marrow in a heparinized syringe or heparinized green top tube. Store at room temperature. Samples should be <30 h old

Usually includes CD45 with myeloid markers CD13, CD33, CD117, and CD65; monocytic marker CD14; T- cell markers CD2, CD5, and CD7; B- cell markers CD19, and CD20; non-lineage- specific markers HLA-DR, CD10, CD34, and TdT; NK cell marker CD56; megakaryocyte marker CD61; may also include CD79a, cCD22, cCD3, cIgM, sIgM, and MPO

Determine lineage of blasts and evaluate for aberrant antigen expression; rule out precursor T or precursor B acute lymphoblastic leukemias and acute leukemia of ambiguous lineage. Baseline pheno-type of blasts may be helpful for excluding relapse or monitoring minimal residual disease following treatment

Bone marrow aspirate is preferred. Helps in recognition of minimally differentiated AML; immunophenotypic patterns can help identify AML with t(8;21) and t(15;17). Blast percentage by flow cytometry may not correlate with aspirate smear if hemodilute sample submitted

Cytogenetics

2-3 mL bone marrow in 4 mL heparinized (sodium) green top tube. Peripheral blood may be alternate sample if circulating immature cells present (>1,000/|L). Store at room temperature; do not refrigerate or freeze

GTG-banded chromosome analysis; minimum 20 metaphases analyzed (when available)

Gives global information about cell karyotype; identifies nonrandom abnormalities with prognostic significance

First pull of aspirate preferred. Do not collect specimen in lithium heparin or EDTA. Excess cells from cytogenetics may be used for FISH analysis

Molecular analysis by FISH

8 mL whole blood in two 4 mL (EDTA) lavender top tubes; 4 mL bone marrow from (EDTA) lavender top tube

May include BCR-ABL, t(15;17), t(8;21), inv(16), MLL rearrangement, and RARa rearrangement

Gives specific information about presence or absence of a particular genetic abnormality

Helpful at diagnosis, especially when cytogenetic sample not submitted or no growth

Molecular analysis by polymerase chain reaction (PCR)

5 mL whole blood in (EDTA) lavender top tube; 2-3 mL bone marrow in (EDTA) lavender top tube; refrigerate

May include t(9;22), t(15;17), inv(16), t(8;21), MLL rearrangement, and FLT3 rearrangement, as necessary

Gives specific information about presence or absence of a particular genetic abnormality. Helpful for minimal residual disease monitoring

Heparinized tube unacceptable due to interference with PCR

circumstances, 20% blasts are not needed for the diagnosis. For example, cases with inv(16), t(r,21) or t(15;17) are diagnosed as acute myeloid leukemia regardless of blast percentage according to WHO guidelines. In acute monocytic and myelomonocytic leukemias, promono-cytes are also counted as monoblasts for the diagnosis.101181 In acute erythroleukemia, blasts are counted as a percentage of non-erythroid cell if erythroid precursors marrow comprise >50% of the differential count.

On morphologic review, myeloblasts in patients with AML typically have delicate nuclear chromatin, three to five nucleoli, and a variable number of fine myeloperoxidase granules in the cytoplasm82 (Figure 3.1). Auer rods (azurophilic granules within lysozymes) are pathognomonic for AML3 (Figure 3.2). "Faggot cells," blast with bundles of Auer rods, are also characteristic in certain AML subtypes (AML with maturation and APL).36 Phi bodies, fusiform or spindle-shaped rods, which are similar to Auer rods and stain with myeloperoxidase, may also be present.483

Other AML subtypes may have distinct morphologic features in the blood and bone marrow. In the monocytic/monoblastic subtype of AML, the nucleus of the monoblasts is often indented and contains one to four large nucleoli.36 A moderate amount of cytoplasm is present (Figure 3.3). Patients with acute myelomonocytic leukemia often have a significant monocytosis in the peripheral blood.10 Acute myeloid leukemia with inv(16), is characterized by abnormal eosinophils.36 84 85 In acute erythroleukemia, abnormal erythroblasts with giant multinucleate forms, nuclear budding, and nuclear fragmentation are found in the bone marrow.36 (Figure 3.4). On review of a peripheral blood film, nucleated red blood cells are often pre-sent.3686 87 Bone marrow fibrosis, which can make bone marrow aspiration difficult, is typical of acute megakaryoblastic leukemia and acute panmyelosis with myelofibrosis.1047 Megakaryoblasts in acute megakaryoblastic leukemia typically have a high nuclear/cytoplasmic ratio, varying size, and pale agranular cytoplasm.36 Malignant proliferation of all three myeloid cell lines is present in acute panmyelo-sis with myelofibrosis.10 Finally, acute basophilic leukemia is characterized by striking basophilic granularity in myelocytes, and cells stain strongly with tolu-idine blue.36

MDS is differentiated from AML on the basis of percentage of blasts in the bone marrow. AML arising from MDS is characterized by dysplastic maturation of the hematopoietic precursors and certain chromosomal abnormalities (such as loss of part or all of chromosome 5 or 7).3 In addition, patients may have a preceding history of low blood counts. AML can often be differentiated from ALL on morphologic grounds. Lymphoblasts tend to be smaller in size, with little cytoplasm and indistinct nucleoli.77

Figure 3.1 Acute myeloid leukemia with t(8;21)(q22;q22): Peripheral blood smear shows a blast with an Auer rod (arrow) and a neutrophil with characteristic salmon-colored cytoplasmic granules (upper left); bone marrow aspirate shows blasts admixed with maturing myeloid cells (bottom left); and bone marrow biopsy is hypercellular with blasts admixed with maturing myeloid cells and eosinophils. (Wright-Giemsa stain, left panels; hematoxylin-eosin stain, right panel)

Figure 3.1 Acute myeloid leukemia with t(8;21)(q22;q22): Peripheral blood smear shows a blast with an Auer rod (arrow) and a neutrophil with characteristic salmon-colored cytoplasmic granules (upper left); bone marrow aspirate shows blasts admixed with maturing myeloid cells (bottom left); and bone marrow biopsy is hypercellular with blasts admixed with maturing myeloid cells and eosinophils. (Wright-Giemsa stain, left panels; hematoxylin-eosin stain, right panel)

Figure 3.2 Acute myeloid leukemia with t(15;17)(q22;q21): Features of blasts include multiple Auer rods (upper left), hypergranular cytoplasm (lower left), and bilobed nuclei (upper and lower right panels). This leukemia is often associated with a low white blood cell count, and characteristic blasts containing multiple Auer rods may require a careful search to identify. (Wright-Giemsa stain)

Figure 3.2 Acute myeloid leukemia with t(15;17)(q22;q21): Features of blasts include multiple Auer rods (upper left), hypergranular cytoplasm (lower left), and bilobed nuclei (upper and lower right panels). This leukemia is often associated with a low white blood cell count, and characteristic blasts containing multiple Auer rods may require a careful search to identify. (Wright-Giemsa stain)

Figure 3.3 Acute monoblastic leukemia: Blasts have moderate amounts of cytoplasm and nuclei with one to two nucleoli (left panel). Monocytic differentiation is demonstrated in the blasts by positive cytochemical stain for nonspecific esterase as shown by red-brown cytoplasmic staining (right panel). (Wright-Giemsa stain, left panel; nonspecific esterase stain [a-naphthyl butyrate], right panel)

Figure 3.3 Acute monoblastic leukemia: Blasts have moderate amounts of cytoplasm and nuclei with one to two nucleoli (left panel). Monocytic differentiation is demonstrated in the blasts by positive cytochemical stain for nonspecific esterase as shown by red-brown cytoplasmic staining (right panel). (Wright-Giemsa stain, left panel; nonspecific esterase stain [a-naphthyl butyrate], right panel)

Figure 3.4 Acute erythroleukemia: An increase in erythroid elements with dysplastic features including megaloblastoid chromatin and nuclear fragmentation is evident in this bone marrow aspirate smear (left panel). PAS stain is useful to identify abnormal "blocklike" cytoplasmic staining in immature erythroid precursors (right panel). (Wright-Giemsa stain, left panel; PAS stain, right panel)

Figure 3.4 Acute erythroleukemia: An increase in erythroid elements with dysplastic features including megaloblastoid chromatin and nuclear fragmentation is evident in this bone marrow aspirate smear (left panel). PAS stain is useful to identify abnormal "blocklike" cytoplasmic staining in immature erythroid precursors (right panel). (Wright-Giemsa stain, left panel; PAS stain, right panel)

However, immunophenotyping and immunohisto-chemistry are used to make a definitive diagnosis. By flow cytometry, myeloblasts usually express the cell surface antigen markers CD13 and CD33. Cells of myeloid origin usually will be myeloperoxidase positive by cytochemical or immunostains.10 In minimally differentiated AML (M0), blasts do not express myeloperoxidase cytochemically; however, they are myeloid antigen positive (CD13 and CD33) based on flow cytometry.82

When performing a bone marrow aspirate, a small amount of the first pull of the aspirate should be placed on a slide, as hemodilution may make it difficult to interpret the aspirate. At least 5 mL of aspirate should be sent in heparinized tubes for flow cytometry and cytogenetics. If an aspirate cannot be obtained secondary to fibrosis or a "packed" marrow, these studies can be performed on the peripheral blood if enough peripheral blasts are present. Flow cytometry can also be attempted on a biopsy specimen by performing an extra biopsy, placing the sample in saline, and "teasing" the cells from the marrow. Although bone marrow aspirates are usually obtained from the posterior iliac crest, a sternal aspirate can be performed if a sample cannot be obtained. Immunophenotyping by flow cytometry is helpful in diagnosing and subclas-sifying AML.88 The most commonly used monoclonal antibodies in the diagnosis of AML are CD13, CD14, CD15, CD33, CD34, and HLA-DR47: CD13 and CD33 both are myeloid markers; CD34 and HLA-DR both are stem cell markers; CD14, monocytic; and CD15, myeloid-granulocytic.89 Myeloid antigens such as CD14, CD15, and CD11b expressed on more differentiate myeloid cells may be found in AMLs with myeloid or monocytic maturation.90 CD14 and CD64 are the best markers for monocytic differentiation.1091 The platelet glycoproteins CD41, CD42, and CD61 are usually present in acute megakaryocytic leukemia.4792 Flow cytometry may be particularly helpful in diagnosing the microgranular variant of APL. With the microgranular variant, granules are not readily seen by light microscopy77 (Figure 3.5). However, unlike the other AMLs, APL is both CD34 negative and HLA-DR negative by flow cytometry.47 In certain instances, flow cytometry may also provide prognostic information. The presence of CD56 in both M2 and M3 AML appears to correlate with adverse prognosis.88'93-96

Immunostains used in evaluating bone marrow biopsies include myeloperoxidase (myeloid), hemoglobin A (erythroid), and CD34, CD79a, CD61, TdT (PAS, non-specific esterase are cytochemical stains, not immunostains). Biopsies can be stained for these markers if an aspirate for flow cytometry cannot be obtained to better subclassify the AML.

Figure 3.5 Acute myeloid leukemia with t(15;17)(q22;q21): The hypogranular variant is often associated with an elevated white blood count with most blasts having lightly granular to almost agranular cytoplasm, but still retaining the typical bilobed nucleus (left panel). A cytochemical stain for myeloperoxidase (right panel) demonstrates intense cytoplasmic positivity as shown by blue-black granules. (Wright-Giemsa stain, left panel; myeloperoxidase stain, right panel)

Figure 3.5 Acute myeloid leukemia with t(15;17)(q22;q21): The hypogranular variant is often associated with an elevated white blood count with most blasts having lightly granular to almost agranular cytoplasm, but still retaining the typical bilobed nucleus (left panel). A cytochemical stain for myeloperoxidase (right panel) demonstrates intense cytoplasmic positivity as shown by blue-black granules. (Wright-Giemsa stain, left panel; myeloperoxidase stain, right panel)

Cytogenetics are needed to assess prognosis, risk-adapt therapy, as well as to subclassify the AML. Cytogenetic abnormalities most commonly involve translocations and inversions of genes encoding transcription regulators.97 Particular cytogenetic abnormalities are associated with specific clinical features and prognosis. Based on the new WHO classification, AML with specific cytogenetic abnormalities [t(8;21) (q22;q22), t(15;17)(q22;q21), 11q23 abnormalities, and variants inv(16)(p13q22) and t(16;16)(p13;q22)] are classified as AML independently of the proportion of blasts in the bone mar-row12 (Figure 3.6). Fluorescence in situ hybridization (FISH) for t(15;17) inv(16), t(8;21) or 11q23 rearrangement should be performed if there is suspicion for one of these rearrangements, especially if cytogenet-ics are normal or unsuccessful since identification of an abnormality can affect both prognosis and therapy. FISH can be more rapid and sensitive than routine cytogenetics. In the future, other molecular markers, such as the presence or absence of fms-like tyrosine kinase 3 (FLT3) and assessment of multidrug resistance markers may become a standard part of the initial evaluation.

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