Acute Leukemia and Special Stains PDF

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Summary

- Leukemia, a malignant disease of hematopoietic tissue. - Chronic Leukemia, associated with mature, well-differentiated cells. - Acute Leukemia, associated with primitive cells. Many similar looking blasts are seen. - Leukemias are classified according to their cell types, lineage, and maturity. - ...

Description

MLSC-3121U, Clinical Hematology II -

Leukemia, a malignant disease of hematopoietic tissue. Chronic Leukemia, associated with mature, well-differentiated cells. Acute Leukemia, associated with primitive cells. Many similar looking blasts are seen. Leukemias are classified according to their cell types, lineage, and maturity. Acute Myeloid Leukemia (AML), affect every hematopoietic cells except B and T lymphocytes. Have Auer rods. Acute Lymphoblastic Leukemia (ALL), affect B and T lymphocytes. Do not have Auer rods. Abnormal expression induced by translocation and genetic fusion or mutation often results in unregulated proliferation. Leads to leukemia. ALL is more common in children and AML is more common in adults.

Lab Evaluation of Acute Leukemia -

Normochromic and normocytic with low platelets. WBC count is variable from decreased to markedly increased. Blood smears show blasts or other immature cells. Occasionally circulating nRBCs. Pseudo-pelger-huet cells and hypo-granular neutrophils are seen.

Leukemia Classification -

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French-American-British Classification, a leukemia classification system that was proposed in 1976. Based on this, leukemia is classified by a blast count of 30% in the bone marrow. WHO Classification (1995), WHO developed another classification system based on morphology, immunophenotyping, genetic features including karyotype and molecular testing and clinical features. This classification system also reduced the required 30% blast count in the bone marrow to 20%. Leukemia is classified in the lab using o Cellular morphology o Cytochemical testing o Immunological cell marker studies o Chromosome analysis o Molecular genetic studies o DNA flow cytometry o Electron Microscopy Auer Rods, present in AML. They are abnormal fusions of primary granules. Seen as pink or purple staining rods or splinter-shaped inclusions in lymphoblast cytoplasm. They can be rare but are diagnostically important.

MLSC-3121U, Clinical Hematology II

AML Classification -

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M0, myeloid without cytologic maturation. Presence of primitive leukemic blasts that show no distinctive morphological features and lack reactivity with cytochemical analysis. Have immunologic reactivity to at least one myeloid marker. M1, myeloid with minimal maturation. Poorly differentiated myeloblasts with less than 10% of nonerythroid cells being differentiating granulocytes or monocytes. The peroxidase reaction demonstrates 3% or more positivity and M1 is nonspecific esterase positive in less than 20% of cells. Caused by a translocation of chromosomes 9 and 22. M2, myeloid with maturation. Resembles M1 except there is evidence of maturation beyond the promyelocyte. Greater than 3% positive for peroxidase and nonspecific esterase does not exceed 20%. Caused by a translocation of chromosome 8 and 21 in 18% of cases. M3, promyelocytic. Abnormal promyelocytes with heavy granulation. Auer rods may be present in the cytoplasm. Strongly positive with peroxidase. Also associated with DIC (granules in the cytoplasm are rich in thromboplastic substances and are released). Caused by a translocation of the chromosomes 15 and 17. M3m, microgranular variant of M3. Primary granules not readily seen on Romanowski stained smear. Has a high incidence of DIC. Peroxidase positive staining. M4, myelomonocytic. Granulocytic and monocytic differentiation. Monocyte count of greater than 5 x 10^9/L is seen. Peroxidase and nonspecific esterase positive in 20-80% of cells. Caused by a translocation of t(6;9)(q23;q34). M4eo, M4 with bone marrow eosinophilia. Some cases of M4 associated with eosinophilia. Stain positive for specific esterase and periodic acid Schiff. Caused by an inverted chromosome 16. M5a, monocytic and poorly differentiated. Predominance of monoblasts (counted as blasts). Non-specific esterase is greater than 80% for positivity. WBC count is elevated past 60.0 x 10^9/L. Caused by a translocation for chromosome 11. AML has clinical manifestations associated with monocytes ability to migrate to extramedullary sites. DIC is common in this disorder. M5b, monocytic and well differentiated. Spectrum of monocytic differentiation, including promonocytes and monocytes. Non-specific esterase shows greater than 80% positivity. Caused by a 11q23 translocation. M6, erythroleukemia. Abnormal proliferation of erythroid and myeloid precursors. Hypercellular bone marrow with megaloblastoid changes seen. Myeloblasts and promyelocytes make up around 30% of non-erythroid cells. Peripheral blood may show nRBCs and myeloblasts. M7, megakaryoblastic. Uncommon form of leukemia. Involves the infiltration of megakaryoblasts and atypical megakaryocytes. This leukemia is recognized with Platelet Peroxidase Studies (PPO). Has a presence of megakaryocytic fragments in peripheral blood.

MLSC-3121U, Clinical Hematology II

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8 and 12 Translocation AMLs, myeloblasts, Auer rods and some maturation. Inverted 6/16 Translocation, increased in myeloid and monocytic cell lines. Occasionally eosinophilia. 15 and 17 Translocation, Acute promyelocytic leukemia (M3). Granules lead to DIC. 11q23 Translocation, increase in monoblasts and immature monocytes. Treatment-Related AML, treatment with alkylating, radiation, topoisomerase II inhibitors can cause this. May develop into secondary leukemia.

AML not Otherwise Categorized -

Do not fit conventional classification groups. Grouped according to morphology, flow cytometry phenotyping and cytochemical reactions. AML Minimally Differentiated, no Auer rods seen and negative for peroxidase and Sudan black. AML Without Maturation, Auer rods present with a positive peroxidase and Sudan Black. AML with Maturation, maturation goes beyond Auer rods and dysplasia is present. Acute Myelomonocytic Leukemia, Myeloid and monocytic cells present. Monocytes are 20% of bone marrow cells. Acute Monoblastic Leukemia, 80% monocytic cells in the bone marrow. Acute Erythroid Leukemia, acute erythroleukemia showing 50% or more bone marrow nucleated cells are normoblasts with 20% being myeloblasts (M6). Acute Megakaryocytic Leukemia, 20% megakaryoblasts. Platelet antigens expressed are CD41, CD42, and CD61.

Acute Leukemia of Ambiguous Lineage -

May be of bilineage (myeloid or lymphoid expression) or Biphenotypic (expression of both myeloid and lymphoid cells).

Treatment of Acute Leukemia -

Aims to eradicate the leukemic cell mass (cytoreduction) and give supportive care. Antileukemic Therapy, involves chemotherapy and radiotherapy but bone marrow transplants are the most effective cure afterwards.

Antigens -

Antigen testing has begun to replace cytochemistry for lineage determination. A panel is chosen that includes antibodies to several myeloid associated antigens as well as T and B lineage markers.

MLSC-3121U, Clinical Hematology II

Acute Lymphoblastic Leukemia -

L1, small uniform lymphoblasts with scant cytoplasm. Clefting may be present in cells. L2, large pleomorphic lymphoblasts. Shows cellular heterogeneity. Nuclear clefting and indentation are characteristic. L3, Burkitt’s lymphoma. Uniform population of large blasts with moderate to abundant, deeply basophilic, vacuolated cytoplasm. Childhood ALL, most able to achieve complete remission and potentially be cured. However, therapy has toxic effects on growing children.

Special Stains Myeloperoxidase Stain (MPX) -

In the presence of hydrogen peroxide, MPX oxidizes dye substrates to create a black staining at the site of activity. Shows Auer rods and cytoplasmic granular staining. Stains myeloid cells but does not stain lymphoid cells.

Sudan Black B (SBB) -

Staining results from the solubility of Sudan Black B in the lipid particles. Shows more a more definitive and localized positive reactions in blast cells and prominently shows Auer rods. Stains myeloid cells but does not stain lymphoid cells.

Esterases -

Used to differentiate myeloblasts and neutrophilic granulocytes from cells of monocytic origin. Esterase stains hydrolyze esters. The product reacts with naphthol compounds to create a brightly coloured stain at the site of reaction. o Specific Esterase, not as sensitive and negative in eosinophils and monocytes. o Non-Specific Esterase, used to identify monocytic cells. Positive in monocytes and negative in granulocytes and lymphocytes (except for T lymphocytes which will show a focal dot-like cytoplasmic staining pattern).

Periodic Acid Schiff -

Stains for glycogen and related compounds including mucoprotein, glycoprotein, glycolipid and polysaccharides. Used for M6 or identifying lymphoid blasts. Periodic acid hydrolyzes glycogen, mucoproteins and other high molecular weight carbs to aldehydes.

MLSC-3121U, Clinical Hematology II -

Stain used for acute erythroid leukemia. The marrow film is stained with Periodic acid Schiff reagent. Shows an intense PAS-positive staining of leukemic erythroblasts. Factor VIII Antibodies -

Immunocytochemical staining for megakaryoblast leukemia (M7). Mono or polyclonal Abs against the FVIII-related antigen on the megakaryoblast surface.

Leukocyte Alkaline Phosphatase (LAP) -

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Differentiates CML from a leukemoid reaction. LAP enzyme found in secondary granules of neutrophils. Substrate is hydrolyzed by the enzyme when added at an alkaline pH. The hydrolyzed substrate is then combined with dye to produce a colour change at the site of reaction. LAP Scoring, 100 consecutive mature neutrophils are counted and staining intensity is graded. Normal range is 11 to 95 and heparin is used as the anticoagulant. Requires a control. Increased LAP, leukemoid reaction or pregnancy (3rd trimester). Decreased, CML, PNH, sideroblastic anemia or myelodysplastic disorders. Normal, secondary polycythemia.

Leukocyte Acid Phosphatase Stain -

Acid phosphatase hydrolyzes the substrate naphthol AS-BI phosphoric acid. The substrate will combine with the dye at the site of activity. When I-tartaric acid is added, all isoenzymes except isoenzyme 5 are inhibited (isoenzyme 5 is tartrate resistant). Isoenzyme 5 is present in hairy cells, therefor staining will occur....