Practical Clinical Hematology Leukemia 13

Cytochemical Reactions in Acute Leukemia Blasts Identified Cellular Element Stained Cytochemical Reaction Myeloblasts strong positive; monoblasts faint positive Neutrophil primary granules Myeloperoxidase (MPO) Phospholipids Sudan Black B (SBB) Myeloblasts strong positive Cellular enzyme Specific esterase Monoblasts strong positive Nonspecific esterase (NSE) Variable, coarse or block-like positivity often seen in lymphoblasts and pronormoblasts, myeloblasts usually negative although faint diffuse reaction may occasionally be seen Glycogen and related substances Periodic acid-Schiff

Leukocyte Alkaline phosphatase (LAP) Alkaline phosphatase is located in the specific granules of neutrophils. . Negative LAP reaction Positive LAP reaction

Myeloperoxidase (MPO) Peroxidase is an enzyme from oxydoreductase group. It is found in myeloid cells and is then called myeloperoxidase. Myeloid cells can destroy microorganisms with the help of this enzyme. Peroxidase is present in primary granules of neutrophils, and in the granules of eosinophils and monocytes. Red brown precipitate Bluish-black granules

Myeloperoxidase stain, bone marrow aspirate The red granular staining peroxidase activity.

Sudan Black B Sudan staining is the use of Sudan dyes to stain sudanophilic substances, usually lipids. Sudan lysochromes (Sudan II, Sudan III, Sudan IV, Oil Red O, and Sudan Black B) are used. Sudan dyes have high affinity to fats, therefore they are used to demonstrate triglycerides, lipids, and lipoproteins. Sudan black B stain is the most sensitive stain for granulocyte precursors. Brownish‑black cytoplasmic granules occur in myeloid precursors. Monocytes have a few small brownish‑black granules. Eosinophilic granules are brown and usually show a central pallor. Lymphoid cells rarely stain Positive sudan black B (SBB) stain in a patient with AML , Not the black staining cytoplasmic granules in the myeloblasts

Acid phosphtase ( with tartarate resistance) Hairy cell leukemia, TRAP stain. Acid phosphatase reaction after incubation with tartaric acid. Granular staining is seen in the lymphocytes. The acid phosphatase within the cell hydrolyzes the substrate naphthol AS‑BI phosphoric acid. The hydrolyzed substrate then couples with the dye (hexagotized pararosaniline) and because the colored complex is insoluble, it precipitates out at the site of enzyme activity. Tartaric acid when added to the incubation mixture, will not inhibit the enzyme fraction found in hairy cell leukemia.  

Non Specific Esterase: {with fluoride inhibition} NSEs α-naphthyl acetate positivity in M5b. Not the granular positiviy in the monoblasts and immature monocytes

Periodic Acid – PAS (Schiff Reaction) PAS stains glycogen. Periodic acid oxidizes glycols to aldehydes. The aldehydes react with Schiff's reagent. Giant multinucleate late normoblasts (left). Granular PAS positivity in proerythroblasts and homogeneous positivity in the later normoblasts

Positive PAS stain acute megakaryocytic leukemia AML, M7. PAS positvity in M6. Not the intense staining of the large abnormal erythroblast. Positive PAS stain acute megakaryocytic leukemia AML, M7. Positive PAS stain in ALL

Specific esterase or chloroacetate Naphthol (AS-D) Chloroacetate Esterase stain in a patint with AML,M2. Not the bright red satining indicating that these two blasts are of myeloid origin.

Maturation and Morphology of Immature Granulocytes myeloblast promyelocyte myelocyte metamyelocyte band neutrophil MATURATION

Maturation and Morphology of Immature Granulocytes Myeloblast: The first and earliest granulocyte (15 μm) High nucleus to cytoplasm (N:C) ratio (5:1) Round or oval nucleus with loose light staining euchromatin 1-2 nucleoli Has minimal light blue cytoplasm Contains no cytoplasmic granules Begins to produce myeloperoxidase granules (MPO) Comprises 1% of the nucleated cells in the bone marrow

Promyelocyte: larger than a myeloblast (20 μm) High N:C ratio (3:1) Loose chromatin with nucleoli Dark blue cytoplasm Contains large nonspecific cytoplasmic granules Containing myeloperoxidase (MPO) Comprises 3-4% of nucleated bone marrow cells

Neutrophilic Myelocyte: medium cell size (12 μm) High N:C ratio (3:1) Round, oval, or slightly indented nucleus with darker blue heterochromatin Last stage of cell division Has active RNA, therefore, the cytoplasm is blue Contains MPO and secondary granules containing leukocyte alkaline phosphatase Comprises 12% of bone marrow nucleated cells

Neutrophilic Metamyelocyte: size (11 μm) N:C ratio (2:1) last mononuclear stage, no mitosis Nucleus is kidney or horseshoe shaped, and has condensed heterochromatin Has a prominent Golgi apparatus – clear area located at the indentation site of the nucleus Cytoplasm is similar to the mature cell Comprises 18% of bone marrow cells

Band Same size as a mature neutrophil (10-12 μm) N:C ratio has reversed (1:2) Nucleus is band- or sausage-shaped without segmentation Cytoplasm is filled with small neutrophilic granules Last immature stage Comprises 11% of bone marrow cells and 0-3% of peripheral WBCs Stored in the bone marrow and released when there is an increased demand for neutrophils • Shift to the left is an increase in immature cells indicating increased demand for WBCs in peripheral blood

Neutrophils Also known as segmented neutrophils, segs, polymorphonuclear cells, polys, and PMNs N:C ratio is 1:3, and the size is 10-12 μm Average nucleus contains 3-5 segments connected by narrow filaments Hyposegmented is less than 3 segments, and may indicate a shift to the left or an anomaly Hypersegmented is more than 5 segments and may indicate infection or megaloblastic anemia Cytoplasm contains very small neutral granules Makes up 55-75% of all peripheral WBCs • Granules can become larger upon bacterial infection producing toxic granulation, which are numerous, large, basophilic granules

Eosinophils Average size is 13 μm Nucleus is generally bilobed Cytoplasm is bright red or orange which is due to large specific, secretory granules containing peroxidase, acid phosphatase, aryl sufatase, beta-glucuronidase, etc. that stain red with the eosin component of Wright’s stain

Basophils Is the smallest granulocyte at 10 μm The nucleus is difficult to see due to heavy granulation Cytoplasm contains large specific, secondary granules that contain heparin and histamines, which stain purple with Wright’s stain. These granules are water soluble and sometimes appear as holes in the cell if the cells are not fixed well during staining. Makes up to 0.5% of peripheral WBCs • Note: Tissue mast cells are similar to basophils but are larger and have no developmental relationship with basophils. Mast cells have a mesenchymal (connective tissue) origin and have granules containing serotonin (basophilic granules contain no serotonin).

Lymphoid maturation MATURATION

ALL AML Hematopoietic stem cell Neutrophils Eosinophils Basophils Monocytes Platelets Red cells Myeloid progenitor Lymphoid B-lymphocytes T-lymphocytes Plasma cells germinal center naïve AML

Morphological Features of Blasts in Acute Myeloid and Acute Lymphoblastic Leukemias ALL AML Feature Variable, small to medium size Larger, usually uniform Blast size Coarse to fine Usually finely dispersed Nuclear chromatin Absent or 1 or 2, often indistinct 1-4, often prominent Nucleoli Usually scant, coarse granules sometimes present (~7%) Moderately abundant, fine granules often present Cytoplasm Not present Present in 60%-- 70% of cases Auer rods Usually not dysplastic Often dyspastic changes in maturing myeloid cells Other cell types

Acute Non-Lymphoblastic Leukaemia Diagnosis Alternative Bone Marrow Appearance M0 Identified by ultrastructural myeloperoxidase activity or immunophenotyping. M1 AML without maturation Monomorphic with one or more distinct nucleoli, occasional auer rod and at least 3% myeloperoxidase positivity. M2 AML with maturation 50% OR > myeloblasts & promyelocytes and common single auer rod. Dysplastic myeloid differentiation may also be present. M3 APL Dominant cell type is promyelocyte with heavy azurophilic granulation. Bundles of Auer rods confirm diagnosis. Microgranular variant exist (M3v) M4 AMMoL As M2 but > 20% promonocytes & monocytes. M5 AMoL > 80% monoblasts is poorly differentiated (M5a) > 80% monoblasts, promonocytes or monocytes is well differentiated (m5b) M6 AEL >50% bizzar, dysplastic nucleated red cells with multinucleate forms and cytoplasmic bridging. Myeloblasts usually > 30%. M7 AMegL Fibrosis, heterogeneous blasts population with cytoplasmic blebs. Platelet peroxidase positive.

AML M0 the blasts are undifferentiated by morphology and cytochemistry The cytoplasm is usually scant, and grey to light blue in color without granules, and Auer rods are not seen. N:C ratio is high The nucleus is round to oval or irregular and usually eccentrically placed The nuclear chromatin ranges from being finely granular and evenly dispersed to being slightly clumped One or more nucleoli may be visible

AML M0 the enzyme MPO demonstrated by immunocytochemistry analysis Bone marrow smear, May-Giemsa stain, x1000

AML M1 The blasts vary in size, but are approximately the size of mature segmented neutrophils The nuclei are round to slightly oval or irregular, and the nuclear chromatin ranges from finely granular and evenly distributed to slightly clumped. One to two nucleoli are usually visible The cytoplasm is scant and grey-blue to light blue in color, and agranular. N:C ratio is high Auer rods are seen in a minority of blasts

M1

M1

Immunostain antibody: CD33 Bone marrow smear, Peroxidase stain

M2

M2

M2 with eosinophilia Peroxidase stain

Hairy cell leukemia. Lymphocytes with filamentous cytoplasmic projections

Prolymphocytic leukemia.

Morphological Features of Acute Lymphoblastic Leukemia large, homogeneous large, heterogeneous small to intermediate Cell size uniform pleiomorphic indentations common Nuclear shape often single large, prominent small or absent Nucleoli moderate (basophilic) scanty Cytoplasm prominent none to few Cytoplasmic vacuoles

Peripheral blood film in L1 (acute lymphoblastic leukaemia) (ALL) showing lymphoblasts and one nucleated red blood cell (NRBC). The lymphoblasts vary in size but are relatively uniform in morphology. The smaller blast cells show some chromatin condensation, which can be a feature of lymphoblasts but not of myeloblasts. This case was shown on immunophenotyping to be of B lineage.

Peripheral blood film in L2 acute lymphoblastic leukaemia (ALL) Peripheral blood film in L2 acute lymphoblastic leukaemia (ALL). The blast cells are larger and more pleomorphic than in LI ALL and in this case have a more diffuse chromatin pattern; one of the blasts has a hand-mirror conformation. This case was shown on immunophenotyping to be of T lineage

(A)and (B), Hypergranular acute promyelocytic leukemia, promyelocytes with prominent azurophilic granules. (C) Hypergranular APL with multiple Auer rods. (D) Microgranular APLv. These abnormal promyelocytes have lobulated nuclei and absent or fine azurophilic granules.

Platelet satellitism. Platelets adhering to the surface of a neutrophil. In this case, the reported platelet count was only slightly decreased.

Platelet clumping (pseudothrombocytopenia) Platelet clumping (pseudothrombocytopenia). Large platelet clumps on the blood smear from a 21-year-old asymptomatic woman. The hematology analyzer reported a platelet count <11,000/L. Specimen anticoagulated with citrate showed a normal platelet count.