Practical Clinical Hematology Leukemia 11

THE LEUKEMIAS malignancies involving lymphoid or hematopoietic cell lines increase in circulating leukocytes (not invariable) bone marrow always involved replacement/hypoproliferation of normal hematopoietic cells anemia, infection, hemorrhage leukemic infiltration in liver, spleen, lymph nodes, and other tissues

THE LEUKEMIAS acute leukemias chronic leukemias

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

THE LEUKEMIAS acute leukemias chronic leukemias acute lymphoblastic leukemia (ALL) acute myeloblastic leukemia (AML) chronic leukemias chronic lymphoid leukemia (CLL) chronic myelogenous leukemia (CML)

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

AML

The original FAB-Classification system of Acute Leukemias, Chronic leukemias Myeloid Lymphoid L1 L2 L3 M0 M1 M2 M3 M4 M5 M6 M7 Leukemias The original FAB-Classification system of Acute Leukemias, heavily based on morphologic findings

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 sulfatase, 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).

Acute Non-Lymphoblastic Leukaemia Class Alternative Bone Marrow Appearance M0 AML with minimal differentiation 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. myeloblastic leukemia with minimal differentiation; CD13 or CD33 positive

AML

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

FAB M1

FAB M1 AUER ROD

M2

M2

M2 with eosinophilia Peroxidase stain

FAB M2

FAB M2

FAB M3

FAB M3

FAB M3

FAB M4

FAB M4 Double esterase stain

FAB M5

FAB M5

FAB M5

FAB M6

FAB M6

FAB M6

FAB M6 PAS Stain

ALL

ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) lymphoblast predominant cell type most frequent in children better prognosis than AML FAB classification L1-L3 not used as a diagnosis, just as a descriptor entirely based on morphology

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

FAB L1 type of ALL

FAB L2 type of ALL

FAB L2 type of ALL

FAB L3 type of ALL

FAB L3 type of ALL

FAB L3 type of ALL

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

Other Lymphoid Leukemia

Hairy cell leukemia. Lymphocytes with filamentous cytoplasmic projections

Hairy cell leukemia

Hairy cell leukemia

Hairy cell leukemia (positive TRAP stain)

Prolymphocytic leukemia.

(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.

Chronic Leukemia

CHRONIC LEUKEMIAS longer clinical course chronic lymphocytic (lymphatic) leukemia (CLL) mature-appearing lymphocytes chronic myelocytic (myeloid, myelogenous, granulocytic) leukemia (CML, CGL) segs, bands, metamyelocytes, and myelocytes

CLL

CML

CML

CML

CML

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