LeukemiaLeukemia

Practical Clinical Hematology

Cytochemical Cytochemical Reactions in Acute Reactions in Acute

LeukemiaLeukemiaCytochemical Cytochemical ReactionReaction

Cellular Element Cellular Element StainedStainedBlasts IdentifiedBlasts Identified

Myeloperoxidase Myeloperoxidase (MPO)(MPO)

Neutrophil Neutrophil primary granulesprimary granules

Myeloblasts strong positive; Myeloblasts strong positive; monoblasts faint positivemonoblasts faint positive

Sudan Black B Sudan Black B (SBB)(SBB)PhospholipidsPhospholipidsMyeloblasts strong positive; Myeloblasts strong positive;

monoblasts faint positivemonoblasts faint positive

Specific esteraseSpecific esteraseCellular enzymeCellular enzymeMyeloblasts strong positiveMyeloblasts strong positive

Nonspecific Nonspecific esterase (NSE)esterase (NSE)Cellular enzymeCellular enzymeMonoblasts strong positiveMonoblasts strong positive

Periodic acid-SchiffPeriodic acid-Schiff Glycogen and Glycogen and related related substancessubstances

Variable, coarse or block-like Variable, coarse or block-like positivity often seen inpositivity often seen inlymphoblasts and lymphoblasts and pronormoblasts, myeloblasts pronormoblasts, myeloblasts usuallyusuallynegative although faint diffuse negative although faint diffuse reaction mayreaction mayoccasionally be seenoccasionally be seen

Leukocyte Alkaline Leukocyte Alkaline

phosphatase (LAP)phosphatase (LAP)

Positive LAP reactionPositive LAP reactionNegative LAP reactionNegative LAP reaction

Alkaline phosphatase is located in the specific granules of neutrophils.

Myeloperoxidase (MPO)Myeloperoxidase (MPO)

Bluish-black granulesBluish-black granules Red brown precipitateRed brown precipitate

Myeloperoxidase Myeloperoxidase stain, bone marrow stain, bone marrow

aspirateaspirate

The red granular staining The red granular staining

peroxidase activity.peroxidase activity.

Sudan Black Sudan Black BB

• Positive sudan black B (SBB) stain in a patient with Positive sudan black B (SBB) stain in a patient with AML ,AML ,

• Not the black staining cytoplasmic granules in the Not the black staining cytoplasmic granules in the myeloblastsmyeloblasts

Acid phosphtase Acid phosphtase

( with tartarate resistance)( with tartarate resistance)• Hairy cell leukemia, TRAP stain. Acid

phosphatase reaction after incubation with tartaric acid. Granular staining is seen in the lymphocytes.

Non Specific Esterase: Non Specific Esterase: {with fluoride inhibition}{with fluoride inhibition}

NSEs α-naphthyl acetate positivity in M5b.Not the granular positiviy in the monoblasts and immature monocytes

Periodic Acid – PASPeriodic Acid – PAS(Schiff Reaction)(Schiff Reaction)

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 .

Positive PAS stain in ALL

PAS positvity in M6. Not the intense staining of the large abnormal erythroblast.

Specific esterase or Specific esterase or chloroacetatechloroacetate

• 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 Maturation and

Morphology of Morphology of

Immature GranulocytesImmature Granulocytes

myeloblast promyelocyte myelocyte metamyelocyte band neutrophil

MATURATIONMATURATION

Maturation and Maturation and Morphology of Immature Morphology of Immature

GranulocytesGranulocytes

• 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: o larger than a myeloblast (20 μm)o High N:C ratio (3:1)o Loose chromatin with nucleolio Dark blue cytoplasmo Contains large nonspecific

cytoplasmic granuleso Containing myeloperoxidase

(MPO)o Comprises 3-4% of nucleated

bone marrow cells

• Neutrophilic Myelocyte:

o medium cell size (12 μm)o High N:C ratio (3:1)o Round, oval, or slightly indented

nucleus with darkero blue heterochromatino Last stage of cell divisiono Has active RNA, therefore, the

cytoplasm is blueo Contains MPO and secondary

granules containing leukocyte alkaline phosphatase

o 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

Bando Same size as a mature

neutrophil (10-12 μm)o N:C ratio has reversed (1:2)o Nucleus is band- or sausage-

shaped without segmentationo Cytoplasm is filled with

small neutrophilic granuleso Last immature stageo 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

• 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

NeutrophilsNeutrophils

• Eosinophilso Average size is 13 μmo Nucleus is generally

bilobedo 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

• Basophilso Is the smallest granulocyte at

10 μmo The nucleus is difficult to

see due to heavy granulation

o 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.

o 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 Lymphoid maturationmaturation

MATURATIONMATURATION

Hematopoieticstem cell

Neutrophils

Eosinophils

Basophils

Monocytes

Platelets

Red cells

Myeloidprogenitor

Lymphoidprogenitor

B-lymphocytesB-lymphocytes

T-lymphocytes

Plasmacells

naïve

ALLALL

AMLAML

Morphological Features of Blasts in Acute Morphological Features of Blasts in Acute Myeloid and Acute Lymphoblastic Myeloid and Acute Lymphoblastic LeukemiasLeukemias

Feature AMLALL

Blast sizeLarger, usually uniform

Variable, small to medium size

Nuclear chromatin Usually finely dispersed

Coarse to fine

Nucleoli1-4 ,often prominentAbsent or 1 or 2, often indistinct

Cytoplasm

Moderately abundant, fine granules often present

Usually scant, coarse granules sometimes present (~7%)

Auer rodsPresent in 60%-- 70% of cases

Not present

Other cell typesOften dyspastic changes in maturing myeloid cells

Usually not dysplastic

Acute Non-Lymphoblastic LeukaemiaDiagnosisAlternativeBone Marrow Appearance

M0Identified by ultrastructural myeloperoxidase activity or immunophenotyping.

M1AML without maturation

Monomorphic with one or more distinct nucleoli, occasional auer rod and at least 3%

myeloperoxidase positivity.M2AML with

maturation50% OR > myeloblasts & promyelocytes and common single auer rod. Dysplastic myeloid

differentiation may also be present.M3APLDominant cell type is promyelocyte with heavy

azurophilic granulation. Bundles of Auer rods confirm diagnosis. Microgranular variant exist

(M3v) M4AMMoLAs M2 but > 20% promonocytes & monocytes.M5AMoL> 80% monoblasts is poorly differentiated (M5a)

> 80% monoblasts, promonocytes or monocytes is well differentiated (m5b)

M6AEL>50% bizzar, dysplastic nucleated red cells with multinucleate forms and cytoplasmic bridging.

Myeloblasts usually > 30%.M7AMegLFibrosis, heterogeneous blasts population with

cytoplasmic blebs. Platelet peroxidase positive.

AML M0AML 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

the enzyme MPO demonstrated by immunocytochemistry analysis

Bone marrow smear, May-Giemsa stain, x1000

AML M0AML M0

AML M1 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

M1M1

M1M1

M1M1Immunostain Immunostain

antibody: CD33antibody: CD33Bone marrow smear, Bone marrow smear,

Peroxidase stainPeroxidase stain

M2M2

M2M2

M2M2

Peroxidase stainPeroxidase stain with eosinophiliawith eosinophilia

Hairy cell leukemia. Lymphocytes with Hairy cell leukemia. Lymphocytes with

filamentous cytoplasmic projectionsfilamentous cytoplasmic projections

Prolymphocytic Prolymphocytic leukemia.leukemia.

Morphological Features of Acute Morphological Features of Acute

Lymphoblastic LeukemiaLymphoblastic Leukemia

L1L2L3

Cell sizesmall to intermediate

large, heterogeneous

large, homogeneous

Nuclear shapeuniformpleiomorphic indentations common

uniform

Nucleolismall or absent

large, prominent

often single

Cytoplasmscantylarge, prominent

moderate (basophilic)

Cytoplasmic vacuoles

none to fewnone to fewprominent

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 Peripheral blood film in L2 acute lymphoblastic leukaemia (ALL). The blast cells are larger and more leukaemia (ALL). The blast cells are larger and more pleomorphic than in LI ALL and in this case have a pleomorphic than in LI ALL and in this case have a more diffuse chromatin pattern; one of the blasts more diffuse chromatin pattern; one of the blasts has a hand-mirror conformation. This case was has a hand-mirror conformation. This case was shown on immunophenotyping to be of T lineageshown 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 Platelet satellitism. Platelets adhering to the surface of a neutrophil. In this case, the surface of a neutrophil. In this case, the reported platelet count was only slightly reported platelet count was only slightly decreased.decreased.

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