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.