1. Practical Clinical Hematology
Leukemia 13

2. 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

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

4. 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

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

6. 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

7. 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.

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

9. 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

10. 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

11. 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.

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

13. 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

14. 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

15. 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

16. 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

17. 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

18. Neutrophils
Also known as segmented neutrophils, segs, polymorphonuclear cells, polys, and PMNs
N:C ratio is 1:3, and the size is 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

19. 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

20. 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).

21. Lymphoid maturation
MATURATION

22. 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

23. 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

24. 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.

25. 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

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

27. 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

28. M1

29. M1

30. Immunostain antibody: CD33 Bone marrow smear, Peroxidase stain

31. M2

32. M2

33. M2
with eosinophilia
Peroxidase stain

34. Hairy cell leukemia. Lymphocytes with filamentous cytoplasmic projections

35. Prolymphocytic leukemia.

36. 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

37. 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.

38. 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

39. (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.

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

41. 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.