Leukocytes, or white blood cells, are divided into myelocytes and lymphocytes. Myelocytes include granulocytes like neutrophils, eosinophils, and basophils, as well as monocytes. Lymphocytes include B and T lymphocytes. Leukopoiesis is the process by which these cells develop from hematopoietic stem cells in the bone marrow. Morphological abnormalities that can occur include alterations in neutrophil nuclei like the Pelger-Huet anomaly and cytoplasmic changes such as May-Hegglin anomaly. Lymphocytes and monocytes can also demonstrate abnormal morphologies like Reed-Sternberg cells.

2. Ahmad A. Al-Qudah
LM751 - ADVANCED HEMATOLOGY
Leukocytes

3. Leukocytes
Part I
- What is Leukocytes.
- Leukopoiesis .
- Myelocytes :
- Neutrophil .
- Eosinophil .
- Basophil .
- Monocyte .
- Lymphocytes :
- B Lymphocyte
- T Lymphocyte

4. Leukocytes
- The second category of cellular blood elements
- Leukocytes ≈ white cells (Leuko: white / cyte: cell)
- Got their name from the white buffy coat obtained upon
centrifugation of whole blood.

6. - Leukocyte are categorized into:
- Myelocytes: Derived from the Myeloid stem cell .
- Lymphocytes: Derived from the Lymphoid stem cells .
Leukocytes
50-70% 0-4% 0-3%
20-40% 3-8%

7. Leukopoiesis
Haematopoietic stem cells (HSCs)
HSC
committed progenitors
neutrophil
NK cell
erythrocytes
dendritic cell
plateletsmegakaryocyte
macrophage
eosinophil
basophil
B cell
T cell
specialized cells
CFU-GEMM

8. Leukopoiesis

9. - Myelocytes are generated and maturate in the BM.
- These cells are divided into:
1. Granulocytes:
- Are granulated cells with poly or bi segmented nuclei
- Include three types of cells
A. Polymorphoneutrophile (PMN)
B. Basophile (Baso)
C. Eosinophile (Eos)
2. Monocytes: Large cells with an asegmented nucleus
Leukopoiesis
Myelocytes

10. Leukopoiesis
Neutrophils
HSC CFU-GEMM CFU-GM
GM-CSF
IL-3
G-CSF
IL-3
Myeloblast
- They have a high N:C ratio , and scanty to moderate amounts of basophilic
cytoplasm.
- The nucleus is approximately round, nuclear chromatin is diffuse and
nucleoli may be apparent.
- It’s the earliest recognizable cell of the neutrophilic series.
- Less than 1% of normal bone marrow cell’s.

11. Leukopoiesis
Neutrophils
HSC CFU-GEMM CFU-GM
GM-CSF
IL-3
G-CSF
IL-3 Myeloblast
Promyelocyte
- Promyelocytes are larger than myeloblasts with lower nucleocytoplasmic ratio.
- The cytoplasm is more basophilic than that of a myeloblast and contains
azurophilic (pinkish-purple) primary granules.
- The nucleus is approximately round, nuclear chromatin is diffuse and nucleoli
may be apparent.
- 1-5 % in the Bon Marrow .

12. Leukopoiesis
Neutrophils
HSC CFU-GEMM CFU-GM
GM-CSF
IL-3
G-CSF
IL-3 Myeloblast Promyelocyte
Myelocyte
- Myelocytes are smaller than promyelocytes.
- They have both azurophilic primary and secondary granules that
are characteristic of specific lineages, i.e. neutrophilic,eosinophilic
or basophilic granules.
- The myelocyte nucleus is round or oval and
shows chromatin condensation;
no nucleolus is apparent.
- Less than 10 % of the total Bone Marrow cells .

13. Leukopoiesis
Neutrophils
HSC CFU-GEMM CFU-GM
GM-CSF
IL-3
G-CSF
IL-3 Myeloblast Promyelocyte
Myelocyte
Metamyelocyte
- Metamyelocytes have similar characteristics to myelocytes but
differ in that the nucleus is indented, U-shaped or C-shaped and
the primary granules are usually no longer apparent.
- 13-22% of the normal Bone Marrow cells .

14. Leukopoiesis
Neutrophils
HSC CFU-GEMM CFU-GM
GM-CSF
IL-3
G-CSF
IL-3 Myeloblast Promyelocyte
MyelocyteMetamyelocyte
Neutrophilic Band
- Band cells are intermediate in characteristics between mature cell. and
metamyelocytes.
- The nucleus has an irregular shape with some parallel edges .
It differs from a mature or segmented neutrophil
in that the nucleus is not divided into
distinct lobes or segments. ( 40 % )

15. Leukopoiesis
Neutrophils
HSC CFU-GEMM CFU-GM
GM-CSF
IL-3
G-CSF
IL-3 Myeloblast Promyelocyte
MyelocyteMetamyelocyteNeutrophilic Band
Polymorphonuclear Neutrophil
- Morphology :
- Multi-lobulated (3 – 4 segments) nucleus where lobes are
connected by thin filament of nuclear material.
- The cytoplasm of neutrophils is very pale blue and is packed
with fine neutrophilic lysosomal granules .

16. Leukopoiesis
Neutrophils
- Granules:
1. Primary: contains myeloperoxidase, acid
phosphatase and acid hydrolases
2. Secondary / specific (predominant):
contains collagenase lactoferrin and
lysozyme
- Drumstick: inactive X-chromosome
- Function:
Chemotactic and phagocytic
- Differential:
50-70% of circulating leukocytes.

17. Leukopoiesis
Eosinophils
HSC CFU-GEMM CFU-Eo
CSF
IL-3 IL-5 Myeloblast Promyelocyte
MyelocyteMetamyelocyte
Eosinophil
- Morphology:
- Bi-lobed (2 segments) nucleus
- Pale blue cytoplasm, which is packed with large orange–red
granules.

18. Leukopoiesis
Eosinophils
- Granules:
1. Large, crystalloid granules:
contain cationic proteins, neurotoxins,
peroxidase, antihistamin
and a variety of lysosomal enzymes.
2. Small granules:
contain aryl sulphatase, Gelatinase and
acid phosphatase
- Function:
- Defense against parasitic infection
- Allergic reactions.
- Removal of fibrin (inflammation)
- Differential:
3 -4 % of circulating leukocytes.

19. Leukopoiesis
Basophils
HSC CFU-GEMM CFU-Bas
CSF
IL-3 IL-6 Myeloblast Promyelocyte
MyelocyteMetamyelocyte
Basophil
- Morphology:
- have a lobulated nucleus, large purple-staining granules ,
very pale blue cytoplasm.
- Cirulate in blood and migarate to the tissues where they become
“mast cells”.

20. - Granules:
- contains heparin and histamine
- Function:
- Immediate hypersensitivity reactions,
- Allergic and inflammatory responses
- Control of parasitic infections
- Differential:
1 -2 % of circulating leukocytes.
Leukopoiesis
Basophils

21. Leukopoiesis
Monocyte
HSC CFU-GEMM CFU-GM
GM-CSF
IL-3
M-CSF
IL-3 MonoBlast Promonocyte
MonocyteMacrophage
Respond to Chemotaxis
Morphology:
- Are the largest normal blood cells.
- Lobulated nuclei and huge greyish- bluecytoplasm
- Cytoplasm is sometimes opaque and may be vacuolated and/or
granulated.

22. Leukopoiesis
Monocyte
- Function:
- Monocytes have an intravascular life span of
several days.
- They function mainly in tissues where they
differentiate into long-lived macrophages
- Antigen presenting cells (APC)
-Influential role of other immune cells
- Differential:
2 -8 % of circulating leukocytes.

23. Leukopoiesis
Lymphocyte
HSC CFU-L
IL-4,7
ProlymphocyteLymphoblast B Lymphocyte
ProlymphocyteLymphoblast
IL-1,2,7
T Lymphocyte
Bone Marrow
Thymus

24. Leukopoiesis
Lymphocyte
- Morphology:
- The majority are small lymphocytes
- High nuclear: cytoplasmic ratio
- dense chromatin clumping (purplish stained)
- Lymphocytes are divided into three morphological
categories, depending on their size, the amount of
cytoplasm and the presence or absence of
cytoplasmic granules.
- Differential:
25 – 35 % of circulating leukocytes.

25. Leukopoiesis
Lymphocyte
- Function:
1. T lymphocytes:
- Recognition of foreign Ag’s on the context of MHC molecules on surfaces of APC
- Cytotoxic T cells (CD8+) : mediate the destruction of their targets
- T-helper cells (CD4+) : influence the innate and adaptive response
- Natural killer (NK) cells: CD8+ T cells express HLA receptor that are required to mediate
the killing of their targets.
2. B-lymphocytes: maturate in BM
Differentiate into antibody-producing cells (Plasma cells)

29. Part II
Leukocytes
Morphological abnormalities

30. Morphological abnormalities
- Normal Morphology .
- Morphologic Alterations of Neutrophil Nuclei :
- Pelger – Huet Anomaly .
- Hyper-segmented neutrophils
- Drumsticks
- Morphologic Alterations of Neutrophil Cytoplasm :
- Alder – Reilly Anomaly
- Chediak – Higashi Syndrome
- May – Hegglin Anomaly
- Dohle Bodies , Toxic Garnulation
- Morphologic Alterations of Lymphocyte & Monocyte :
- Reed-Sternberg cells
- Hand-mirror cells
- Sezary cells
- Smudge cells
- Atypical lymphocyte , Activated monocytes

31. Normal Morphology
Neutrophils

32. Normal Morphology
Neutrophils

33. Normal Morphology
Eosinophils

34. Normal Morphology
Basophils

35. Normal Morphology
Lymphocytes

37. Normal Morphology
Monocytes

38. Morphologic Alterations of Neutrophil Nuclei
Pelger – Huet Anomaly
- A neutrophil with a hypolobulated, rounded nuclei and
condensed chromatin.
- A thin strand of chromatin may connect the lobes, creating a
pince-nez (spectacle) shape, or a larger bridge can give the nucleus
a peanut appearance.

39. Morphologic Alterations of Neutrophil Nuclei
Hyper-segmented neutrophils
- Neurophils with abnormally increased number of nuclear lobes
- > 5% of PMN with 5 lobes or any appearance of 6-loops PMN
- Very common in cases of megaloblastic anemia (B12 or folate def.)

40. Morphologic Alterations of Neutrophil Nuclei
Drumsticks
- Inactive X Chromosome in females .

41. Morphologic Alterations of Neutrophil
Cytoplasm
Alder – Reilly Anomaly
- Recessive disorder
- Deposition of Mucopolysaccharides ( Lipids ) in cytoplasm.
- Appear as metachromatic granules .

42. Morphologic Alterations of Neutrophil
Cytoplasm
Chediak – Higashi Syndrome
- Rare Autosomal Recessive state .
- Abnormally large Peroxidase-Positive lysosomes are seen in the PMN
(and most cells of the body) results in Albinism.

43. Morphologic Alterations of Neutrophil
Cytoplasm
May – Hegglin Anomaly
- Rare Autosomal Dominant condition .
- Presence of Large Dohle Body-Like formation ( combination of
rods and granules that are ribosomal in origin )

44. Morphologic Alterations of Neutrophil
Cytoplasm
Dohle Bodies
- Small blue-gray (single or multiple) inclusions in the cytoplasm of
neutrophils, often at the margins (eccentric).
- Composed of rough endoplasmic reticulum and glycogen granules.
- Associated with inflammatory disorders, burns, MPD and MDS

45. Morphologic Alterations of Neutrophil
Cytoplasm
Toxic Garnulation
- Neutrophils that are characterized by an increased numbers of granules that
are larger and more basophilic than normal.
- May appear in severe bacterial infections, burns, malignancies,
drug reactions.

46. Morphologic Alterations of Lymphocyte &
Monocyte
Reed-Sternberg cells
The Mirror Nuclei

47. Morphologic Alterations of Lymphocyte &
Monocyte
Hand-mirror cells
- Characteristic “hand mirror” shape of T cells in a patient
with T-cell acute lymphocytic leukemia.

48. Morphologic Alterations of Lymphocyte &
Monocyte
Sezary cells
- Lymphocytes with frequently convoluted nuclei (Sezary cells) in a
patient with advanced mycosis fungoides.

49. Morphologic Alterations of Lymphocyte &
Monocyte
Smudge cells
- Fragile lymphocytes rupture (during film preparation)
- Nucleus appears spread out with hazy borders and absent
cytoplasm.

50. Morphologic Alterations of Lymphocyte &
Monocyte
Atypical lymphocyte
- Common in viral infections (e.g. Herpes infection and HIV)
- Large lymph with prominent foamy/vaculated cytoplasm and
irregular nucleus (kidney shaped or lobulated)
- Basophilic cytoplasm and coarse chromatin

51. Morphologic Alterations of Lymphocyte &
Monocyte
Activated monocytes
- Associated with inflammatory reaction to bacteremia
- Macrophages with increased granulation

52. Morphologic Alterations of Lymphocyte &
Monocyte
Auer rods
- Are red, needle-like structures thought to be Accumulation of
primary granules.
- Characterestic of acute myeloid leukemia

54. Part III
Leukocytes
Special stains

55. Special stains
- Myeloperoxidase ( MPO )
- Sudan Black B (SBB)
- Leukocyte alkaline phosphatase (LAP)
- Specific esterase
- Non-specific esterase
- Acid phosphatase
- Periodic acid schiff (PAS)

56. Myeloperoxidase ( MPO )
- Myeloperoxidase is present in the primary granules of neutrophils
and the secondary granules of eosinophils
- Principle: Benzidine or diaminobenzidine are converted (oxidized)
inside the granules into brownish precipitate.
- Interpretation:
- PMN’s/Eosinophils and the progenitors (from the
promyelocytic stage on) are positively stained.
- Monocytes lysosomal granulocytes are faintly positive
- Lymphocytes and NRBC’s lack the enzyme
- Purpose: To differentiate a myelogenous or monocytic leukemia
from acute lymphocytic Leukemia.

57. Myeloperoxidase ( MPO )
Leukemic myeloblasts stained with peroxidase

58. Myeloperoxidase ( MPO )
Acute lymphocytic leukemia stained with peroxidase, the blast cells are
unreactive (unstained) while the neutrophil is positively stained

59. Sudan Black B (SBB)
- SBB is a fat soluble stains that stains intracellular lipids as well as
phospholipids.
- Staining pattern is parallel to myeloperoxidase (MPO) staining
- SBB can be used to stain old blood or BM sample and the stain does
not faid with time (MP is sensitive to light; therefore fresh samples are
recommended/ enzymatic activity may diminish on samples older
than 3 weeks)
- Interpretation: Balck/grayish-black staining of the cytoplasm

60. Sudan Black B (SBB)

61. Sudan Black B (SBB)
Positive sudan black
B (SBB) stain in a
patient with AML.

62. Leukocyte alkaline phosphatase (LAP)
- AP activity is found in the cytoplasm of PMN’s, osteoclasts and
some lymphocytes
- Based on the determination of LAP score
- Differential test for CML from leukemoid reactions and other
MPD
- Sodium -naphtyl phsophate (or naphtol-AS-BI phosphate)is
used as a substrate to produce a bright red products
- Interpretation:
- Stain intensity is determined for 100 counted PMN or band
and scored from 0 - 4
- The sum of the scores reflects LAP score (index)
- Normal LAP score is 15 - 130

63. Leukocyte alkaline phosphatase (LAP)
- Interpretation:
- Low LAP score (<15)
- Chronic myeloid leukemia, PNH, Myelodysplastic
syndrom, rare infections or toxic exposure
- High LAB score (>130)
- Leukomoid reactions in response to infections
and MPD other than CML, inflammatory disorders ,
stress, certain drugs (including lithium, corticosteroids
and estrogen)

64. Leukocyte alkaline phosphatase (LAP)
Grading:
- (0) No stain
- (+1) Faint stain
- (+2) Moderate stain
- (+3) Strong stain
- (+4) Strong stain without cytoplasmic background
Positive LAP reactionNegative LAP reaction

65. Specific esterase
- Also called Leder stain
- Is used to identify cells of granulocytic series ONLY.
- Cellular esterase hydrolyze naphtol AS-D chloroacetate substrate
to produce a bright red (red-pink) product at the site of enzymatic
activity
- Neutrophilic granulocyte show a positive reaction from the
promyelocytes stage on

66. Specific esterase

67. Non-specific esterase
- This is performed using -naphtyl butyrate or -naphtyl
acetate as a substrate
- Stain positive for monocytic cells but not granulocyteic cells
- Mature T lymphocytes stain positively with a characterestic
focal dot-like pattern.

68. Acid phosphatase
- Acid phosphatase is found in all hematopoietic cells with the
highest levels in macrophages and osteoclast.
- A localized dot-like pattern is seen in many T-lymphoblasts.
- Tartar-resistance acid phosphatase (TRAP) is an isoenzyme that is
found at high levels in cells of hairy cell leukemia.
- To test for TRAP: In the presence of tartaric acid, Naphtol-AS-BI
phosphate coupled to fast garent GBC salt are used to produce a
bright red homogenous or granular precipitate in the cytoplasm of
cells with enzymatic activity (if a resistance isoenzyme is present)

69. Acid phosphatase

70. Periodic acid schiff (PAS)
- Detects intracellular glycogen and neutral mucopolysaccharides
that are found in the majority of hematopoietic cells at variable
quantities.
- Detection is based on the oxidation of -glycols in carbohydrates
and CHO-containing compounds resulting in the formation of
polyaldehyde that can be detected by schiff reagent.
- Products of the staining is a diffuse red stain or pink-to-red
granules or even clumps of varying size.

71. Periodic acid schiff (PAS)

72. Periodic acid schiff (PAS)
Marrow film stained with periodic acid Schiff reagent. Intense PAS-positive
staining of leukemic erythroblasts (Acute erythroid leukemia)

73. Special stains
Blasts Identified
Cellular Element
Stained
Cytochemical Reaction
Myeloblasts strong positive;
monoblasts faint positive
Neutrophil primary
granules
Myeloperoxidase (MPO)
Myeloblasts strong positive;
monoblasts faint positive
PhospholipidsSudan Black B (SBB)
Myeloblasts strong positiveCellular enzymeSpecific esterase
Monoblasts strong positiveCellular enzyme
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