Leukocytes - Advance Hematology
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Ahmad A. Al-Qudah
LM751 - ADVANCED HEMATOLOGY
Leukocytes
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LeukocytesPart I
- What is Leukocytes.- Leukopoiesis .- Myelocytes : - Neutrophil . - Eosinophil . - Basophil . - Monocyte .- Lymphocytes :
- B Lymphocyte - T Lymphocyte
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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.
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- 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%
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LeukopoiesisHaematopoietic stem cells (HSCs)
HSC
committed progenitors
neutrophil
NK cell
erythrocytes
dendritic cell
plateletsmegakaryocyte
macrophage
eosinophil
basophil
B cell
T cell
specialized cells
CFU-GEMM
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Leukopoiesis
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- 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
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LeukopoiesisNeutrophils
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.
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LeukopoiesisNeutrophils
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 .
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LeukopoiesisNeutrophils
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 .
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LeukopoiesisNeutrophils
HSC CFU-GEMM CFU-GM
GM-CSF
IL-3
G-CSF
IL-3 Myeloblast Promyelocyte
Myelocyte
Metamyelocyte
- Metamyelocytes have similar characteristics to myelocytes butdiffer in that the nucleus is indented, U-shaped or C-shaped andthe primary granules are usually no longer apparent.- 13-22% of the normal Bone Marrow cells .
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LeukopoiesisNeutrophils
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 % )
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LeukopoiesisNeutrophils
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 .
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LeukopoiesisNeutrophils
- 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.
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LeukopoiesisEosinophils
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.
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LeukopoiesisEosinophils
- 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.
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LeukopoiesisBasophils
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”.
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- Granules: - contains heparin and histamine
- Function: - Immediate hypersensitivity reactions,
- Allergic and inflammatory responses
- Control of parasitic infections
- Differential: 1 -2 % of circulating leukocytes.
LeukopoiesisBasophils
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LeukopoiesisMonocyte
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.
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LeukopoiesisMonocyte
- 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.
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LeukopoiesisLymphocyte
HSC CFU-L
IL-4,7
ProlymphocyteLymphoblast B Lymphocyte
ProlymphocyteLymphoblast
IL-1,2,7
T Lymphocyte
Bone Marrow
Thymus
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LeukopoiesisLymphocyte
- 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.
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LeukopoiesisLymphocyte
- 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)
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Part IILeukocytes
Morphological abnormalities
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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
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Normal MorphologyNeutrophils
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Normal MorphologyNeutrophils
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Normal MorphologyEosinophils
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Normal MorphologyBasophils
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Normal MorphologyLymphocytes
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Normal MorphologyMonocytes
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Morphologic Alterations of Neutrophil NucleiPelger – 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.
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Morphologic Alterations of Neutrophil NucleiHyper-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.)
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Morphologic Alterations of Neutrophil Nuclei
Drumsticks
- Inactive X Chromosome in females .
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Morphologic Alterations of Neutrophil Cytoplasm
Alder – Reilly Anomaly- Recessive disorder - Deposition of Mucopolysaccharides ( Lipids ) in cytoplasm.- Appear as metachromatic granules .
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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.
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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 )
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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
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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.
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Morphologic Alterations of Lymphocyte & Monocyte
Reed-Sternberg cellsThe Mirror Nuclei
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Morphologic Alterations of Lymphocyte & Monocyte
Hand-mirror cells
- Characteristic “hand mirror” shape of T cells in a patient with T-cell acute lymphocytic leukemia.
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Morphologic Alterations of Lymphocyte & Monocyte
Sezary cells
- Lymphocytes with frequently convoluted nuclei (Sezary cells) in a
patient with advanced mycosis fungoides.
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Morphologic Alterations of Lymphocyte & Monocyte
Smudge cells
- Fragile lymphocytes rupture (during film preparation)
- Nucleus appears spread out with hazy borders and absent cytoplasm.
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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
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Morphologic Alterations of Lymphocyte & Monocyte
Activated monocytes
- Associated with inflammatory reaction to bacteremia
- Macrophages with increased granulation
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Morphologic Alterations of Lymphocyte & Monocyte
Auer rods
- Are red, needle-like structures thought to be Accumulation of
primary granules.
- Characterestic of acute myeloid leukemia
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Part IIILeukocytes
Special stains
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Special stains
- Myeloperoxidase ( MPO )
- Sudan Black B (SBB)
- Leukocyte alkaline phosphatase (LAP)
- Specific esterase
- Non-specific esterase
- Acid phosphatase
- Periodic acid schiff (PAS)
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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.
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Myeloperoxidase ( MPO )
Leukemic myeloblasts stained with peroxidase
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Myeloperoxidase ( MPO )
Acute lymphocytic leukemia stained with peroxidase, the blast cells are unreactive (unstained) while the neutrophil is positively stained
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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
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Sudan Black B (SBB)
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Sudan Black B (SBB)
Positive sudan black B (SBB) stain in a patient with AML.
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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
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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)
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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
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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
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Specific esterase
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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.
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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)
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Acid phosphatase
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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.
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Periodic acid schiff (PAS)
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Periodic acid schiff (PAS)
Marrow film stained with periodic acid Schiff reagent. Intense PAS-positive staining of leukemic erythroblasts (Acute erythroid leukemia)
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Special stainsBlasts Identified Cellular Element
Stained Cytochemical Reaction
Myeloblasts strong positive; monoblasts faint positive
Neutrophil primary granules Myeloperoxidase (MPO)
Myeloblasts strong positive; monoblasts faint positive Phospholipids Sudan Black B (SBB)
Myeloblasts strong positive Cellular enzyme Specific esterase
Monoblasts strong positive Cellular enzyme Nonspecific esterase (NSE)
Variable, coarse or block-like positivity often seen inlymphoblasts and pronormoblasts, myeloblasts usuallynegative although faint diffuse reaction mayoccasionally be seen
Glycogen and related substances Periodic acid-Schiff
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