leukocytes - advance hematology

Ahmad A. Al-Qudah

LM751 - ADVANCED HEMATOLOGY

Leukocytes

LeukocytesPart I

- What is Leukocytes.- Leukopoiesis .- Myelocytes : - Neutrophil . - Eosinophil . - Basophil . - Monocyte .- Lymphocytes :

- B Lymphocyte - T Lymphocyte

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.

- 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%

http://en.wikipedia.org/wiki/File:Neutrophil.png

http://en.wikipedia.org/wiki/File:Basophil.png

http://en.wikipedia.org/wiki/File:Eosinophil2.png

http://en.wikipedia.org/wiki/File:Lymphocyte.png

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

Leukopoiesis

- 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

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.


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 .


HSC CFU-GEMM CFU-GM

GM-CSF

IL-3

G-CSF


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 .


HSC CFU-GEMM CFU-GM

GM-CSF

IL-3

G-CSF


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 .


HSC CFU-GEMM CFU-GM

GM-CSF

IL-3

G-CSF


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 % )


HSC CFU-GEMM CFU-GM

GM-CSF

IL-3

G-CSF


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 .


- 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.

LeukopoiesisEosinophils

HSC CFU-GEMM CFU-Eo

CSF

IL-3 IL-5 Myeloblast Promyelocyte


Eosinophil

- Morphology: - Bi-lobed (2 segments) nucleus - Pale blue cytoplasm, which is packed with large orange–red granules.

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.

LeukopoiesisBasophils

HSC CFU-GEMM CFU-Bas

CSF

IL-3 IL-6 Myeloblast Promyelocyte


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”.

- Granules: - contains heparin and histamine

- Function: - Immediate hypersensitivity reactions,

- Allergic and inflammatory responses

- Control of parasitic infections

- Differential: 1 -2 % of circulating leukocytes.

LeukopoiesisBasophils

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.

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.

LeukopoiesisLymphocyte

HSC CFU-L

IL-4,7

ProlymphocyteLymphoblast B Lymphocyte

ProlymphocyteLymphoblast

IL-1,2,7

T Lymphocyte

Bone Marrow

Thymus


- 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.


- 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)

Part IILeukocytes

Morphological abnormalities

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

Normal MorphologyNeutrophils

Normal MorphologyEosinophils

Normal MorphologyBasophils

Normal MorphologyLymphocytes

Normal MorphologyMonocytes

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.

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.)

Morphologic Alterations of Neutrophil Nuclei

Drumsticks

- Inactive X Chromosome in females .

Morphologic Alterations of Neutrophil Cytoplasm

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


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.


May – Hegglin Anomaly- Rare Autosomal Dominant condition .- Presence of Large Dohle Body-Like formation ( combination of

rods and granules that are ribosomal in origin )


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


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.

Morphologic Alterations of Lymphocyte & Monocyte

Reed-Sternberg cellsThe Mirror Nuclei


Hand-mirror cells

- Characteristic “hand mirror” shape of T cells in a patient with T-cell acute lymphocytic leukemia.


Sezary cells

- Lymphocytes with frequently convoluted nuclei (Sezary cells) in a

patient with advanced mycosis fungoides.


Smudge cells

- Fragile lymphocytes rupture (during film preparation)

- Nucleus appears spread out with hazy borders and absent cytoplasm.


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


Activated monocytes

- Associated with inflammatory reaction to bacteremia

- Macrophages with increased granulation


Auer rods

- Are red, needle-like structures thought to be Accumulation of

primary granules.

- Characterestic of acute myeloid leukemia

Part IIILeukocytes

Special stains

Special stains

- Myeloperoxidase ( MPO )

- Sudan Black B (SBB)

- Leukocyte alkaline phosphatase (LAP)

- Specific esterase

- Non-specific esterase

- Acid phosphatase

- Periodic acid schiff (PAS)

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.

Myeloperoxidase ( MPO )

Leukemic myeloblasts stained with peroxidase

Myeloperoxidase ( MPO )

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

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

Sudan Black B (SBB)

Sudan Black B (SBB)

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

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

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)

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

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

Specific esterase

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.

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)

Acid phosphatase

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.

Periodic acid schiff (PAS)

Periodic acid schiff (PAS)

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

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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leukocytes - advance hematology

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