The white cells granulocytes and their benign disorders

The White Cells
Granulocytes and Their
Benign Disorders
Usman Ali Shams
MLS M-Phil HaematoTechnology
University of Health Sciences Lahore

Learning Outcome
At the end of this lecture students will be to
• Describe Granulcyte and granulopoeisis as well as
its growth factor,
• Elaborate Clinical Application of G-CSF.
• Neutrophil and monocyte function i-e adherence,
chemotaxis, phagocytosis, and killing.
• Defects in Phagocytic Cell Function.
• Benign Disorders of Neutrophils.

Contents
• Introduction to leucocytes.
• Granulcytes
• Granulopoeisis as well as its growth factor
• Clinical Application of G-CSF.
• Neutrophil and monocyte function
• i-e adherence, chemotaxis, phagocytosis, and
killing.
• Defects in Phagocytic Cell Function.
• Benign Disorders of Neutrophils.
• Pelger–Huët anomaly
• May–Hegglin anomaly
• Chédiak–Higashi syndrome

Leukocytes Introduction
►The terms leukocyte and white blood cell (WBC) are the synonymous names given to the
nucleated blood cells that are involved in the defense against foreign pathogens or
antigens.
►Leukocytes develop from the pluripotential hematopoietic stem cell in the bone marrow
► Leukocytes are classified as granulocytes (neutrophils, eosinophils, basophils), and
Agranulocytes i-e monocytes, and lymphocytes.

Leukocytes Introduction CONT…
►With the Exception of T lymphocytes, leukocyte precursors proliferate, differentiate, and
mature in the bone marrow.
►Mature leukocytes are released into the peripheral blood where they circulate briefly until
they move into the tissues in response to stimulation.
►They perform their function of host defense primarily in the tissues.

Granulocytes
►Granulocytes are a type of white blood cell that has small granules.
►Granulocytes and monocytes are formed in the bone marrow from a common precursor
cell.
►Typically, granulocytes have a role both in innate and adaptive immune responses in the
fight against viral and parasitic infections.
►The specific types of granulocytes are neutrophils, eosinophils, and basophils.

Granulopoiesis
►In the granulopoietic series progenitor cells, myeloblasts,
promyelocytes and myelocytes form a proliferative or mitotic pool of
cells.
►while the metamyelocytes, band and segmented granulocytes make
up a post‐mitotic maturation compartment.

Granulopoiesis
►Following their release from the marrow, granulocytes spend only 6–10 hours
in the circulation before entering tissues where they perform their phagocytic
function.
►They spend on average 4–5 days in the tissues before they are destroyed
during defensive action or as the result of senescence.

Control of Granulopoiesis.
Growth Factor
►Many growth factors are involved
in this maturation process.
► interleukin‐1 (IL‐1), IL‐3, IL‐5
►IL‐6, IL‐11, granulocyte
macrophage colony‐stimulating
factor (GM‐CSF).
►granulocyte CSF (G‐CSF) and
monocyte CSF (M‐CSF).

Control of Granulopoiesis.
Growth Factor
►Increased granulocyte and
monocyte production in response
to an infection is induced by
increased production of growth
factors from stromal cells and T
lymphocytes,
►Stimulated by endotoxin, and
cytokines such as IL‐1 or tumour
necrosis factor (TNF).

Clinical Application of G-CSF
►Clinical administration of G‐CSF intravenously or subcutaneously
produces a rise in neutrophils.
►Short‐acting G‐CSF is given daily.
►A longer‐acting PEGylated G‐CSF can be given once in 7–14 days)

Clinical Application of G-CSF CONT……
1)Post‐chemotherapy, radiotherapy or stem cell transplantation (SCT):
►In these situations, G‐CSF accelerates granulocytic recovery and shortens
the period of neutropenia.
►This may translate into a reduction of length of time in hospital, antibiotic
usage and frequency of infection.
►Periods of extreme neutropenia after intensive chemotherapy cannot be
prevented.

2) Myelodysplasia and aplastic anaemia:
►G‐CSF has been given alone or in conjunction with erythropoietin in an
attempt to improve bone marrow function and the neutrophil count.
3) Severe benign neutropenia:
►Both congenital and acquired neutropenia, including cyclical and
drug‐induced neutropenia, often respond well to G‐CSF.

4) Peripheral blood stem cell mobilisation:
►G‐CSF is used to increase the number of circulating multipotent
progenitors from donors or the patient.
►improving the harvest of sufficient peripheral blood stem cells for
allogeneic or autologous transplantation.

Granulocytes
1)Neutrophil (polymorph):
► This cell has a characteristic dense nucleus consisting
of between two and five lobes and a life span of 6-
10hrs in blood.
► Having a pale cytoplasm with an irregular outline
containing many fine pink–blue (azurophilic) or grey–
blue granules.
► The granules are divided into primary, which appear
at the promyelocyte stage.
► Secondary (specific), which appear at the myelocyte
stage and predominate in the mature neutrophil

Granulocytes Neutrophil CONT…….
► Both types of granule are lysosomal in origin.

Granulocytes…..CONT
2) Eosinophils:
► These cells are like neutrophils, except that the cytoplasmic
granules are coarser and more deeply red staining and there
are rarely more than three nuclear lobes .
► Eosinophil myelocytes can be recognized but earlier stages
are indistinguishable from neutrophil precursors.
► The blood transit time for eosinophils is longer than for
neutrophils.
► Have a special role in allergic responses, defence against
parasites and removal of fibrin formed during inflammation

Granulocytes…..CONT
3) Basophils:
► These are only occasionally seen in normal
peripheral blood.
► They have many dark cytoplasmic granules
which overlie the nucleus and contain
heparin and histamine.
► In the tissues they become mast cells.
► They have immunoglobulin E (IgE) attachment
sites and their degranulation is associated
with histamine release.

Agranulocytes
1)Monocyte:
► These are usually larger than other peripheral blood
leucocytes.
► possess a large central oval or indented nucleus with
clumped chromatin.
► The abundant cytoplasm stains blue and contains
many fine vacuoles, giving a ground‐ glass
appearance.
► Cytoplasmic granules are also often present.

Neutrophil Maturation/Precursor
► The Earliest recognizable precursor is
MyeloBlast:
• Bone marrow percentage is 0.2-1.5%. Size
vary from 14-20μm.
• High N:C ratio.
• Has round or Oval Nucleus, delicate Lacy
Chromatin, Nucleoli is present.
• Light Blue Cytoplasm.
• Granules are absent.
• CD 13,33,34,38 are the Markers.
• Has almost One day Maturation Transit Time.

►The Myeloblast upon Maturation Leads to.
Promyelocyte:
• Bone marrow percentage is 2-4%. Size vary from 15-21μm.
• High N:C ratio but less than Myeloblast.
• Has round or Oval Nucleus, delicate Lacy Chromatin but more condensed than
Blasts, Nucleoli is present.
• Deep Blue Cytoplasm.
• Large, reddish-purple (azurophilic) primary or nonspecific granules.
• CD 33,38 are the Markers.
• Has almost 1-3 days Maturation Transit Time.

Promyelocyte:

►The Promyelocyte upon Maturation Leads to.
Myelocyte:
• Bone marrow percentage is 8-16%. Size varies from 12-18μm.
• Decreased N:C ratio from Promyelocyte.
• Has round to Oval Nucleus, More condensed Chromatin, Nucleoli is often Absent.
• Light Blue, More mature tan to pink color cytoplasm.
• Small pinkish-red to specific granules, azurophilic Granules, secretory Vesicles.

Myelocyte:

►The myelocyte upon Maturation Leads to.
Metamyelocyte:
• Bone marrow percentage is 9-25%. Size varies from 10-18μm.
• Decreased N:C ratio from myelocyte.
• Has kidney to Bean shaped Nucleus, More condensed Chromatin stains dark
purple.
• Pinkish-tan color cytoplasm.
• Predominance of Small pinkish-lavender to specific granules, azurophilic
Granules, secretory Vesicles.
• Has almost 0.5-4 days Maturation Transit Time.

Metamyelocyte:

►The Metamyelocyte upon Maturation Leads to.
Band cells (Non segmented or stab):
• Bone marrow percentage is 9-15%. slightly smaller in diameter than the
metamyelocyte.
• Decreased N:C ratio from metamyelocyte.
• Has horseshoe shaped Nucleus, More condensed Chromatin stains dark purple.
• Pinkish-tan to clear cytoplasm.
• Abundant small, pinkish lavender specific granules, azurophilic Granules,
secretory Vesicles, tertiary Granules.
• Has almost 0.5-4 days Maturation Transit Time.

Band cells (Non segmented or stab):

►The Band Cells upon Maturation Leads to.
Segmented Neutrophil (Polymorphonuclear):
• Although similar in size to the band form, the neutrophil, or PMN, is recognized,
as its name implies, by a segmented nucleus with two or more lobes connected
by a thin nuclear filament.
• Decreased N:C ratio.
• Nucleus segmented into 2-4 lobes, Chromatin condensed, stains Deep
Purple/Black.
• Pinkish or tan color cytoplasm.

Segmented Neutrophil (Polymorphonuclear): CONT……
• Abundant small, pinkish lavender specific granules, azurophilic Granules,
secretory Vesicles, tertiary Granules.
• Fewer than three lobes are considered hypo segmented.
• A cell with more than five lobes is considered abnormal and referred to as a
hyper segmented neutrophil.
• CD markers for neutrophils are CD15,CD16, CD11b/CD18

Segmented Neutrophil (Polymorphonuclear): CONT……

Neutrophil &
Monocyte
Function

Neutrophil & Monocyte
• To be effective in its role in host defense, the neutrophil must move to the site of
the foreign agent, engulf it, and destroy it.
• Thus, neutrophils function primarily in the tissues where microbial invasion
typically occurs.
• The four steps in the innate immune response can be described as
• Adherence.
• Migration (chemotaxis).
• Phagocytosis.
• Bacterial killing

Neutrophil Function
1: Adherence:
• Neutrophils flow freely along the vascular endothelium when neither the
neutrophil nor VEC is activated,
• Neutrophil adherence and migration to the site of infection begin with a series of
interactions between the neutrophils and VEC when these cells are activated by a
variety of inflammatory mediators (cytokines).

Neutrophil Function Adherence:CONT….
• Several different families of cell adhesion molecules (CAM) and their ligands play
a major role in the adherence process.
• Adhesion molecules and their ligands located on the leukocytes and VEC act
together to induce activation-dependent adhesion events.
• Adhesion molecules are transmembrane proteins with three domains:
• Extracellular.
• Transmembrane.
• Intracellular.

Neutrophil Function
The most common Adhesion molecules are,
• β2 (CD18) family of leukocyte integrins and their ligands (immunoglobulin-like
CAM)
•Selectins and their ligands.
•Intercellular adhesion molecules (ICAMs)

Neutrophil Function
Adherence will be Completed in 4 stages i-e
Stage 1:
Involves the activation of VECs that allows for a loose association of VECs
with neutrophils. Inflammatory cytokines induce the expression of E- and P-selectins
and L-selectin ligands on VEC.

Neutrophil Function
Stage 2:
• Is the activation of neutrophils.
• Chemokines (cytokines with chemotactic activity) or other chemo-attractants bind
to the endothelial cell surface where they interact with the loosely bound
neutrophils and result in activation of neutrophil integrins.

Neutrophil Function
Stage 3:
• Involves arrest of neutrophil rolling because the activated neutrophils are more
tightly bound to the VECs.
• Expression of activation-dependent b2-integrin adhesion molecules on
neutrophils mediates firm adherence to ICAMs expressed by activated VECs near
the site of infection or inflammation.
• Stage 3 ends with a strong, sustained attachment of the leukocyte to the VEC.

Neutrophil Function
Stage 4:
• involves the transendothelial migration phase that occurs when neutrophils
move through the vessel wall at the borders of VECs by the process of diapedesis.
• As neutrophils pass out of the vessel and into the tissue, VECs modify their cell-to-
cell adherent junctions.
• The neutrophils use pseudopods to squeeze between endothelial cells, leaving
the vascular space and passing through the subendothelial basement
membranes and peri endothelial cells.

Neutrophil Function
2: Migration (chemotaxis):
• Once in the tissues, neutrophil migration (chemotaxis) is guided by
chemoattractant gradients released from damaged tissues .
• Neutrophils continue their migration through the extravascular tissue, moving by
directed ameboid motion toward the infected site.
• Locomotion of neutrophils (and other leukocytes) is a process of “crawling,” not
“swimming.”

Neutrophil Function Migration (chemotaxis):CONT….
• There is an extension of a broad pseudopodium (protopod) at the anterior of the
cell (containing the nucleus and organelles) and a narrow knoblike tail (uropod) at
the rear of the cell.
• Neutrophil migration through the tissues requires b1 and b2 integrins with the
continuous formation of new adhesive contacts at the cell front and detachment
from the adhesive substrate at the rear of the cell.

Neutrophil Function Migration (chemotaxis): CONT……..
Chemotaxis is induced by a variety of chemoattractant molecules including
• bacterial formyl peptides (fMLP),
• C5a,
• IL-8,
• PAF
• many of the same molecules that activated the neutrophil during Stage 2.

Neutrophil Function
3: Phagocytosis:
• After arriving at the site of infection, phagocytosis by neutrophils can begin.
• Phagocytes must recognize the pathogen as foreign before attachment occurs
and phagocytosis is initiated.
• Once a pathogen is recognized, ingestion of the particle, fusion of the neutrophil
granules with the phagosome (degranulation), and finally the process of bacterial
killing and digestion occur.

Neutrophil Function Phagocytosis:CONT….
• Phagocytosis is an active process that requires a large expenditure of energy by
the cells. The energy required for phagocytosis can result from anaerobic
glycolysis or aerobic processes.
• Phagocytes recognize unique molecular characteristics of the pathogen’s surface
(Pathogen-associated molecular pattern (PAMP) and bind to the invading
organism via specific PRR(Pattern recognition receptors).
• Enhancement of phagocytosis through the process of opsonization speeds the
ingestion of particles.

Neutrophil Function Phagocytosis:CONT…..
• Two well-characterized opsonin's are immunoglobulin G (IgG) and complement
component C3b.
• The antibody IgG binds to the microorganism/particle by means of its Fab region.
• The Fc region of the antibody attaches to three classes of Fc receptors on the
neutrophil membrane.
• Thus, the antibody forms a connecting link between the microorganism/particle
and the neutrophil. (FcgRI[CD64], FcgRII[CD32], FcgRIII[CD16]).
• The neutrophil also has receptors for activated complement component C3b
(CR1/CD35, CR3/CD11b/CD18, CR4/CD11c/ CD18).
• Following recognition and attachment, the particle is surrounded by neutrophil
cytoplasmic extensions or pseudopods.

• As the pseudopods touch, they fuse, encompassing the particle within a
phagosome that is bound by the cytoplasmic membrane turned “inside out.”
• A plasma membrane–bound oxidase is activated during ingestion, which plays an
important role in microbicidal activities.
• Some bacteria with polysaccharide capsules avoid recognition, thus reducing the
effectiveness of phagocytosis.
Neutrophil Function Phagocytosis:CONT…..

Neutrophil Function
Phagocytosis:CONT…..

Neutrophil Function
4: Bacterial Killing and/or digestion:
• After formation of the phagosome, neutrophil granules migrate toward and fuse
with the phagosome membrane, discharging their granule contents into the
phagocytic vesicle (degranulation), forming a phagolysosome.
• Microbicidal mechanisms that follow ingestion can be divided into
1. Oxygen-dependent/Oxidative
2. Oxygen-independent/Nonoxidative Activities.

Neutrophil Function Bacterial Killing and/or digestion: CONT…
1: Oxygen Dependent Pathway: (MPO dependent Pathway)
• In the oxygen‐dependent reactions, superoxide (𝑂2
−
), hydrogen peroxide (𝐻2𝑂2)
and other activated oxygen (𝑂2) species, are generated from 𝑂2 and reduced
nicotinamide adenine dinucleotide phosphate (NADPH).
• Once assembled, the oxidase produces superoxide anion (O2-) and NADP+.
• The NADP+ activates the HMP shunt ,generating more NADPH.
• 𝑂2
−
is further metabolized to produce additional ROS with increasing microbicidal
potency

Oxygen Dependent Pathway: CONT…….
• The second oxygen-dependent microbicidal system involves the neutrophil’s
primary granule enzyme myeloperoxidase (MPO).
• Myeloperoxidase catalyzes the interaction of hydrogen peroxide produced during
the Previous Pathway with intracellular halide ions (e.g., chloride/Cl-)
• giving rise to oxidized halogens (e.g., hypochlorous acid/HOCl) that increase
bacterial killing

2: Oxygen Independent Pathway: (MPO Independent Pathway)
• Oxygen-independent granule proteins present in primary, secondary, and tertiary
granules of neutrophils can successfully kill and degrade many strains of both
gram-negative and gram-positive bacteria.
• Initially, the pH within the phagolysosome decreases and inhibits bacterial
growth, but this alone is insufficient to kill most microbes.
• These may act alone (e.g. cathepsin G) or in conjunction with 𝐻2𝑂2 (e.g. lysozyme,
elastase).
• Lactoferrin, an iron‐binding protein, is bacteriostatic by depriving bacteria of iron
and generating free radicals.

Oxygen dependent and independent factors

Defects in
Phagocytic Cell
Function

Defects in Phagocytic Cell Function
1: Chemotaxis:
• These defects occur in rare congenital abnormalities (e.G. ‘Lazy leucocyte’
syndrome).
• Common acquired abnormalities, either of the environment.
• Corticosteroid therapy or of the leucocytes themselves.
• In acute or chronic myeloid leukaemia, myelodysplasia and the myeloproliferative
syndromes.

2: Phagocytosis:
• These defects usually arise because of a lack of opsonization
• which may be caused by congenital or acquired causes of
hypogammaglobulinaemia or lack of complement components.

3: Killing:
• This abnormality is clearly illustrated by the rare X‐linked or autosomal recessive
chronic granulomatous disease that results from abnormal leucocyte oxidative
metabolism.
• (CGD) is an inherited disorder that occurs when a type of white blood cell
(phagocyte) that usually helps your body fight infections doesn't work properly.
• As a result, the phagocytes can't protect your body from bacterial and fungal
infections.
• People with chronic granulomatous disease may develop infections in their lungs,
skin, lymph nodes, liver, stomach and intestines, or other areas.

Defects in Phagocytic Cell Function Killing:CONT..
• In addition, a failure to switch off the inflammatory response leads to the
formation of granulomas, the distinctive hallmark of the disorder.
• They may also develop clusters of white blood cells in infected areas.
• Most people are diagnosed with CGD during childhood, but some people may not
be diagnosed until adulthood.

Defects in Phagocytic Cell Function Killing:CONT..
Causes/Pathophysiology of CGD:
• All the subtypes of X-linked CGD are caused by mutations in the gene for the
gp91-phox subunit of cytochrome b (CYBB) located at Xp21.1.
• The genes normally produce proteins that form an enzyme that helps your
immune system work properly.
• The enzyme is active in white blood cells (phagocytes) that catch and destroy
fungi and bacteria to protect you from infections.
• The enzyme is also active in immune cells that help your body heal.
• When there are mutations to one of these genes, the protective proteins are not
produced, or they're produced but they don't function properly.

Benign
Disorders of
Neutrophils

Benign disorders
A number of the hereditary conditions may give rise to changes in granulocyte
morphology
That include
• Pelger–Huët anomaly
• May–Hegglin anomaly
• Chédiak–Higashi syndrome

Pelger Huet Anomaly
• It is a benign anomaly inherited in an autosomal dominant fashion and occurs
in about 1 in 5000 individuals.
• The neutrophil nucleus does not segment beyond the two-lobed stage and
can appear as a single, round nucleus with no segmentation.
• The bilobed nucleus has a characteristic dumbbell shape with the two lobes
connected by a thin strand of chromatin.
• Rod-shaped and peanut-shaped nuclei can also be found.
• Cells with this appearance are often called pince-nez cells.

Pelger Huet Anomaly CONT….
• Individuals heterozygous for Pelger-Huët anomaly have neutrophil nuclei that
are primarily bilobed.
• Whereas most neutrophils in homozygous individuals have a round or oval
nucleus with only a few cells having the classic bilobed shape.
• The cell is functionally normal, and individuals with hereditary Pelger-Huët
anomaly do not display increased susceptibility to bacterial infections.

Pelger Huet Anomaly CONT….
(a) Note pince-nez, or eyeglass-shaped,
nuclei. These mature cells could easily
be confused for bands if not for the
highly clumped chromatin.
(b) The nucleated neutrophil is the
nonsegmented or round nucleus form
that can be found

May–Hegglin Anomaly
• May-Hegglin anomaly is a rare, inherited, autosomal dominant trait in which
granulocytes contain inclusions consisting mainly of RNA from rough
endoplasmic reticulum, which are similar in appearance to Döhle bodies.
• The inclusions can be distinguished from true Döhle bodies because they are
usually larger and rounder in shape.
• Variable thrombocytopenia with giant platelets is characteristic.
• The only apparent clinical symptom patients can exhibit is abnormal bleeding
related to the low platelet count.

May–Hegglin Anomaly
• There is a neutrophil with a Döhle-like
structure in the cytoplasm and a large
platelet

Chédiak–Higashi syndrome
• Chédiak-Higashi syndrome is a rare autosomal recessive disorder in which
death usually occurs in infancy or childhood because of recurrent bacterial
infections.
• Giant gray-green peroxidase-positive bodies and giant lysosomes are found in
the cytoplasm of leukocytes as well as most granule-containing cells of other
tissues.
• Chediak–Higashi syndrome neutrophils also have a deficiency of antimicrobial
proteins as well as disordered degranulation and chemotaxis
• These bodies are formed by fusion of primary, nonspecific and secondary,
specific neutrophilic granules.

Chédiak–Higashi syndrome CONT…
• This abnormal fusion of cytoplasmic membranes prevents the granules from
being delivered into the phagosomes to participate in killing of ingested
bacteria.
• Neutropenia and thrombocytopenia are frequent complications as the
disease progresses.
• The patients have skin hypopigmentation, silvery hair, and photophobia from
an abnormality of melanosomes.
• Lymphadenopathy and hepatosplenomegaly are characteristic.

Chédiak–Higashi syndrome CONT…
• (a) Neutrophil from Chédiak-
Higashi syndrome.
• (b) Lymphocyte from the
same patient as in a.

References
• Hoffbrand’s essential haematology / A. Victor Hoffbrand, Paul A. H. Moss. — eight edition.
• Wintrobe's clinical hematology / editors, John P. Greer, Daniel A. Arber,Bertil Glader, Alan
F. List, Robert T. Means Jr., Frixos Paraskevas, George M. Rodgers ; editor emeritus, John
Foerster. —13th edition.
• Clinical laboratory hematology / Shirlyn B. McKenzie, J. Lynne Williams, Kristin Landis-
Piwowar. — Third edition.
• Postgraduate haematology / edited by A. Victor Hoffbrand, Douglas R. Higgs, David M.
Keeling, Atul B. Mehta. – Seventh edition.

Thank you
For Comments/Suggestions:
isuasbe@gmail.com

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