• Granulopoiesis in embryo
• Granulocytes morphology
• Diseases of granulocytes
• Quantitative defects
• Qualitative defects
• Inflammation pathway in brief
• Morphological variations
John Hunter 1761
• Cellular (buffy coat) component of blood could retard the
“spoilage” of blood; we now would recognize these
effects as reflecting the antibacterial properties of
leukocytes. In these classic studies, Hunter observed that
when blood was allowed to stand, a “buff colored” layer
was visible on top of the red cells.
• He noted that blood from patients with infected wounds
had a thicker “buff coloured inflammatory crust” than
observed in blood from healthy subjects. With time, he
noted that blood would “spoil,” as determined by the
development of an odour typical of spoiled food.
• Hunter found that the addition of the “buff coloured
inflammatory crust” to a blood sample would delay the
time to “spoilage,” now understood to reflect the
antibacterial abilities of neutrophils.
Granulopoiesis in embryo theories
• There are many studies done (1960s and 70s), few of
them are validated, but still the theories for
haematopoiesis remains controversial.
• First haematopoietic cell arise during late gastrulation
stage, in extra-embryonic yolk sac in a structure called
as blood islands.
• Primitive haematopoiesis: transient and de novo
process- Day 19 to 8 weeks. Begins prior to the
development of circulatory system and goes on.
• Definitive haematopoiesis occurs 1-2 days latter, in
AGM and in spleen as CFU-S.
• Further from AGM cells- arises LTR-HSCs. These are
adult type of definitive stem cells.
• Newer theories suggested placenta being an individual
source(de novo) of definitive haematopoiesis with
proportion of LTR-HSC being 25 fold more than that in
• Accepted theory is that, yolk sac though had an early
contribution in haematopoiesis, the HSCs developed
closer to the birth are exclusively derived from
band or stab cells
Lobulated, dark nucleus,
mature grey cytoplasm with small
Horse-shoe shaped, darkening
nucleus, grey mature cytoplasm
Oval to kidney bean-shaped,
indented nucleus, grey mature
cytoplasm with a weak blue tent
Large, oval, non-indented nucleus,
large amount of cytoplasm with
specific and non-specific granules
Large, oval, non-indented nucleus,
large amount of sky blue cytoplasm
with non-specific granules
White blood cell
• Most abundant circulating leukocyte.
• Neutrophil homeostasis is maintained by a fine
Bone marrow storage and release,
Intravascular margination, clearance and
Neutrophil production in bone marrow
• Neutrophils are produced within haematopoietic
cords interspersed within the venous sinuses of the
• Neutrophil population in the bone marrow can be
subdivided into three pools: the stem cell pool, the
mitotic pool and the post-mitotic pool
• Stem cell pool undifferentiated haematopoietic
stem cells (HSCs),
• Mitotic pool committed granulocytic progenitor
cells that are undergoing proliferation and
• Post-mitotic pool Fully differentiated mature
neutrophils which forms the bone marrow reserve,
available for release
Neutrophil production in bone marrow
• Principal regulator of physiological granulopoiesis
is granulocyte colony stimulating factor (G-CSF)
whose effects include-
• Commitment of progenitor cells to the myeloid
lineage, proliferation of granulocytic precursors,
reduction of transit time through the
granulocytic compartment, and release of
mature cells from the bone marrow.
• Lack the G-CSF receptor and humans who
express a dominant negative receptor mutation
are profoundly neutropenic.
Neutrophil release from bone marrow
• Journey upto the tissue is needed for proper functioning.
• CXC chemokine receptor 4 (CXCR4), a G-protein coupled
receptor, is also expressed at low levels on the cell surface
of mature neutrophils and important for release of
• Interacts with stromal- derived factor 1 (SDF-1), a CXC
chemokine that is produced constitutively by bone
marrow stromal cells. The interaction between CXCR4 and
SDF-1 retains neutrophils within the marrow environment
• G-CSF exerts its multiple effects on neutrophil homeostasis
is by inhibiting the CXCR4-SDF-1 axis. Treatment of mice
with G-CSF decreases stromal cell SDF-1 production, which
correlates with an increase in neutrophil release.
Neutrophil release from bone marrow
• α4 intergrin very late antigen-4 (VLA-4) is expressed by
neutrophils- adhesion to bone marrow stromal cells and
• Expression of VLA-4 is downregulated during neutrophil
maturation in the bone marrow, and α4 blockade increases
mobilization of neutrophils from the bone marrow
Circulating and marginated pools of
• Recoverable portion of granulocytes is termed the
• 49% of cells resides in the circulating pool and the
remaining 51% in the marginated pool
Neutrophil uptake and removal by the
liver, spleen and bone marrow
• In the promyelocyte stage: Primary granules,
large peroxidase-positive granules that stain
metachromatically (reddish-purple) with a
polychromatic stain such as Wright stain, are
formed. Most of the granules are spherical
and have a diameter of 500 nm, but ellipsoid,
crystalline forms and small granules
connected by filaments also are present.
• The specific or secondary granules, which are
peroxidase negative, are formed.
• At the end of the promyelocyte stage, peroxidase
abruptly disappears from rough endoplasmic
reticulum and Golgi cisternae, and the production
of azurophilic granules ceases.
• The myelocyte stage begins with production of
peroxidase-negative specific granules. Typically
are spherical (approximately 200 nm) or rod
shaped (130 x 1000 nm).
• Two types of granules, azurophilic and specific,
are distributed in fairly equal numbers.
Metamyelocyte, band, and mature
• Nonproliferating (no mitosis) cells that precede
the development of the mature neutrophil.
• The mature, segmented neutrophilic cells contain
primary, peroxidase-positive granules and specific
peroxidase-negative granules in a 1:3 ratio.
• The late stages of maturation consist of
nondividing cells that can be distinguished by
their nuclear morphology, mixed granule
populations, small Golgi regions, and
accumulations of glycogen particles
• On average, an electron micrograph of a
neutrophil displays 200 to 300 granules.
• The size of most of the peroxidase-negative,
specific granules (approximately 200 nm) is at the
limit of resolution of the light microscope. The
granules cannot be distinguished individually but
are responsible for the pink background color of
neutrophil cytoplasm during and after the
• Some mature neutrophils in women have
drumstick- or club-shaped nuclear appendages.
These appendages contain the inactivated X
Primary (Azurophilic) Granules
Formed at promyelocyte stage
Last to be released
• Acid β-glycerophosphatase
Secondary (Specific) Granules
Formed during myelocyte and
Third to be released
• • β2-Microglobulin
• • Collagenase
• • Gelatinase
• • Lactoferrin
• • Neutrophil gelatinase-
• Tertiary Granules
• Formed during metamyelocyte
and band stages
• Second to be released
• • Gelatinase
• • Collagenase
• • Lysozyme
• • Acetyltransferase
• • β2-Microglobulin
• CD11b/CD18 integrin (MAC-1) is known to interact in
cis fashion with glycosylphosphatidylinositol (GPI)-
anchored membrane proteins.
• Interaction with CD11b/CD18 promotes antibody-
dependent phagocytosis, whereas CD14 interaction
with CD11b/CD18 occurs in the presence of LPS and
LPS-binding protein to generate proinflammatory
• Toll-like receptors (TLRs):different pathogen-associated
• They contain plasma proteins, seemingly without any
selectivity. Albumin thus serves as a marker for secretory
vesicles and has allowed the identification of these as
small intracellular vesicles that are scattered throughout
the cytoplasm of neutrophils just like granules.
• The plasma proteins inside secretory vesicles show no
sign of degradation, thus no fusion takes place with
lysosomal structures. The secretory vesicles behave like
the traditional neutrophil granules.
• First identified marker of secretory vesicles, latent
alkaline phosphatase. The best marker for secretory
vesicles is CD35.
• Eosinophil myelocytes are characterized by the
presence of large (resolvable at the light
microscope level), pale, reddish orange
secondary granules along with azure granules
in blue cytoplasm. The nucleus is similar to
that described for neutrophil myelocytes.
• Eosinophil metamyelocytes and band forms
resemble their neutrophil counterparts with
respect to their nuclear shape.
• Committed eosinophil progenitor expresses high levels
of interleukin (IL)-5 receptor and is negative for MPO.
• Mature eosinophilic leukocyte has a bilobed nucleus,
and its cytoplasm is filled with large eosinophilic
granules. Susceptible to mechanical damage during
preparation of blood films.
• Approximately 3% of nucleated bone marrow cells are
• Of these, slightly more than a third are mature, a
quarter are metamyelocytes, and the remainder are
promyelocytes or myelocytes.
• The time from the last myelocyte mitotic division to
the emergence of mature eosinophils from the marrow
is about 3.5 days.
• Once in the circulation, half-life of roughly 18 hours;
however is prolonged when eosinophilia occurs.
• The tissue destinations of eosinophils under normal
circumstances appear to be underlying columnar
epithelial surfaces in the respiratory, gastrointestinal,
and genitourinary tracts.
• Functions of eosinophils:
• Hallmark of allergic disorders
• They are also implicated in the initiation of either type1
or type 2 immune responses due to their ability to
rapidly secrete preformed cytokines in a stimulus-
• Eosinophils regulate mast cell function through the
release of major basic protein that causes mast cell
degranulation as well as cytokine production.
• They transmigrate into the thymus of the newborn and
are believed to be involved in the deletion of double-
positive thymocytes. (immune regulation)
• Eosinophils are capable of acting as antigen presenting
cells and promoting the proliferation of effector T cells
• Primary Granules
• Formed during promyelocyte stage
• • Charcot-Leyden crystal protein
• Secondary Granules
• Formed throughout remaining maturation
• • Major basic protein (core)
• • Eosinophil cationic protein (matrix)
• • Eosinophil-derived neurotoxin (matrix)
• • Eosinophil peroxidase (matrix)
• • Lysozyme (matrix)
• • Catalase (core and matrix)
• • β-Glucuronidase (core and matrix)
• • Cathepsin D (core and matrix)
• • Interleukins 2, 4, and 5 (core)
• • Interleukin-6 (matrix)
• • Granulocyte-macrophage colony-stimulating factor (core)
• Basophils are the least numerous of the circulating
• making up between 0.5% and 1.5% of circulating
• Basophil development is similar to eosinophil
development in that it requires the cytokines IL-3, IL-5,
and GM-CSF; however, transforming growth factor β
suppresses eosinophil differentiation and enhances
• Basophil Kinetics
• Basophil kinetics is poorly understood because of their
very small numbers.
• According to an early study, the time for bone marrow
development and storage of basophils is 4.3 days +/-11
hours, and their mean transit time in the peripheral
blood is 3.7 days +/- 21 hours.
• The life span of basophils is significantly longer than
that of the other granulocytes.
• This has been attributed to the fact that, when they are
activated by the cytokine IL-3, antiapoptotic pathways
are initiated which cause the prolongation of the
basophil life span.
• “poor relatives” of mast cells and minor players in
allergic inflammation because, like mast cells, they
have immunoglobulin E (IgE) receptors on their surface
membrane that, when cross-linked by antigen, result in
• Mast cells are the effectors of IgE-mediated chronic
allergic inflammation, basophils function as initiators
of the allergic inflammation through the release of
• Regulate immune response: releasing large quantities
of subtype 2 helper T cell (TH2) cytokines such as IL-4
and IL-13 that regulate the TH2 immune response.
• Stimulate B lymphocytes to produce IgE.
• Basophils are evidently capable of synthesizing granule
proteins based on activation signals. For example,
basophils can be induced to produce a mediator of
allergic inflammation known as granzyme B.
• Mast cells can induce basophils to produce and release
retinoic acid, a regulator of immune and resident cells
• in allergic diseases.
• Basophils may also play a role in angiogenesis through
the expression of several forms of vascular endothelial
growth factor and their receptors.
• The term granulocytopenia,
reduced numbers of blood
eosinophils, and basophils),
sometimes are imprecisely
used as synonyms for
• Agranulocytosis literally
means a complete absence
of blood granulocytes, but
this term often is used to
indicate severe neutropenia,
that is, counts less than 0.5 x
103/L (0.5 x 109/L).
Absolute neutrophil count > 7000/cu
Neutropenia Absolute neutrophil count < 1800/cu
Eosinophilia Absolute eosinophil count > 700/cu
Basophilia Absolute basophil count > 110/cu mm
Absolute lymphocyte count > 4000/cu
mm or >8000/cumm in children
Distinguishing Between Chronic
Myelogenous Leukemia and Leukemoid Reaction
• Chronic Myelogenous Leukemia
1. Increases in all granulocytes including eosinophils and
basophils and their immature form
2. Dyspoietic morphology such as mixed granulation or
pseudo Pelger-Huet, Involvement of platelets including
giant, hypogranular forms.
3. Leukocyte alkaline phosphatase score markedly decreased
• Leukemoid Reaction
1. Increases in neutrophils and their immature forms; possible
increased monocytes but eosinophils and basophils are
2. No dyspoietic morphology; however, reactive morphology
is usually present
3. No abnormal platelet morphology
4. Leukocyte alkaline phosphatase score markedly increased
• For newborns ranges:
• At birth: 1.1 x 109/L.
• Birth to 7 days : 0.15-1x 109/L
• 7 – 14 days : 0.1-0.95 x 109/L
• For children from 1 month to 10 years old.
• Blood neutrophil count less than 1.5 x 103/L (1.5
• For individuals older than age 10 years, count
less than approximately 1.8 x 109/L.
Neutropenia• Decreased production
• Reticular dysgenesis
• Dyskeratosis congenita
• Schwachman – Diamond – Oski syndrome
• Cyclic neutropenia
• Kostmann syndrome
• Hyper - IgM syndrome
• Chronic idiopathic neutropenia
• Increased peripheral destruction
• Immune mediated
• Drug induced
• Associated with collagen vascular disease
(Felty syndrome, SLE)
• Complement mediated (haemodialysis,
• Altered distribution
• Aplastic anaemia
• Bone marrow infiltration (leukaemia,
lymphoma, tumours, tuberculosis, etc.)
• Severe infection
• Drug induced (cytotoxic chemotherapy,
radiation, chloramphenicol, penicillins,
phenylbutazone, gold, antithyroid
drugs, quinidine, anticonvulsants,
• Myelodysplastic syndrome
• Vitamin B 12 or folate defi ciency
• Pure white cell aplasia
• T - γ lymphocytosis and neutropenia
• Neutropenia associated with metabolic
• Acute leukaemia
• Neutropenia occurs because of
(1) hypoplastic neutropoiesis,
(2) ineffective neutropoiesis (resulting from
exaggerated apoptosis of late precursors),
(3) accelerated removal or utilization of circulating
(4) shifts of cells from the circulating to the marginal
blood pools, or a combination of these mechanisms
• Some mutations and acquired defects shorten the
survival of the precursor cells, that is, they accelerate
apoptosis. This form of cell loss now is thought to be
the mechanism for "maturation arrest" in several
• Examples : Apoptosis causing neutropenia include
vitamin B12 deficiency, clonal cytopenias
(myelodysplasia), congenital and cyclic neutropenia
and the Shwachman-Diamond syndrome.
• Extrinsic factors such antineutrophil antibodies and
toxic cytokines generated by other cells.
• Neutrophil function, such as glycogen storage disease
type 1b, Chédiak-Higashi syndrome and HIV.
• Subjects who are not otherwise compromised: Gram-
positive cocci and usually are superficial, involving skin,
oropharynx, bronchi, anal canal, or vagina. However,
any site can become infected, with gram-negative
organisms, viruses, or opportunistic organisms.
• Drug-induced neutropenia is distinguished by the
rapidity of onset. Abrupt-onset neutropenia more likely
is severe and leads to symptoms. If the neutrophil
count approaches zero (agranulocytosis), high fever;
chills; necrotizing, painful oral ulcers (agranulocytic
angina), and prostration may occur, presumably as a
result of sepsis. As the disease progresses, headache,
stupor, and rash may develop.
• Decrease in pus formation- Severe neutropenia. The failure
to suppurate can mislead the clinician and delay
identification of the infection site because minimal physical
or radiographic findings develop.
• lack of pneumonic consolidation in granulocytopenic
• An exudate, swelling, heat, and regional adenopathy are
much less prevalent in granulocytopenic patients.
• Fever is common, and local pain, tenderness, and erythema
nearly always are present despite a marked reduction in
Chronic idiopathic (benign)
• The mechanism of neutropenia and the severity of the
deficiency of cells play roles in clinical manifestations.
• Apparent normal granulopoiesis in the marrow and is
asymptomatic even when the neutropenia has been
present for prolonged periods, sometimes in the face
of neutrophil counts approaching zero. Presumably the
delivery of neutrophils from marrow to tissues is
sufficient to prevent infection despite the low blood
• Monocyte counts are normal, which may aid in host
defenses because monocytes are effective phagocytes.
Chronic cyclic neutropenia
• Autosomal dominant or sporadically occurring , regularly recurring
episodes of severe neutropenia, usually every 21 days. Oscillations of
other white cells, reticulocytes, and platelets are sometimes
observed. Mutations in the gene for neutrophil elastase (ELA-2) at
• The diagnosis usually is made in the first year of life. The neutropenic
periods last for 3 to 6 days. A few cases of acquired cyclic neutropenia
in adults, some of whom have an associated clonal proliferation of
large granular lymphocytes have been reported.
• Diagnosis : Serial differential white cell counts, at least two or three
times per week for a minimum of 6 weeks. Sequencing of the gene.
Most affected children survive to adulthood.
• Treatment: G-CSF. G-CSF does not abolish cycling, but it shortens the
neutropenic periods sufficiently to prevent symptoms and infections.
Kostmann Syndrome and Related
• Autosomal recessive disease (sweedish -family)
• Neutropenia is caused by mutations in the HAX-1 gene.
HAX-1 is a mitochondrial protein, and the mutations
lead to accelerated apoptosis of myeloid cells, as well
as neurologic abnormalities.
• Mutations in the gene for glucose-6-phosphatase
catalytic subunit 3 (G6PC3) : Neutrpenia as well as
congenital cardiac and urogenital abnormalities.
• Mutations in the gene for the receptor for G-CSF also
occur in patients with severe congenital neutropenia.
• At diagnosis, the neutrophil count usually is less than
0.2 x 103/L (0.2 x 109/L).
• Monocytosis, mild anemia, thrombocytosis, and
splenomegaly frequently are present.
• Characteristically, the marrow shows early neutrophil
precursors (myeloblasts, promyelocytes) but few or no
myelocytes or mature neutrophils. Marrow
eosinophilia is common.
• Treatment: G-CSF, 5% cases unresponsive: BMT.
• Autosomal recessive disorder is characterized by partial
occulocutaneous albinism, giant granules in many cells
(including granulocytes, monocytes, and lymphocytes),
neutropenia, and recurrent infections.
• Chromosomal mutation at 1q43 affecting the LYST
gene. The product of this gene regulates lysosomal
• In Chediak-Higashi syndrome, the neutropenia usually
is mild, and susceptibility to infection is attributed to
neutropenia and defective microbiocidal activity of the
Myelokathexis, WHIM, and Related
• Autosomal dominant or sporadically occurring .
• Severe neutropenia and lymphocytopenia, leukopenia
• WHIM syndrome, characterized by warts,
hypogammaglobulinemia, infections, and myelokathexis.
• Mutation in the gene encoding the receptor for stromal
cell-derived factor-1 (SDF-1)/CXCR-4.In these syndromes,
the marrow usually shows abundant precursors and
developing neutrophils. Neutrophils in the marrow and the
blood show hypersegmentation with pyknotic nuclei and
• Favorable responses to G-CSF and GM-CSF occur, as does
evolution to the myelodysplastic syndrome.
• A myelokathexis-like variant of myelodysplastic syndrome
has been reported.
Glycogen Storage Diseases
• Autosomal recessive disorders are characterized by
hypoglycemia, hepatosplenomegaly, seizures, and
failure to thrive in infants.
• Only type 1b is associated with neutropenia. The
genetic defect in type 1b maps to chromosome 11q23
and is attributed to a defect in an intracellular
transport protein for glucose.
• The marrow appears normal despite severely reduced
blood neutrophils. The neutrophils have a reduced
oxidative burst when stimulated and defective
• Treatment with G-CSF is effective for correcting the
neutropenia and improving the associated
inflammatory bowel disease.
• Inflammation caused by bacterial or fungal organisms, and
a variety of cancers, especially if metastatic.
• Certain drugs, such as glucocorticoids or hematopoietic
growth factors and minocycline, can induce neutrophilia, as
can ethylene glycol intoxication.
• Acute hemolysis or acute hemorrhage may also result in
• A notable cause of neutrophilia is cancers that elaborate
granulocyte-colony stimulating factor (G-CSF). Numerous
cancers have been associated with neutrophilia and in
many cases, elaboration of very high concentrations of G-
CSF has been documented. In these cases, neutrophil
counts exceeding 100,000 L (100 x 109/L) are common
Noninflammatory aspects of
• can transiently occlude capillaries.
• such occlusions may reduce local blood flow transiently and
contribute to the development of ischemia. Impairment of
reperfusion of the coronary microcirculation has been
thought to be dependent, in part, on neutrophil plugging of
myocardial capillaries, but these effects can occur at
normal neutrophil concentrations.
• An elevated neutrophil count is a feature of sickle cell
disease and is a prognostic variable, increasing the
likelihood of vasocclusive events.
• In patients with ischemic vascular disease, an increased
neutrophil count is associated with an increased probability
of acute thrombotic episodes and the severity of chronic
Causes of neutrophilia.
• Chronic idiopathic
• Familial myeloproliferative disease
• Leukaemoid reaction associated with congenital anomalies
• Leucocyte adhesion deficiency (LAD) types I and II
• Familial cold urticaria and leucocytosis
• Chronic inflammation
• Drugs (steroids, lithium, tetracycline)
• Non- haematological neoplasms
• Asplenia and hyposplenism
• Chronic myeloid leukaemia
• Other myeloproliferative disorders (myelofibrosis, polycythaemia
• vera, essential thrombocythaemia)
• Pelger-Huet Anomaly
• An autosomal dominant disorder resulting in
decreased nuclear segmentation.
• Most common genetic disorder of leukocytes.
• Mutation of the lamin B receptor. The lamin B receptor
is an integral protein in the inner nuclear membrane.
PHA is one of a large number of so-called
laminopathies ranging from a type of muscular
dystrophy to premature aging.
• Heterozygous PHA (segmented only once)
• Homozygous PHA(no segmentation)
• Homozygous : impaired cognitive development, skeletal
abnormalities, and heart defects. Leukocyte differential
counts performed on blood samples from patients with either
heterozygous or homozygous PHA may mistakenly identify the
abnormal cells as immature granulocytes if the highly
clumped, dense chromatin pattern is not noticed
Pseudo pelger huet anomaly
• Must distinguish inherited PHA from an acquired form
of nuclear hyposegmentation
• Pseudo–Pelger-Huët anomaly (pseudo-PHA) that may
be seen in a variety of malignant myeloproliferative
neoplasms. Two distinctions between inherited PHA
and pseudo-PHA are the following:
(1) The percentage of affected neutrophils is much higher
in the inherited form (usually over 80%), whereas in the
acquired form fewer than 50% are usually affected; and
(2) Pseudo-PHA alterations are usually accompanied by
other morphologic indications of malignancy, such as the
presence of blast forms.
Hereditary Neutrophil Hypersegmentation
• Hereditary neutrophil hypersegmentation designates a
group of at least two disorders.
• The first is a benign autosomal dominant condition:
• hypersegmented neutrophils with no clinical signs or
1) In the hereditary form, a large majority of neutrophils are
hypersegmented and there is no macrocytic anemia.
2) In megaloblastic anemia, fewer than 50% of neutrophils are
• Myelokathexis an inherited form of neutropenia
characterized by hypersegmented neutrophils and bone
marrow hyperplasia of myeloid cells. There is increased
programmed cell death (apoptosis) of granulocyte precursors.
The neutrophil nuclei, in addition to being hypersegmented,
are pyknotic and the intersegmental filaments are longer than
• Alder-Reilly Anomaly
• Alder-Reilly anomaly: autosomal recessive trait.
• Granulocytes with large, darkly staining metachromatic
granules that may resemble toxic granulation.
• The granules are composed of mucopolysaccharides
and are sometimes referred to as Alder-Reilly bodies or
Reilly bodies. In more severe forms, the granules may
also be found in monocytes and lymphocytes.
• The basic defect is the incomplete degradation of
complexes), and there may be a structural abnormality
in the myeloperoxidase gene.
Hypersegmented neutrophils and
• Rare and fatal autosomal recessive : Enlarged
• All cells with lysosomal organelles are affected,
including the melanosomes of melanocytes in the skin,
the dense granules of platelets, and leukocyte
• The platelet abnormality usually results in a prolonged
bleeding time, and granule release by thrombin is
Giant lysosomal granules in granulocytes and monocytes
• It is one of the four overlapping autosomal dominant
platelet disorders: May-Hegglin anomaly (MHA),
Sebastian syndrome (SBS), Fechtner syndrome (FS),
and Epstein syndrome (EPS).
• All are caused by mutations of the nonmuscle myosin
heavy-chain type IIA gene referred to as MYH9.
• MHA, SBS, and FS are characterized by large basophilic
inclusions in leukocytes, thrombocytopenia and giant
• Clinically, individuals with MHA are asymptomatic;
however, a few have had bleeding episodes, possibly
related to the thrombocytopenia
• The leukocyte inclusions in MHA have been described
• True Dohle bodies are made up of lamellar rows of
rough endoplasmic reticulum, whereas the MHA
inclusions consist of randomly placed rods in an
• In addition, Dohle bodies are found only in neutrophils,
whereas MHA inclusions are seen in neutrophils,
eosinophils, basophils, and monocytes.
• MHA inclusions may stain a very pale color with
Wright stain and may be missed in monocytes whose
cytoplasm is also blue-grey.
• Chronic granulomatous disease (CGD): Inability to
produce superoxide and reactive oxygen species.
• One or more mutations in any of four genes responsible
for proteins that make up a complex known as NADPH
• ( 60% to 65%) X-linked recessive > (35% to 40%)
autosomal recessive. The X-linked : higher mortality rate.
• Bacterial and fungal infections: The formation of
granulomas that can obstruct hollow organs such as the
stomach, intestines, and urinary tract.
• Catalase-negative bacteria are rarely involved, they
generate their own hydrogen peroxide within the
lysosome and can thus be killed by the neutrophil
Diagnosis of CGD
• Nitroblue tetrazolium reduction test, normal
neutrophils, when stimulated, reduce the yellow
water-soluble nitroblue tetrazolium to a dark blue
• CGD neutrophils cannot perform this reduction.
• Flow cytometry uses a fluorescent probe, such as
dihydrorhodamine- 123, to measure intracellular
production of reactive oxygen species.
Leukocyte Adhesion Disorders
• Leucocyte adhesion deficiency (LAD) is a
• that presents with persistent leucocytosis,
delayed separation of the umbilical cord,
recurrent infections, impaired wound healing
and defects of neutrophil activation.
• The condition is caused by defects in adhesion
of neutrophils to blood vessel walls.
Leukocyte Adhesion Disorders
• LAD-I is caused by a mutation in the gene(s)
responsible for β2 integrin subunits (CD11/
• LAD-II : the basic defect is faulty ligands for
selectin adhesive molecules.
• LAD-III: A mutation in KINDLIN3, a gene
responsible for a protein that binds to the β2
Flow in LAD
• We have flow cytometric panel for LAD-1.
• CD 18, CD11a, CD11b, CD11c
• 2005-2014 : study done in CMC, we got 27
cases of LAD1.
• Out of which 24 were diagnosed severe LAD,
with CD18 expression in less than 2% of the
neutrophils and 2 were diagnosed as
moderate LAD with CD18 expression in around
30% of the neutrophils.
Miscellaneous Granulocyte Disorders
• Hyperimmunoglobulinemia E/ Jobs syndrome:
Neutrophils in these patients have poor
• Lazy leukocyte syndrome: Neutropenia and poor
neutrophil response to chemotactic agents.
• Myeloperoxidase (MPO) deficiency: read most
• Diabetes mellitus with abnormal oxidative burst
• Nuclear abnormalities caused by reaction to infection,
inflammation or stress include nuclearhypersegmentation
• Hypersegmentation is often seen in chronic infections:
reflect borderline folate deficiency caused by longterm
• Pyknotic nuclei= Imminent cell death.
• In a pyknotic nucleus, nuclear water has been lost and the
chromatin becomes very dense and dark; however,
filaments can still be seen between segements.
• Necrotic nuclei are rounded fragments of nucleus with no
filaments and no chromatin pattern.
• 1.Dohle bodies, 2.toxic granulation, 3. vacuolation,
• Cytoplasmic inclusions consisting of ribosomal
ribonucleic acid (RNA) arranged in parallel rows.
• Found in band and segmented neutrophils
• With staining, they appear as pale blue, round or
elongated bodies between 1 and 5 μm in diameter.
• They are usually located in close apposition to
• Delay in preparing the
• EDTA tube can affect
their appearance so that
they may look more gray
than blue and in some
cases may not be visible.
• associated with a wide
range of conditions,
• ??Physiological condition
2. Toxic granulation reflects an increase in
mucosubstance within primary granules.
• There is a positive correlation between levels of
C-reactive protein and the percentage of
neutrophils with toxic granulation; therefore,
toxic granulation is considered an important
marker of inflammation.
• Toxic granulation may be induced by increases in
granulocyte colony stimulating factor.
• Toxic granules are larger than specific granules
and stain blue-black with Romanowsky stains.
• It is important to distinguish between true toxic
granulation, poor staining, and the
metachromatic granules found in inherited MPS.
• True toxic granules tend to cluster within the neutrophil
cytoplasm, and not all neutrophils are equally affected
3. Cytoplasmic vacuolation:
• Autophagocytosis or of extracellular material.
• Autophagocytosis can be caused by drugs such as
sulfonamides and chloroquine, by prolonged storage in
EDTA, by autoantibodies, by acute alcoholism, and by
exposure to high doses of radiation.
• Autophagocytic vacuoles tend to be small
(approximately 2 μm) and distributed throughout the
• Phagocytic vacuoles are often seen in septic processes
caused by either bacteria or fungi.
• Phagocytic vacuoles are large (up to 6 μm) and are
frequently outlined by visible toxic granules.
• ?True vacuolation / ? excessive storage in EDTA.
• Allergic reactions (e.g.,
• fever, drug sensitivity)
• Parasitic infestations
(especially with tissue-
• Hypersensitivity reactions
(e.g., Loeffler syndrome)
• Skin diseases (e.g.,
• Tropical eosinophilia
• Certain infections (e.g.,
• Infections or inflammation
in which neutrophilia occurs
• Thymoma and
• Rare inherited forms
• Autoimmune disorders with
• Unknown causes
• HES is characterized by
• sustained eosinophilia of 30–70% of total leucocyte count (
> 1.5 × 10 9 /L) for longer than 6 months,
• absence of other underlying causes of eosinophilia
• evidence of organ dysfunction due to eosinophilic tissue
• Presenting features include anorexia, weight loss, fever,
sweating, thromboembolic episodes, heart failure,
splenomegaly, and skin and central nervous system (CNS)
• bone marrow eosinophils are increased (30 – 60%) but
myeloblasts are usually not.
• It has been difficult to assess the clonality of HES.
• Clonal karyotypic abnormalities and the performance of
restriction polymorphism analysis in females, are now
reclassified by WHO (2008) as chronic eosinophilic
• The most common cause of basophilia is the presence
of a malignant myeloproliferative neoplasm
• such as chronic myelogenous leukemia.
• Nonmalignant causes of basophilia are relatively rare
• ulcerative colitis,
• Some bee stings, and
• some types of nephrosis.
• Because the normal number of basophils is so low,
basopenia is difficult to establish.
• Basopenia has been reported in chronic Urticaria.