GRANULOPOIESIS

PRESENTED BY
DR ETU-EFEOTOR T. P.
 OUTLINE

 INTRODUCTION
HAEMATOPOIESIS
BONE MARROW OVERVIEW
GRANULOPOIESIS
 REGULATION OF GRANULOPOIESIS
 CLINICAL CORRELATES
 THERAPEUTIC TARGETS
 DISORDERS OF GRANULOPOIESIS
 CONCLUSION
 REFERENCE
 INTRODUCTION

HAEMATOPOIESIS
 Haematopoiesis (from Ancient Greek: haima
blood; poiesis to make) (or hematopoiesis in
the United States; sometimes also
haemopoiesis or hemopoiesis) is the
formation of blood cellular components. All
blood cellular components are derived from
haematopoietic stem cells in the bone
marrow.
 Introduction Contd.

Components Of Haematopoiesis
 Erythropoiesis- red cell formation
 Myelopoiesis- granulocytes(granulopoiesis)
and monocyte(monopoiesis) formation
 Thrombopoiesis- formation of platelets
 Lymphopoiesis- formation of lymphocytes
Haematopoiesis
 Introduction Contd.

Sites Of Haematopoiesis
 Fetus - 0-2months (Yolk sac)
2-7months (Liver, spleen)
5-9months (Bone marrow)
 Infants - Bone marrow (practically all bones)
 Adults - Vertebrae, ribs, sternum, skull,
sacrum and pelvis, proximal ends of femur
GRANULOPOIESIS
 GRANULOPOIESIS

 Granulopoiesis is the formation of

granulocytes ( neutrophils, eosinophils and
basophils) primarily in the bone marrow.
 It is the process of terminal differentiation
from pluripotent haematopoietic stem cells
through a common myeloid progenitor and
granulocyte-macrophage progenitor stage to
a committed mature granulocytes.
 Granulopoiesis contd

 Granulopoiesis involves the following stages:

 Pluripotential haematopoietic stem cells
 Myeloblasts
 Promyelocyte
 Myelocyte
 Metamyelocyte
 Band cells
 Mature granulocytes
Pluripotential haematopoietic stem
cells
Features:
 Extensive cell renewal
 Extensive proliferative potential
 Capacity to differentiate into the progenitors
of all the blood cell lineages-multipotency
 Phenotypic markers-CD34+; Thy-1+; AC133+;
c-KIT+; CD33-;CD38-; Lineage markers-; HLA-
DR-
CD34 Positive Stem Cells
 Myeloblasts

 Earliest precursors of granulopoiesis that can be identified by

light microscopy
 Size: 14-18micronmeter
 N:C: 4:1
 Cell shape: round to oval
 Nuclear shape: round to oval
 Chromatin: composed of very fine non aggregated chromatin ie
finely reticular
 Nucleoli: distinct, 2 to 5
 Cytoplasm: scanty, basophilic character and is devoid of
granules.
Myeloblast
 Promyelocytes

 Rare in the blood of healthy people

 Larger than myeloblasts
 Size: 20-25micronmeter
 N:C : 3:1
 Cell shape: round to oval
 Nuclear shape: round to oval, usually eccentric
 Cytoplasm: more basophilic than myeloblast; contains
azurophilic(primary granules) and lacks secondary granules
 Chromatin: finely reticular, slight clumping
 Nucleoli: distinct, 1 to 3
Promyelocyte
 Myelocytes

 Uncommon in the blood of healthy subjects except in the

neonatal period and during pregnancy.
 Smaller than promyelocytes
 Size: 14-20micronmeter
 N:C: 2:1
 Cell shape: round to oval
 Nuclear shape: oval, slightly indented or flattened
 Chromatin: variable amount of clumping
 Cytoplasm: blue-pink. Have both primary(azurophilic) and
secondary granules characteristic of specific lineages
 Nucleoli: absent
Neutrophil myelocyte
 Metamyelocytes
 Also known as JUVENILE FORMS
 N:C: 1:1
 Size: 10-15micronmeter
 Cell shape: round to oval
 Nuclear shape: indented, kidney shaped
 Chromatin: clumping with distinct regions
 Cytoplasm: pink, plentiful, contains many specific granules, rare
primary granules
 Nucleoli: absent
 Small numbers of neutrophil metamyelocytes are present in the blood
of healthy subjects
 Basophil and eosinophil metamyelocytes are not seen in the blood of
healthy subjects.
Neutrophil metamyelocyte
 Band Cells

 Neutrophil band forms are present as a minor population in the blood of healthy
people
 Size: 9-15micronmeter
 N:C: 1:1.5 to 1:2
 Cell shape: round to oval
 Nuclear shape: S, C, U or lobated with visible chromatin in isthmus
 Chromatin: coarse, clumpy
 Cytoplasm: pink, plentiful, contains mainly specific granules, rare primary granules
 Nucleoli: Absent
 They are intermediate in character between metamyelocytes and mature
neutrophils
 Eosinophil and basophil band forms are quite uncommon in the blood of healthy
subjects.
Neutrophil band
 MATURE GRANULOCYTES
Neutrophils
 Size: 9 to 15micronmeter
 N:C: 1:3
 Cell shape: round to oval
 Nuclear shape: segmented, 2 to 5 lobes connected by filaments
 Chromatin: clumped
 Nucleoli: Absent
 Cytoplasm: Pale pink with many specific (lilac) granules.
 45-75 percent WBC
 Acute inflammatory response cell
 Phagocytic
 Lifespan in the blood is only about 6-10hours
 Spend 4-5 days in the tissues
A neutrophil with a segmented nucleus at the
center surrounded by erythrocytes.
Neutrophil granules
 Eosinophils

 Size: 10 to 16 micronmeter
 N:C: 1:3
 Cell shape: round to oval
 Nuclear shape: segmented with 2-3 lobes connected by a thin
filament of chromatin
 Chromatin: dense and compact
 Nucleoli: Absent
 Cytoplasm: large, coarse, spherical, uniform red-orange refractile
granules with orange-pink cytoplasm
 1-6 percent of all leukocytes
 Defends against helminthic and protozoan infections
Eosinophil
Eosinophil granules
 Basophils

 Size: 10 to 14micronmeter
 N:C: 1:3
 Cell shape: round to oval
 Nuclear shape: segmented, often obscured by granules
 Chromatin: coarse, clumped, bilobed
 Cytoplasm: many large specific purple-black granules
 Nucleoli: Absent
 Occasionally seen in normal peripheral blood.
 In the tissues they become mast cells
 Have IgE attachment sites
 Degranulation leads to histamine release.
Basophil surrounded by
erythrocytes
Basophil and mast cell granules
 Granulocytes contd

 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
POSTMITOTIC MATURATION COMPARTMENT.
 Large number of band and segmented
neutrophils are held in the bone marrow as a
RESERVE POOL or STORAGE COMPARTMENT.
Transit time(hour) of neutrophil and
precursors in the bone marrow

MITOTIC COMPARTMENT:
 Myeloblast- 23
 Promyelocyte- 26-78
 Myelocyte- 17-126
MATURATION STORAGE COMPARTMENT:
 Metamyelocyte- 8-108
 Neutrophil band- 12-96
 Mature neutrophil- 0-120
REGULATION OF GRANULOPOIESIS

 Regulation of Granulopoiesis is achieved by the

Myeloid growth factors and interleukins which are
glycoprotein hormones that regulate the proliferation
and differentiation of haematopoietic progenitor cells
and the function of mature blood cells.
 Myeloid growth factors- GM-CSF; G-CSF;
 Interleukins- IL-1; IL-3; IL-5; IL-6; IL-11 and cytokine
Flt3 ligand
 Transcription factors- PU.1; CBF; C/EBP-alpha;
RARalpha
 Myeloid Growth Factors

GM-CSF
 GM-CSF- It stimulates colonies of neutrophils,
macrophages,
early multipotential stem cells,
megakaryocyte progenitors
eosinophil progenitors
erythroid progenitors
 GM-CSF contd

 Human GM-CSF contains 127 aminoacids;

 it’s a glycoprotein
 It’s cloned from leukaemic cell line
 Mol. wt of 14KD;
 it’s gene is located on chromosome 5.
Myeloid Growth Factors contd

G-CSF
 It’s a polypeptide with a 30 aminoacid signal
sequence followed by mature G-CSF
sequence of 177 aminoacids.
 The protein stimulates predominantly
neutrophil colony formation.
 It’s mol wt is 19KD
 It’s gene is located on chromosome 17.
Indications Of Myeloid Growth Factors

 Postchemotherapy, radiotherapy or bone

marrow transplantation
 Acute myeloid leukaemia
 Myelodysplasia
 Severe neutropenia
 Severe infection
 Peripheral blood stem cell transplant
Control of granulopoiesis by growth factors
Control Of Granulopoiesis By Growth
Factors contd
Generalized diagram of signal transduction pathways
activated by cytokines and their receptors
Sites of action of myeloid growth factors
and interleukins
Transcription factors active at various
stages of granulopoiesis contd
Regulation of granulopoiesis and
AML(impaired differentiation) by
C/EBPalpha-miR34a-E2F3 axis
Regulation of granulopoiesis and AML(impaired
differentiation) by CBF complex
Regulation of granulopoiesis and AML(Proliferative and
survival advantage) by FLT3
Regulation of granulopoiesis and AML(impaired
differentiation) by RARalpha
 CLINICAL CORRELATES
 In haematologic malignancies, mutations are present in genes
regulating intracellular signalling.
 These include:
1)Cytokine receptors –mutation in FLT-3 and KIT genes in AML
2)Intracellular signalling components-
+Mutation in JAK-2 in Tcell ALL, Myeloproliferative disorders
+Constitutive activation of ABL kinase by the BCR-ABL fusion gene in
CML
+ Mutation in RAS and PTP11 gene in AML
3)Nuclear retinoid signalling
+ Balanced translocation involving RARalpha gene (17q21) and the
PML gene (15q22) found in AML M3(APML) in >90 percent of cases.
Therapeutic Targets
Therapeutic targets contd

Farnesyltransferase inhibitors:
 Tipifarnib
 Lonafarnib
FLT-3 inhibitors
 SU 5416
 SU11248
 CEP-701
Therapeutic Targets contd
Therapeutic targets contd

JAK 2 Inhibitors
 Pacritinib(SB1518)
 CYT387
 Lestaurtinib
BCR/ABL inhibitors
 Imatinib
Therapeutic targets contd
DISORDERS OF GRANULOPOIESIS

 Disorders of granulopoiesis can be classified

under the following headings:
 Quantitative disorders
 Qualitative disorders
 Neutrophils: Quantitative disorders include-
 Neutropenia-neutrophil count< 0.5 x 109/L
 Neutrphilia-neutrophil count> 7.5 x 109/L
 QUALITATIVE
DISORDERS OF
NEUTROPHILS
Qualitative disorders of
neutrophils
 Qualitative disorders of neutrophils can classified into:
A) Variations in neutrophil morphology
-Barr body
-Pelger-Huet anomaly
-Alder-Reilly anomaly
-May-hegglin anomaly
-Hypersegmented neutrophils
-Chediak-Higashi syndrome
-Hypogranular neutrophils
-Dohle bodies
-Toxic granulations
B) Disorders of neutrophil motility, migration and
chemotaxis
-Lazy leukocyte syndrome
-Leukocyte adhesion deficiency
C) Disorders of bacterial killing by neutrophils
-Myeloperoxidase deficiency
-Chronic granulomatous disease
-Chediak-Higashi syndrome
D) Disorders of neutrophil opsonization
-Complement deficiency
Variations in neutrophil morphology

Neutrophil barr body

 In women, 2-3% of neutrophils show an
appendage at a terminal nuclear segment.
 This ‘drumstick’ appendage is about 1.5µm
and is connected to the nucleus by a short
stalk.
 It represents the inactive X-chromosome.
Drum stick appendage in neutrophil
Variations in neutrphil morphology contd

Pelger-Heut anomaly
Uncommon condition
Inheritance-Autosomal dominant.
Bilobed neutrophils are found in peripheral
blood.
Occasional unsegmented neutrophils with
round nuclei are also seen particularly
during infection.
Pelger-Heut anomaly
Variations in neutrophil morphology contd

Alder-Reilly anomaly
Neutrophils contain prominent purple granules(
also present in monocytes and lymphocytes)
Carriers of this anomaly frequently have
associated bone and joint deformities.
This anomaly is known to occur in
mucopolysaccharide storage disorders
Alder-Reilly anomaly
Variation in neutrophil morphology contd

May-Hegglin anomaly
 Rare condition.
 Neutrophils contain basophilic inclusions of
RNA (resembling Doehle bodies) in the
cytoplasm.
 Inheritance-Autosomal dorminant
 There is an associated mild
thrombocytopenia with giant platelets
May-Hegglin anomaly with Dohle
body in cytoplasm
Variation in neutrophil morphology contd

Hypersegmented Neutrophil
Hypersegmentation exists when >5% of
neutrophils have five nuclear lobes or
when any quantity has six or more lobes.

Such hypersegmentation is common in

folate and vitamin B12 deficiency, MDS
and MPD
Hypersegmented neutrophils
Variation in neutrophil morphology contd

Chediak-Higashi syndrome
 AR
 Disordered coalescence of lysosomal granules
 Chromosomal mutation at 1q43 affecting the LYST gene
Impaired function:
 Decreased neutrophil chemotaxis, degranulation,bactericidal activity and
impaired natural killer cell function
Features:
 Partial oculocutaneous albinism
 Neutropenia
 Recurrent infections
 Giant granules in granulocytes; monocytes; lymphocytes
 Hepatosplenomegaly
 Thrombocytopenia
Chediak-Higashi with giant granules
Variation in neutrophil morphology contd

Hypogranular Neutrophil
A decrease in granules within the
neutrophil cytoplasm is commonly seen in
MDS, MPD, reflecting the abnormal cell
maturation that is a hallmark of these
disorders.
Hypogranular neutrophil
Variation in neutrophil morphology contd

Dohle Bodies
Composed of rough endoplasmic reticulum and
glycogen granules.
They are single or multiple, small blue-gray
inclusions in the cytoplasm of neutrophils, often
at the periphery.
Seen in pregnancy, bacterial infections,
inflammatory disorders, burns, MPD, MDS,
pernicious anaemia, and cancer chemotherapy.
Dohle body in the neutrophil cytoplasm
periphery
Variation in neutrophil morphology contd

Toxic Granulation
Toxic granulation indicates presence of
increased numbers of granules that are larger
and more basophilic than normal.

May occur from Rx with CSF, in pregnancy,

severe infections (especially bacterial), burns,
malignancies, drug reactions, aplastic
anemia, and the hypereosinophilic syndrome.
Toxic granulations
Disorders of neutrophil motility, migration
and chemotaxis

 A) Lazy leukocyte syndrome

 It was first discovered by Miller et al in 1971 in two children
with recurrent infection.
 Findings:
 Normal humoral and cellular immunity
 Neutropenia
 Adequate numbers of neutrophils in bone marrow
 Intact intracellular killing and phagocytosis
 Defect in the mobilization of functionally normal neutrophils
in response to chemical and inflammatory stimuli.
 B) Corticosteroid therapy
Disorders of neutrophil motility,
migration and chemotaxis contd

Leukocyte Adhesion Deficiency 1 (LAD1)

 It’s characterized by deficiency of glycoprotein CD11b, which
forms the alpha subunit of the Mac-1 beta integrin (CD11b/CD18)
on the cell surface of the neutrophils.
 Intracellular molecule 1( ICAM-1) expressed on the vascular
endothelium serves as ligand to Mac-1 for the neutrophil
adhesion.
 Because of the mutation and absence of Mac-1, neutrophils in
LAD type 1 are not able to attach to the endothelium and undergo
transendothelial migration.
 Additionally, their ability for chemotaxis,
phagocytosis,degranulation and respiratory burst activity is also
impaired.
Disorders of neutrophil motility,
migration and chemotaxis

Leukocyte Adhesion Deficiency 2

 Extremely rare condition caused by the
absence of neutrophil Sialyl Lewis X structure
that binds to E and P-selectins on vascular
endothelium for rolling and adhesion of
neutrophils.
Disorders of bacterial killing by
neutrophils
Chronic granulomatous disease
 It’s an inherited deficiency in the production of one of
several subunits of NADPH oxidase.
 This defect eliminates the neutrophil’s ability to
produce many critical oxygen-dependent intracellular
metabolites (.O2-, .OH, O2 and H2O2).
 The two other intracellular killing mechanisms remain
intact eg myeloperoxidase and lysosomal contents.
 Patients are infected by catalase positive organisms
eg Staphylococcus,klebsiella, Serratia and Aspergillus
Disorders of bacterial killing by
neutrophils contd

Myeloperoxidase Deficiency
Rare autosomal recessive inheritance
Myeloperoxidase in the lysosomes acts on
hydrogen peroxide and chloride ions to
produce hypochlorite ( the active ingredient
in household bleach), which is microbicidal.
Infections generally mild but with more
susceptibility for candida infections
DISORDERS OF BASOPHIL PRODUCTION
 Basophilia

 Basophilia- Basophil count > 0.1 x 109/l

 Causes-
 Allergy/ inflammation-Ulcerative colitis, food, drugs,
urticaria, erythroderma,
 Endocrinopathy- DM, Estrogen administ, Hypothyroidism
 Infection- Chicken pox, influenza, small pox
 Iron deficiency
 Exposure to ionizing radiation
 Basophilic leukemia
 Myeloproliferative disorders-PRV, CML, Myelofibrosis,
 Basopenia

 Basopenia-peripheral blood basophils< 0.1

x109/L
 Causes-
 Hereditary absence of basophils
 Elevated levels of glucocorticoids
 Hyperthyroidism or treatment with thyroid
hormones
 Ovulation
DISORDERS OF EOSINOPHIL PRODUCTION
 Eosinophilia
 Eosinophilia-Eosinophil count > 0.4 x 109/l
 Causes-
 Allergic diseases-bronchial asthma, hay fever, urticaria, food sensitivity
 Parasitic diseases-amoebiasis, hookworm, ascariasis, tapeworm
infestation, filariasis, schistomiasis and trichinosis
 Recovery from acute infection
 Certain skin diseases- psoriasis, pemphigus and dermatitis herpetiformis
 Drug sensitivity
 Polyarthritis nodosa
 Hodgkin’s disease
 Eosinophilic leukemia(rare)
 Treatment with GM-CSF
 CONCLUSION

 The understanding of how regulatory

proteins interact to regulate granulopoiesis
has become increasingly important to
hematology researchers and clinicians with
the discovery that many of the genes that
encode regulatory activities in granulopoiesis
are targets for alterations that underlie the
development of cancer.
THANK YOU
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