Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Granulocytes in health and
disease
Dr.Kiran
Contents
• Granulopoiesis in embryo
• Granulocytes morphology
• Diseases of granulocytes
• Quantitative defects
• Qualitat...
John Hunter 1761
• Cellular (buffy coat) component of blood could retard the
“spoilage” of blood; we now would recognize t...
Granulopoiesis in embryo theories
• There are many studies done (1960s and 70s), few of
them are validated, but still the ...
• Further from AGM cells- arises LTR-HSCs. These are
adult type of definitive stem cells.
• Newer theories suggested place...
Normal haematopoiesis
Neutrophilic
metamyelocytes
Neutrophils
Neutrophilic
myelocytes
Promyelocytes
Neutrophilic
band or stab cells
Lobulated, d...
`
Bone marrow
32583
Developing granulocytes
32583
White blood cell
entering a
blood vessel
Bone marrow
Blood
vessel
Blood
vessel
Neutrophil
• Most abundant circulating leukocyte.
• Neutrophil homeostasis is maintained by a fine
balance between
Granul...
Neutrophil production in bone marrow
• Neutrophils are produced within haematopoietic
cords interspersed within the venous...
Neutrophil production in bone marrow
• Principal regulator of physiological granulopoiesis
is granulocyte colony stimulati...
Neutrophil release from bone marrow
• Journey upto the tissue is needed for proper functioning.
• CXC chemokine receptor 4...
Neutrophil release from bone marrow
• α4 intergrin very late antigen-4 (VLA-4) is expressed by
neutrophils- adhesion to bo...
Circulating and marginated pools of
granulocytes
• Recoverable portion of granulocytes is termed the
marginated pool
• 49%...
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-pu...
Myelocyte stage
• The specific or secondary granules, which are
peroxidase negative, are formed.
• At the end of the promy...
Metamyelocyte, band, and mature
neutrophil
• Nonproliferating (no mitosis) cells that precede
the development of the matur...
• On average, an electron micrograph of a
neutrophil displays 200 to 300 granules.
• The size of most of the peroxidase-ne...
Neutrophil Granules
Primary (Azurophilic) Granules
Formed at promyelocyte stage
Last to be released
Contain:
• Myeloperoxi...
Phagocytosis
• CD11b/CD18 integrin (MAC-1) is known to interact in
cis fashion with glycosylphosphatidylinositol (GPI)-
an...
Secretory vesicles
• They contain plasma proteins, seemingly without any
selectivity. Albumin thus serves as a marker for ...
Eosinophils
• Eosinophil myelocytes are characterized by the
presence of large (resolvable at the light
microscope level),...
• Committed eosinophil progenitor expresses high levels
of interleukin (IL)-5 receptor and is negative for MPO.
• Mature e...
Eosinophil Kinetics
• Approximately 3% of nucleated bone marrow cells are
eosinophils.
• Of these, slightly more than a th...
• Functions of eosinophils:
• Hallmark of allergic disorders
• They are also implicated in the initiation of either type1
...
• Primary Granules
• Formed during promyelocyte stage
• Contain:
• • Charcot-Leyden crystal protein
• Secondary Granules
•...
Basophils
• Basophils are the least numerous of the circulating
WBCs.
• making up between 0.5% and 1.5% of circulating
leu...
• Basophil Kinetics
• Basophil kinetics is poorly understood because of their
very small numbers.
• According to an early ...
Function
• “poor relatives” of mast cells and minor players in
allergic inflammation because, like mast cells, they
have i...
• Basophils are evidently capable of synthesizing granule
proteins based on activation signals. For example,
basophils can...
Basophil Granules
• Secondary Granules
• Histamine
• Platelet activating factor
• Leukotriene C4
• Interleukin-4
• Interle...
Inflammation
Neutrophils pathology
• Quantitative
• Qualitative
• The term granulocytopenia,
reduced numbers of blood
granulocytes (neutrophils,
eosinophils, and basophils),
sometimes ar...
Distinguishing Between Chronic
Myelogenous Leukemia and Leukemoid Reaction
• Chronic Myelogenous Leukemia
1. Increases in ...
Neutropenia
• 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 1...
Neutropenia• Decreased production
• Inherited
• Reticular dysgenesis
• Dyskeratosis congenita
• Schwachman – Diamond – Osk...
General Mechanisms
• Neutropenia occurs because of
(1) hypoplastic neutropoiesis,
(2) ineffective neutropoiesis (resulting...
• Some mutations and acquired defects shorten the
survival of the precursor cells, that is, they accelerate
apoptosis. Thi...
Clinical scenario
• Subjects who are not otherwise compromised: Gram-
positive cocci and usually are superficial, involvin...
• Decrease in pus formation- Severe neutropenia. The failure
to suppurate can mislead the clinician and delay
identificati...
Chronic idiopathic (benign)
neutropenia
• The mechanism of neutropenia and the severity of the
deficiency of cells play ro...
Chronic cyclic neutropenia
• Autosomal dominant or sporadically occurring , regularly recurring
episodes of severe neutrop...
Kostmann Syndrome and Related
Disorders
• Autosomal recessive disease (sweedish -family)
• Neutropenia is caused by mutati...
Kostmann syndrome
• At diagnosis, the neutrophil count usually is less than
0.2 x 103/L (0.2 x 109/L).
• Monocytosis, mild...
Chediak-Higashi Syndrome
• Autosomal recessive disorder is characterized by partial
occulocutaneous albinism, giant granul...
Myelokathexis, WHIM, and Related
Syndromes
• Autosomal dominant or sporadically occurring .
• Severe neutropenia and lymph...
Glycogen Storage Diseases
• Autosomal recessive disorders are characterized by
hypoglycemia, hepatosplenomegaly, seizures,...
Neutrophilia
• Inflammation caused by bacterial or fungal organisms, and
a variety of cancers, especially if metastatic.
•...
Noninflammatory aspects of
neutrophilia
• can transiently occlude capillaries.
• such occlusions may reduce local blood fl...
Causes of neutrophilia.
• Primary
• Hereditary
• Chronic idiopathic
• Familial myeloproliferative disease
• Leukaemoid rea...
Nuclear Abnormalities
• Pelger-Huet Anomaly
• An autosomal dominant disorder resulting in
decreased nuclear segmentation.
...
• Homozygous : impaired cognitive development, skeletal
abnormalities, and heart defects. Leukocyte differential
counts pe...
Pseudo pelger huet anomaly
• Must distinguish inherited PHA from an acquired form
of nuclear hyposegmentation
• Pseudo–Pel...
Hereditary Neutrophil Hypersegmentation
• Hereditary neutrophil hypersegmentation designates a
group of at least two disor...
Cytoplasmic Abnormalities
• Alder-Reilly Anomaly
• Alder-Reilly anomaly: autosomal recessive trait.
• Granulocytes with la...
Hypersegmented neutrophils and
Alder-Reilly granules
Chediak-Higashi Syndrome
• Rare and fatal autosomal recessive : Enlarged
lysosomal vesicles.
• All cells with lysosomal or...
Giant lysosomal granules in granulocytes and monocytes
May-Hegglin Anomaly
• It is one of the four overlapping autosomal dominant
platelet disorders: May-Hegglin anomaly (MHA),
...
• True Dohle bodies are made up of lamellar rows of
rough endoplasmic reticulum, whereas the MHA
inclusions consist of ran...
May hegllin anomaly
Functional Abnormalities
• Chronic granulomatous disease (CGD): Inability to
produce superoxide and reactive oxygen specie...
Diagnosis of CGD
• Nitroblue tetrazolium reduction test, normal
neutrophils, when stimulated, reduce the yellow
water-solu...
Leukocyte Adhesion Disorders
• Leucocyte adhesion deficiency (LAD) is a
congenital disorder
• that presents with persisten...
Leukocyte Adhesion Disorders
• LAD-I is caused by a mutation in the gene(s)
responsible for β2 integrin subunits (CD11/
CD...
Flow in LAD
• We have flow cytometric panel for LAD-1.
• CD 18, CD11a, CD11b, CD11c
• 2005-2014 : study done in CMC, we go...
Miscellaneous Granulocyte Disorders
• Hyperimmunoglobulinemia E/ Jobs syndrome:
Neutrophils in these patients have poor
di...
Qualitative (Morphologic)
Abnormalities
• Nuclear abnormalities caused by reaction to infection,
inflammation or stress in...
Pyknosis vs apoptosis
Cytoplasmic abnormalities
• 1.Dohle bodies, 2.toxic granulation, 3. vacuolation,
degranulation, 4.swelling.
• Cytoplasmic ...
• Delay in preparing the
film
• EDTA tube can affect
their appearance so that
they may look more gray
than blue and in som...
2. Toxic granulation reflects an increase in
mucosubstance within primary granules.
• There is a positive correlation betw...
• 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 suc...
• Eosinophilia
• Allergic reactions (e.g.,
asthma, hay
• fever, drug sensitivity)
• Parasitic infestations
(especially wit...
• HES is characterized by
• sustained eosinophilia of 30–70% of total leucocyte count (
> 1.5 × 10 9 /L) for longer than 6...
• The most common cause of basophilia is the presence
of a malignant myeloproliferative neoplasm
• such as chronic myeloge...
•Thank you
Granulocytes in health and disease
Granulocytes in health and disease
Granulocytes in health and disease
Granulocytes in health and disease
Granulocytes in health and disease
Granulocytes in health and disease
Granulocytes in health and disease
Upcoming SlideShare
Loading in …5
×

Granulocytes in health and disease

Hematopoiesis, Nonneoplastic myeloid diseases

  • Login to see the comments

Granulocytes in health and disease

  1. 1. Granulocytes in health and disease Dr.Kiran
  2. 2. Contents • Granulopoiesis in embryo • Granulocytes morphology • Diseases of granulocytes • Quantitative defects • Qualitative defects • Inflammation pathway in brief • Morphological variations
  3. 3. 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.
  4. 4. 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.
  5. 5. • 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 AGM. • Accepted theory is that, yolk sac though had an early contribution in haematopoiesis, the HSCs developed closer to the birth are exclusively derived from intraembryonic tissue.
  6. 6. Normal haematopoiesis
  7. 7. Neutrophilic metamyelocytes Neutrophils Neutrophilic myelocytes Promyelocytes Neutrophilic band or stab cells Lobulated, dark nucleus, mature grey cytoplasm with small granules 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 Granulopoiesis
  8. 8. ` Bone marrow 32583 Developing granulocytes
  9. 9. 32583 White blood cell entering a blood vessel Bone marrow Blood vessel Blood vessel
  10. 10. Neutrophil • Most abundant circulating leukocyte. • Neutrophil homeostasis is maintained by a fine balance between Granulopoiesis, Bone marrow storage and release, Intravascular margination, clearance and destruction
  11. 11. Neutrophil production in bone marrow • Neutrophils are produced within haematopoietic cords interspersed within the venous sinuses of the bone marrow • 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 differentiation. • Post-mitotic pool  Fully differentiated mature neutrophils which forms the bone marrow reserve, available for release
  12. 12. 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.
  13. 13. 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 neutrophils • 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.
  14. 14. Neutrophil release from bone marrow • α4 intergrin very late antigen-4 (VLA-4) is expressed by neutrophils- adhesion to bone marrow stromal cells and endothelium • Expression of VLA-4 is downregulated during neutrophil maturation in the bone marrow, and α4 blockade increases mobilization of neutrophils from the bone marrow
  15. 15. Circulating and marginated pools of granulocytes • Recoverable portion of granulocytes is termed the marginated pool • 49% of cells resides in the circulating pool and the remaining 51% in the marginated pool
  16. 16. Neutrophil uptake and removal by the liver, spleen and bone marrow
  17. 17. • 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.
  18. 18. Myelocyte stage • 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.
  19. 19. Metamyelocyte, band, and mature neutrophil • 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
  20. 20. • 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 myelocyte stage. • Some mature neutrophils in women have drumstick- or club-shaped nuclear appendages. These appendages contain the inactivated X chromosome.
  21. 21. Neutrophil Granules Primary (Azurophilic) Granules Formed at promyelocyte stage Last to be released Contain: • Myeloperoxidase • Acid β-glycerophosphatase • Cathepsins • Defensins • Elastase • Proteinase-3 Secondary (Specific) Granules Formed during myelocyte and metamyelocyte stages Third to be released 2ndary Contain: • • β2-Microglobulin • • Collagenase • • Gelatinase • • Lactoferrin • • Neutrophil gelatinase- associated lipocalin • Tertiary Granules • Formed during metamyelocyte and band stages • Second to be released • Contain: • • Gelatinase • • Collagenase • • Lysozyme • • Acetyltransferase • • β2-Microglobulin
  22. 22. Phagocytosis • 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 mediators. • Toll-like receptors (TLRs):different pathogen-associated molecular patterns
  23. 23. Secretory vesicles • 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.
  24. 24. Eosinophils • 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.
  25. 25. • 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.
  26. 26. Eosinophil Kinetics • Approximately 3% of nucleated bone marrow cells are eosinophils. • 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.
  27. 27. • 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- specific manner. • 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
  28. 28. • Primary Granules • Formed during promyelocyte stage • Contain: • • Charcot-Leyden crystal protein • Secondary Granules • Formed throughout remaining maturation • Contain: • • 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)
  29. 29. Basophils • Basophils are the least numerous of the circulating WBCs. • making up between 0.5% and 1.5% of circulating leukocytes. • 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 differentiation
  30. 30. • 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.
  31. 31. Function • “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 granule release. • Mast cells are the effectors of IgE-mediated chronic allergic inflammation, basophils function as initiators of the allergic inflammation through the release of preformed cytokines. • 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.
  32. 32. • 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.
  33. 33. Basophil Granules • Secondary Granules • Histamine • Platelet activating factor • Leukotriene C4 • Interleukin-4 • Interleukin-13 • VEGF A • VEGF B • Chondroitin sulfates (e.g., heparin)
  34. 34. Inflammation
  35. 35. Neutrophils pathology • Quantitative • Qualitative
  36. 36. • The term granulocytopenia, reduced numbers of blood granulocytes (neutrophils, eosinophils, and basophils), sometimes are imprecisely used as synonyms for neutropenia. • 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). Neutrophilic leukocytosis Absolute neutrophil count > 7000/cu mm Neutropenia Absolute neutrophil count < 1800/cu mm Eosinophilia Absolute eosinophil count > 700/cu mm Basophilia Absolute basophil count > 110/cu mm Lymphocyto sis Absolute lymphocyte count > 4000/cu mm or >8000/cumm in children
  37. 37. 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 frequently absent 2. No dyspoietic morphology; however, reactive morphology is usually present 3. No abnormal platelet morphology 4. Leukocyte alkaline phosphatase score markedly increased
  38. 38. Neutropenia • 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 x 109/L). • For individuals older than age 10 years, count less than approximately 1.8 x 109/L.
  39. 39. Neutropenia• Decreased production • Inherited • Reticular dysgenesis • Dyskeratosis congenita • Schwachman – Diamond – Oski syndrome • Cyclic neutropenia • Kostmann syndrome • Hyper - IgM syndrome • Chronic idiopathic neutropenia • Increased peripheral destruction • Hypersplenism • Immune mediated • Drug induced • Associated with collagen vascular disease (Felty syndrome, SLE) • Complement mediated (haemodialysis, cardiopulmonary bypass) • Altered distribution • Drugs • Stress • Acquired • Aplastic anaemia • Bone marrow infiltration (leukaemia, lymphoma, tumours, tuberculosis, etc.) • Severe infection • Drug induced (cytotoxic chemotherapy, radiation, chloramphenicol, penicillins, cephalosporins, phenothiazine, phenylbutazone, gold, antithyroid drugs, quinidine, anticonvulsants, alcohol) • Myelodysplastic syndrome • Vitamin B 12 or folate defi ciency • Pure white cell aplasia • T - γ lymphocytosis and neutropenia • Neutropenia associated with metabolic disorders • Acute leukaemia
  40. 40. General Mechanisms • Neutropenia occurs because of (1) hypoplastic neutropoiesis, (2) ineffective neutropoiesis (resulting from exaggerated apoptosis of late precursors), (3) accelerated removal or utilization of circulating neutrophils, (4) shifts of cells from the circulating to the marginal blood pools, or a combination of these mechanisms
  41. 41. • 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 diseases. • 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.
  42. 42. Clinical scenario • 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.
  43. 43. • 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 subjects. • 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 neutrophils.
  44. 44. Chronic idiopathic (benign) neutropenia • 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 pool size. • Monocyte counts are normal, which may aid in host defenses because monocytes are effective phagocytes.
  45. 45. 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 locus 19q3. • 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.
  46. 46. Kostmann Syndrome and Related Disorders • 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.
  47. 47. Kostmann syndrome • 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.
  48. 48. Chediak-Higashi Syndrome • 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 trafficking. • In Chediak-Higashi syndrome, the neutropenia usually is mild, and susceptibility to infection is attributed to neutropenia and defective microbiocidal activity of the phagocytes.
  49. 49. Myelokathexis, WHIM, and Related Syndromes • 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 cytoplasmic vacuoles. • 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.
  50. 50. 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 chemotaxis. • Treatment with G-CSF is effective for correcting the neutropenia and improving the associated inflammatory bowel disease.
  51. 51. Neutrophilia • 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 neutrophilia. • 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
  52. 52. Noninflammatory aspects of neutrophilia • 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 atherosclerosis.
  53. 53. Causes of neutrophilia. • Primary • Hereditary • Chronic idiopathic • Familial myeloproliferative disease • Leukaemoid reaction associated with congenital anomalies • Leucocyte adhesion deficiency (LAD) types I and II • Familial cold urticaria and leucocytosis • Secondary • Infection • Stress • Chronic inflammation • Drugs (steroids, lithium, tetracycline) • Non- haematological neoplasms • Asplenia and hyposplenism • Neoplastic • Chronic myeloid leukaemia • Other myeloproliferative disorders (myelofibrosis, polycythaemia • vera, essential thrombocythaemia)
  54. 54. Nuclear Abnormalities • 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)
  55. 55. • 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
  56. 56. 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.
  57. 57. 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 symptoms. 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 usually hypersegmented. • 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 normal.
  58. 58. Cytoplasmic Abnormalities • 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 mucopolysaccharides (protein-carbohydrate complexes), and there may be a structural abnormality in the myeloperoxidase gene.
  59. 59. Hypersegmented neutrophils and Alder-Reilly granules
  60. 60. Chediak-Higashi Syndrome • Rare and fatal autosomal recessive : Enlarged lysosomal vesicles. • All cells with lysosomal organelles are affected, including the melanosomes of melanocytes in the skin, the dense granules of platelets, and leukocyte granules. • The platelet abnormality usually results in a prolonged bleeding time, and granule release by thrombin is impaired.
  61. 61. Giant lysosomal granules in granulocytes and monocytes
  62. 62. May-Hegglin Anomaly • 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 platelets. • 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 as“Dohle body–like”.
  63. 63. • True Dohle bodies are made up of lamellar rows of rough endoplasmic reticulum, whereas the MHA inclusions consist of randomly placed rods in an amorphous background. • 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.
  64. 64. May hegllin anomaly
  65. 65. Functional Abnormalities • 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 oxidase. • ( 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 phagocytes.
  66. 66. Diagnosis of CGD • Nitroblue tetrazolium reduction test, normal neutrophils, when stimulated, reduce the yellow water-soluble nitroblue tetrazolium to a dark blue insoluble formazan. • CGD neutrophils cannot perform this reduction. • Flow cytometry uses a fluorescent probe, such as dihydrorhodamine- 123, to measure intracellular production of reactive oxygen species.
  67. 67. Leukocyte Adhesion Disorders • Leucocyte adhesion deficiency (LAD) is a congenital disorder • 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.
  68. 68. Leukocyte Adhesion Disorders • LAD-I is caused by a mutation in the gene(s) responsible for β2 integrin subunits (CD11/ CD18). • 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 integrin subunit.
  69. 69. 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.
  70. 70. Miscellaneous Granulocyte Disorders • Hyperimmunoglobulinemia E/ Jobs syndrome: Neutrophils in these patients have poor directional motility. • Lazy leukocyte syndrome: Neutropenia and poor neutrophil response to chemotactic agents. • Myeloperoxidase (MPO) deficiency: read most common. • Diabetes mellitus with abnormal oxidative burst activity
  71. 71. Qualitative (Morphologic) Abnormalities • Nuclear abnormalities caused by reaction to infection, inflammation or stress include nuclearhypersegmentation and pyknosis. • Hypersegmentation is often seen in chronic infections: reflect borderline folate deficiency caused by longterm hyperproliferation. • 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.
  72. 72. Pyknosis vs apoptosis
  73. 73. Cytoplasmic abnormalities • 1.Dohle bodies, 2.toxic granulation, 3. vacuolation, degranulation, 4.swelling. • 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 cellular membranes.
  74. 74. • Delay in preparing the film • 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, including bacterial infections, sepsis. • ??Physiological condition
  75. 75. 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.
  76. 76. • True toxic granules tend to cluster within the neutrophil cytoplasm, and not all neutrophils are equally affected
  77. 77. 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 cytoplasm. • 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.
  78. 78. • Eosinophilia • Allergic reactions (e.g., asthma, hay • fever, drug sensitivity) • Parasitic infestations (especially with tissue- invading parasites) • Hypersensitivity reactions (e.g., Loeffler syndrome) • Skin diseases (e.g., dermatitis herpetiformis) • Tropical eosinophilia • Malignant myeloproliferative disorders • Certain infections (e.g., scarlet fever) • Eosinopenia • Infections or inflammation in which neutrophilia occurs • Thymoma and hypogammaglobulinemia • Rare inherited forms • Autoimmune disorders with antieosinophil antibodies • Unknown causes
  79. 79. • 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 infiltration. • Presenting features include anorexia, weight loss, fever, sweating, thromboembolic episodes, heart failure, splenomegaly, and skin and central nervous system (CNS) disease. • 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
  80. 80. • 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 and include • hypothyroidism, • 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.
  81. 81. •Thank you

×