W4L8 - Reactive & Non-Neoplastic Disorders of Leukocytes

Question 1

Local Infection

Answer 1

Localised bacterial infections are typically contained by neutrophil recruitment and extravasation into the infected tissues
Bidirectional migration of neutrophils is possible enabling them to re-enter the vasculature
The majority undergo cell death in the inflamed tissue as a consequence of their antibacterial effector functions
- phagocytosis
- degranulation
‘Steady state’ granulocytopoiesis is maintained

Question 2

Systemic Infection

Answer 2

Failure to control an infection locally leads to systemic bacterial dissemination, resulting in further neutrophil ‘consumption’ (& demand)
To counterbalance neutrophil depletion & meet the demand for neutrophils during infection, ‘steady state’ granulocytopoiesis is switched to ‘emergency granulocytopoiesis’.
Characterised by
- enhanced production of neutrophils
- accelerated cellular turnover
- release of immature and mature neutrophils from the bone marrow into the peripheral blood

Question 3

Emergency Granulocytopoiesis

Answer 3

Can be regarded as an example of demand adapted haematopoiesis
Orderly production of neutrophils results from increased myeloid progenitor cell proliferation
Aims to increase neutrophil output in order to meet the higher demand for neutrophils during the innate response to severe infection when these cells are consumed in large quantities
The presence of pathogens is translated into enhanced neutrophil production through the increased release of granulopoietic cytokines
The major granulopoietic growth factor is G-CSF with further contributions from GM-CSF and IL-6

Question 4

Emergency Granulocytopoiesis - Direct Activation

Answer 4

Haematopoietic stem and progenitor cells can sense microorganisms through the expression of pattern recognition receptors
- such as Toll-like receptors
These detect different classes of PAMPs
Upon ligation, PAMPs stimulate proliferation and differentiation into neutrophils

Question 5

Emergency Granulocytopoiesis - Indirect Activation

Answer 5

Depends on the existence of a specialised cell type that can function as a microbial sensor
This cell type stimulates myeloid progenitor cell proliferation and granulocytic differentiation indirectly through the direct or indirect release of granulopoietic cytokines

Question 6

Neutrophil NETs

Answer 6

‘Neutrophil extracellular traps’ (NETs) are large, extracellular, web-like structures composed of cytosolic and granule proteins that are assembled on a scaffold of de-condensed (‘unwound’) chromatin
NETs trap, neutralise and kill and prevent dissemination of microbial organisms

Question 7

NET Formation

Answer 7

Triggered by innate immune receptors through downstream intracellular mediators that include reactive oxygen species
- promoted by NADPH oxidase & mitochondria
ROS then activate myeloperoxidase (MPO), neutrophil elastase (NE) and protein-arginine deiminase type 4 (PAD4) to promote chromatin decondensation

Question 8

NET Formation - 2 Pathways

Answer 8

1. A cell death pathway that begins with nuclear delobulation and the disassembly of the nuclear envelope
   Continues with loss of cellular polarisation, chromatin decondensation and plasma membrane rupture
2. A form independent of cell death that involves the secreted expulsion of nuclear chromatin that is accompanied by the release of granule proteins through degranulation

Question 9

WBC - Automated Analyser

Answer 9

Separates WBC using side scatter and fluorescence
RBC, platelets lysed
Polymethine dye enters WBC, binds nucleic acids
Fluoresces at 633 nm
Organic acid, ‘Stromatolyser-4DL’ binds to eosinophil granules

Question 10

Morphological Variation in Leukocytes

Answer 10

Morphology associated with reactive granulocytopoiesis
Morphology associated with reactive lymphocytosis
Morphology associated with systemic disorders
Morphology associated with therapy
Morphology associated with aetiological agents

Question 11

Morphological changes in Reactive Granulocytopoiesis

Answer 11

Morphological changes may reflect cellular immaturity
Döhle bodies
- aggregates of RNA
- blue-grey irregularly shaped structures in cytoplasm
Toxic granulation
- prominent basophilic cytoplasmic granules
Cytoplasmic vacuolation

Question 12

Morphological changes in Reactive Lymphocytosis

Answer 12

Morphological changes:

• increased size
• increased cytoplasm
• increased basophilic cytoplasm
• irregularly shaped nucleus
• indented/cleaved nucleus
• plasmacytoid appearance

Question 13

Reactive Lymphocytosis Causes

Answer 13

Viral infections
- EBV, CMV, influenza, herpes,
Bacterial infections
- many; eg brucellosis, tuberculosis
Other infections
- toxoplasmosis, malaria, babesiosis
Non-infectious
- autoimmune, drug hypersensitivity, immunisation, trauma, extreme exercise

Question 14

Systemic Disorders with Morphological Atypia of Leukocytes Associated

Answer 14

Pelger-Huët anomaly
Psuedo Pelger-Huët anomaly
Chediak-Higashi granules
May Hegglin anomaly
Storage disorders:
- mucopolysaccharidosis/ Alder Reilly granules
- Morquio syndrome
- Hurler syndrome

Question 15

Pelger-Huët Anomaly

Answer 15

Granulocytes exhibit hypo-segmented nucleus
Lack toxic granulation/Döhle bodies
Autosomal dominant inheritance

Question 16

Psuedo Pelger-Huët Anomaly

Answer 16

A.k.a pelgeroid
Acquired disorder
Result in ‘immature’ appearance of leukocytes
Reflects disorders affecting bone marrow production of leukocytes
Causes include:
- myelodysplastic disorder
- myeloproliferative disorders
- acute leukemia
- certain drugs
- occasional acute infections

Question 17

Chediak-Higashi Granules

Answer 17

Grey granules in neutrophil cytoplasm
- (also platelets melanocytes)
Autosomal recessive disorder of storage/secretory granules
=> dysfunctional bactericidal activity

Question 18

May Hegglin Anomaly

Answer 18

Characterised by:
- giant platelets
- thrombocytopenia
- neutrophil cytoplasmic inclusions
Similar morphology to Döhle bodies
Different structure; composed of ribosomes & microfilaments

Question 19

Storage Disorders

Answer 19

Accumulation of material within cytoplasm of leukocytes due to metabolic atypia
Many recognised disorders
Alder-Reilly anomaly
- round azurophilic, cytoplasmic inclusions of leukocytes
- composed of mucopolysaccharide
Morquio syndrome
- type II mucopolysaccharidosis
- large basophilic granules in leukocytes
Hurler syndrome
- type I mucopolysaccharidosis
- azurophilic granules in leukocytes

Question 20

Morphology Associated with Therapy

Answer 20

A wide range of therapeutic agents may have an effect on the morphology of leukocytes
This may occur by a direct action on the mature leukocyte or during stages of myelopoiesis
May affect the morphology of the nucleus/cytoplasm
For example therapeutic haematopoietic growth factors affecting leukocytes such as G-CSF & GM-CSF may induce morphology similar to ‘toxic granulation’

Question 21

Morphology Associated with Aetiological Agents

Answer 21

A wide range of aetiological agents may be phagocytosed whilst in the peripheral blood => altered cell morphology
Include:
- bacteria
- fungi
- protozoa
Need to be recognised
DDX post collection contamination and proliferation