Testing of phagocyte function

Question 1

Define phagocytosis:

Answer 1

non-specific immune mechanism playing an essential role in host defence mechanisms through the uptake and destruction of infectious pathogens.

Question 2

Phagocytosis is the process in which:

Answer 2

specialised cells of the immune system engulf and destroy foreign cells as well as organisms own damaged or dead cells, and so participate in the clearance of microbes and the restitution of damaged tissues

Question 3

Which are the most effective phagocytic cells?

Answer 3

neutrophils and monocytes - immature cells circulating in the bloodstream, and
macrophages - present in various organs and tissues

Question 4

Which other cells can also phagocytose?

Answer 4

eosinophils - small particles
dendritic cells - some particles

Question 5

What is the most important role of eosinophils?

Answer 5

Kill multicellular parasites by release of the biologically active components on the surface of these parasites

Question 6

Why is phagocytosis important for dendritic cells?

Answer 6

Elaboration of a specific immune response rather than for directly destroying the pathogens

Question 7

What are the first inflammatory cells to enter damaged tissue from the blood after tissue damage?

Answer 7

neutrophils (short-lived cells)

Question 8

What phases of inflammation does macrophages participate in?

Answer 8

sub-acute and chronic phases of inflammation, tissue restoration, wound healing, antigen presentation, cytokine production and phagocytosis

Question 9

What are the stages of phagocytosis?

Answer 9

1. Chemotaxis
2. Adhesion
3. Engulfment
4. Destruction
5. Indigestible and waste material is finally discharged from the phagocytic cell

Question 10

What happens during chemotaxis? (1st step)

Answer 10

migration of leukocytes along the concentration gradient of chemoattractants, produced in damaged tissues or as a result of microbial invasion

Question 11

What happens in adhesion (2nd step)?

Answer 11

Recognition of the particles to be phagocytosed by receptors expressed on the leukocyte’s surface.
Coating the particle - opsonisation of opsonins (antibodies(IgG), complement fragments, mannose-binding lectin, C-reactive protein etc.)
Receptors are coated with various proteins and give signals to activate phagocytes.

Question 12

What happens during engulfment (3rd step)?

Answer 12

Ingestion.
Binding of a particle to phagocytic leukocyte receptors initiates the process of active phagocytosis of the particle.
Engulfment result in phagosome (a vacuole), which fuses with granules (lysosomes) containing antimicrobial substances to form a phagolysosome.

Question 13

What happens during destruction (4th step)?

Answer 13

Antimicrobial substances digest foreign material, destroy bacteria or inhibit their growth.
These substances include products of the lysosome, acidic hydrolases, proteases, lactoferrin, bactericidal permeability-increasing protein, defensins, reactive oxygen and nitrogen intermediates.

Question 14

The role of phagocytosis is crucial in the…?

Answer 14

Innate immune defence

Question 15

What does defects in phagocyte functions result in?

Answer 15

Impaired protection against bacteria and fungi.
Pyogenic or granulamatous bacterial and fungal infections located in the skin, respiratory or gastrointestinal tract.

Question 16

What are primary immunodeficiencies?

Answer 16

diseases with abnormal phagocyte number or impaired ability to migrate, engulf and destroy pathogens

Question 17

What is the first step in diagnosis of failure of phagocytosis?

Answer 17

evaluation of neutrophil and monocyte numbers and then subsequent functional studies of the individual steps and mechanism of phagocytosis:
- ingestion
- metabolic activation (resp. burst)
- microbial activity

Question 18

What is the maximum concentration of heparin for phagocytic cell function test?

Answer 18

10 IU per 1 ml of the blood

Question 19

What is used during evaluation of ingestion?

Answer 19

Full-heparinised blood or isolated leukocytes using various microorganisms or microscopic hydrophilic particles as a substrate

Question 20

What are the functional methods for evaluation of ingestion called?

Answer 20

PhA - phagocytic activity
PhI - phagocytic index

Question 21

What is used for quantitative evaluation of ingestion?

Answer 21

Flow cytometric assays, to measure neutrophil or monocyte-associated fluorescence after incubation of whole blood with FITC-labelled E. coli

Question 22

What is added to distinguish between internalised and membrane bound bacteria in evaluation of ingestion?

Answer 22

Trypan blue

Question 23

How do you calculate PhA and PhI?

Answer 23

PhA = number of phagocytes with ingested particles / number of counted phagocytes x 100

PhI = number of phagocytosed C. albicans cells in 100 phagocytes / 100

Question 24

Define the term ‘‘respiratory burst’’

Answer 24

when the consumption of oxygen dramatically increases following ingestion of microbes and activation of phagocytes, in order to produce free superoxide anion and other reactiove species

Question 25

What is chronic granulomatous disease?

Answer 25

A type of primary immunodeficiency caused by mutations in genes encoding for subunits of NADPH-oxidase.

Question 26

What assays can be used for quantitative evaluation of oxygen radical productions?

Answer 26

spectrophotometry or flow cytometry

Question 27

What is the most widely used quantitative colorimetric assay?

Answer 27

iodonitro-tetrazolium salt (INT) test

Question 28

What happens during iodonitro-tetrazolium salt test?

Answer 28

Tetrazolium salt penetrates into the cell during phagocytosis and is subsequently reduced into formazan (insoluble and purple)

Question 29

Which steps are involved in the INT test?

Answer 29

• incubation of purified PMNLs with tetrazolium salt + particles, or diffusible activators like phorbol myristate acetate (PMA)
• protein kinase K in the cell is activated
• NADPH-cytochrome B system is activated
• respiratory burst is directly induced

Question 30

What is the index of metabolic burst?

Answer 30

the metabolic activity of examinated PMNL measured by the absorbance of solution of formazan using spectrophotometric method

Question 31

Other tests for evaluation of oxygen radical production are

Answer 31

Flow cytometric assays
- Dihydrorhodamine 123 (DHR)

Question 32

Describe the dihydrorhodamine 123 test

Answer 32

• incubation with phorbol myristate acetate-activated PMNLs
• penetrates into mitochondria
• oxidised to rhodamin 123
• emission of a bright red fluorescent signal upon excitation by light beams, detected in a flow cytometer

Question 33

Another way of evaluating metabolic activation:

Answer 33

measurement of chemiluminescent activity

Question 34

What is the principle of measurement of chemiluminescent activity?

Answer 34

Hydrogen peroxide produced during respiratory burst serves as substrate for the subsequent production of HCLO and CLO-, catalyzed by myeloperoxidase

Question 35

How do you calculate index of metabolic burst?

Answer 35

IMB = absorbance of stimulated cells (test sample) / absorbance of un-stimulated cells (negative control)

Question 36

Which stain is used for evaluation of microbial activity?
and for what purpose?

Answer 36

Methylene blue
- differentiation between dead and viable candida
- viable cells are unstained

Question 37

How is the candical activity calculated?

Answer 37

% of cidia = Deac C.albicans in sample - Dead C.albicans in control / Number of all C.albicans cells x100