First Aid, Chapter 4 Laboratory Tests, Chemotaxis, Phagocytosis and cell killing

Question 1

How do chemotactic assays work?

Answer 1

Chemotaxis is the ability of a protein to direct the traffic and migration patterns of a specific cell. Basically, an assay creates a gradient of the chemotactic agent and allows cells to migrate through a membrane toward said agent. If the agent is not chemotactic for the cell, then the cells will remain on the membrane. If the agent is chemotactic, then the cells will move through the membrane and settle on the chemotaxis plate.

Question 2

Which surface markers are not present in leukocyte adhesion deficiency 1?

Answer 2

CD18, which is part of CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (p150/95)

Question 3

What cell are chemotactic assays most useful to determine defects in? What disorders are they used to diagnose?

Answer 3

Neutrophils.

Chediak-Higashi Syndorme, Leukocyte adhesion deficiency 1 (LAD1), and Ras-related C3 botulinum toxin substrate 2 (RAC2) deficiency

Question 4

Describe the findings and clinical manifestations of Chediak-Higashi syndrome. How is diagnosis made?

Answer 4

Neutropenia, recurrent skin, sinus, pulmonary infections, oculocutaneous albinism, mitral regurgitation (MR), neuropathy. Diagnosis made with detection of giant granules seen on a manual smear, and mutations in LYST 1q42

Question 5

Describe the findings and clinical manifestations of Leukocyte adhesion deficiency 1 (LAD1). How is diagnosis made?

Answer 5

Missing CD18 protein; chemotaxis and adhesion of leukocyte to wall of endothelium and diapedesis impaired; recurrent necrotizing infections of lung, GI tract and skin; umbilical stump separation delayed beyond 1 month of age; poor wound healing; cigarette paper scarring. Diagnosis made through impaired chemotaxis, but can also be made with flow cytometry that shows abnormal CD18

Question 6

Describe the findings and clinical manifestations of Ras-related C3 botulinum toxin substrate 2 (RAC2) deficiency. How is diagnosis made?

Answer 6

RAC facilitates actin cytoskeleton regulation; therefore deficiency causes adhesion problems and impaired chemotaxis. Clinically resembles LAD 1. Transmission autosomal dominant.

Question 7

How large are particles that are phagocytosed?

Answer 7

solid material >500nm in diameter

Question 8

What receptors do phagocytes express?

Answer 8

• Phagocytic cells express various receptors, such as mannose receptors, scavenger receptors, and Toll-like receptors, that can recognize pathogens.
• They also express opsonin receptors, which increase phagocytosis of bacteria coated with IgG antibodies or with complement.

Question 9

How does a phagocyte phagocytose material and then internalize it?

Answer 9

Bound pathogen is surrounded by the phagocyte cell membrane and then internalized in a membrane-bound vesicle known as a phagosome (Figure 418).

Question 10

What happens after a phagosome is internalized?

Answer 10

• The phagosome becomes acidified, killing most pathogens.
• Macrophages and neutrophils have lysosomal granules that contain microbicidal enzymes, proteins, and peptides.
• The phagosome fuses with the lysosome, producing the phagolysosome, in which lysosomal contents are released to destroy the ingested pathogen.

Question 11

Describe how NADPH oxidase is assembled.

Answer 11

Ingestion of microorganisms activates the phagocyte to assemble the multisubunit enzyme NADPH oxidase from its components. The enzyme consists of several subunits and converts molecular oxygen to oxygen free radicals, which are toxic to bacteria (Figure 4-19).

Question 12

What does myeloperoxidase do? What does a deficiency in MPO cause?

Answer 12

Myeloperoxidase (MPO) uses hydrogen peroxide to convert normally unreactive halide ions (Cl– and Br–) into reactive hypohalous acids [i.e., hypochlorite (HOCL) and hybromite] that are toxic to bacteria. o MPO deficiency is a phagocytic disorder that is asymptomatic in most individuals. Recurrent candida infections (skin, bones, blood) have been described rarely in patients with diabetes mellitus and partial/complete MPO deficiency.

Question 13

What cell produces nitric oxide? What is its function?

Answer 13

Macrophages produce reactive nitrogen intermediates, mainly nitric oxide, by the action of inducible nitric oxide synthase (iNOS), which can combine with hydrogen peroxide to form peroxynitrite radicals, which are toxic to bacteria.

Question 14

Describe the steps involved in respiratory burst and intracellular killing.

Answer 14

• The NAPDH oxidase system makes moleculer O2 into Superoxide ion. The superoxide dismutase turns it into hydrogen peroxide.
• MPO uses hydrogen peroxide to convert unreactive halides CL- and Br- into hypohalous acids that are toxic to bacteria.
• Nictric oxide and reactive nitrogen intermediates combine with hydrogen peroxide to form peroxynitrite radicals which are toxic to bacteria.
• Hydrogen peroxide is converted to hydroxy radicals via peroxidase and iron

Question 15

What is CGD? What is the defect? What are the manifestations?

Answer 15

The importance of the respiratory burst is highlighted in patients with chronic granulomatous disease (CGD), which is caused by mutations in the genes encoding the NADPH oxidase system. Individuals with CGD have defective intracellular killing of bacteria and fungi that predispose them to infections with catalasepositive organisms (e.g., Staphylococcus aureus, Serratia marcescens, Pseudomonas, and Salmonella spp.).

Question 16

What do bactericidal killing assays measure? What are the three tests that measure it?

Answer 16

This assay measures the respiratory burst, which is responsible for the generation of reactive oxygen species and intracellular microbial killing. Respiratory burst activity can be measured by several methods, including nitro blue tetrazolium (NBT) reduction test, dihydrorhodamine 123 (DHR) oxidation test, and chemiluminescence assay.

Question 17

Describe the NBT (nitro blue tetrazolium) reduction test. Is it the gold standard?

Answer 17

Neutrophils are mixed with NBT and stimulated with phorbol myristate acetate. Normal neutrophils reduce the NBT to formazan, which changes color from yellow to blue. (PAM) Patients with CGD lack a component of the oxidase system that produces superoxide anion and, thus, cannot reduce NBT to formazan. It is no longer the gold standard.

Question 18

How do X-linked CGD patients react to the NBT (nitro blue tetrazolium) reduction test? How do autosomal recessive CGD patients react? How do x-linked carrier states react?

Answer 18

• Most patients with X-linked CGD will have nearly absent reduction of the NBT, so mostly negative cells (yellow).
• Patients with autosomal recessive CGD may have some positive cells, leading to a missed diagnosis.
• Carriers of the autosomal recessive form of CGD usually have normal NBT results.
• In the X-linked carrier state, usually between 30–80% of the cells will be negative (yellow). The results are variable and can range between 10–90% negative cells.

Question 19

What is the DHR (Dihydrorhodamine 123) oxidation test? How does it work? Why is it prefered?

Answer 19

Dihydrorhodamine 123 (DHR) Oxidation Test—In this test, the nonfluorescent rhodamine derivative, DHR, is taken up by phagocytes and oxidized to a green fluorescent compound by products of the NADPH oxidase. The fluorescence emitted is detected by flow cytometry. The DHR test is preferred because it is a quantitative assay, offers relative ease of use, enables distinction between X-linked and autosomal forms of CGD, as well as providing the ability to detect gp91phox carriers. Other tests can provide reliable diagnosis of CGD, but cannot distinguish carrier status or they require significant operator experience. Figure 4-21 shows the normal and abnormal results with a DHR assay.

Question 20

Which test can distinguish among X-linked, autosomal, AND X-linked carrier forms of CGD?

Answer 20

Dihydrorhodamine 123 assay (DHR)

Question 21

Describe how a chemiluminescence assay works? How is the light measured? How sensitive is it? How is it interpreted?

Answer 21

Chemiluminescence Assay—In this assay, light is generated from the interaction of reactive oxygen species (generated during respiratory burst) with ingested microorganisms. The light can be detected using a scintillation counter, with the amount released reflecting the respiratory burst activity of the cells. It is more sensitive than the NBT test.
Interpretation:
-Patients with CGD lack a chemiluminescence response.
-Maternal carriers of X-linked CGD have intermediate values.
-Patients with CD11 or CD18 surface glycoprotein deficiency may have normal chemiluminescence after stimulation with soluble stimulators, such as PMA; but, they may have delayed and diminished chemiluminescence after stimulation with opsonized particles.