Pyrogen and LAL test

Question 1

Define pyrogen

Answer 1

A substance that produces fever when introduced into the body

Question 2

Define endotoxin

Answer 2

A pyrogen of bacterial origin, specifically LPS from gram -ve bacteria

Question 3

TRUE or FALSE

All pyrogens are endotoxins.

Answer 3

FALSE

All endotoxins are pyrogens but not all pyrogens are endotoxins

Question 4

FACT:

Answer 4

Endotoxins contaminate most aqueous solutions

Question 5

Endotoxins can cause what condition?

Answer 5

septic shock (sepsis) - systemic immunological response to infection (which in extreme cases may be fatal)

Question 6

Define sepsis

Answer 6

A systemic, immunological response to infection, which in extreme cases, may be fatal

Question 7

Sepsis may cause these symptoms

Answer 7

• tachypnoea (abnormally rapid breathing)
• tachycardia (abnormally rapid heart rate)
• altered body temperature
• hypotension

Question 8

TRUE or FALSE
Endotoxins are responsible for many of the symptoms of active infection, BUT endotoxin molecules which are no longer part of a living cell can still cause these symptoms.

Answer 8

TRUE
Dead Gram-negative bacteria still contain heat-stable endotoxins regardless of whether the dead cells are intact or broken into fragments. So an injection may be sterile but still contaminated with dangerous levels of endotoxin

Question 9

Endotoxin levels are measured in ______ _____ (EU).

Answer 9

endotoxin units (EU)

Question 10

What are the two tests that can be done to test for pyrogens or endotoxins? Which is preferred and why?

Answer 10

Rabbit pyrogen test
Bacterial endotoxin test (BET)
- BET preferred; less limitations

Question 11

Endotoxin testing is described in the pharmacopoeias as Bacterial Endotoxin Test (BET) which is also known as?

Answer 11

Limulus Amebocyte Lysate (LAL)

Question 12

How does the LAL test work?

Answer 12

Horseshoe crabs have blood cells called amoebocytes which contain a clotting factor (coagulogen) that is very sensitive to endotoxin. Even after extensive dilution the coagulogen clots when mixed with endotoxin.

Question 13

What are the three LAL methods?

Answer 13

• Gel clot (+ve or -ve)
• Turbidimetric assay
• Chromogenic assay

Question 14

How does a LAL gel clot test work?

Answer 14

• Proenzyme
• Addition of endotoxin
• Converted to active enzyme
• Active enzyme + coagulogen —–> clot formed

Question 15

How does a LAL turbidimetric test work?

Answer 15

Proenzyme

• Addition of endotoxin
• Converted to active enzyme
• Active enzyme + coagulogen —–> increase in turbidity (early clot)

Question 16

How does a LAL chromogenic test work?

Answer 16

Proenzyme

• Addition of endotoxin
• Converted to active enzyme
• Active enzyme + chromogenic substrate —-> colour change

Question 17

Which LAL method is the simplest and cheapest?

Answer 17

Gel clot