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Renin Angiotensin Mechanism.

Pyrogen Tests

 

Introduction:

  • A pyrogen can be defined as “Fever Producing Substance”, which induces “Fever (Hyperpyrexia)”.

  • Bacterial endotoxins are the most common type of “Exogenous Pyrogen”.

  • Bacteria, viruses, malaria parasites and fungi on entering in the body releases certain chemicals that act as Pyrogen.

  • During the sterilization process these microorganisms die but presence of pyrogens in the products may cause a febrile reaction.

  • Test for sterility ensures absence of microorganisms while the Pyrogen test ensures absence of pyrogens in the pharmaceutical products.

  • The lipopolysaccharide (LPS) from gram negative bacterias is a very common example of pyrogen.

  • The common pyrogen tests are,

    • LAL Test (Limulus Amoebocyte Lysate Test).

    • Sham Test (Rabbit Test).

Pyrogen Test Methods.

  • Pyrogen tests can be carried out using one of the following methods,

    • Rabbit Test.

    • LAL Test.

Rabbit Test.

  • For this test, three healthy rabbits are selected each weighing at least 1.5 kg. 

  • No rabbit should be selected if:

    • 1.  It has a normal temperature greater than 49.8°C.

    • 2.  It was used in a positive test during the last two weeks.

Method for Pyrogen Test:

  • The pyrogen testing is performed in an air-conditioned room. 

  • The food and water is withheld from the rabbit overnight. 

  • A clinical thermometer is inserted in the rectum of each rabbit to a depth of not less than 7.5 cm. 

  • Two readings of the temperature of rabbit in normal conditions should be taken at the interval of half an hour before the start of the test and the mean of both should be calculated to determine the initial temperature.

  • The equipment, injectors and needles used in the test should be pyrogen-free. 

  • These should be washed with water for injection and then heated at 260°C for two hours. 

  • The injection is warmed to 38°C before injecting to the rabbits. 

  • 0.5 to 1.0 ml per kg dose should be injected through the ear vein. 

  • Six readings of temperature are recorded at an interval of half an hour.

Pyrogen Test Results:

  • The response of each rabbit is detected by the difference of initial temperature and the highest temperature recorded. 

  • The response of all three rabbits gives the sum of responses and can be concluded as:

    • i)  If the sum of responses does not exceed 1.4°C and any rabbit shows the response less than 0.6° C, the product passes the test.

    • ii)  If the sum of responses is greater than 1.4 °C or any rabbit shows the response 0.6 or greater, continue the test using 5 rabbits.

    • iii)  If the test is done using 5 rabbits, then if the sum of responses of all 5 rabbits is greater than 3.7°C and the individual response of not more than three rabbits is greater than 0.6°C, the product passes the test.

LAL Test.

  • Also called “Limulus Amoebocyte Lysate Test” and “Bacterial Endotoxin Test”.

  • The LAL test reagent is prepared by lysing amoebocyte blood cells from 

  • American Horseshoe crab Limulus polyphemus and other species like Tachyleus tridentus, Tachypleus gigas and Carcinoscropius rotundicauda.

  • The horseshoe crab has blue colored blood.

  • The amebocyte blood cells of horseshoe crab show defensive clotting in presence of bacterial endotoxins.

  • When a test material containing a pyrogen is added to the LAL reagent it forms turbidity or a gel consistency indicating presence of endotoxins.

  • The test is 100 times more sensitive than the rabbit test.

  • LAL reagent is available commercially and is a popular alternative to animal tests.
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