PYROGEN TESTING
Pyrogens produce symptoms of fever, chill, joint pain, malaise, headache and other complaints following IV injection within 30-120 minutes which may subside within 10-12 hours. Pyrogens are the heat stable, filterable and soluble substances of 0.05-1.0 micrometer size and arise from microbial contamination. Chemically these are lipopolysaccharides from the outer cell wall of the bacteria, thus, the term endotoxins is also used interchangeably but not correct entirely. Both G+ and G- bacteria produce pyrogens, however, the pyrogens of G- bacteria are more potent. The pyrogens are heat stable up to some extent, thus, withstand normal sterilization temperatures.
Depyrogenation
Depyrogenation is the removal of pyrogen. This is achieved by the following methods.
Inactivation - Application of very high dry heat (2500P) for not less than 30 minutes is the desired method for rendering material pyrogen free.
Removal of pyrogen by distillation
Detection and quantification of Pyrogens
1) In-vivo pyrogen (rabbit) test
In-vivo pyrogen test involves the evaluation of the presence of pyrogens in parenteral sample by quantitative fever response produced in rabbits. The principle is based on the fact that the human and rabbits are equally responsive to pyrogen injected intravenously on a dose per weight basis. This test requires the following.
Test animals: healthy adult rabbits (of either sex) weighing not less than 1500 gm
(1.5kg). The animals have been properly maintained in terms of environment and diet prior to the performance of test. The animals are screened for their temperature.
Their control temperature must not differ more than 1°C from each other. Any individual animal having temperature 39.8°C or less than 38.0°C is excluded from the test.
The rabbit-retaining boxes are required to house the rabbits. These boxes "hold" the rabbits so that the temperature can be noted easily during test. The specific directions given in the individual monograph must be followed for the products.
The sample to be tested is injected with a slower rate to the animals. The dose of the sample if not specified should be smaller than 10 ml/kg. Special preliminary steps are required and thus, consideration must he given for the products requiring; 1) dilution, 2) pH adjustment, and 3) isotonicity adjustment.
PROCEDURE
The control (baseline) temperature of three rabbits is determined. The sample is injected into the ear vein of each of three rabbits which are held in the retaining boxes. A dose of 10ml/kg of body weight is used unless specified in the individual monograph. The temperature of each rabbit is determined at 1, 2, and 3 hours subsequent to the injection of sample. The difference between the initial and final temperatures of each rabbits is noted. Any increase in temperature is taken to be the response of sample injected.
Interpretation of the results
The material under examination meets the requirements for apyrogenicity if no rabbit shows an individual rise in temperature of 0.6°C or more above its respective control temperature OR the sum of the temperature rise of 3 rabbits does not exceed 1.4°C. If the results are not within the limits, the test is repeated for additional 5 rabbits and the result is considered for the eight rabbits. After repeating, the material under examination meets the requirements if not more than 3 out of eight rabbits show individual rise in temperature of 0.6 °C OR the sum of rise in the temperature in eight rabbits does not exceed 3.7°C.
Sometimes the difference of initial and the final temperature is negative. If the difference is negative, the result of the rabbit test is counted as zero response and the sample is considered apyrogenic.
Advantages of Rabbit Test
The human and rabbits are equally responsive to threshold levels of the pyrogens.
2) Limulus Amebocyte Lysate Test
The limulus amebocyte lysate test is also called as in-vitro pyrogen test (USP XXI Specified new test). Officially it is termed as bacterial endotoxin test (BET). The test principle is based on the clotting of lysate of amebocyte (an enzyme obtained from the horse shoe crab) in the presence of pyrogens. The extract from the blood cells of horse shoe crab, Limulus Polyphemus contains an enzyme and protein system called "Limulus- Amebocyte Lysate" (LAL) which reacts with pyrogens so that an assay mixture increases in viscosity and opacity until an opaque gel is formed.
Amebocyte + Pyrogen ~ Opaque gel
The reaction accomplishes within 15-60 minutes, depending on concentration of pyrogens after mixing. The concentrated pyrogens make the gel more turbid and thick.
Requirements
Limulus-Ambocyte Lysate is prepared by bleeding healthy mature specimens by heart puncture. The amebocytes are carefully concentrated, washed and lysed by osmotic effects. Prior to perform the LAL test, lysate assay is carried out with purified endotoxins and are accepted if it detects 0.001ug/ml or less concentration of the purified endotoxins.
The glassware, such as glass test tubes (10 x 75mm) used in the test must be thoroughly cleaned, dry and heat sterilized. A buffer solution of potassium phosphate 2mEq/ml is used to adjust the pH of test sample at 7. The alcoholic content in sample is to be removed as it causes precipitation of lysate. If the sample contains proteins, it produces gel thus the proteins must be diluted to appropriate concentration before the test.
Similarly other interfering substances present in sample must also be removed before the test.
Procedure
The pH of test sample if specified is adjusted. The test solution and standardized LAL are separately mixed in equal parts (0.05-0.2ml). The mixture is incubated immediately at 36-38°C for 1 hour in assay tube. The assay tube must be remained undisturbed completely because agitation may irreversibly destroy the gel leading to a false negative result. The test tube is observed after the specified time and is examined for the formation of opaque gel. Formation of gel represents a positive test endpoint reaction. The test is performed using a commercial LAL test kit. This kit contains a lyophilized LAL, and E. coli endotoxin and pure water as standards and these later two are used to check the sensitivity of the test.
Advantage of LAL test
1. It is in-vitro and does not require animal handling, thus is more convenient
2. It is 10 times more sensitive than that of the in-vivo rabbit test
3. It is economical
4. It consume less time, i.e., 1 vs 3 hours required by rabbits test
5. It requires less laboratory facilities and minimum equipments
6. It requires less test volume
7. It is more accurate
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