Pyrogen/Endotoxin (LAL) Testing

Purpose: Pyrogens are fever-producing materials that most often originate from gram-negative bacterial cell walls, but can also originate as leachates from some chemical materials. Pyrogens from bacterial cell walls (the most commonly encountered type of pyrogen) are referred to as bacterial endotoxin and are readily detected by Limulus Amebocyte Lysate (LAL) testing systems. LAL is an aqueous extract of the blood cells of horseshoe crabs which forms a clot or change in color, depending on the technique, in the presence of bacterial endotoxin. Validated test methods include both the gel clot and chromogenic methods. MicroMed Labs performs kinetic chromogenic testing in accordance to the FDA, AAMI, and USP guidelines. USP <85>, AAMI ST72:2002

The Kinetic Chromogenic LAL method provides direct quantification of the detected endotoxin level and is especially useful for very low-level detection, determining endotoxin reduction of various production processes, monitoring the quality of water systems, and providing endotoxin levels for lot release of products.

Each time a new device/product is produced, or significant change in material formulation is made on an existing device/product, a validation must be performed on samples from three production lots. The purpose of this is to ensure that the materials used in the construction of the device do not impart an inhibiting or enhancing effect on the LAL test system. Other changes, such as change in the testing laboratory, may only require a single lot validation.

Sample requirements

Sample requirements for both the validation testing and routine testing are typically determined by the size of the production lots from which the samples are selected.

Approximate Turnaround time 2 – 4 days (Expedited testing available for additional fee)

 

LAL Test Validation Validation of the inhibition or enhancement properties of the materials.Sample Requirements: [FDA and AAMI Guidelines]

  • Samples from three (3) production lots should be tested for inhibition and enhancement before this test is considered validated for use with the test product.
LAL Limit Test-Finished Product Testing Quantitative determination of endotoxin level for finished devices or other materials.Sample Requirements: [FDA Guidelines]

  • For lots of less than 30 units – 2 sample devices
  • For lots of 30 – 100 units – 3 sample devices
  • For lots of greater than 100 units – 3% of lot, up to a maximum of 10 [recommended that samples are sterile]
LAL Liquid Test Endotoxin testing of water system samples or other liquids.Sample Requirements:

  • 5 mL in sealed endotoxin-free polystyrene or glass container.

 

Kinetic Chromogenic Limulus Amebocyte Lysate Test Summary
The test articles are either immersed in or flushed with USP Sterile Water for Injection (SWFI) at room temperature for 60 ± 2 minutes. Test article extracts are assayed in duplicate at the neat concentration. A standard curve of endotoxin is prepared with concentrations of 0.005, 0.05, 0.5, and 5 EU/mL. A positive product control (PPC) is prepared containing 0.09 mL of the test article and 0.01 mL of a 5 EU/mL endotoxin standard to give a final concentration of 0.5 EU/mL. LAL Reagent (endotoxin-free) water and SWFI serve as the negative controls. The microtiter plate is pre-incubated in the plate reader at 37 ± 1°C for ≥10 minutes. After incubation, LAL (0.1 mL) was added to each well and the absorbance of each well at 405 nm was read every 150 seconds for a total of 40 data points or until the concentration reaches 0.2 absorbance units. The reader uses the initial reading of each well as its own blank. The absolute value of the correlation coefficient (r) must be ≥0.980 in order for the test to be valid.


Turn Around Time (TAT)

 

Code Test Description TAT
PY-002 Pyrogen (LAL) Chromogenic 2 – 4 days
PY-002A Dilution Setup fee (If required for new samples) 2 – 4 days
PY-004 Endotoxin spiked vials or components 2 – 4 days
PY-005 LAL Product Validation-Inhibition & Enhancement (3 lots)-One Time Test 2 – 4 days
PY-007 Liquid Pyrogen Chromogenic Routine 2 – 4 days
PY-007A Liquid Pyrogen Chromogenic New samples (3 dilutions) 2 – 4 days
SF-001 Stat Fee (tests performed on the day they are received) Add 50% ***

***All stat testing must be started same day unless received after 2pm
***Reports for Stat LAL testing will be sent on the same day as well