LAL is a reagent derived from the blood cells of the horseshoe crab, unlike a mammal the crab does not have a developed immune system, however the LAL component in its blood will bind to and inactivate endotoxins, in the crab the resulting clot also forms a protective barrier against bacterial infection.

At its simplest the LAL test consists of adding LAL reagent to the sample in a test tube, incubating at 37°C for 1 hour. The tube is then gently inverted - if a gel or clot has formed then a positive result is recorded.

A variety of LAL assay options are available - gel-clot; turbidimetric; and chromogenic measurement. The latter 2 can be measured either as an endpoint or kinetic reaction. The primary requirement for all methods is that the test reagents, containers, pipettes etc. are pyrogen free.

The Gel-clot is the simplest and most widely used, it is also fully described by most pharmacopaeis. The method can be qualitative or semi-quantitative when comparing a sample against a dilution series of an endotoxin standard. Can be used to provide a pass/fail for a certain limit and is best used with low sample numbers. The method may be performed manually with little or no requirement for instrumentation.

Tubidimetric systems depend upon an increasing concentration of insoluble coagulin released as the test progresses, this changes the turbidity of the sample solution. The change in turbidity can be measured either as transmission or absorbance requiring at least a spectrophotometer.

Chromogenic assays depend upon a chromogen which changes color if endotoxin is present in the sample. The higher the concentration of endotoxin then the more chromgen is released. Optical reading device operating at the chromgen wavelength is required.

Both the turbidimetric and chromogenic assays can be performed either as endpoint or kinetic assays.

Endpoint assays involve the measurement of the turbidity or color after incubation at a fixed temperature and time period. Instrumentation requirements can be more basic than those needed for kinetic assays.

Kinetic assays measure the rate of change in turbidity or color during the assay. Instrumentation is used to incubate at a fixed temperature, take measurements, compare those measurements and generate the results. Kinetic assays can provide a greater sensitivity over a wider range than endpoint assays but more sophisticated instrumentation is usually required.

Because the potency of an endotoxin to cause pyrogenic reaction will vary according to the nature of the toxin, the FDA developed Endotoxin Units (EU) for result comparisons.

Choice of LAL technique may be proscribed by the pharmacopaies but may also be affected by other factors such as the inherent turbidity or color of the sample as well as required sensitivities. Electronics technology has also advanced to the stage that portable endotoxin systems with results available in 15 minutes are now on the market, which are more suited for lower sample numbers with the advantage of little operator training being required. If higher sample numbers are to be processed more sophisticated instrumentation in the form of dedicated systems or incubated microplate readers with appropriate software programs can be used, automated robotic systems are also available.

Recently there have been developments intended to decrease the reliance on horseshoe crabs. Examples of these Alternative methods include:

- genetically engineered recombinant factor C - the initial element of the LAL clotting cascade is linked to fluorogenic substrates;

- cell lines sensitive to LPS linked to color changes in the test medium;

Although a postive LAL test indicates the presence of pyrogens a negative result does not necessaryily mean the product is pyrogen free, as non-endotoxin pyrogens may be present but not picked up by the LAL test. But this test does enable products that are unsuitable for the rabbit test to be screened.