Endotoxin Testing - Frequently Asked Questions

 

Which LAL method is the best?

In reality, there is no “best method.” In order to decide which method can be more suitable to your needs, consider the type of product to be tested, its interference characteristics, and the endotoxin limit that you want to detect. The gel-clot method is the simplest and most widely used. This method can provide a sensitivity up to 0.03 EU/mL. The turbidimetric and chromogenic methods do not have a labeled sensitivity. The sensitivity of the test is defined to be the lowest endotoxin concentration in the standard curve. These methods require spectrophotometric instrumentation. As long as the product can be tested by any method, the decision becomes one of personal choice.

 

When should Glucashield Buffer be used?

The LAL cascade reaction mechanism is a complex series of reactions that are dependent on one another. Many different points in the reaction can be affected by interfering factors either showing a false positive or a false negative. The LAL reagent as well as being sensitive to endotoxin also reacts with (1→3) -β-glucan. Glucan initiates the cascade mechanism which leads to a false positive. Glucashield Buffer prevents the interference from glucan by inhibiting the reaction pathway making the assay endotoxin specific.

Glucashield Buffer should be used to reconstitute Pyrochrome and Pyrotell-T reagent whenever interference in the LAL cascade reaction is suspected.

 

When should Pyrosol Buffer be used?

The Pyrosol Buffer is for reconstituting Pyrotell Multi-Test vial or Pyrotell-T reagents. It is used for testing electrolytes, strongly buffered solutions (especially bicarbonate buffers), and solutions for which it is difficult to adjust pH into the required range.

 

What is potency?

The potency of a Control Standard Endotoxin, CSE, is a measure of its activity relative to the activity of the Reference Standard Endotoxin, RSE, which is labeled in Endotoxin Units per mL (EU/mL). Potency of a CSE is expressed as EU of RSE per nanogram of CSE (EU/ng).

 

Why do I need an LAL and CSE lot number to obtain Certificate of Analysis?

The Certificate of Analysis provides the results of the potency determination for a combination of an endotoxin lot with a particular LAL lot. Potency is a measurement of biological activity and can change with each lot of LAL or CSE. It is important not to assume that potency will remain the same with different LAL lots, even if the same CSE lot is being used.

Reference: 1. Endotoxin Standards and CSE Potency. Associates of Cape Cod, Inc. LAL Update, Vol. 11, No. 3.

 

How do I convert from µg/vial of CSE to EU/mL?

Associates of Cape Cod, Inc., ACC, control standard endotoxin, CSE is sold in vials labeled in micrograms or nanograms per vial. The conversion of this value to EU/mL is quite simple.  For example: CSE labeled as 0.5µg/vial.  0.5 µg/vial = 500 ng/vial

This vial of CSE is reconstituted with 5mL of LRW (LAL Reagent Water), therefore, 500 ng/vial = 500 ng/5mL = 100 ng/mL

We obtain a potency value from the Certificate of Analysis (C of A). These potencies can be anywhere from 2-50EU/ng. For this example, we will use a potency of 10EU/ng, 100 ng/mL X 10 EU/ng = 1,000 EU/mL

Therefore we are starting with 1000EU/mL in the vial of CSE. The C of A, in this case, with a potency of 10 EU/ng also indicates that we have 5,000 EU/vial or 5,000 IU/vial. If we reconstitute with 5mL of LRW then we have 5,000 EU/5mL or 1,000 EU/mL.

CSE that is packaged as part of a kit containing CSE and lysate is labeled in EU/vial (for example the CSE in the ACC Pyrochrome kit). We label CSE from a kit in EU because the CSE/lysate combination is defined and the CSE is specific to the kit reagent combination.

 

How do I convert EU/mL to ng/mL of endotoxin?

Endotoxin Units (EU) are a measure of the activity of the endotoxin. Endotoxins differ in their biological activity or potency; the pyrogenicity or LAL reactivity of one endotoxin preparation may be very different from that of another of the same weight. Conversely, two endotoxin molecules may be different sizes and different weights but may have the same reactivity in an LAL test. The potency of an endotoxin determined with one LAL reagent lot may differ from that determined with another lot. Expressing endotoxin concentrations in EUs avoids the issues of different potencies of different endotoxins and allows us to compare results of different LAL tests performed in different labs. Consequently, we do not usually convert results of an LAL test in EU/mL to units of weight of endotoxin per mL.

References: Associates of Cape Cod, Inc. LAL Update, Vol. 15, No. 4, December 1997. Gould, M.J. 1998. Performing the LAL gel-clot test in facilities. Nephrology News & Issues 2(11):26-27,29.

 

How do I convert from EU to IU?

The conversion of results of an LAL test expressed in EU/mL into IU/mL is very easy: 1 EU=1 IU. The United States Pharmacopoeia, the World Health Organization and the European Pharmacopoeia have adopted a common standard. Various names and lot numbers have been given to this standard (e.g. lot G, EC-6), but the content of the vials is identical and the units are equivalent. This standard consists of endotoxin extracted from the cells of Escherichia coli O113; it is assigned an activity of 10,000 EU or 10,000 IU per vial. Therefore, results in EU can equally be reported in IU and vice versa.

References:
1. Associates of Cape Cod, Inc. LAL Update, Vol. 15, No. 4, December 1997.
2. 2. Poole, S., Dawson, P., and Gaines Das, R.E. 1997. Second international standard for endotoxin: calibration in an international collaborative study. Journal of Endotoxin Research 4(3):221-231.

 

How do I know that disposables (plastic tubes, pipet tips, etc.) used in my laboratory are adequate for LAL testing?

Anything that comes in contact with samples or reagents for LAL testing must be free of detectable endotoxin and also free of extractable substances that may interfere with the test.

Because some types of materials adsorb endotoxin, and containers used to collect/store endotoxin may be made of various substances, containers should be qualified by doing a test for adsorption as well as tests for endotoxin and extractable substances.

Materials need to be extracted in LRW for a given period of time to obtain a sample for the endotoxin assay and to test for extractable substances. Use any LAL method to test for endotoxin and include the positive product control as an indicator of interference.

To test for an extractable, use the extract to dilute endotoxin for a standard curve. Confirm that the sensitivity of the gel-clot test or the parameters of the curve generated in a photometric test are the same (within the error) as the sensitivity or parameters obtained using LRW as the diluent.

To test for adsorption, store a known quantity of endotoxin under the same conditions as you would expect to store samples (same time and temperature). Test the sample at "time zero" and again at the end of the storage period. If the concentration is recovered, then adsorption should not be an issue.

Refer to the manufacturer's product insert for recommended materials. ACC recommends polystyrene plastic containers. For glass containers that adsorb (not all do), ACC recommends washing, drying, and depyrogenating several times to block further adsorption and make the containers suitable for use.

References:
1. The Problems with Plastics. Associates of Cape Cod, In. LAL Update, Vol. 6, No. 3.
2. Plastics, Endotoxins, and the Limulus Amebocyte Lysate Test. Journal of Parenteral Science and Technology Vol. 45, No. 2 pp. 83-87