The impact of non-endotoxin LAL-reactive materials on Limulus amebocyte lysate analyses.

Limulus amebocyte lysate (LAL) is activated by bacterial endotoxins and certain glucans (beta-D-glucan, LAL-RM). The potential for conflicting inter-laboratory results for LAL tests exists because commercial LAL reagents are highly variable in response to LAL-reactive glucans. The nature of beta-D-glucan activation of LAL and means for rendering LAL non-responsive to glucan are reviewed to provide a background for resolving conflicting data. Kinetic LAL methods are particularly useful for screening materials potentially contaminated with glucan. The presence of beta-D-glucan in parenterals is uncommon and is likely limited to products exposed to microbial or cellulosic materials. A scheme is suggested for identifying LAL-reactive glucans and for LAL release-testing without glucan interference.

Understanding and setting endotoxin limits.

From time to time, biotechnology and other parenteral drug companies must validate LAL pyrogen tests for raw materials or new drug entities. Since there usually are no established endotoxin limits for these items, quality control personnel must be prepared to determine, and defend, pass/fail LAL pyrogen test limits for these articles. An explanation of the FDA/USP approach to setting endotoxin limits is given, and suggestions are made for devising appropriate in-house LAL test limits for new drug raw materials and finished products.

Determination of endotoxin levels and their impact on interleukin-1 generation in continuous ambulatory peritoneal dialysis and hemodialysis.

Endotoxins represent a family of ubiquitous bacterial lipopolysaccharides found in water and raw materials. These substances have the ability to generate interleukin-1 (IL-1) and induce fever, as well as other acute phase phenomena. A study was undertaken to determine levels of background endotoxin in (1) continuous ambulatory peritoneal dialysis solution, (2) spent dialysate subsequent to overnight dwell, (3) hemodialysis solution, and (4) Limulus amebocyte lysate-reactive material (LAL-RM) in hemodialyzers and patient plasma. Levels of endotoxin in all of the above cases were less than thought to be required to induce biological activity, such as pyrogenicity, through IL-1 generation. Although nanogram amounts of LAL-RM are associated with some hollow-fiber membranes as well as the plasma of patients on those membranes, this material per se does not appear to produce IL-1 in vitro.

Detection of endotoxin in the plasma of patients with gram-negative bacterial sepsis by the Limulus amoebocyte lysate assay.

A total of 120 Limulus amoebocyte lysate (LAL) determinations were made on plasma obtained from normal, healthy human blood donors. Results demonstrated a mean endotoxin level in blood of 0.02 to 1.57 pg/ml. The amount of Escherichia coli endotoxin added to human plasma samples can be quantitated by both nephelometry and turbidimetry. Endotoxin-spiked samples were shown to be significantly different from unspiked samples. When plasma samples were collected from 45 patients hospitalized at three centers, a strong association was demonstrated between a positive Limulus amoebocyte lysate assay and a septic condition. Sensitivity, specificity, and false-positive and false-negative rates for the Limulus amoebocyte lysate assay as a diagnostic test for gram-negative bacteremia were estimated.

Relative potencies of four reference endotoxin standards as measured by the Limulus amoebocyte lysate and USP rabbit pyrogen tests.

Four commonly used reference endotoxin standards, Escherichia coli O113:H10:K0, E. coli O55:B5, Salmonella abortusequi, and Shigella dysenteriae were compared by the USP rabbit pyrogen and the Limulus amoebocyte lysate tests. By the rabbit pyrogen test, S. abortus equi was identified as the most potent endotoxin, followed closely by E. coli O113:H10:K0 and E. coli O55:B5.

Comparison of several control standard endotoxins to the National Reference Standard Endotoxin--an HIMA collaborative study.

A collaborative study, initiated under the auspices of the Health Industry Manufacturers Association (HIMA), was designed to establish the relationship of Escherichia coli O55:B5 endotoxin (the control standard endotoxin of HIMA and the Food and Drug Administration's Office of Medical Devices) to the U.S. National Reference Standard Endotoxin and to two internationally used control standard endotoxins. By using two Limulus amoebocyte lysate test systems, it was established that the E. coli O55:B5 endotoxin lot originally used by HIMA and the Office of Medical Devices to establish Limulus amoebocyte lysate release test criteria for pyrogen testing of medical devices contains approximately 4.5 endotoxin units (EU) per ng. Thus, the 1.0-ng/kg endotoxin dose limit currently established for medical devices is approximately the same as the 5.0-EU/kg endotoxin limit (on an activity basis) established by several other Food and Drug Administration agencies for human and animal parenteral drugs and biological products.