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PubMed1985

Cross comparison of the Limulus test between laboratories. Two European collaborative studies.

The results of two independent collaborative interlaboratory surveys evaluating limulus amebocyte lysate assays are presented. We investigated the variation of the results from laboratories all using the same reference endotoxin, but working with different gel-clot techniques. In the first study, four lysates were compared in quantitative gel-clot assays using either a macro- or a microtechnique. In the second study, the performance of a proposed limit test was checked under the auspices of the European Pharmacopoeia Commission. A generally good agreement was reached with the limit test; in the quantitative test, however, considerable interlaboratory variation and significant differences among the lysates were observed. In order to obtain consistent results, a thorough validation of all reagents and test parameters is essential. Collaborative studies are an important tool to detect discrepancies between laboratories and to decrease interlaboratory variability.

Endotoxins↗
PubMed1985

[Limulus test using a chromogenic method: application to the control of pyrogens in blood derivatives].

We report here the application of the LAL Test to a chromogenic substrate to detect endotoxins in Human Blood Products. In order to reduce the cost, we used a microplate procedure with the Multiskan Reader. Quantitative results in the range of 0,01 to 0,1 ng/ml allowed for a good correlation with the Rabbit Pyrogen test. For 20% albumins and 4% albumins, the mean endotoxins levels of non pyrogenic lots were 0,38 +/- 0,18 and 0,09 +/- 0,03 ng/ml. All the lots which passed the Rabbit Pyrogen test had endotoxins levels lower than 1 ng/ml and 0,2 ng/ml, respectively. We can use this test for other plasma derivatives; Gammaglobulines and PPSB are easily tested. Dried Concentrated Antihemophilic Factor and Dried plasma contain citrate which inhibits the reaction. Dilution and Addition of Calcium Chloride overcome this inhibition. For dried plasma, we should destroy plasma inhibitors by heating at 75 degrees C. This sensitive and reproductible in vitro assay improves the control of pyrogenecity in Human Blood Products.

Blood Transfusion↗
PubMed1987

Removal of endotoxin from culture media by a polymyxin B sepharose column. The activity of contaminating endotoxin in culture media measured by the interleukin 1 inducing effect on human monocyte cultures and by the Limulus test.

The in vitro study of monocytes (Mo) poses several problems. Minor contamination with endotoxin (ET) of media and utensils as well as adherence to glass or plastic surfaces may activate the cells and cause pronounced production of monokines. Many commercially liquid culture media were found to contain ET in concentrations above 25 X 10(-12) g/ml. A simple system for the removal of ET from media and solutions was established by use of a commercially available Polymyxin B Sepharose gel. To measure the lipopolysaccharide (LPS) binding capacity of the gel, known concentrations of LPS were added to culture media, which were passed through a column consisting of the Polymyxin B Sepharose gel. The content of ET and added LPS in media was measured by the Limulus amoebocyte lysate (LAL) test before and after passage of the column. The LPS-binding capacity of the gel was approximately 2.4 X 10(-6) g/10 ml. The biological activity of contaminating ET and added LPS in media, before and after passage of the column, was also characterized by the capacity of the media to induce interleukin 1 (IL-1) secretion in human Mo cultures. The content of IL-1 in Mo culture supernatants was determined by the mouse thymocyte costimulatory (LAF) assay. By comparison of the activity of ET in these different biological systems, it was demonstrated that 15-20 X 10(-12) g/ml of ET stimulate human Mo cultures to IL-1 secretion.

Adult↗
PubMed1996

Application of a new perchloric acid treatment method to measure endotoxin in both amniotic fluid and cord blood by an endotoxin-specific chromogenic Limulus test in intra-amniotic infection.

Endotoxin in both amniotic fluid and cord blood was measured to detect intra-amniotic fetal infection. Both amniotic fluid and cord blood plasma were pretreated by a perchloric acid treatment, and the endotoxin level was measured by Endospecy test. Cut off values for endotoxin in amniotic fluid and cord blood were 8.5 pg/mL and 7.6 pg/mL, respectively. Escherichia coli intra-amniotic infection caused respiratory distress syndrome (RDS)-mimicking pneumonia. Abnormally high values of endotoxin in both amniotic fluid and cord blood were detected. Intra-amniotic infection caused by Gram-positive bacteria (group B streptococci, Enterococcus fecalis) was shown to be endotoxin negative in both amniotic fluid and cord blood. In cases of negative amniotic fluid culture, measurement of the value of endotoxin in the amniotic fluid is useful in identifying intra-amniotic fetal infection.

Amniotic Fluid↗
PubMed1997

[Validation of three limulus tests for the determination of endotoxin in dialysate/substitute used in on-line hemodiafiltration].

The on-line hemodiafiltration, a newly developed technic of artificial kidney, requires a large mount of dialysate as a substitute for filtered plasma. The dialysate is prepared in the hospital, and is liable to be contaminated. To avoid adverse effects from contaminants, the dialysate needs to be checked at least for endotoxin (ET) with a highly sensitive, accurate and precise method. Three commercial kits, Endospecy (Seikagaku, Tokyo), ES-single (Wako, Osaka) and QCL (BioWhittacker, Maryland), were tested to see if they receive any interference from dialysate. The recoveries of Escherichia coli and Salmonella ET spiked in dialysate at final concentrations of 50, 100 and 150 EU/l were evaluated against those spiked in distilled water using simple linear regression analysis. The validation criteria was that the coefficient of a regression line which was forced to pass through the origin should fall between 0.75 and 1.25. No interference was observed with Endospecy. The QCL showed no interference with E. coli ET but enhancement with Salmonella ET. The ES-single was least sensitive for either ET. Dilution of dialysate affected the recoveries by QCL and ES-single but not those by Endospecy. The Endospecy was thus the only reagent that could detect ET level as low as 1.0 EU/l without interference from dialysate. The rise in body temperature after 4-hour on-line hemodiafiltration was correlated with ET level in the substitution fluid (r = 0.48, p < 0.05), and was significant when the level was 1.0 EU/l or more. These results suggest that the ET level of substitution fluid should be monitored with Endospecy and that the level should be kept below 1.0 EU/l.

Dialysis Solutions↗
PubMed1981

Evaluation of the Limulus test for endotoxemia in neonates with suspected sepsis.

A simplified test for endotoxemia was evaluated in parallel with 198 blood cultures from 115 neonates requiring transitional or intensive care. The Limulus assay disclosed endotoxemia in seven of eight patients with gram-negative bacteremia tested on one or two occasions. It was not specific for bacteremia, especially during the first week of life, when 37 of 112 tests from nonbacteremic infants (33%) were positive. In older infants, positive tests were obtained in only ten of 55 without bacteremia (18%) (P less than 0.07), six of whom had necrotizing enterocolitis as the likely source of endotoxemia. Gram-negative bacteremia existed in 5% of infants (two of 39) less than or equal to 7 days with positive tests and in 37% of older infants (six of 16) (P less than 0.01). Endotoxemia appears to be frequent among infants appearing to have sepsis and may contribute to neonatal morbidity. The Limulus assay may be a useful diagnostic test for coliform bacteremia and necrotizing enterocolitis beyond the first week of life.

Endotoxins↗
PubMed1986

[Detection of endotoxins in radiopharmaceutical preparations--III. Limulus test assessment using radiopharmaceutical preparations; correlation with the rabbit pyrogen test].

Experiments using 17 radiopharmaceuticals containing known amounts of added endotoxin show that none of them inhibits the pyrogenic reaction of the rabbit. Gelation of the Limulus amoebocyte lysate (LAL) is inhibited by 4 of them: colloidal erbium 169Er citrate, colloidal rhenium 186Re sulfide, colloidal technetium 99mTc (Re) sulfide for liver scintigraphy and the colloidal technetium 99mTc (Re) sulfide for lymphography. This inhibition is cancelled, either by dilution or after neutral pH adjustment. Both controls were performed on 313 batches of various radiopharmaceuticals, 95% of results were identical (93% negative, 2% positive). The remaining 5% correspond to positive LAL tests vs negative rabbit tests on the same batches. No negative LAL test vs positive rabbit test was observed.

Animals↗
PubMed1974

Detection of endotoxemia with the Limulus test: preliminary studies in severely malnourished children.

Eighteen Peruvian children with protein-calorie malnutrition were studied to evaluate the usefulness of the limulus assay for endotoxin in detecting gram-negative sepsis. Nine of the children had at least one positive Limulus assay, and eight of this group had bacteriologic cultures indicative of gram-negative infection. Four of these nine children with positive Limulus assays died, whereas only one of the nine children with negative assays died. This preliminary study suggests the usefulness of the Limulus assay as an adjunct to bacteriologic cultures in the early detection of significant gram-negative infection in severely malnourished children.

Bacteria↗