Biological activities of tritiated endotoxins: correlation of the Limulus lysate assay with rabbit pyrogen and complement-activation assays for endotoxin.

Tritiated endotoxins were prepared by three different methods. The biological activities of the tritiated endotoxins were determined by the Limulus amebocyte lysate assay, a rabbit pyrogen assay, and a complement-activation assay and were compared to native, unlabeled endotoxin. All three tritiated endotoxin preparations manifested adequate biological activity in each of the three assay systems, and all three assays ranked the biological activity of the different endotoxin preparations in the same order. Endotoxin tritiated by the Wilzbach procedure retained most of its biological activity and also had the highest specific radioactivity. The good correlation between the Limulus lysate, rabbit pyrogen, and complement-activation assays suggests that the same active site of the endotoxin molecule is identified by the three different assays.     

COMPARISON OF CULTURED HUMAN MONONUCLEAR CELLS, LIMULUS AMEBOCYTE LYSATE and RABBITS IN THE DETECTION OF PYROGENS

Isolated human mononuclear cells exposed to either lipopolysaccharide or Staphylococcus aureus secreted interleukin-1 like material. The secretion was concentration dependent. The minimal detectable level in the test solution of lipopolysaccharide and Staphylococcus aureus was 200 pg/ml and 10(5) cells/ml respectively. The sensitivity and specificity of the Limulus Amebocyte Lysate test, the rabbit pyrogen test and the monocyte test are compared. The monocyte test is proposed as an alternative in-vitro test to the rabbit pyrogen test.     

A new sensitive microplate assay of plasma endotoxin.

We have developed a microplate method for determining endotoxin in platelet-rich plasma-using Endospecy, an endotoxin-specific chromogenic Limulus test reagent. Nonspecific activators and inhibitors of the test were eliminated by exposing samples (5 microliters) to the alkali reagent consisting of KOH, CaCl2, Triton X-100, ethyleniminepolymer and N,N-bis(2-hydroxyethyl)glycine. The recoveries of various endotoxins were almost complete and not enhanced by dilution. The dose-response curve was linear over endotoxin concentrations of 2-400 pg/ml with good precision (C.V. less than 5.0%). Normal human plasmas (n = 30) contained less than 5.0 pg/ml of endotoxin in reference to that of Escherichia coli 0111: B4. All plasma samples with high concentration of endotoxin by a conventional method showed high values by the microplate assay as well. Since it does not require centrifugation, the new treatment allows the whole reactions to proceed on the same microplate. This permits us to apply the Limulus test to an automated assay system, making plasma endotoxin determination simpler and more rapid than a conventional test tube method.     

Pyrogen reactions to human serum albumin during plasma exchange

Reactions to human serum albumin (HSA) in therapeutic plasma exchange (TPE) are rare. Nevertheless, older literature describes possible adverse effects, including specific immune responses to albumin or other proteins, and reactions due to contaminating organisms or pyrogen. During an eight day period three patients in our unit had unusual reactions after infusion of 1.5–2 L of HSA. Patient 1 had trembling that persisted for 20 min. Patient 2 had shaking for 40 min despite calcium gluconate infusion, and fever to 100.8° F. Patient 3 had severe rigors that subsided after 90 min when meperidine was finally given, and fever to 103.5°F. Record reviews revealed that all three patients had received HSA from the same lot, and that only one other TPE patient had received HSA from that lot. Neither our pharmacy nor the manufacturer was aware of other reactions associated with that lot. Material from a bottle only partially infused to patient 3 was negative in culture and was negative for pyrogen when retested by the manufacturer. Nevertheless, because patients 1 and 2 had each had multiple previous uneventful TPEs and because all three patients tolerated subsequent TPEs without incident when another brand of HSA was used, we conclude that these patients had pyrogen reactions to the implicated HSA lot. This experience illustrates the value of cluster recognition in arousing suspicion of unusual reactions to HSA and the value of recorded lot numbers in pursuing such suspicions. Apheresis personnel should be aware of the potential for pyrogen reactions with HSA and should record lot numbers of all fluids infused during TPE.     

An improved in vitro pyrogen test: to detect picograms of endotoxin contamination in intravenous fluids using limulus amoebocyte lysate.

A method for in vitro pyrogen testing using Limulus amoebocyte lysate (LAL) has been described. The method is based upon the measurement of endotoxin-precipitable protein and can be used to measure picogram quantities equivalent to E. coli endotoxin in unknown solutions. When increasing concentrations of E. coli endotoxin are added to a constant amount of LAL and the reaction is allowed to proceed to completion, there is a proportional increase in the protein precipitated by endotoxin. Therefore, by measuring the amount of protein precipitated from LAL, it is possible to determine the equivalent E. coli endotoxin concentration in unknown solutions, when samples of the unknowns are run simultaneously with E. coli endotoxin standards and negative controls. The endotoxin proportional precipitation of protein occurs in reaction mixture showing gelation as well as in reaction mixture where the levels of endotoxin are lower than required for gelation. Determination of precipitated protein provides greater sensitivity for endotoxin detection than the gelation methods currently in use.     

A modified Limulus amebocyte lysate test with increased sensitivity for detection of bacterial endotoxin

We have developed a simple modification of the chromogenic Limulus amebocyte lysate test that increases the sensitivity for the detection of bacterial endotoxins. In this assay, free paranitroaniline, cleaved from synthetic chromogenic substrates by proteases that were generated by Limulus lysate after incubation with endotoxin, was then derived. Derivation was with p-dimethylaminocinnamaldehyde in the presence of strong acid, forming a stable Schiff base end product with much greater molar absorbancy than the parent chromogen. Conditions (times and temperatures of incubations, concentrations of reagents) for the augmented chromogenic procedure were optimized. A ten-fold or greater increase in sensitivity for bacterial endotoxin was obtained with the modified assay as compared with the standard chromogenic Limulus test, with unequivocal detection of endotoxin concentrations of less than 100 pg/ml. The greater sensitivity of this modified Limulus test increases its usefulness for a wide range of research applications and clinical investigations.     

Detection of endotoxin in triglyceride-rich lipoproteins in vitro

Numerous investigations have been performed in which volunteers have received infusions of triglyceride-rich lipoproteins without apparent screening of the infusates for bacterial endotoxin. This study was designed to examine the capacity of triglyceride-rich lipoproteins to mask their endotoxin content in vitro as measured by a chromogenic modification of the standard Limulus assay. Lipoproteins and lipoprotein-deficient plasma were isolated from normal human plasma by sequential ultracentrifugation under apyrogenic conditions. Individual lipoproteins and a synthetic lipid emulsion were suspended in 10% lipoprotein-deficient plasma. Samples were then incubated at 37 degrees C for 4 hours with increasing concentrations of E. coli (055:B5) endotoxin and assayed for detectable endotoxin activity. The capacity to inhibit detection of endotoxin in 10% lipoprotein-deficient plasma was significantly increased (10 to 100 times) by the addition of VLDL (1.0 mg triglyceride/ml), chylomicrons (1.0 mg triglyceride/ml), or the synthetic lipid emulsion (2.5 mg triglycerides/ml). These data demonstrate that triglyceride-rich lipoproteins, and the synthetic lipid emulsion, can markedly inhibit the detection of endotoxin by the Limulus assay in vitro. In addition to the potential of harm to experimental subjects, infusion of endotoxin could vitiate kinetic studies by direct alteration of lipoprotein metabolism and by inducing changes in hepatic blood flow. Thus experimental protocols that involve the infusion of humans with triglyceride-rich lipoproteins should include detailed testing for the presence of endotoxin.     

THE RATE OF RELEASE OF LEUKOCYTE PYROGEN FROM RABBIT BLOOD INCUBATED WITH ENDOTOXIN

The time course of leukocyte pyrogen liberation from whole rabbit blood incubated at 37°C with various concentrations of proteus endotoxin was studied. The kinetics of the process provide further evidence for the view that leukocyte pyrogen is an independent entity. It was shown that complete liberation of all available leukocyte pyrogen from blood is too slow a process to account for the fevers seen when rabbits are injected with endotoxin. However, very small quantities of leukocyte pyrogen were demonstrated after short periods of incubation which are adequate to account for endotoxin fevers. It was also found that small, but definite, quantities of leukocyte pyrogen are liberated from rabbit blood incubated without endotoxin.     

IN ACTIVATION OF B. SUBTILIS SPORES AND E. COLI ENDOTOXIN BY ETHYLENE OXIDE

Exposure of Bacillus subtilis spores to ethylene oxide (EO) showed correlation between the killing rate and the EO concentration, when the temperature was kept at 55 degrees C and the relative humidity at 100%. The co-efficient of dilution was calculated to be 0.9. The effect of EO on Escherichia coli endotoxin was investigated by the chromogenic Limulus Amebocyte Lysate (LAL) test. A solution of endotoxin was dried on glass tubes and exposed to 450 or 900 mg EO/l during 1-46 h under the same conditions as the spore inactivation. The LAL activity of the endotoxin was reduced to about 30%. The EO-treated endotoxin was tested in the rabbit pyrogen test. The summed temperature increase for three rabbits was 0.9 degrees C, while the same assay using untreated test pieces showed an increment of 3.7 degrees C. Administration of the same quantity of EO-treated and untreated endotoxin to the rabbits, as adjusted by the LAL-test, produced the same temperature increment. The addition of polymyxin B (PB) to an endotoxin solution reduced the LAL activity by 75%. Had the endotoxin been exposed to EO, thereby reducing the LAL activity by 70%, addition of PB further reduced the activity by 99%. The reaction of EO on the endotoxin reduced the LAL activity as well as the pyrogenic response and increased the affinity to PB.     

Detection of endotoxins in human blood and plasma. An improved in-vitro pyrogen test.

We describe an improved in-vitro procedure for detection of endotoxin in human blood and plasma by use of Limulus amoebocyte lysate. Increasing concentrations of Escherichia coli endotoxin added to a constant amount of the lysate cause a proportional increase in protein precipitated by the endotoxin. By measuring the amount of protein precipitated, it was possible to determine the equivalent E. coli endotoxin concentration in unknown samples, when samples were run with E. coli endotoxin standards and negative controls. The E. coli endotoxin, present in human whole blood and platelet-rich plasma, failed to react with the lysate. However, the concentration of endotoxin in whole blood and platelet-rich plasma could be measured with this Limulus test after lysing the platelets to release the endotoxin and subsequently removing the inhibitory proteins by chloroform precipitation. With this procedure it was possible accurately and repeatedly to determine E. coli equivalent endotoxin concentrations as low as 195 ng per liter of whole blood or 49 ng per liter of platelet-rich plasma.     

Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1

Recombinant human tumor necrosis factor (rTNF alpha) injected intravenously into rabbits produces a rapid-onset, monophasic fever indistinguishable from the fever produced by rIL-1. On a weight basis (1 microgram/kg) rTNF alpha and rIL-1 produce the same amount of fever and induce comparable levels of PGE2 in rabbit hypothalamic cells in vitro; like IL-1, TNF fever is blocked by drugs that inhibit cyclooxygenase. At higher doses (10 micrograms/kg) rTNF alpha produces biphasic fevers. The first fever reaches peak elevation 45-55 min after bolus injection and likely represents a direct action on the thermoregulatory center. During the second fever peak (3 h later), a circulating endogenous pyrogen can be shown present using passive transfer of plasma into fresh rabbits. This likely represents the in vivo induction of IL-1. In vitro, rTNF alpha induces the release of IL-1 activity from human mononuclear cells with maximal production observed at 50-100 ng/ml of rTNF alpha. In addition, rTNF alpha and rIFN-gamma have a synergistic effect on IL-1 production. The biological activity of rTNF alpha could be distinguished from IL-1 in three ways: the monophasic pyrogenic activity of rIL-1 was destroyed at 70 degrees C, whereas rTNF alpha remained active; anti-IL-1 neutralized IL-1 but did recognize rTNF alpha or natural cachectin nor neutralize its cytotoxic effect; and unlike IL-1, rTNF alpha was not active in the mitogen-stimulated T cell proliferation assay. The possibility that endotoxin was responsible for rTNF alpha fever and/or the induction of IL-1 was ruled-out in several studies: rTNF alpha produced fever in the endotoxin-resistant C3H/HeJ mice; the IL-1-inducing property of rTNF alpha was destroyed either by heat (70 degrees C) or trypsinization, and was unaffected by polymyxin B; pyrogenic tolerance to daily injections of rTNF alpha did not occur; levels of endotoxin, as determined in the Limulus amebocyte lysate, were below the minimum rabbit pyrogen dose; and these levels of endotoxin were confirmed by gas chromatography/mass spectrometry analysis for the presence of beta-hydroxymyristic acid. Although rTNF alpha is not active in T cell proliferation assays, it may mimic IL-1 in a T cell assay, since high concentrations of rTNF alpha induced IL-1 from epithelial or macrophagic cells in the thymocyte preparations. These studies show that TNF (cachectin) is another endogenous pyrogen which, like IL-1 and IFN-alpha, directly stimulate hypothalamic PGE2 synthesis. In addition, rTNF alpha is an endogenous inducer of IL-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Endotoxin concentration in neutropenic patients with suspected gram-negative sepsis: correlation with clinical outcome and determination of anti-endotoxin core antibodies during therapy with polyclonal immunoglobulin M-enriched immunoglobulins.

We carried out a study in patients with severe neutropenia from hematologic malignancy and suspected gram-negative sepsis to evaluate the clinical significance of endotoxin concentrations in plasma before and during a therapeutic intervention with a human polyclonal immunoglobulin M (IgM)-enriched immunoglobulin preparation (Pentaglobin; Biotest, Dreieich, Germany). Twenty-one patients with acute leukemia or non-Hodgkin's lymphoma entered the study upon the development of clinical signs of gram-negative sepsis and received the IgM-enriched immunoglobulin preparation every 6 h for 3 days (total dose, 1.3 liter with 7.8 g of IgM, 7.8 g of IgA, and 49.4 g of IgG), in addition to standardized antibiotic treatment. Concentrations of endotoxin and IgM and IgG antibodies against lipid A and Re lipopolysaccharide (LPS) in plasma were determined by a modified chromogenic Limulus amebocyte lysate test and semiquantitative enzyme linked immunosorbent assay, respectively, before each immunoglobulin infusion and during the following 25 days. Seventeen patients were endotoxin positive; in five of these patients, gram-negative infection was confirmed by microbiologic findings. Prior to therapy, endotoxemia correlated significantly with the occurrence of fever, and a quantitative correlation between the endotoxin concentration and body temperature was found during the individual course of infection in 8 of the 17 patients. Overall mortality from endotoxin-positive sepsis was 41% (7 of 17) and 64% (7 of 11) in patients with symptoms of septic shock. Nonsurvivors had significantly higher maximum concentration of endotoxin in plasma compared with those of survivors at the first study day (median of 126 versus 34 pg/ml; P < 0.05) and during the whole septic episode (median of 126 versus 61 pg/ml; P < 0.05). In survivors, immunoglobulin therapy resulted in a significant decrease in endotoxin levels in plasma within the initial 18-h treatment period, from a pretreatment median value of 28 pg/ml to a value of 8 pg/ml (P< 0.05). In the seven patients who died from uncontrollable infection, no effect of therapy on endotoxin levels in plasma was observed. IgM and IgG antibodies against lipid A and Re LPS increased significantly under immunoglobulin treatment, with significant correlations between antibodies against lipid A and Re LPS. These data strongly suggest a prognostic significance of the endotoxin levels in plasma and a potential effect of treatment with a polyclonal IgM-enriched immunoglobulin preparation. Further studies are needed to substantiate these findings and to assess the impact on the clinical course by way of a prospective placebo-controlled clinical trial.     

新鲜蒸馏水和储留蒸馏水冲洗清除热原效果比较

目的了解新鲜蒸馏水和储留蒸馏水冲洗去热原的效果。方法用鲎试验方法对三效热原灭活剂浸泡去热原洗涤的玻璃注射器进行了检测。结果经用新鲜过滤流动蒸馏水清洗的注射器,热原检测合格率为98.3%。用储存24 h的蒸馏水清洗后的注射器热原检测合格率65.0%;储存48h的蒸馏水清洗后的注射器热原检测合格率为45.0%。结论经去热原处理后的玻璃器材应该用新鲜流动蒸馏水进行清洗,储存24 h以上的蒸馏水清洗会影响最终去热原效果。     

STUDIES ON THE PATHOGENESIS OF FEVER : VI. THE INTERACTION OF LEUCOCYTES AND ENDOTOXIN IN VITRO

Study in vitro of the interaction of bacterial endotoxin with rabbit polymorphonuclear leucocytes has resulted in the following findings: 1. Incubation of endotoxin and leucocytes in saline results in: (a) the release of leucocytic pyrogen, and (b) the inactivation of endotoxin. 2. Cell-free extracts of leucocytes also inactivate endotoxin. 3. Incubation of leucocytes in "physiological" saline causes rapid discharge of leucocytic pyrogen. In contrast, relatively little pyrogen is released by leucocytes incubated in fresh serum. 4. The release of leucocytic pyrogen in serum is markedly stimulated by the presence of endotoxin. 5. Leucocytes obtained from tolerant rabbits interact with endotoxin in essentially the same manner as leucocytes from normal rabbits. The pertinence of these findings to the pathogenesis of fever and to related information concerning human leucocytes has been discussed.     

Anaphylaxis after treatment with recombinant factor VIII

BACKGROUND: Treatment of hemophilia patients with recombinant factor VIII concentrates has not previously been associated with anaphylaxis. STUDY DESIGN AND METHODS: A 5-week-old boy with severe hemophilia A developed dyspnea, cyanosis, hypotension, and a diffuse urticarial rash following treatment with a recombinant factor VIII (Recombinate). To identify the cause of anaphylaxis in this patient, the vial lot was examined for the presence of endotoxin, and a checkerboard immunoblotting technique was used to test serum and/or plasma samples from the patient and mother for the presence of antibodies (IgA, IgG, IgE, and IgM) to Recombinate-related antigens (recombinant factor VIII, von Willebrand factor, human serum albumin, Chinese hamster ovary proteins, bovine serum albumin, mouse monoclonal anti-human factor VIII, polyethylene glycol 3350), and to ethylene oxide, the agent used to sterilize the infusion equipment. RESULTS: No immune response directed against the Recombinate-related antigens or ethylene oxide that could be associated with the anaphylactic reaction was identified. Endotoxin was not present upon rabbit pyrogen testing of the therapeutic product. CONCLUSION: These studies failed to show any association between Recombinate and the onset of the allergic reaction. This seems to be the first reported case of anaphylaxis following the infusion of a recombinant form of factor VIII concentrate.     

The effect of the number of withdrawals on the sterility of multidose medication vials.

Policy development with regard to the shelf life of multidose medication vials is difficult because of a lack of sufficient data. The purpose of this study was to evaluate one factor that may play a role in the contamination of multidose vials. We evaluated the effect that the number of withdrawals had on the potential contamination of multidose medication vials. Thirty multidose vials of heparin and 30 multidose vials of lidocaine (lignocaine) were used in the study. Ten vials from each group (heparin and lidocaine) were used as controls and were deliberately contaminated with Staphylococcus aereus at various concentrations (10 colony forming units per millilitre (CFU/ml) to 10(8) CFU/ml). The 20 test vials remaining from each group were then subjected to varying rates of 1-ml fluid withdrawal. Five test vials were sampled every 2 h, five were sampled twice per day, five were sampled once per day, and five were sampled every other day. Each sample was incubated in trypticase soy broth (24 h at 37 degrees C) and Columbia agar with 5% sheep blood (48 h at 37 degrees C). No multidose vials became contaminated regardless of the rate of withdrawals. Some of the deliberately contaminated multidose vials became sterile with time. Lightly contaminated vials (10(1)-10(2) CFU/ml) generally cleared within 10-15 h after initial contamination. The implications of these findings are discussed.     

Albumin absorption and catabolism by isolated perfused proximal convoluted tubules of the rabbit.

Overall characteristics and kinetics of tubular absorption of albumin (Alb) were studied in isolated perfused proximal convoluted tubules of the rabbit. The fate of absorbed Alb was determined in tubules perfused with low [Alb]. Alb was labeled with tritium by reductive methylation ( [3H3C]Alb). At [Alb] = 0.03 mg/ml, approximately 80% of the absorbed [3H3C]Alb was released to the peritubular bathing solution as catabolic products. Transcellular transport of intact [3H3C]Alb was negligible. Iodoacetate (IAA, 4 mM) inhibited albumin absorption (JAlb) by greater than 95% and fluid reabsorption (JV) by 55%. At [Alb] = 0.1 mg/ml the absorption rate of a derivatized cationic Alb (pI = 8.4) was fivefold greater (P less than 0.01) than that of anionic Alb. Higher cationic [Alb] had deleterious effects on tubular functions. Overall Alb absorption was of high capacity and low affinity (JmaxAlb = 3.7 ng/min per mm tubule length, apparent Michaelis constant (Km) = 1.2 mg/ml). A low capacity system that saturates at near physiological loads was also detected (JmaxAlb = 0.064 ng/min per mm, apparent Km = 0.031 mg/ml). High [Alb] did not alter the rate of endocytic vesicle formation as determined by the tubular uptake of [14C]inulin. Results show that Alb absorption is a saturable process that is inhibited by high IAA concentrations and is affected by the charge of the protein. Absorbed Alb is hydrolyzed by tubular cells and catabolic products are readily released to the peritubular side. The dual kinetics of Alb absorption may be due to a combination of adsorptive endocytosis (low capacity system) and fluid endocytosis of albumin aggregates (high capacity system). Results indicate that albuminuria occurs much before albumin absorption is saturated. The kinetic characteristics of the process of tubular absorption of albumin helps to explain the concomitance of albuminuria, increased renal catabolic rates of albumin, and renal cell deposition of protein absorption droplets in severe glomerular proteinurias.     

QUANTITATIVE ASPECTS OF THE RELEASE OF LEUKOCYTE PYROGEN FROM RABBIT BLOOD INCUBATED WITH ENDOTOXIN

This paper describes the relations between the endotoxin concentration and leukocyte pyrogen yield when endotoxin is incubated with whole rabbit blood for 24 hr. The results provide strong evidence for the view that leukocyte pyrogen is not modified endotoxin, and also show that the simplified assay method described in the previous paper works reasonably well.     

Correlation between endotoxin level and bacterial count in bronchoalveolar lavage fluid of ventilated patients

OBJECTIVE: To assess the predictive value of the endotoxin level in the bronchoalveolar lavage (BAL) and to propose to the clinician a guide in the diagnosis of gram-negative bacterial (GNB) pneumonia. DESIGN: Retrospective and prospective studies to investigate the relation between endotoxin level and quantitative bacterial culture of BAL and to test the predictive value of a defined threshold. SETTING: University hospital general intensive care unit. PATIENTS: In the first part of the study, 77 consecutive ventilated patients with clinical suspicion of nosocomial pneumonia between January 1995 and January 1996. In the second part of the study, 93 consecutive ventilated patients studied prospectively between February 1996 and April 1997. MEASUREMENTS AND MAIN RESULTS: Quantitative cultures for aerobic bacteria were performed directly from the fluid. Bacterial species were determined with standard techniques. The detection of endotoxin in BAL was made using a quantitative chromogenic Limulus assay. In the retrospective analysis, a significant correlation between quantitative GNB cultures and BAL endotoxin levels was observed (r2 = 0.60, p < .0001). An endotoxin level > or = 4 endotoxin units/mL (EU/mL) distinguishes patients with a significant GNB count from colonized patients with a sensitivity of 92.6%, a specificity of 81.4% and a correct classification rate of 84.9%. In the prospective analysis, the 4 EU/mL threshold permits identification of infected patients with a sensitivity of 82.2%, a specificity of 95.6%, and a correct classification rate of 90.3%. The receiver operating characteristic curve analysis showed that the Limulus assay still had a good discrimination power in the prediction of significant bacterial count in BAL fluid. CONCLUSIONS: Endotoxin detection immediately after bronchoscopy is a distinct advantage to the clinician because antimicrobial gram-negative therapy may be immediately justified according to the results.     

Optimization of detection of bacterial endotoxin in plasma with the Limulus test

Detection and quantification of bacterial endotoxin in plasma by the Limulus amebocyte lysate test (or other assays for endotoxins) is hindered by the presence of inhibitors. Treatment of plasma to overcome inhibitory activities is required before plasma can be successfully assayed for endotoxin. We have conducted an investigation comparing the three most commonly used procedures (dilution-heating, trifluoroacetic acid oxidation, and chloroform extraction) for treatment of plasma before its assay for endotoxin with the chromogenic Limulus test. Initially, conditions were optimized for treatment of plasma by each of these methods. Subsequently, a direct comparison of the three plasma treatment procedures was performed with plasma spiked with known concentrations of endotoxin. The optimized dilution-heating procedure resulted in the most sensitive detection of endotoxin, with sensitivity approximately 10 times greater than the optimized trifluoroacetic acid oxidation procedure and approximately 100 times greater than treatment of plasma by chloroform extraction. Maximal detection of low concentrations of endotoxin by the chromogenic Limulus test was obtained by dilution of plasma fourfold with 0.15 mol/L NaCl followed by heating at 60 degrees C for 30 minutes. This procedure was simple, rapid, and did not involve addition of any reagents to plasma that could potentially add contaminating endotoxin.