Measurement of endotoxin activity in critically ill patients using whole blood neutrophil dependent chemiluminescence

Comprehensive clinical and laboratory data were collected on the day of ICU admission. Survival status was assessed at 30 days, and at ICU and hospital discharge. Admission multiple organ dysfunction score [23] and Acute Physiology and Chronic Health Evaluation II score [24] were calculated. Systemic inflammatory response syndrome (SIRS) was defined in accordance with the criteria of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference [25]. Sepsis was defined as SIRS plus infection. Infection was diagnosed by the presence of microorganisms invading an otherwise sterile body fluid or site. Coagulase-negative staphylococci grown in one of two bottles from a blood culture was considered a contaminant. Blood for endotoxin assay was collected within 12 hours of admission in endotoxin-free tubes, with EDTA as an anticoagulant. Aliquots of platelet-poor plasma were stored at -70°C for subsequent LAL assay in pyrogen-free tubes. Endotoxin assays were compared with cultures drawn within 8 hours of the endotoxin sample.

Hypotension was defined as a systolic blood pressure below 90 mmHg for at least 1 hour, despite adequate filling pressures (pulmonary artery wedge pressure >12 mmHg), or as administration of an intravenous fluid bolus greater than 500 ml or the use of inotropic support (dopamine >5 g/kg per min, or phenylephrine, epinephrine or norepinephrine at any dose to maintain systolic blood pressure >90 mmHg).

The EAA assay is described in detail elsewhere [22]. Briefly, a 2-ml sample of whole blood was drawn through an indwelling arterial line into an endotoxin-free K3EDTA blood collection tube (Vacutainer systems; Becton Dickinson, Franklin Lakes, NJ, USA). Samples were immediately transported to a laboratory adjacent to the ICU for assay. Blood samples were maintained at room temperature and all samples were assayed within 30 min of collection.

To assay levels of endotoxin, a 10 l aliquot of whole blood was placed in each of three tubes containing luminol buffer (300 μl/tube). The control tube contained blood and buffer only, whereas a positive control contained a maximum stimu-latory concentration of endotoxin (2 ng/ml); the final tube contained the test sample. All three tubes were incubated at 37°C for 5 min and assayed in triplicate. Chemiluminescence was initiated by the addition of 20 l/tube human complement opsonized zymosan. Continuous measurements were made of light emissions at 30-s intervals over a total period of 20 min in a reciprocating tube luminometer (Autolumat LB 953; E. G. & G. Berthold, Wildbad, Germany).

Quantitation of endotoxin in whole blood was based on a standard curve of averaged light emission versus endotoxin concentration [22]. For each patient, a normalized response factor was calculated by subtracting the averaged 20-min light integral of the control from the assay tube and the maximally stimulated tube. The response factor was the difference light integral of the test sample divided by the difference integral of the maximally stimulated tube. The endotoxin concentration was interpolated from the dose–response curve of response factor versus endotoxin concentration.

Determination of endotoxin using the LAL technique was conducted with the Associates of Cape Cod LAL assay (Pyrochrome Assay Kit; Associates of Cape Cod, Falmouth, MA, USA), with the sample pretreatment protocol of Tamura [26]. The assay was performed according to the manufacturer's instructions, using a 96-well microplate format (Pyrochrome 96-well plates; Associates of Cape Cod). All dilutions of standards and reagents were performed in glass vessels that had been depyrogenated by heating to 220°C for a minimum of 4 hours. All pipette tips were endotoxin free (Diamed Laboratory Supplies Inc., Mississauga, Ontario, Canada). Solutions were prepared with endotoxin-free water, as verified by LAL assay.

Continuous variables are described as means and standard deviations, unless otherwise specified. Normality was ensured using normal probability plots, and continuous biological variables were compared using Student's t-test. Dichotomous variables were compared using a χ² test or Fisher's exact test, where appropriate. By convention, an α level of P < 0.05 was considered to be statistically significant.

Lipopolysaccharide was added to whole blood of four ICU patients with no detectable endotoxin levels, in graded doses of 100, 400 and 800 pg/ml. The resulting dose–response curve was indistinguishable from one constructed from the blood of nonhospitalized ambulatory normal control individuals [22]. In a parallel series of studies (Fig. 1), patient samples with elevated endotoxin levels were treated with polymyxin B to chelate lipopolysaccharide (300 g/ml blood for 20 min at 37°C) and reassayed using the EAA. This treatment resulted in complete abrogation of the endotoxin-dependent signal in the majority of samples.

Using the EAA, 43 out of 74 patients (58%) had endotoxin levels greater than 50 pg/ml at the time of ICU admission. The association of endotoxin levels with reason for admission is shown in Table 2. Endotoxin levels by EAA were highest in patients with an admission diagnosis of sepsis, and endotoxin levels greater than 50 pg/ml significantly correlated with an admission diagnosis of sepsis (P < 0.01). This association was not evident when blood samples were assayed using the LAL.

Culture-proven infection was present in 19 (26%) patients at the time of ICU admission: eight patients had pneumonia, eight had positive blood cultures, two had positive cultures from an intra-abdominal source, and one had a positive urine culture. Ten patients had culture-proven Gram-negative infections (five pneumonias, two bacteraemias, two intra-abdominal infections and one urinary tract infection). All patients with Gram-negative infections had endotoxin levels greater than 50 pg/ml using the EAA (Tables 3,4,5). As shown in Fig. 2, patients with Gram-negative infection had higher mean endotoxin levels than did those without Gram-negative infection when measured using the EAA (P < 0.05); no difference in endotoxin levels was observed when the same samples were assayed using the whole-blood LAL method.

There were no statistically significant associations between an ICU admission endotoxin level greater than 50 pg/ml and ICU or hospital mortality (Table 6). Patients who had endotoxin levels below 50 pg/ml, as measured using the EAA method, had a trend toward a shorter duration of ICU stay (3.5 ± 6.2 days) than did patients with levels greater than 50 pg/ml (8.1 ± 18.8 days; P = 0.082, by Mann–Whitney U test). Acute Physiology and Chronic Health Evaluation II scores were not significantly different between patients who were endotoxaemic and those who were not.

SIRS, as defined by the presence of two or more criteria [25], was present in 60 out of 74 patients (80%) at the time of admission to the ICU; however, its presence was not associated with endotoxaemia. Mean white blood cell counts for patients with endotoxaemia were significantly higher (15.7 ± 9.1 × 10⁹ cells/l for endotoxaemic patients versus 10.8 ± 6.2 × 10⁹ cells/l for patients without endotoxaemia; P = 0.014). Using the EAA method, patients who were hypotensive on admission had a mean endotoxin level of 440 ± 350 pg/ml, versus 320 ± 330 pg/ml for normotensive patients; however, the difference was not statistically significant (P = 0.18).