Myocardial necrosis in ICU patients with acute non-cardiac disease: a prospective study.

OBJECTIVE: To ascertain if, after an episode of hypotension, unnoticed myocardial necrosis could occur in critical care patients with acute non-cardiac illness and to search for signs of cardiac necrosis. DESIGN: A prospective observational study. SETTING: General intensive care unit (ICU) at a tertiary level hospital. PATIENTS: Thirty-one patients in two groups. Group 1 included 19 patients with severe sepsis/septic shock (ACCP/SCCM Consensus Conference). Group 2 included 12 patients with hypovolemic shock. INTERVENTIONS: Biochemical markers of myocardial necrosis (cardiac troponin I (cTnI), creatine kinase (CK), creatine kinase MB mass (CKMB) and myoglobin) were measured at 12 h (T1), 24 h (T2) and 48 h (T3) after enrollment. A standard 12-lead ECG was recorded upon enrollment (T0) and at T2. Anomalous Q-waves or ST segment depression or elevation was considered diagnostic for acute myocardial infarction (AMI). A hypotensive episode (arterial systolic pressure < 90 mmHg at heart rate > 100 bpm) was considered moderate if it lasted 30-60 min or severe if longer than 60 min. MEASUREMENTS AND RESULTS: At T0 none of the patients had AMI on ECG. At T2 a non-Q AMI developed in five patients. Increased levels of troponin I, myoglobin, CK and CKMB were found in 74.2 %, 96.8 %, 74.2 % and 67.7 % of the patients, respectively. Cardiac troponin I increased in 11 out of 19 septic patients and in all hypovolemic patients. There was a significant difference between the groups (p < 0.05). All biochemical markers increased in relationship to the degree of hypotension with cTnI again showing a significant difference. The longer the hypotensive episode was, the greater was the increase (moderate hypotension: median 1.16; quartiles 0.55-3.44 ng/ml, severe hypotension: median 8.53; quartiles 1.1-20.7 ng/ml; p < 0.05). Abnormal levels of cTnI were more frequent in non-survivors than in survivors (p < 0.05). CONCLUSIONS: Hypotension may cause cardiac damage in critically ill patients with acute non-cardiac diseases as shown by abnormal levels of cTnI. It is likely that a high number of these myocardial necroses may go unnoticed on the ECG.

Accuracy of cholesterol measurements in Italian clinical laboratories. Joint project GISSI prevention--Italian Society of Clinical Biochemistry. SIBioC GISSI Prevenzione Group.

We report the results of an external quality assessment scheme for serum total cholesterol measurement involving about 100 Italian laboratories participating in an epidemiological study of post myocardial infarction. Two frozen human serum pools with Abell-Kendall assigned values are distributed quarterly at the laboratories (up to now seven events occurred); the obtained results are evaluated and discussed. In one exercise (# 5) duplicated measurements were repeated on three different days. Eighty-five to 98% of the laboratories obtained results within the total error limits (+/- 8.9%). But, while precision (calculated on the six replicates of exercise # 5) is good (90% of the laboratories obtained CV < 3%), inaccuracy problems are evident in every event. Indeed the mean bias from the reference method value ranged from 1.54 and 3.49% in the various events.

A step forward in enzymatic measurement of creatinine.

We describe an improved enzymatic ultraviolet absorbance method for assaying creatinine in serum, plasma, and urine. Creatinine is hydrolyzed by creatinine iminohydrolase (EC 3.5.4.21) to ammonia and N-methylhydantoin. The ammonia produced combines with 2-oxoglutarate and NADPH in the presence of glutamate dehydrogenase to yield glutamate and NADP+. The consumption of NADPH, measured by a two-point fixed-time assay, is proportional to the amount of creatinine in the sample. The assay is carried out in two steps: The first step eliminates background absorbance in hyperlipemic samples and endogenous ammonia through a "clearing system" and an isocitrate dehydrogenase-based "ammonia scavenger system"; the second step starts creatinine measurement. The method affords a simple, rapid, and sensitive assay with good precision and extended linearity; it employs working solutions stable at least 4 months. Test results compare closely with those of the isotope dilution-mass spectrometry Definitive Method, the HPLC procedure, and the fuller's earth method. The proposed method is not subject to interference from several metabolites or from the 72 drugs tested. Because it is easily automated, the method is suitable for routine work in clinical laboratories.

Axon clinical chemistry analyzer evaluated according to ECCLS protocol.

We assessed the analytical performance of the Axon system (Bayer Diagnostici), according to the European Committee for Clinical Laboratory Standards guidelines, for assay of 12 analytes: cholesterol, creatinine, glucose, total protein, urea, uric acid, alkaline phosphatase, alpha-amylase, aspartate aminotransferase, creatine kinase, sodium, and potassium. The field evaluation lasted approximately 5 months and involved the collection of approximately 10,000 data points with the Axon. The following results were obtained: The highest CVs for controls and human sera at different concentration/activity values were 2.2% for within-run imprecision (n = 60; 3 days, pooled estimate) and 3.5% for the between-day imprecision (n = 20 days). Close correlation was found with results for patients' specimens assayed with comparative instruments (Hitachi 717 for substrates and enzymes, Beckman Synchron EL/E4A for electrolytes). No drift was observed during 8 h of operation. The linearity range was broad, sometimes exceeding the manufacturer's claims. No sample-, reagent-, or cuvette-related carryover was found. Measurement of control sera gave results within +/- 5% of the assigned values. We conclude that good reliability and practicability make the Axon system suitable for laboratories with various needs.

Multicentre evaluation of the Boehringer Mannheim/Hitachi 717 analysis system.

We conducted an European multicentre trial to assess the performance of the new Boehringer Mannheim/Hitachi 717 analysis system. The photometer response was linear up to an absorbance of 2.8. The maximal CV of photometric imprecision was 0.5% for the wavelength pair 340/405 nm within the absorbance range 0.9 to 2.4. For the 13 analytes in our study, mean within-run imprecision was less than 2%, and mean between-day imprecision less than 2.5%. The results obtained with the Hitachi 717 instrument correlated closely with those of comparison instruments. Linearity for the various tests was high and exceeded the manufacturer's claims. No drift was detected during an 8-hour work period; carry over could not be detected under the chosen experimental conditions. The new instrument was readily accepted by the evaluators because of its ease of handling and simple daily maintenance.

One-step protocol for assays of total and direct bilirubin with stable combined reagents.

We describe an improved colorimetric method for assays of total and direct bilirubin in serum. Bilirubin reacts with diazotized sulfanilic acid in an acidic medium to form a blue azopigment. Total bilirubin is assayed in the presence of reaction accelerators (caffeine, urea, and citric acid), direct bilirubin in their absence. The azo compound so formed is read at the same wavelength (570 nm) in both assays. A sample blank is run in parallel. Standard curves are linear for total and direct bilirubin concentrations up to 513.0 and 256.5 mumol/L, respectively. The method is characterized by (a) use of the same protocol for both assays, i.e., a one-step procedure with short reaction time (5 min at room temperature), and (b) use of a single working solution, which, refrigerated, is stable for one month. The method is reliable, yields results that compare closely with those of the classical Jendrassik--Gróf method, is suitable for routine use, and lends itself to automation.

A multicentre evaluation of the Boehringer Mannheim/Hitachi 704 analysis system.

The selective multitest Boehringer Mannheim/Hitachi 704 analysis system was examined according to the ECCLS guidelines in a multicentre evaluation involving four laboratories. Ten routine parameters, covering most of the application settings of the instrument, were measured in the respective laboratory at temperatures 25, 30 or 37 degrees C. The trial lasted four months and gave more than 40,000 data. It yielded the following results: 1. Within the four laboratories the mean coefficients of variation for three control sera at different concentrations were found to be equal to or better than 1.6% for the within-run imprecision and 2.8% or better for the between-day imprecision. 2. No drift was observed during eight hours. 3. Because of the high linear measuring range a re-run analysis was seldom necessary. 4. Sample-related carry-over was not seen. Reagent-dependent carry-over was measured from cholesterol to uric acid and from triacylglycerols to lipase. Through modification of the cholesterol and triacylglycerol reagents, the carry-over effect was practically eliminated. 5. The recovery of the assigned values of control sera showed average values between 99 and 104%. For bilirubin, creatinine, creatine kinase and alanine aminotransferase some control sera showed deviations greater than 10%. 6. In all cases, regression analysis of the results obtained in comparisons of the present instrument with the Hitachi 705 or 737 yielded slopes close to unity with extreme values of 0.95 and 1.06. 7. During the entire evaluation period there was no malfunction or breakdown of the instruments. The evaluators came to the conclusion that the analytical performance as well as the reliability and practicability of the Hitachi 704 can be rated as excellent.

Assessment of an automatic liquid chromatograph for foetal and abnormal haemoglobins.

The DIAMAT TM (Bio-Rad) analyser is a microprocessor-operated HPLC system using a silica-based weak cation exchange column with three phosphate buffers of increasing ionic strength as the step gradient mobile phase. A dual wavelength detector measures absorbance at 415 and 690 nm; each sample is completely processed in 8 minutes. The instrument effectively separates and quantifies HbF in a discrete peak. We have verified that the HbF assay is linear up to about 65% values. We calculated within-run imprecision for n = 20 in 3 different haemolysed blood samples; the results are shown below as percent of total haemoglobin: A) Mean = 1.06 SD = 0.048 CV% = 4.5 B) Mean = 1.90 SD = 0.041 CV% = 2.1 C) Mean = 8.93 SD = 0.047 CV% = 0.5 Between-run imprecision for similar samples (n = 18) was: D) Mean = 3.74 SD = 0.20 CV% = 5.5 E) Mean = 9.94 SD = 0.15 CV% = 1.5 Accuracy was assessed in different series by correlating Hb values (y) with those obtained by the alkali denaturation test (x). The regression line equation was y = 1.03 x - 0.33 (r = 0.999, n = 62). The DIAMAT TM instrument also reveals the presence of any HbS and calculates its peak area correctly, as we found by electrophoretic reassay in heterozygous subjects. We also noted the presence of other abnormal haemoglobins characterized by specific retention times.

Enzymic ethanol assay: a new colorimetric method based on measurement of hydrogen peroxide.

We describe a new colorimetric method for measuring ethanol in plasma by use of a peroxidase-coupled assay system and alcohol oxidase (EC 1.1.3.13) from Pichia species. Absorptivity is low enough to give useful results without sample dilution. The procedure is also applicable to saliva samples and utilizes only one working reagent. The absorbance of the blue dye that is formed is measured at 600 nm. The standard curve for the method is linear for ethanol concentrations up to 4 g/L. Average analytical recovery of ethanol in human plasma exceeded 99%. Within-run and between-run CVs were less than 2.45% and less than 1.92%, respectively. Results correlate very well with those by gas chromatography (r = 0.9977). The method is adaptable to automation.

A kinetic colorimetric assay of gamma-glutamyltransferase.

We have explored a kinetic colorimetric method for measuring gamma-glutamyltransferase (EC 2.3.2.2) activity in serum, using L-gamma-glutamyl-3,5-dibromo-4-hydroxyanilide and glycylglycine as donor and acceptor substrates. The released product, 3,5-dibromo-4-hydroxyaniline, reacts with 2,5-dimethylphenol to produce a blue quinone monoimine in the presence of ascorbate oxidase (EC 1.10.3.3). This dye has peak absorption at 610 nm, whereas the donor substrate shows negligible absorption throughout the visible spectrum. The reaction can be run with all the reagents in a single working solution with serum as starter, or with the substrate solution as starting reagent. The sample/reagent volume ratio is 1:24. Adaptation of the method to several automated instruments gave good precision in all cases. Comparison with a method in which L-gamma-glutamyl-3-carboxy-4-nitroanilide is the donor substrate showed good correlation of results (r greater than or equal to 0.987). The dynamic range of the method exceeds the upper limits of the reference intervals for men (9-33 U/L) and women (8-25 U/L) by at least 18-fold.

Enzymic creatinine assay: a new colorimetric method based on hydrogen peroxide measurement.

We describe a new colorimetric method for measuring creatinine in serum and urine. Creatinine hydrolysis is catalyzed by creatinine amidohydrolase, and the creatine so produced is assayed in reactions catalyzed sequentially by creatine amidinohydrolase and sarcosine oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is measured at 510 nm in a reaction catalyzed by horseradish peroxidase, with 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone as the chromogen. This series of reactions is complete in 30 min at room temperature. A blank sample measurement corrects for endogenous creatine. The standard curve is linear for creatinine concentrations as great as 2.21 mmol/L. Analytical recovery of creatinine in human sera and urine averaged 99.8%. Within-run and between-run precision studies gave CVs of less than or equal to 3.3 and less than or equal to 4.3% for a serum with a creatinine concentration of 69 mumol/L. Results by this method agree well (r greater than 0.99) with those by both the enzymic ultraviolet method of Wahlefeld and the fuller's earth/Jaffé method.

Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide.

In this direct colorimetric procedure, serum triglycerides are hydrolyzed by lipase, and the released glycerol is assayed in a reaction catalyzed by glycerol kinase and L-alpha-glycerol-phosphate oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is monitored in the presence of horseradish peroxidase with 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone as the chromogenic system. The high absorbance of this chromogen system at 510 nm affords useful results with a sample/reagent volume ratio as low as 1:150, and a blank sample measurement is not needed. A single, stable working reagent is used; the reaction is complete in 15 min at room temperature. The standard curve is linear for triglyceride concentrations as great as 13.6 mmol/L. Average analytical recovery of triglycerides in human sera is 100.1%, and within-run and between-run precision studies showed CVs of less than or equal to 1.6 and less than or equal to 3.0%, respectively. The method is suitable for automation.

Enzymic assay of creatinine in serum and urine with creatinine iminohydrolase and glutamate dehydrogenase.

We describe an assay for creatinine in which it is converted by creatinine iminohydrolase (EC 3.5.4.21) into ammonia and N-methylhydantoin. The ammonia is subsequently assayed by use of alpha-ketoglutarate and glutamate dehydrogenase (EC 1.4.1.3). Use of NADPH as coenzyme eliminates all interferences from endogenous reactions. Endogenous ammonia in the sample is eliminated during a preincubation. The reaction reaches the endpoint in 15 min at working temperatures of 20-37 degrees C. No sample blank or reagent blank is needed. The standard curve is linear at least to 884 mumol (100 mg) of creatinine per liter. Average analytical recovery of creatinine in serum and urine is 99%. Within-run and between-run CVs are less than or equal to 2% and less than or equal to 6% for creatinine values of 335 mumol/L (38 mg/L) and 80 mumol/L (0 mg/L), respectively. Results by the described method (y) compare well with those by Jaffé's kinetic test (y = 1.01x -- 12.8), Berthelot/AutoAnalyzer method after treatment with immobilized creatinine iminohydrolase (y = 0.987x -- 13.2), Jaffé's test run on the SMA 12/60 (y = 1.011x -- 5.8), the Wahlefeld method (y = 1.014x -- 0.88), and Jaffé's test after deproteinization and absorption on fuller's earth (y = 0.985x -- 3.08). The method may be suitable for discrete, including centrifugal, automation.

Use of 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine.

A new direct colorimetric procedure for uric acid assay in serum or urine is described, utilizing a 3,5-dichloro-2-hydroxybenzene sulfonic acid/4-aminophenazone chromogenic system in the presence of horseradish peroxidase and uricase from Aspergillus flavus. This chromogen system has a high absorptivity, affording useful results with sample/reagent volume ratios as low as 0.025. The procedure is applicable to serum, plasma, or diluted urine. A single working reagent is used; the reaction is complete in less than 15 min at room temperature. The red dye formed is measured at 520 nm; a blank sample measurement is not needed. The standard curve for the method is linear for uric acid concentrations up to 1500 mumol/L. Average analytical recovery of uric acid in human sera and urine exceeded 99%; within-run and between-run precision studies showed CV's of less than or equal to 1.2 and less than or equal to 2.2%, respectively. The new procedure correlated well with the uricase/catalase and uricase/ultraviolet methods. The method is suitable for automation.