1052 Technical Briefs

Transcription

1 1052 Technical Briefs analyzed in a single run for HDL-C using these three homogeneous HDL-C reagents. Mean concentrations of HDL-C in group 1 were 508 mg/l for the direct HDL-C assay, 517 mg/l for the N-geneous TM HDL-C assay, and 531 mg/l for the EZ-HDL TM -C assay. However, mean concentrations of HDL-C in group 2 were 253 mg/l for the direct HDL-C assay, 352 mg/l for the N-geneous TM HDL-C assay, and 253 mg/l for the EZ-HDL TM -C assay. Marked differences were noted between assays in individual patients. Sugiuchi et al. (6) and Harris et al. (12) reported precisions slightly better (CV 4%) than the results we obtained for direct HDL-C assay and N-geneous TM HDL assay. This may be due to use of different analyzers or differences in the methods of calibration. The data in our study agree with Okamoto et al. (7), who reported that higher HDL-C concentrations were obtained by the direct method than by the precipitation method. Our finding of no statistically significant interference from bilirubin in any of the three homogeneous HDL-C assays agrees with the findings of Sugiuchi et al. (6) and Harris et al. (12).We found that hemoglobin produced a negative interference ( 10%) with the N-geneous TM HDL-C assay but had little effect on direct HDL-C assay and EZ-HDL TM -C assay. Nauck et al. (5) reported that hemoglobin produced a positive interference ( 10%) and bilirubin produced a negative interference ( 10%) with a different homogeneous HDL-C assay, which uses PEG and antibodies against apo B and apo C-III (5). We found that there was a negative interference ( 10%) by Intralipid at triglyceride concentrations mg/l for the N-geneous TM HDL-C and EZ-HDL TM -C assays but not for the direct HDL-C assay. However, we found inconsistent HDL-C bias among high triglyceride concentration serum samples, suggesting that interference measurements using Intralipid may not truly represent the clinical situation. The negative bias of HDL-C concentration observed in serum of high triglyceride concentration ( mg/l to mg/l) using EZ-HDL TM -C assay was much more apparent than with the other two homogeneous HDL-C assays. Harris et al. (12) reported a positive bias for N-geneous TM assay with high triglyceride concentration (4000 mg/l to mg/l) serum samples. We are unable to explain this discrepancy. We conclude that these three homogeneous HDL-C assays are acceptable (total error 22%) for clinical laboratory use according to NCEP performance goals. These assays can shorten turnaround time and save labor costs. Overall, the direct HDL-C assay demonstrated slightly less interference from bilirubin, hemoglobin, and lipemia than the N-geneous TM and EZ-HDL TM -C assays. For accurate determination of HDL-C results in samples with lipemia and/or containing high concentrations of triglyceride, analysis by the ultracentrifugation reference method is indicated. Boehringer Mannheim Corp. provided the direct HDLcholesterol reagent kit and funded the ultracentrifugation HDL-C testing; Genzyme Diagnostics provided the N- geneous TM HDL cholesterol reagent kit; and Sigma Diagnostics provided the EZ-HDL TM cholesterol reagent kit. The Medical Editing Department, Kaiser Foundation Research Institute, provided editorial assistance. References 1. Gordon T, Castelli WP, Hjortland MC, Kannel WB, Dawber TR. High density lipoprotein as a protective factor against coronary heart disease: the Framingham Study. Am J Med 1977;62: Summary of the second report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II). JAMA 1993;269: Musto JD, Lawlor JF. HDL-cholesterol: online separation and analysis utilizing an automated chemistry analyzer [Abstract]. Clin Chem 1993;39: Kakuyama T, Kimura S, Hashiguchi Y. Fully automated determination of HDL-cholesterol from human serum with Hitachi 911 [Abstract]. Clin Chem 1994;40: Nauck M, März W, Haas B, Wieland H. Homogeneous assay for direct determination of high-density lipoprotein cholesterol evaluated. Clin Chem 1996;42: Sugiuchi H, Uji Y, Okabe H, Irie T, Uekama K, Kayahara N, et al. Direct measurement of high-density lipoprotein cholesterol in serum with polyethylene glycol-modified enzymes and sulfated -cyclodextrin. Clin Chem 1995; 41: Okamoto Y, Tanaka S, Nakano H. Direct measurement of HDL cholesterol preferable to precipitation method [Letter]. Clin Chem 1995;41(12 Pt 1): Okazaki M, Sasamoto K, Muramatsu T, Hosaki S. Evaluation of precipitation and direct methods for HDL-cholesterol assay by HPLC. Clin Chem 1997; 43: NCCLS. Tentative guideline EP5 T2. Evaluation of precision performance of clinical chemistry devices, 2nd ed. Villanova, PA: National Committee for Clinical Laboratory Standards, March, 1992: Warnick GR, Wood PD. National Cholesterol Education Program recommendations for measurement of high-density lipoprotein cholesterol: executive summary. The National Cholesterol Education Program Working Group on Lipoprotein Measurement. Clin Chem 1995;41: Glick MR, Ryder KW, Glick SJ. Interferographs: user s guide to interferences in clinical chemistry instruments, 2nd ed. Indianapolis, IN: Science Enterprises, 1991: Harris N, Galpchian V, Thomas J, Iannotti E, Law T, Rifai N. Three generations of high-density lipoprotein cholesterol assays compared with ultracentrifugation/dextran sulfate-mg 2 method. Clin Chem 1997; 43: Effects of Anticoagulants in Amino Acid Analysis: Comparisons of Heparin, EDTA, and Sodium Citrate in Vacutainer Tubes for Plasma Preparation, Chih-Kuang Chuang, 1 Shuan-Pei Lin, 1,2* Yu-Ting Lin, 1 and Fu-Yuan Huang 2 (Division of Genetics and Metabolism, Departments of 1 Medical Research and 2 Pediatrics, Mackay Memorial Hospital, No. 92, Chung-San N. Rd., Sec. 2, Taipei 10449, Taiwan; *address for correspondence: Departments of Medical Research and Pediatrics, Mackay Memorial Hospital, No. 92, Sec. 2, Chung-San N. Rd., Taipei 10449, Taiwan; fax , zsplin@ ms2.mmh.org.tw) Measurements of the concentrations of free amino acids in physiological fluids such as blood, cerebrospinal fluid (CSF), and urine are used mainly as biochemical indicators of inborn errors of metabolism (aminoacidopathies), nutritional status, and as monitors of therapy (1). Amino acids can be measured by thin-layer chromatography, electrophoresis, mass spectrometry, and ion-exchange

2 Clinical Chemistry 44, No. 5, chromatography (2). The last method has many advantages: it provides good resolution, is very accurate, can be automated, and is easy to perform. The long analytic time is the only drawback in this method. The steps in plasma free amino acid analysis, including sample pretreatment, deproteinization, and instrument operation, are easily performed. However, the use of different anticoagulants for plasma sample preparation can increase the potential for analytic errors (2, 3). Currently, heparin and EDTA are widely used as anticoagulants, but sodium citrate-treated plasma and serum samples are occasionally used. We investigated the analytic variation among samples treated with these anticoagulants. We used a System 6300 high performance amino acid analyzer (Beckman Instruments), which uses a single-column, ion-exchange method that uses four lithium buffers (Li-ADEF method), a ninhydrin reagent, and a 12-cm lithium high performance column. Four different types of Vacutainer Tubes were purchased from Becton Dickinson, including a plasma tube with heparin (sodium heparin), a whole blood tube with liquid EDTA (K 3, 15%), a whole blood tube with mol/l buffered sodium citrate (3.8% sodium citrate), and a serum separation tube. Centrifuge filtration was performed to eliminate the residual protein in sulfosalicylic acid dihydrate (SSA) supernatants. The microspin filter units (0.2 m) were from Lida Manufacturing. All lithium buffers, ninhydrin reagents, and calibrators were purchased commercially from Beckman Instruments. Four lithium buffers with various phs were used in this experiment: Li-A buffer (ph 3.0), Li-D buffer (ph 3.0), Li-E buffer (ph 3.7), and Li-F buffer (ph 4.0). The ninhydrin reagent was prepared by mixing 18.0 g of ninhydrin and 0.7 g of hydrindantin with a reagent containing (per liter) 767 ml of dimethyl sulfoxide, 225 ml of water, 7.0 g of lithium acetate, and 1 ml of concentrated acetic acid; the reagent was kept at room temperature for 4 h. Calibrators and quality control materials were made by mixing 400 L of STD Calibration Standard, 400 L of AN Calibration Mixture, and 400 L ofb Calibration Mixture (Beckman Instruments) with 400 L of 25.0 mmol/l glutamine asparagine, 400 L of 5.0 mmol/l tryptophan (Trp), and 400 L of 5.0 mmol/l d-glucosaminic acid (an internal standard) (all from Sigma Chemical) in a 10-mL volumetric flask and bringing the volume to 10 ml with Li-S sample dilution buffer. To deproteinate the plasma, we used an aqueous solution of 35 g of SSA (Sigma Chemical) in a total volume of 100 ml. To help separate and identify the amino acids, the plasma samples treated with SSA had the same ph as the amino acid calibration mixture used to calibrate the amino acid analyzer (2). Blood samples were taken from 43 healthy adults (15 men and 28 women; years of age) after a minimum of 12 h fasting. All subjects gave signed informed consent. The results of all biochemistry tests, such as aspartate aminotransferase, alanine aminotransferase, total protein, and albumin, were within the reference intervals in these 43 adults. No patients with aminoacidopathies were included in this study. Blood samples were drawn in 4.5- or 7-mL heparin, EDTA, sodium citrate, and serum separation Vacutainer Tubes. The plasma was separated from the blood cells immediately after the blood was drawn by centrifugation at 2000g for 15 min. A plasma sample of 200 L was placed in a 0.6-mL centrifuge tube, and 20 L of SSA solution (350 g/l) was then added. The tube was capped, agitated for a few seconds, allowed to stand for 5 min at room temperature, and then centrifuged at g for 5 min. The SSA supernatant was transferred to a microspin filter reservoir (0.2 m), capped, and centrifuged at 1800g for 30 min. The centrifuged aliquot was diluted with an equivalent volume of Li-S sample dilution buffer containing mol/l of d-glucosaminic acid, which was used for quality control. Finally, the prepared sample (50 L) was automatically injected into the analyzer for free amino acid analysis. We used the Li-ADEF method (Beckman) with care to keep the flow rate (20 ml/h), temperatures (33.0 C, 65.5 C, and 73.0 C), and times of buffer transitions constant to avoid fluctuations in the retention times of the amino acids. The mean amino acid concentrations ( SD) for adults varied among sample types (Table 1). The concentrations of most free amino acids were higher in serum than in plasma. The sodium citrate-treated samples yielded the lowest values of most free amino acids. Results from both of these sample groups differed significantly from the heparin-treated control group, based on paired t-tests (t-values: serum, 2.88; sodium citrate, 2.38; P 0.005). No statistically significant differences were seen between EDTA-treated samples and the heparin-treated controls. The CVs for most free amino acids of the four sample types (n 172) were between 12% and 42%, except for the following amino acids: phosphoethanolamine (PTEN), aspartic acid (Asp), hydroxyproline (Hyp), citrulline (Cit), -alanine, ethanolamine, and Trp. The high CV found in the above amino acid quantitations came mainly from anticoagulant effects, which might have produced relatively poor resolution and incongruous retention times. Serum samples gave an average increase of 27.8% (range, 10 54%) in most free amino acids, except for taurine, Asp, Hyp, and Cit, for which the degrees of variation were all 100%. Remarkably, we found the same cystine (Cys) concentration in serum as in the heparin-treated control. This was not true of either the EDTA- or sodium citrate-treated samples. Sodium citrate-treated plasma had an average decrease of 21.3% (range, 8 40%) in most free amino acids, except for the dicarboxylic amino acids, Asp and glutamic acid, which were increased 38% and 23%, respectively. The concentration of Cys was about 10% of that of the control. EDTA-treated samples showed few variations from the control, except for PTEN, Cit, Cys, and Trp. The PTEN concentration was 20 times higher than in any of the other samples. The concentration of Cit was also markedly increased in EDTA-treated samples, and the variation was

3 1054 Technical Briefs too large to be accurately determined. A very low concentration of Cys was also observed in EDTA, about 3% of the concentration of the control group. This was very close to the concentration measured in the sodium citrate-treated samples. In the free amino acid analysis, the most variable retention time was the interval between Asp and serine, ranging from ( 2.5) min to ( 1.0) min. When serum or sodium citrate-treated plasma was compared with control heparin-treated plasma, a delayed retention time of, on average, min was observed between Hyp and threonine, respectively. Nevertheless, there was no substantial difference when EDTA-treated plasma was used (Fig. 1). The average within-run imprecision (CV) under these four conditions was 2.0% (range, %) for all free amino acid (n 16) multiple measurements. Although serum is the most commonly used sample in many biochemical tests, plasma is preferred over serum for amino acid analysis. When blood clots, amino acids may be released from or metabolized by erythrocytes, leukocytes, or platelets and are then dispersed into the Table 1. Normal values for plasma free amino acids ( mol/l) for four anticoagulants. Heparin EDTA Sodium citrate Serum Amino acid Mean Range a Mean Range a Mean Range a Mean Range a Phosphoserine Taurine Phosphoethanolamine Aspartic acid Hydroxyproline Threonine Serine Asparagine Glutamic acid Glutamine Sarcosine Aminoadipic acid Proline Glycine Alanine Citrulline Amino- -butyric acid Valine Cystine Methionine Cystathionine Isoleucine Leucine Tyrosine Phenylalanine Alanine Aminoisobutyric acid Homocystine Aminobutyric acid Ethanolamine Tryptophan Hydroxylysine Ornithine Lysine Methylhistidine Histidine Methylhistidine Anserine Carnosine Arginine n 43 healthy adults, 15 men and 28 women; ages y. a Range, mean SD.

4 Clinical Chemistry 44, No. 5, serum. Thus, serum has relatively higher and more variable concentrations of most free amino acids than does plasma (1 2, 4 5). According to our investigation, taurine, Hyp, Asp, Cit, and arginine showed a wide fluctuation of concentration in serum. In addition, the concentrations of glutamine and asparagine were slightly increased. This latter finding did not match the results of Scriver et al. (1), which showed decreased concentrations of glutamine and asparagine. The results from EDTA-treated samples were fairly comparable with the results from heparin-treated samples. However, we found that caution must be exercised in evaluating results from these samples. EDTA can react with the ninhydrin reagent and produce a ninhydrinpositive contaminant that is eluted from the column at min. This contaminant might be added to the concentration of any amino acid with the same retention time by mistake, giving a falsely increased value. We found a large increase in the quantity of PTEN in EDTAtreated plasma, about a 20-fold increase over the other sample groups. The retention time appeared to have a 0.4-min delay on average. This possible source of error should be kept in mind if EDTA-treated samples are used. Heparin contains sodium metabisulfite as a preservative. This interferes with the quantitation of sulfur-containing amino acids, such as cystine and homocystine, by adding sulfocysteine to the sample (6). In our study, the cystine measured in heparin-treated samples was much higher than in sodium citrate- and EDTA-treated samples. No substantial difference in cystine concentrations was Fig. 1. Retention time evaluations comparing serum, sodium citrate-, and EDTA-treated plasma with heparin-treated plasma as a control. (A) A delayed retention time was found between threonine and serine in the serum and sodium citrate-treated groups. (B) No substantial differences were found between EDTA and heparin-control groups.

5 1056 Technical Briefs observed between serum and heparin-treated plasma. Thus, sodium citrate- and EDTA-treated samples may yield more accurate values for cystine. In summary, the anticoagulant used in sample preparation may markedly affect the results of amino acid analysis by automated ion exchange chromatography. Sample preparation should be standardized, and clinicians should be aware of the possible sources of error. We recommend the use of either heparin or EDTA exclusively, with careful attention to potential inaccuracies in cystine concentrations with the former and PTEN with the latter. We greatly appreciate the Department of Medical Research, Mackay Memorial Hospital, which provided the necessary instrumentation and financial assistance to complete this project. This project was partly supported by Research Grant No. 8519, Mackay Memorial Hospital, Taipei, Taiwan. References 1. Scriver CR, Clow CL, Lamm P. Plasma amino acids: screening, quantitation, and interpretation. Am J Clin Nutr 1971;24: Slocum RH, Cummings JG. Amino acid analysis of physiological samples. In: Hommes FA, ed. Techniques in diagnostic human biochemical genetics: a laboratory manual. Somerset, NJ: Wiley-Liss Inc., 1991: Parvy PR, Bardet JI, Kamoun PP. EDTA in Vacutainer Tubes can interfere with plasma amino acid analysis. Clin Chem 1983;29: Armstrong MD, Stave U. A study of plasma free amino acid levels. I. Study of factors affecting validity of amino acid analysis. Metabolism 1973;22: DeWolfe MS, Baskurt S, Cochrane WA. Automatic amino acid analysis of blood serum and plasma. Clin Biochem 1967;1: Parvy PR, Bardet JI, Babier D, Bonnefont JP, Kamoun P. A new pitfall in plasma amino acid analysis. Clin Chem 1989;35:178. Plasma S-100b Protein Concentration in Healthy Adults Is Age- and Sex-Independent, Martin Wiesmann, 1* Ulrich Missler, 1 Daniela Gottmann, 1 and Svante Gehring 2 ( 1 Neuroradiology, Klinikum Grosshadern, Ludwig Maximilian University, Munich, Germany, and 2 Immunology and Transfusion Medicine, University of Luebeck, School of Medicine, 2400 Luebeck, Germany; *address for correspondence: Abteilung fuer Neuroradiologie, Klinikum Grosshadern, Ludwig-Maximilians-Universitaet, Marchioninistr. 15, Muenchen, Germany; fax 0049 (89) , wiesmann@ikra.med.uni-muenchen.de) S-100 protein (S-100) is a calcium-binding protein found predominantly in the cytosol of glial cells in all parts of the central nervous system (CNS). Three different subtypes, designated S-100a, S-100b, and S-100a0, are known. S-100b predominates in the brain. The concentration of S-100 can be measured in cerebrospinal fluid (CSF) and in blood. For methodological reasons, most studies reported in the literature measured S-100 concentrations in CSF, and an increased concentration of S-100 in the CSF has been found to be a sensitive although nonspecific indicator of nervous system damage in patients with various neurological disorders (1). Increased concentrations of S-100 have also been found in the blood of patients suffering from CNS tumors or cerebrovascular insults, and maximal concentrations of S-100 in the blood are correlated with the infarct volume after acute ischemic stroke (1 6). It has recently been reported that concentrations of S-100 in blood may be of prognostic value in patients with minor head injury (7) and may indicate acute exacerbation of multiple sclerosis (8). Van Engelen et al. (9) reported that S-100 concentrations in CSF increase with age. Nygaard et al. (10) confirmed this and also found a difference in the mean concentration of S-100 in the CSF of male and female subjects. For routine clinical use, these findings would necessitate ageand sex-corrected reference intervals. We have described a method for determining the concentration of S-100 in blood that is sensitive enough to detect the concentration of S-100 in the plasma of healthy subjects (1). We believe that this method could be useful clinically, but the dependency on age or sex of S-100 concentrations in blood has not been studied yet. We therefore conducted a study to determine whether S-100 concentrations in blood change with age or depend on the individual s sex. Blood samples were obtained from 200 healthy blood donors between 18 and 65 years of age who had no history of previous neurological deficit or any other serious disorder. Subjects were receiving no medications, and the results of physical examination and routine laboratory tests were normal. The study was in accordance with the current revision of the Helsinki Declaration of 1975, and all subjects gave informed consent to the procedure. Subjects were grouped according to age: years, years, years, years, and years. Each group was composed of 40 individuals, 20 men and 20 women. Blood was collected in heparin-containing tubes, centrifuged within 12 h of collection, and stored at 70 C until analysis. S-100 concentrations were determined with an immunofluorometric sandwich assay as described earlier (1). In brief, all measurements were set up in duplicate. Microtiter plates coated with 10 L of anti-s-100 -chain (Sigma Chemical) in 20 ml of phosphate buffer (0.05 mol/l, ph 8.6) were incubated with 200 L per well of S-100 calibrators, controls, or samples for 120 min. Biotin-labeled rabbit anti-s-100 antibody (DAKO) in a Tris (0.05 mol/l), NaCl (0.15 mol/l), CaCl 2 (10 mmol/l), NaN 3 (0.15 mmol/l) buffer was added, and the plates were incubated for another hour. After the plates had been washed, 200 L of streptavidin-europium in assay buffer (0.05 mol/l Tris; 0.15 mol/l NaCl; 1 g/l bovine serum albumin; 0.5 g/l bovine -globulin, both from Sigma; and 0.15 mmol/l NaN 3 ) was added to each well, and the plates were incubated for 30 min. As a last step, 200 L of enhancement solution (0.01 mol/l acetic acid, 38 mg/l tri-n-octylphosphine oxide, 1.3 g/l potassium phthalate,