Multicenter Analytical Evaluation of an Automated Immunoassay for Total Plasma Homocysteine
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1 Annals o f Clinical & Laboratory Science, vol. 30, no. 2, Multicenter Analytical Evaluation of an Automated Immunoassay for Total Plasma Homocysteine Alan H.B. W u,1 Verena H oltm an,1 Fred S. A p p let Vincent Ricchiuti,2 Patricia M. DiBello,3 and Donald Jacobsen3 1 Department of Pathology and Laboratory Medicine, Hartford Hospital, Hartford, Connecticut 2 Department of Laboratory Medicine and Pathology, Hennepin County Medical Center, Minneapolis, Minnesota 3 Cleveland Clinic Foundation, Cleveland, Ohio Abstract. A fully automated immunoassay for total plasma homocysteine assay was evaluated at four centers. To measure total homocysteine, oxidized forms of homocysteine in serum and plasma were reduced by dithiothreitol and assayed by a competitive fluorescence polarization technique. The assay had within-run precision from 0.9 to 3.0% and total precision from 2.8 to 4.1% for control materials with homocysteine concentrations of approximately 7, 12.5, and 25 JLmol/L, a sensitivity of 0.35 lmol/l, good parallelism upon dilution, and analytical recovery ranging from 97.4 to 103.8%. The immunoassay correlated with four different HPLC assays for homocysteine, yielding a slope of 0.98, an intercept of jlmol/l, and a correlation coefficient of for 440 paired samples. The reference range, determined with plasma samples from 609 males and 600 females, yielded a mean of 9.17i2.86 (Omol/L, with a central 95% range of (Xmol/L. The immunoassay is a suitable alternative to HPLC and may be useful in screening persons with high risk of coronary artery disease. Keywords: Plasma homocysteine, fluorescence polarization immunoassay, cardiovascular disease Introduction Hom ocysteine is a dem ethylated product of methionine metabolism and circulates in the plasma in reduced and oxidized forms; the oxidized forms include the disulfide, homocystine, and mixed disulfides containing homocysteine, cysteine, and protein. Total homocysteine (thcy) is a strong independent risk factor for cardiovascular disease [1,2]. Thus, elevated total plasma homocysteine is associated with atherosclerosis [3], stroke [4], peripheral vascular disease [5], venous thrombosis [6], and end-stage renal disease [7]. The causes of hyperhomocysteinemia in subjects with cardiovascular disease are poorly understood and multifactorial, involving genetic and acquired components [8-11]. Address correspondence to Alan H.B. Wu, Ph.D., Hartford Hospital, 80 Seymour St., Hartford, CT 06102; tel ; fax ; awu@harthosp.org. During the past several years, various assays have been developed to measure thcy [12]. These are based on, but not limited to, gas chromatography/mass spectrom etry [13], high pressure liquid chrom atography (HPLC) with electrochemical detection [14], and HPLC with fluorescence detection [15-17]. Although there is generally good agreement among the assays, most are labor intensive and have relatively low throughput. Recently, immunoassays for thcy were introduced [18,19]. In the present study, a fully automated commercial immunoassay with relatively high throughput, using the Abbott IMx analyzer, has been evaluated in four different laboratories by comparisons with established HPLC methods for thcy. Materials and M ethods This study was performed at four laboratories: Hennepin County Medical Center, Minneapolis, MN; /00/ $1.50; 2000 by the Association of Clinical Scientists, Inc.
2 186 Annals o f Clinical dr Laboratory Science Cleveland Clinic Foundation, Cleveland, OH; Oregon Regional Primate Research Center, Beaverton, OR; and Hartford Hospital, Hartford, CT. Samples provided by these sites were serum or plasma (EDTA or lithium heparin), which represented fresh surplus samples that remained after routine analyses or frozen samples that remained from previous analyses. Enrollment of subjects followed protocols that were approved by the Institutional Review Board at each institution. Criteria for enrollment for the normal range study varied from site to site. Age and gender were recorded for >99% of the patients (age information was missing for four samples); the type of collection tube was also recorded. One site excluded volunteers who had a history of coronary artery disease, and one site included outpatients without evidence of disease and with a normal chemistry profile (including lipids and lipoprotein) that did not include specific screening tests for cardiac disease. The two remaining sites used samples from general hospital populations (outpatients and inpatients) which had homocysteine results in the normal range, based on HPLC analyses. For correlation studies, elevated samples were obtained from high risk patients, patients undergoing methionine load testing, and samples known to be elevated from previous HPLC testing. Plasma samples were processed within 20 min of collection. Serum samples were collected in SST tubes and centrifuged within 1 hr. All samples were stored frozen (< -20 C) until assay by the Abbott IMx technique. Samples were tested within 3 mo of storage. For correlation studies, samples were frozen between HPLC and IMx analyses. For the reference range, study site #1 collected 225 serum, 62 EDTA plasma, and 2 lithium heparin plasma samples; site #2 collected 320 EDTA samples; site #3 collected 2 serum and 298 EDTA samples; and site #4 collected 300 serum samples. For the correlation study, site #1 collected 48 serum, 5 EDTA, and 65 heparin samples; site #2 collected 80 EDTA samples; site #3 collected 176 EDTA samples; and site #4 collected 66 serum samples. All sites were provided with IMx homocysteine reagents, calibrators, and controls (Abbott Laboratories, Abbott Park, IL) and the samples for precision studies. The sites provided the samples for correlation with HPLC assays, the normal range study, and the dilution recovery. IM x method. The IMx homocysteine assay is a quantitative, competitive fluorescence polarization im m unoassay (FPIA) which measures total homocysteine (free, bound, oxidized, reduced) in human serum or plasma (EDTA or heparin). The assay method has been described previously [18,20] and is based on the enzymatic conversion of homocysteine to S-adenosyl-homocysteine. The assay utilizes six S- adenosyl-l-homocysteine calibrators (0,2.5, 5.0,10.0, 20.0, and 50.0 Jmol/L) prepared in a buffered solution. The calibration curve is stored and may be used for a minimum of two weeks. Three assay controls (7.0, 12.5, and 25.0 (Xmol/L) are composed of L- homocystine added to a serum-based matrix. Precision. Assay precision was assessed at each site over five days and at two sites over twenty days, according to a modified NCCLS protocol, using the three serumbased controls. Additionally, precision of the automated dilution protocol (1:10) was compared to a manual 1:10 dilution using two L-homocysteinespiked serum samples and the modified five-day NCCLS protocol. Analytical sensitivity. Analytical sensitivity was determined by testing ten replicates of IMx calibrator A (0.0 lmol/l) followed by calibrator B (2.5 fimol/l) in replicates of two. The sensitivity of the IMx homocysteine assay was calculated by determining the concentration (jimol/l) corresponding to two standard deviations from the mean of the rate of calibrator A. Dilution linearity. Five serum samples with elevated total homocysteine concentrations were serially diluted and assayed in duplicate at each site. The sites were requested to analyze two samples with thcy value between 50 and 100 j_lmol/l and three samples between 20 and 50 imol/l. Linear regression analysis of the observed homocysteine concentration was performed as a function of the expected concentration. Statistical analyses. Data were analyzed using SAS computer software, version 6.09, on a Unix operating system. Linear regression was performed using proc reg, which is a least-squares linear regression analysis. The statistical method for establishing the reference range was proc capability, which uses the 2.5 and
3 Evaluation o f the IM x assay for plasma homocysteine percentiles of each sample category to represent the category s expected 95% range of values. borohydride for reduction and HPLC separation with electrochemical detection [14]. Reference range. Samples from approximately 150 males and 150 females were assayed at each site to determine the reference range for thcy using the IMx assay. Samples were collected in serum, lithium heparin, or EDTA plasma tubes. Specimen history forms were completed to record the specimen collection tubes, plus each participant s age and gender. Samples were stored refrigerated (2-8 C) for < 3 days before analysis, or frozen at 20 C for < 6 months before analysis. A total of 1209 samples were tested. Correlation with HPLC. Specimens with thcy values spanning the dynamic range of the IMx assay (0 50 Omol/L) were assayed by both IMx and HPLC. Linear regression analysis was performed with the HPLC assay (x-axis) versus the IMx assay (y-axis) at each site. The four sites used three different HPLC methods. Sites #1 and #4 used tri-n-butylphosphine for reduction, derivitization with 7-fluorobenzo-furazane-4-sulfonic acid, and separation by HPLC [16,21]. Site #2 used sodium borohydride reduction, derivitization with monobromobimane, and HPLC separation with fluorescence detection [17]. Site #3 used sodium Results At the four sites, the precision of the IMx assay for thcy over 5 days ranged from 0.9 to 3.0%, and for thcy over 20 days from 2.8 to 4.1%, for the three control levels. The precision of the automated dilution ranged from 1.9 to 3.1%, compared to the manual dilution, which ranged from 1.7 to 9.9%. The grand means and coefficients of variation (CV) across all sites and reagent lots are shown in Table 1. There were no significant differences among the four sites in analytical precision, based on total CV percentages calculated across the sites (data not shown). The data from 12 runs using two different reagent lots (4 runs with lot A and 8 runs with lot B) were used to calculate analytical sensitivity. The analytical sensitivity was determined to be 0.35 jimol/l. Twenty samples were serially diluted to a final concentration of approximately 5 Umol/L, which corresponded to endpoint dilution factors ranging from Table 2. Homocysteine concentrations in a reference population, categorized by age and gender. Table 1. Precision studies of the IMx homocysteine assay.a Age (yr) & gender N Plasma homocysteine mean ±SD conc ( Jmol/L) log mean Hog SD Control N Mean homocysteine concentration Umol/L Within-run precision data SD %CV Total precision data SD %CV Low Medium High Auto l b Auto 2b Manual Ie Manual 2C a n = 80 for the 5-day study; n =1 6.0 for the 20 day study, k Auto = automated dilution, 1 & 2 are different samples. c Manual = manual dilution, 1 & 2 are different samples. females ± males
4 188 Annals o f Clinical & Laboratory Science 1:4 to 1:16. The recovery values ranged from 97.4% to 103.8%. Linear regression analysis of the observed thcy concentration vs the expected concentration showed linearity (r = , slope = ). The mean thcy concentration in the studied population (n = 1209) was 9.17 imol/lwith a central 95% range of lmol/l; the population distribution was skewed to the right and upon logarithmic transformation was normally distributed (Fig. 1). The mean thcy concentration for females (8.50 jlmol/l, central 95% range Jmol/L) was significantly lower than the mean th cy concentration for males (9.82 jlmol/l, central 95% range lmol/l). The data, analyzed by age and sex (n = 1205), showed an increase in thcy levels with increasing age, with the values for males consistently greater than the corresponding values for females in each age group. The population data are summarized in Table 2. Each site s correlation data for IMx vs HPLC assays are summarized in Table 3. Fig. 2 shows the combined correlation data. Overall, the correlation between HPLC and IMx was 0.966, with a slope of 0.98 and intercept of-0.19 fimol/l. The differences in slope at the various sites can be attributed to the different methods of assay, calibration, and standardization. Discussion Modern analytical methods for the measurement of th cy are largely based on some form of chromatography [13-17]. These methods measure total homocysteine concentration, which comprises the sum of the reduced and oxidized species. The disadvantages of some chromatographic techniques are that they are laborious, time consuming, and not amenable to high volumes of testing or screening of the general population. An enzyme-linked immunoassay has been developed using a microtiter plate format [19,22]. Although this assay compares well to the results by HPLC assay, semi-automated analysis requires m icrotiter plate washers and readers that are uncommon in clinical laboratories in the USA. The H C Y nmol/l I Log H C Y im ol/l Fig. 1. Frequency distribution graphs for plasma homocysteine concentrations in the reference population. In panel A, the abscissa has an arithmetic scale; in panel B, the abscissa has a logarithmic scale.
5 Evaluation o f the IM x assay for plasma homocysteine 189 development of the first fully automated assay for thcy facilitates the routine availability of this assay. A prototype assay of the current system demonstrated excellent precision, calibration curve stability, parallelism, recovery, and correlation with chromatographic methods [18], and the precision of this automated method was equal or superior to other published assays [17,19,21]. In this report, use of the FDA-approved IMx homocysteine assay was validated in a multi-center clinical study. The availability of results on an automated basis enabled verification of the reference range on a large population (n = 1209). Although different assays were used, results in this study were correlated with other reference range reports, as summarized by Ueland et al [23]. Previous studies of paired serum and plasma samples showed significant differences [14,17] due to the synthesis and release of homocysteine from red cells while the serum sample was clotting at room temperature. The preferred sample is EDTA plasma that is either centrifuged immediately to remove the red cells, or stored on ice until centrifugation. Storage of the sample on ice slows the synthesis of the homocysteine, and previous studies have dem onstrated no significant increase in homocysteine in EDTA plasma samples stored on ice for 8 hr [20]. Since this study used excess samples from previous studies, we were unable to collect matched serum and plasma samples. Use of an autom ated assay also provides a convenient means for screening homocysteine in high risk patients, as some have recently recommended [24]. Prospective studies are being conducted to determine if lowering plasma homocysteine concentrations is attended by decreased risks of cardiovascular, peripheral vascular, or cerebrovascular diseases. If such correlation is evident, routine screening may be warranted. Acknowledgments This study was supported by Abbott Laboratories, Abbott Park, IL, USA, and by Axis Biochemicals ASA, Oslo, Norway. Table 3. Correlation statistics for plasm a hom ocysteine concentrations assayed by the IMx and HPLC techniques at the four laboratory sites. Lab Site Homocysteine concentration range by IMx ( Imol/L) N r Slope o f the regression line (95% Cl) [and its y-intercept (95% Cl)] ( )] [0.12 ( )] ( ) [0.17 ( )] ( ) [0.21 ( )] Homocysteine conc. by HPLC assay (fxmol/l) Fig. 2. Combined correlation graph o f plasma homocysteine concentrations assayed by the IMx and HPLC techniques (square = site 1; circle = site 2; inverted Y = site 3; asterisk = site 4) ( ) [1.93 ( )] All ( ) [-0.19 ( )]
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