Cholesterol screening: comparative evaluation of on-site and laboratory-based measurements

We measured cholesterol in capillary blood samples from 9683 volunteers over a four-day on-site community screening program, using the "Reflotron" desk-top analyzer (Boehringer-Mannheim Diagnostics, Indianapolis, IN). We also measured cholesterol in venous blood samples from 3% of those screened (a) with the Reflotron at the screening sites, (b) in a qualified hospital clinical laboratory, and (c) in a Centers for Disease Control standardized lipoprotein research laboratory. The sensitivity (and specificity) of the Reflotron measurements, with use of the lipoprotein laboratory measurements as the point of reference, was 0.95 (0.73) in capillary blood samples and 0.88 (0.93) in venous blood samples, compared with 0.99 (0.87) in the hospital clinical laboratory. The Reflotron measurements correlated less well with the lipoprotein laboratory values in both venous blood (r = 0.91) and capillary blood (r = 0.89) samples than did the clinical laboratory values (r greater than 0.99). Furthermore, the capillary blood measurements averaged 7% higher than venous measurements when both kinds of samples were analyzed in the Reflotron.     

Reliability of the Reflotron in the determination of cholesterol

Measurements of total cholesterol in the field by means of the Reflotron dry-chemistry system (capillary blood) were compared to total cholesterol obtained by a standardized conventional wet-chemistry method in a clinico-chemical laboratory (serum). A total of 1200 people participated in the study. Two identical Reflotron machines were used. In the first period of the study an excellent agreement was found between Reflotron measurements of a reference serum provided by the manufacturer (mean, 4.99 mmol/l; CV, 1.8%) and the stated value (4.97 mmol/l). In the rest of the study higher values and greater variation were found with the Reflotron (mean, 5.32 mmol/l; CV 5.2%). Clearly the Reflotron measurements in the latter period of study were not reliable. In the period with stable instruments most of the values obtained at the two Reflotron machines differed from each other by less than 10%, with a mean difference of 0.08 mmol/l. Reflotron (both machines) and wet-chemistry measurements agreed well for the first 500 participants in the study (mean difference, Reflotron-wet-chemistry, -0.008 mmol/l; 95% confidence interval, -0.035 to 0.019 mmol/l; correlation, 0.967). In this period most Reflotron values differed from wet-chemistry values by less than 9% below to 9% above. With the next 200 participants the Reflotron gave on average slightly higher values than wet-chemistry measurements. The coefficients of variation for measurement variation were higher for Reflotron that for wet-chemistry even in the period with stable instruments. In all parts of the study period a lower HDL-cholesterol level was associated with larger differences between total cholesterol determined by Reflotron and wet-chemistry.     

Revised calibration of the Reflotron cholesterol assay evaluated

We evaluated the Boehringer Mannheim (B.M.) Reflotron Total Cholesterol "dry-chemistry" method after its recalibration in 1987. Reports in the literature up to 1986-1987 of a negative bias (up to -10%) in the method prompted a revision of the factory-set calibration of the Reflotron. For this, B.M. prepared a new set of calibrators with 12 different concentrations of cholesterol. We checked in two ways whether accuracy had been achieved: (a) The values assigned to the calibrators by B.M. were checked with the manual Abell-Kendall Reference Method (MAK) performed in an official Reference Center. These were shown to be correct. (b) Concurrently, a direct comparison was made by analyzing 200 fresh samples of human serum. Reflotron cholesterol values obtained for these samples proved to be accurate, meeting the current World Health Organization/Centers for Disease Control criterion of maximum bias less than or equal to 5%. Orthogonal regression analysis yielded the following correlation: Reflotron = 0.985 MAK + 0.238 mmol/L (y = ax + b). Reflotron mean = 6.26 mmol/L; MAK mean = 6.09 mmol/L. SDa = 0.015 mmol/L; SDb = 0.120 mmol/L, and r = 0.989.     

Comparison of the reflectance method (Reflotron reflectance photometer) with the absorbance method (automatic analysers) for the determination of cholesterol

The European Atherosclerosis Society (1) and the Expert Panel of the US National Cholesterol Education Program (2) have issued detailed guide values for recognition and management of hyperlipidaemia in adults. In these guidelines, the diagnosis of dyslipidaemia based on the measurements of total cholesterol, triacylglycerols, HDL and LDL cholesterol plays an important role. A prerequisite for the desired success of interventive measures is the reliability of the analytical data. The aim of this study was to investigate the precision and accuracy of Reflotron Cholesterol, a method based on the dry chemistry principle. Accuracy was assessed by establishing the correlation with the standardized automated methods used in routine lipid diagnosis. In addition, it was also examined whether the Reflotron Cholesterol results in plasma and blood are comparable. The Reflotron cholesterol (sample: blood) showed a good correlation with the CHOD/PAP method on a Hitachi 737 instrument (sample: plasma). The median value of the differences of the test results was -0.4%. Similarly, the method comparison of Reflotron Cholesterol (sample: blood) versus CHOD/PAP method on a SMAC instrument (sample: plasma) showed that Reflotron produces slightly (1.8%) higher results. The Reflotron Cholesterol values obtained from blood samples were slightly lower than those from plasma samples (median value of the differences: -2.2%). The results suggest that for routine purposes Reflotron Cholesterol provides results which are in good agreement with those obtained by standardized wet chemistry methods.     

Use of the Reflotron system for cholesterol assay in capillary blood, venous blood, and serum--evaluation of accuracy and lot-to-lot reagent comparability

Since its recalibration in 1987, the Reflotron "dry-chemistry" cholesterol method can produce accurate results. Here we report how cholesterol concentrations in venous blood, plasma, and serum determined with Reflotron compare with values determined for fingerstick samples. When no EDTA is used in the latter, accurate results may meet the WHO/CDC criterion of a maximum bias less than or equal to 5%. Reflotron users should avoid use of EDTA. Lot-to-lot variation among the solid-phase reagent carriers was evaluated in four lots of these strips. The largest difference, 0.4 mmol/L, was seen in a commercial quality-control serum with a cholesterol concentration of approximately 7 mmol/L. The maximum differences in results for several serum pools were not always found between the same reagent lots, although the highest results in five of six materials occurred with a single lot of strips. As in all clinical chemical testing, regular quality control is necessary.     

Creatine kinase determination: a European evaluation of the creatine kinase determination in serum, plasma and whole blood with the Reflotron system.

We evaluated a new dry-reagent carrier system for the determination of creatine kinase (EC 2.7.3.2) activity, Reflotron CK, with special attention to analytical performance with whole blood. We found a good within series imprecision. The median coefficient of variation was 3.1% for Reflotron CK (blood, serum and plasma) and 0.9% for the automatic analysers (serum and plasma only). The between-days imprecision with Reflotron CK (median CV: less than or equal to 3%) was similar to that for the comparison method on different analysers. Fresh samples of human blood, plasma and serum were examined by Reflotron CK and by a N-acetylcysteine activated creatine kinase method in six different clinical laboratories and in the Evaluation Department of Boehringer Mannheim GmbH. The correlation between these methods was excellent (r greater than or equal to 0.99), the median systematic deviation (bias) for all samples being smaller than -5%. Haematocrits between 0.25 and 0.50, haemolysis up to 6 g/l haemoglobin, and icteric samples with bilirubin concentrations up to 0.2 g/l showed no interference. No drug in therapeutic concentration was found to affect the Reflotron CK results; ascorbic acid, calcium dobesilate and sulphamethoxazole lowered the values only when present in high concentrations. Reflotron CK may be considered as a suitable alternative for decentralized testing sites, especially in situations where creatine kinase results are needed quickly.     

Direct measurement of high-density lipoprotein cholesterol by the reflotron assay with no manual precipitation step

We evaluated the quantification of high-density lipoprotein (HDL) cholesterol in plasma with the Boehringer Mannheim Reflotron reflectance photometric analyzer. The Reflotron is designed for testing in small to medium-size laboratories and physicians' offices. This HDL method does not require a manual precipitation step because the reagent tab contains dextran sulfate (Mr 50,000) and magnesium acetate. It takes 90 s to complete an analysis of 30 microL of plasma. Within-day standard deviations (SDs) were 0.02-0.04 mmol/L (6-16 mg/L). Total SDs over a three-month period were 0.03-0.06 mmol/L (11-23 mg/L). The Reflotron values averaged 0.02 mmol/L (6 mg/L) or 1.3% lower than the Hitachi 737 values; the standard error of the estimate (Sy.x) was 0.07 mmol/L (29 mg/L).     

Regional interlaboratory standardization of determinations of cholesterol, high-density lipoprotein cholesterol, and triglycerides

The Clinical Chemistry Forum of Central Virginia initiated a lipid standardization program to help ensure that its members meet the current National Cholesterol Education Program guidelines for cholesterol testing, and to standardize assays of high-density lipoprotein (HDL) cholesterol and triglycerides so as to provide accurate lipid profiles. We found that freshly collected, never-frozen human sera must be used to assess interlaboratory accuracy for cholesterol, HDL cholesterol, and triglycerides assays, and that at least 23 samples are required to detect a 3% bias with 90% power when the between-laboratory imprecision (CV) is 3%. After recalibration, all 12 laboratories had a mean HDL cholesterol bias less than or equal to 5%, nine of 10 laboratories had a mean HDL cholesterol bias less than or equal to 40 mg/L for samples with values less than or equal to 570 mg/L, and 10 of 12 laboratories had a mean triglycerides bias less than or equal to 10% for fresh human sera split between participants and the Centers for Disease Control. Pools of frozen human sera were shown to have matrix biases greater than 3% for cholesterol in seven of 11 laboratories, and greater than 40 mg/L for HDL cholesterol in six of nine laboratories.     

Blood cholesterol concentration: fingerstick plasma vs venous serum sampling

To assess whether venous and fingerstick blood samples yield similar cholesterol concentrations, we obtained both types of samples simultaneously in 108 volunteers participating in a cholesterol screening program. All samples were analyzed by the same enzymatic method in a standardized laboratory, and pairs of simultaneous samples were measured in the same laboratory run. Cholesterol concentrations in fingerstick-derived plasma were consistently higher than in the venous serum (P less than 0.0001), by a positive bias averaging 3.6%. Cholesterol values in fingerstick plasma also were higher than cholesterol results for venous serum placed in a capillary collection tube (average bias +2.4%). The positive bias of fingerstick plasma vs venous serum results appears to be at least partly due to specimen handling, although a true physiological difference between venous and fingerstick cholesterol concentrations is probably also involved. If a positive bias of this magnitude from fingerstick blood sampling is left unadjusted, substantial numbers of people will be labeled "at risk" and referred to physicians when their true values were actually within the acceptable range.     

Bias and precision of cholesterol analysis by physician's office analyzers

We studied the bias and precision of serum cholesterol analysis by physician's office analyzers. Total imprecision (CV range, %) for analysis of serum pools was: Abbott Vision 1.5%-1.9%; Ames Seralyzer 3.9%-4.5%; BMD Reflotron 2.3%-3.8%; Chrometrics Cholesterol Test System 2.3%-2.8%; Kodak DT-60 1.6%-2.2%. The Ames Seralyzer exhibited an excessive between-run component of variation. We collected, from 109 volunteers, samples of venous serum, heparin-treated whole blood, heparin-treated plasma, and fingerstick whole blood, and analyzed each type (where possible) with each system; serum was also analyzed in duplicate (by a proposed Reference Method) at the Centers for Disease Control. For assays with serum, the BMD Reflotron and Kodak DT-60 exhibited negative bias. All systems gave lower results for plasma and whole blood than for serum. All systems except the Kodak DT-60 were less precise for analysis of patients' sera than for analysis of serum pools; between-specimen variables may influence the results of these systems.     

Dry chemistry: an assessment of the BCL Reflotron system

The Reflotron dry chemistry analyser was assessed over a period of 9 months for the 11 available analytes: cholesterol, triglycerides, glucose, urate, gamma glutamyl transpeptidase (gamma GT), haemoglobin, aspartate amino transferase (AST), urea, alanine amino transferase (ALT), bilirubin and amylase. In comparison with established laboratory methods, results showed good agreement with end-point methods, but for enzymes there were significant but explainable differences, reflected in the normal ranges quoted for both the Reflotron and laboratory methods. Imprecision observed was between 2% and 6.5% CV (between-day) for enzyme analyses and 1% and 6% CV for end-point methods. Analytical ranges for each method were similar to those expected, with the exception of ALT, where the range was lower, although adequate. Delay between sample application and measurement should not exceed 60 s. Variations in packed cell volume of whole blood samples does not appear to affect results adversely. In the hands of semi-trained staff the instrument produced adequate results.     

Laboratory and field evaluation of three desktop instruments for assay of cholesterol and triglyceride

We evaluated three desktop instruments suitable for decentralizing the assays of blood cholesterol and triglycerides to satellite and physician's office testing. The instruments, Ektachem DT 60 (E), Reflotron (R), and Seralyzer (S), were used according to their manufacturers' instructions to assay serum or capillary blood from outpatients at two physicians offices. Accuracy was assessed in the centralized laboratory by using an automated centrifugal analyzer (A). The bias of A was monitored with an international quality-control material. We found all instruments easy to handle. Regression equations for cholesterol determinations were: E = 0.92A + 0.7 (n = 331, r = 0.94), R (capillary blood) = 0.96A + 0.3 (n = 256, r = 0.95), and S = 0.93A + 0.6 (n = 260, r = 0.92). For triglycerides we obtained E = 1.02A (n = 331, r = 0.97), R (cap. blood) = 0.88A (n = 213, r = 0.97), R = 0.94A + 0.1 (n = 90, r = 0.99), and S = 0.96A (n = 266, r = 0.98). Duplicate and within-day precision was less than 8%. Between-day precision (during a month) was less than 10%. We stress the need of both laboratory and field evaluation and emphasize the benefit of quality control.     

A review of the analytic performance of the Reflotron System for cholesterol testing

The analytic performance of the Reflotron System for cholesterol testing has been assessed in 17 studies since 1987. The precision of the test (total coefficient of variation) ranged from 0.8% to 9.9% and was between 1% and 5% in 23 of the 27 specimen types tested. The correlation coefficient of the Reflotron with that of a reference methods was over .9 in 15 specimen types tested and over .95 in ten of 15. The type of specimen tested and the training of the test personnel affected both precision and accuracy. It is concluded that the Reflotron analyzer provides reliable and accurate cholesterol measurements.     

Alkaline phosphatase activity: new assay for the Reflotron system. Results of the evaluation in eight clinical laboratories.

A new reagent carrier, Reflotron ALP, has been developed for the Reflotron system, allowing easy and rapid measurement (in less than 3 minutes) of alkaline phosphatase (ALP) activity in capillary blood, venous blood, heparinized plasma or serum. The evaluation of the analytical performance of the assay was carried out at eight clinical laboratories. The study of the imprecision using the measurements in human samples resulted in coefficients of variation ranging from 1.3% to 4.6% (within-run) and from 3.2% to 4.0% (day-to-day). The analytical specificity of the Reflotron ALP assay agrees well with ALP methods using a N-methyl-D-glucamine buffer solution. The calibration of the Reflotron ALP assay, however, is related to the reference intervals for ALP methods using a diethanolamine buffer solution. Method comparisons were performed with the ALP method on Hitachi instruments using diethanolamine buffer. Reflotron ALP measurements in blood and plasma in 157 randomly selected split samples showed excellent agreement (slope: 0.99; intercept: 0.7 U/l; median bias: 2.3%; median difference from the comparison method: -0.3%). Specimens from pregnant women and adolescents were excluded from this study. Differing values were obtained in a method comparison using 48 samples containing predominantly the ALP bone isoform (slope: 0.81; intercept: 31.5 U/l; median bias: 5.7%; median difference from the comparison method: -12.2%). Regression analysis of the results from 21 sera with prevailing placental ALP gave a slope of 1.51, and an intercept of -41.1 U/l (median bias: 8.6%; median difference from the comparison method: 35.6%). Reflotron ALP was compared with three different wet chemistry procedures using different buffer compounds: N-methyl-D-glucamine or diethanolamine or 2-amino-2-methyl-1-propanol. In samples containing predominantly ALP isoforms not of liver origin, the measurements with N-methyl-D-glucamine buffer gave the best fit with respect to Reflotron. In an interference study with 18 drugs, no effect on the test results could be detected. Total bilirubin up to 750 micromol/l and hemolysis up to 1.7 g/l free hemoglobin did not influence the test. Reflotron ALP proved to be an easy and rapid method with excellent precision. The accuracy related to an ALP method using diethanolamine buffer was good. The systematic differences for ALP in samples from pregnant women and adolescents have to be taken into account. The assay is well suited for differential diagnosis of hepatic diseases in decentralized testing.     

Calculated values for low-density lipoprotein cholesterol in the assessment of lipid abnormalities and coronary disease risk

Low-density lipoprotein (LDL) cholesterol concentrations are most commonly estimated by the formula LDL cholesterol = total cholesterol - [triglycerides (TG)/5 + high-density lipoprotein cholesterol], although alternative factors such as TG/6 have also been used. Using standardized, automated, enzymatic lipid assays, we analyzed 4797 plasma samples from normal and dyslipidemic adults, to compare LDL cholesterol concentrations obtained after ultracentrifugation with those calculated by several such methods (i.e., TG/4-TG/8). or TG concentrations less than or equal to 0.50 g/L, TG/4 agreed best with the direct assay; for TG of 0.51-2.00 g/L, TG/4.5 was best; and for TG of 2.01-4.00 g/L, TG/5 was best. Differences in estimated values were generally small, however. At TG greater than 4.00 g/L, none of the factors tested allowed a reliable estimate of LDL cholesterol. When TG were less than or equal to 4.00 g/L, 86% of estimated LDL cholesterol values were properly classified according to National Cholesterol Education Program cutpoints when the factor TG/5 was used. We conclude that a convenient direct method for measuring LDL cholesterol is needed but, until one is available, use of the factor TG/5 will assure that most individuals with TG less than or equal to 4.00 g/L, as measured in a standardized laboratory, can be reasonably well classified for risk of coronary artery disease.     

Estimation of VLDL cholesterol in hyperlipidemia

Lipoprotein data from 10947 fasting blood samples drawn between 1968 and 1982 in the Molecular Disease Branch at the National Institutes of Health were used to test the generalizability of estimating very low density lipoprotein cholesterol (VLDL-C) from plasma triglyceride (TG). Patient samples with total cholesterol levels over 500 mg/l and triglyceride values in the 0-100 000 mg/1 range were included in this study. A previously defined linear relationship VLDL-C = 0.20 (TG) was observed in the past by Friedewald and collaborators, allowing estimation of low density lipoprotein cholesterol (LDL-C) without ultracentrifugation for TG values up to 4 000 mg/1. The results from this report extend the use of the Friedewald relationship to higher TG levels, and to various dyslipidemic states. As the VLDL-C estimates become increasingly imprecise for TG values greater than 10 000 mg/l, caution should be exercised using the estimate in the higher TG ranges. Comparisons with an alternative equation VLDL-C = 0.166 (TG) showed equal or improved accuracy with this estimation procedure, particularly at high TG levels.     

Biological variability of lipoproteins and apolipoproteins in patients referred to a lipid clinic.

We determined the physiological variability of total cholesterol, high- (HDL) and low-density lipoprotein (LDL) cholesterol, triglycerides, and apolipoproteins A-I and B in fasting blood samples from patients referred to the Johns Hopkins Lipid Referral Clinic. Samples were taken on each of three occasions during baseline evaluation visits before the patients were treated. The median physiological coefficients of variation (CVp) were as follows: total cholesterol, 5.0%; triglycerides, 17.8%; HDL cholesterol, 7.1%; LDL cholesterol, calculated from the previous three measurements, 7.8%; and apolipoproteins A-I and B, 7.1% and 6.4%, respectively. There were no significant differences in CVp between children (less than or equal to 18 years) and adults (greater than 18 years) for any of the measurements. On the basis of our findings, single measurements in serial samples taken on three occasions suffice to establish the patients' usual values with the following precisions (+/- 1 CV): total cholesterol, +/- 4%; triglycerides, +/- 11%; HDL cholesterol, +/- 8%; LDL cholesterol, +/- 6%; and apolipoproteins A-I and B, +/- 7%.     

Reflotron cholesterol measurement evaluated as a screening technique

We evaluated the analytical performance of Boehringer Mannheim Diagnostics' "Reflotron" analyzer for the measurement of cholesterol. Coefficients of variation (CVs) for whole-blood cholesterol were: within-day 2.0% and 2.2% at 1680 and 2670 mg/L, respectively; between-day 1.8% and 2.4% (n = 9 and 8). Results were similar for serum and heparinized or EDTA-treated single-donor plasma (CV 1.4% to 2.6%). CVs of results for two reconstituted commercial quality-control materials were 3.4% and 4.6%. Heparin and hematocrit were evaluated as interferents, and critical limits for interference were identified for bilirubin, hemoglobin, and triglyceride in blood and plasma or serum. When sample collection and analysis were controlled by trained personnel, results with the Reflotron (y) compared well with those by the Ektachem procedure (x) for both blood and serum samples: r = 0.950, y = 0.944x + 130 mg/L; and r = 0.955, y = 0.93x + 43.5 mg/L, respectively. The same comparability was observed when the analysis was performed by briefly trained high-school students: r = 0.980, y = 0.949x + 23 mg/L. Performance decreased when both collection and analysis were performed by laymen: r = 0.880, y = 0.870x + 186 mg/L.     

Comparison of the ratio HDL-cholesterol/total cholesterol on the Reflotron vs. conventional wet chemistry methods.

A new HDL-cholesterol assay using solid phase reagent chemistry on the Reflotron has become available recently. This raises the question of whether the Reflotron now is suited for the determination of the ratio, HDL-cholesterol/total cholesterol, as a supplement to the lipid profile, viz. cholesterol, triacylglycerol and HDL-cholesterol. We investigated this ratio, which is an additional measure for the risk of coronary heart disease and found a good agreement between the Reflotron and conventional wet chemistry methods. We conclude that the Reflotron can be used for establishing the complete lipid profile of a patient.     

Preliminary studies of diabetic decompensation assessed with bedside glucose-monitoring techniques

Blood glucose-monitoring techniques originally developed to aid outpatient management of diabetic patients are now being used to facilitate hospital care. However, applications in hyperglycemic patients have been limited because many glucose-oxidase strips and meters respond only to glucose values less than or equal to 400 mg/dl. We asked if prior dilution of blood samples would permit reliable estimations. Ten consecutive decompensated diabetic patients (age 35-73, glucose 506-879, HCO3 12-28) had blood glucose determinations done simultaneously by the hospital laboratory and by Chemstrip bG after dilution of heparinized blood 1:2 in saline. Thirty-one samples were obtained before and during insulin therapy. Correlations with laboratory glucose values were 0.95 with strips read by Accu-Chek meter and 0.90 read visually, both P less than 0.001. Average deviations from laboratory values were 7.9% with Accu-Chek and 12.9% with visual readings. Accu-Chek deviations averaged 9.6% for glucose greater than 700 mg/dl, and 6.9% for glucose greater than 400 mg/dl. Over the first hour of insulin therapy, glucose fell 150 +/- 30 mg/dl by Accu-Chek, comparable to 168 +/- 29 by laboratory measurement; the decrement by visual reading was 107 +/- 32, not significantly different. We conclude that dilution of blood samples with glucose greater than 400 allows reliable estimation of elevated values by home glucose-monitoring techniques. This approach is cost-effective and provides the rapid feedback needed for the management of critically ill patients.