Lipemic Specimens Research Articles

A-119 Lipemia Interference

Abstract Background Lipemic specimens is a common problem in clinical laboratories. Analytical results may be perturbed by lipemia, leading to misdiagnosis and unnecessary treatments for patients. Serum index testing is an important preanalytical test for assessing the potential effect of endogenous HIL interferents (hemoglobin (H), bilirubin (I) or lipids (L)) on test results. Automated HIL systems are not standardized and differ in their response. HIL induced interference should be verified by all clinical laboratories. Intralipid, a commercial lipid emulsion with particle size ranges from 200 to 600 nm is often used to verify the L index. However, intralipid lacks particles that mimic large VLDL, as well as the lower and upper ranges for chylomicrons size. In contrast, patient samples contain a complex mixture of macromolecular lipid and protein structures. Therefore, lipemia induced by Intralipid is not identical to lipemia in patient serum samples. The purpose of this study is to increase awareness of lipid interference and demonstrate the relationship of a lipid L Index to intralipid and triglyceride interference. Methods A set of 16 panels were prepared from a commercially available Triglyceride rich Lipoprotein material. These panels spanned a lipid concentration from 0 to 3000 mg/dL. Panels were tested on the Alinity c and ARCHITECT c8000 TRIG2 assays (04U0615 and 04T1016 respectively) on 2 reagent lots in a single run to determine the Triglyceride concentration. Then these samples were assessed for an HIL L score using the Abbott HIL saline protocol. Results The relationship between the Triglyceride rich Lipoprotein and the L Index was linear with a high correlation (r= 0.999) for Alinity and ARCHITECT respectively. The L Index underestimated the Triglyceride Rich Lipoprotein concentration. The reason for the lower estimate of Triglyceride rich lipoprotein specimens on an L Index is likely due to the differences in size of the lipid particles between the two artificial lipid materials, Intralipid and Triglyceride rich lipoprotein. In addition, while the triglyceride assay provides a quantitative measurement of the lipemic concentration, the L Index is designed to provide an estimated result. Conclusions Intralipid is an acceptable material used to verify spectrophotometric HIL systems. The L Index provides an estimate of sample turbidity, not the concentration of Triglyceride. Vendor purchased human derived Triglyceride rich lipoprotein, which contains a heterological mixture of lipid particles, may be regarded as a more appropriate material for assessing lipid interference. Thus, while recovery of the L Index correlates well to the Intralipid concentration, it can have a poorer correlation with vendor purchased samples containing Triglyceride due to the different vesicle sizes in these materials. This study demonstrates the Triglyceride rich material used to assess lipid interference in Sigma Strong assays provides a difference in response between the Triglyceride concentration of this material determined on a Triglyceride assay vs the L Index. The choice of lipemic material to use, (eg. artificial, native), can influence the results. Thus, care should be taken in comparing results for these materials.

Hemolysis, icterus and lipemia interfere with the determination of two oxidative stress biomarkers in canine serum

BackgroundOxidative stress has been proven to play a role in numerous human and canine diseases. Among the biomarkers of oxidative stress, Thiobarbituric Acid Reactive Substances (TBARS) and Total Antioxidant Status (TAS) are two of the most widely used. Preanalytical factors are crucial for obtaining accurate results in these assays. Hemolysis, icterus and lipemia (HIL) are common sources of preanalytical errors in the laboratory; however, limited information is available regarding the considerations for canine specimens. Therefore, the objective of this study was to evaluate the potential interferences of HIL in the determination of TBARS and TAS in canine serum.MethodsSolutions of pooled canine serum samples were prepared by adding increasing concentrations of hemolysate, bilirubin and a synthetic lipid emulsion. TBARS and TAS were determined, and biases from the control value caused by the interfering substances were calculated.ResultsHemolysis, icterus and lipemia induced significant interferences on TBARS and TAS, albeit to varying degrees depending on the specific biomarker and interfering substance. TBARS appeared to be more susceptible to interferences in this study. Slight hemolysis, moderate icterus and slight lipemia caused notable deviations in TBARS values, surpassing the acceptable threshold for interference. TAS assay was also affected by HIL, although to a lesser extent compared to TBARS. Significant biases from TAS control value were observed when icterus was moderate, and when hemolysis and lipemia were more pronounced.ConclusionsIn light of our results, we conclude that hemolyzed, icteric and lipemic specimens are not suitable for TBARS and TAS determination in canine serum. Our findings hold considerable practical utility, as a simple visual inspection would be sufficient for identifying and excluding such specimens.

Effect of Pre-Analytical Errors in Laboratory Testing Facilities: The Way Forward

Pre-analytical errors contribute a significant proportion of errors of all major sources of mistakes made in laboratory testing processes and are responsible for several patient safety risks. It contributes to wrong therapeutic interventions, irrelevant follow up laboratory investigations and diagnostic delays, which impact negatively on the economy and laboratory effectiveness of health services. Pre-analytical phase is directly related to the procedure of specimen collection and is mostly out of the direct control of the laboratory; further, most pre-analytical errors are related to human factors. The aim of the study is to determine the nature and the occurrence of pre-analytical errors and recommendations on possible measures to reduce these errors. A total of 300 specimens were randomly sampled from a study population of 600 patients and analyzed for pre-analytical errors. One hundred and eighty-four (184) samples were found unsuitable for further processing accounting for 1.9% of all samples analyzed for pre-analytical errors and sample rejection. Rejections were due to following reasons: hemolysis 21.7 % (40) wrong tubes 19 % (35); clotted blood 17% (32); inappropriate timing of collection 15.7% (29); mislabeled specimens 15.2% (28); insufficient specimen quantity 6.5 % (12) and lipemic specimens 4.3% (8). The overall percentage of rejection was 1.9% and the substantial numbers of the rejected specimens were re-tested. Efforts aimed to reduce the rates of rejected samples can improve the quality of laboratory-based health care response. Keywords: Error, Laboratory, Pre-analytical, Rejection, Specimen.

Autoverification-based algorithms to detect preanalytical errors: Two examples

The preanalytical phase of testing accounts for the majority of the errors. Software-based quality rules, such as autoverification, can assist in preanalytical error detection; therefore, preventing erroneous results from being reported. Two autoverification rules, turbidity/lipemia, and pseudohypoglycemia/pseudohyperkalemia alarms, are highlighted. Increased sample turbidity may arise from several causes outside of lipemia. Typically, this can be resolved by clarifying the sample with standard centrifugation. Truly lipemic specimens typically require higher centrifugation speeds and greater centrifugation time. At our facility, 96% of direct bilirubin (DBIL), 95% of aspartate transaminase (AST), and 98% of alanine transaminase (ALT) turbidity/lipemia alarms were found to be from sample turbidity versus lipemia. Secondly, a pseudohypoglycemia/pseudohyperkalemia rule was employed for specimens with delayed separation from cellular material. Of the total potassium results >6.0 mmol/L or glucose results <40 mg/dL (2.2 mmol/L), 30% and 50% respectively were noted to have delayed sample separation.

Lipemic Interference in Basic Metabolic Panels: Increasing the Lipemia Index Threshold in Order to Decrease the Frequency of Ultracentrifugation

Abstract Introduction Lipemia in clinical chemistry samples is a problematic form of interference. Clearing these samples for routine testing can be time consuming and increases the turn-around time for these specimens. In our laboratory, samples with a lipemia index &amp;gt;50 (L-index) are manually inspected and visibly lipemic specimens are cleared by ultracentrifugation. Objective The objective of this study was to determine at what L-index ultracentrifugation of lipemic BMP specimens is necessary prior to sample testing to ensure accurate results. Methods Specimens consisted of routinely ordered basic metabolic panels (BMP) that met current criteria for ultracentrifugation, which included an L-index &amp;gt;50 as measured on the Abbott Architect c8000 and visual lipemia. Specimens meeting these criteria were ultracentrifuged and retested. The difference of the pre-ultracentrifuged and post-ultracentrifuged result was evaluated and put into a percent to find the ‘percent difference’ and evaluated against the total allowable error (TEa) for each analyte. If the difference observed following ultracentrifugation was less than or equal to 50% of the TEa, clearance of lipemia by ultracentrifugation was considered unnecessary. Values from all BMP component tests were analyzed in order to find an L-index threshold at which samples need to be ultracentrifuged which could be applied to the entire panel. The report of lipemic indices for BMPs for the month of January 2020 were extracted from the laboratory information system to evaluate the potential impact of altering the L-index threshold for ultracentrifugation. Results Based on the acceptance criteria of ≤50% of TEa, L-index thresholds for Na, K, Cl, calcium, glucose, creatinine, CO2 and BUN were &amp;lt;203, &amp;lt;410, &amp;lt;287, &amp;lt;387, &amp;lt;410, &amp;lt;285, &amp;lt; 153 and &amp;lt;285, respectively. All the calculated differences or percent differences for each analyte did not exceed 50% of the TEa for a given analyte when the L-index was 150 or less. Adjusting the L-index to 150 and applying it to the 195 lipemic BMP samples in January 2020, would have potentially decreased the number of samples requiring ultracentrifugation to 24 lipemic BMPs (88% reduction). Conclusion These data suggest that an L-index greater than 150 can be used for all analytes within a BMP as the threshold for requiring ultracentrifugation. The BMP is one of the most frequently ordered tests in our laboratory and consistently accounts for a substantial portion of the lipemic samples that require ultracentrifugation. Increasing the L-index at which samples will be ultracentrifuged from 50 to 150 would potentially result in an 88% reduction in one month of BMP samples requiring ultracentrifugation.

Lipemia Interferences in Biochemical Tests, Investigating the Efficacy of Different Removal Methods in comparison with Ultracentrifugation as the Gold Standard.

Introduction. As a common interferer in clinical chemistry, lipemic specimens could be a source of significant analytical errors. Ultracentrifugation has been by far the only reliable, but an unavailable and expensive, method to eliminate the lipemic effect. Materials and Methods. Among the daily samples, those with triglyceride >400 mg/dL (4.6 mmol/L) and also turbid were selected, divided into three groups, based on triglyceride concentration, and three pooled serums were made for each group. Then all pooled serums were investigated by using a DIRUI biochemistry analyzer CS-800 for routine chemistry tests in different methods including direct measurement, serum blank, serum dilution, and measurement after ultracentrifugation. Results According to our study, there were significant differences before and after ultracentrifugation in all lipemic levels and for all parameters except for alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, and uric acid. Based on allowable inaccuracy for each parameter, calcium, magnesium, phosphorus, total protein, iron, total iron-binding capacity (TIBC), urea, and chloride are being influenced by all lipemic degree and neither serum dilution nor using serum blank is as effective as ultracentrifuge for elimination. Serum blank was a proper method of lipid removal for the measurement of glucose. Conclusion Lipemia is a well-known interferer in clinical chemistry. One cannot avoid lipemia, but fortunately, severe lipemia is a rare phenomenon in the laboratory, and for assessment of some analytes in a lower degree of lipemia, use of serum blank eliminates the need for ultracentrifuge.

Evaluation of L-index interference limits on Roche cobas c502 and c702 immunoturbidimetric assays using endogenously lipemic specimens and intralipid spiking

ObjectivesSpecimen lipemia is a primary concern with turbidimetric and nephelometric assays due the potential interference caused by light scattering or absorption. The purpose of this study was to evaluate lipemic interference thresholds across seven FDA-cleared assays using patient specimens with varying degrees of endogenous lipemia pre- and post-ultracentrifugation (UC) and with Intralipid spiking. MethodsUsing an IRB-approved protocol, residual human serum specimens (n=42; L-indices, 1-1769; H-index ≤85; I-index ≤2) were obtained. Baseline and post-UC testing was conducted across assays on cobas c502 and c702 instruments (Roche Diagnostics; Indianapolis, IN). Serum indices and triglyceride (TRIG) concentrations were also measured pre- and post-UC. Intralipid spiking studies with human AB serum were also conducted. Lipoprotein subfraction analysis (Lipoprint; Quantimetrix; Redondo Beach, CA) was performed on three additional patient specimens with elevated TRIG post-UC to determine which TRIG-containing lipoprotein fraction(s) remain. ResultsSeveral assays showed L-index thresholds derived from endogenously lipemic specimens that were below previously defined limits from the package inserts (PIs) [new (prior)]: AAT 400 (500); CERU 100 (200); HAPTO 450 (600); TRSF 250 (500). L-index limits derived from Intralipid spiking were generally higher than those listed in PIs. UC did not adversely impact results in non-lipemic or lipemic specimens. UC was effective at clearing lipemic interference, although persistence of residual VLDL was often observed. ConclusionsThis study provides an analysis of L-index thresholds for seven immunoturbidimetric assays. Due to the variety of human lipoproteins, limits defined using endogenously lipemic patient specimens may be different from those derived from spiking studies using Intralipid.

Lipemic interference of ceruloplasmin assays – An evaluation of lipid removal methods

BackgroundThe present studies were conducted to characterize lipemic interference across three FDA-cleared ceruloplasmin (CERU) assays and to evaluate procedures designed to remove lipemic interference. MethodsCERU assays on the Abbott ARCHITECT ci8200, Beckman AU5800, and Roche cobas Integra 400 Plus were evaluated. Precision, linearity with dilution, lipemic interference, and three methods for removing lipemia were assessed on each platform: ultracentrifugation (UC), lipemia-clearing reagent LipoClear (LC), and 1:5 dilution (DIL). Lipemia-index (L-index) thresholds were established using endogenously lipemic specimens and sera spiked with human-derived triglyceride-rich lipoproteins. ResultsThe ci8200 showed greater susceptibility to endogenous lipemic interference than would be expected based on vendor-derived limits established with Intralipid. Endogenous lipemia causes a negative interference on the ci8200 and a positive interference on the Integra. UC was generally the most reliable method of removing lipemic interference without impacting baseline CERU results. ConclusionsCERU assays on different platforms have varying susceptibility to lipemic interference. L-index thresholds derived using Intralipid may not accurately represent interference caused by endogenous lipemia.

Evaluation of the automated coagulation analyzer CS-5100 and its utility in high throughput laboratories

Automated analyzers are an important component of modern laboratories. As a representative of the newest generation of coagulation analyzers, the CS-5100 features several technical refinements including a pre-analytical assessment unit as well as multi-wavelength optical detection units. Therefore, the CS-5100 is supposed to rapidly and accurately perform a broad panel of coagulation tests. In the current study, the CS-5100 was evaluated regarding its precision and practicability in a clinical laboratory setting. The CS-5100 was evaluated regarding its intra- and inter-assay precision using commercially available control samples. RESULTS of patient samples, including hemolytic, icteric and lipemic specimens, measured on the CS-5100 were compared to reference analyzers, which are used in our accredited laboratory. The coefficients of variation, assessed in the intra- and inter-assay precision analyses were below 5% representatively for most parameters. RESULTS, obtained by the CS-5100 showed predominantly a high comparability to used reference analyzers, with correlation coefficients ranging from 0.857 to 0.990. Only minor ranged systemic or proportional differences were found in Passing-Bablok regression between the CS-5100 and reference analyzers regarding most of the tested parameters. Lipemic samples had a tendency to deteriorate correlation coefficients, but an overall effect of the sample's triglyceride level could be ruled out. In a routine setting, the analyzer reached a sample throughput rate of 160 tests per hour. The CS-5100 is able to rapidly and precisely measure patient samples. No considerable influence on test comparability was found for elevated levels of free hemoglobin, bilirubin or triglycerides.

Cyclodextrin Removal of Lipemic Interference: An Attractive Alternative to Ultracentrifugation for Satellite Laboratories

To the Editor.—Interference from lipemia can significantly impede accurate analysis of a variety of laboratory tests independent of instrumentation or manufacturer.1 Conventional treatment of lipemia involves ultracentrifugation of specimens at approximately 199 000g to pellet and clarify interfering lipids. Although effective, this approach requires additional instrumentation, which may be costly for smaller, satellite laboratories offering a limited testing menu. Triaging lipemic samples to larger reference laboratories or core facilities may be impractical because of extended turnaround times and specimen rejection of lipemic samples, thus hindering patient care.Recently, our institution evaluated a lipid-precipitating, nonionic-cyclodextrin polymer, LipoClear (Statspin, Westwood, Massachusetts)2 as an alternative to ultracentrifugation in satellite laboratories as a means of reducing cost and improving turnaround time. Plasma and serum samples (n = 40) with grossly elevated lipemia (≥3+) were collected and refrigerated before analysis. Lipemia indices before treatment ranged from 3 to more than 10 (median = 6+). Specimens were split and treated to conventional ultracentrifugation (15 minutes at 199 000g) or with LipoClear (5 minute incubation of 500 μL of sample and 100 μL of LipoClear, followed by 7 minutes at 3461g). Clarified specimens were transferred to new tubes and analyzed on a Beckman Coulter DxC800 (Fullerton, California).LipoClear-treated specimens showed good agreement with paired ultracentrifuged specimens for all assays tested: sodium, potassium, chloride, carbon dioxide, calcium, glucose, blood urea nitrogen, total protein, albumin, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, total bilirubin, phosphorous, creatinine, and creatine kinase. Linear regression analysis demonstrated acceptable performance across clinical decision points for all analytes. Correlation coefficients ranged from R = 0.92 to R = 0.99 for all analytes, except sodium and total protein, whose coefficients were R = 0.90. Sodium demonstrated a mean positive bias of 3.6 mEq/L (mmol/L) using LipoClear, compared with ultracentrifugation (Figure 1, A and B). This was considered within the limits of acceptability for the assigned total error budget of ±4 mEq/L (mmol/L) at pertinent medical decision points.3 Total protein demonstrated a mean negative bias of 0.65 (g/dL) using LipoClear, compared with ultracentrifugation. Previous work has compared preanalyte and postanalyte measurements on the Beckman Synchron LX-20, after artificial spiking of nonlipemic samples with the synthetic lipid Intralipid (Uppsala, Sweden).4 In this study, no discordance between recovery with ultracentrifugation and LipoClear was observed with sodium or total protein, highlighting potential limitations for Intralipid as a universal substitute for assessing endogenous lipemic interferences. Additionally, 78% of LipoClear-treated samples showed lower lipemia indices after treatment than did their paired ultracentrifuged specimens, suggesting a more-complete removal of interfering lipids. The results here are limited to performance on the DxC800, and it is the responsibility of the laboratory to validate any preanalytic specimen manipulation not covered in the manufacturer's instructions for use.Analytic performance aside, implementation of LipoClear in satellite laboratories reduced cost and drastically improved turnaround time for reporting lipemic specimens. Process mapping identified 11 steps necessary for dealing with lipemic specimens before chemotherapy infusion for our oncology clinic (Figure 2). Before any clinical procedure, an additional waiting time of at least 2 hours was not uncommon because of courier pickup, delivery, ultracentrifugation at the offsite laboratory, and result reporting. Incorporation of the onsite treatment of lipemia reduced the number of steps from 11 to 7 and added only 15 minutes compared with normal specimens. The economic barrier of approximately US $14,000 for purchasing ultracentrifuges for every satellite laboratory, as well as the need for a dedicated air supply for their operations, had previously prevented onsite treatment of affected specimens. Incorporation of this protocol returned results for lipemic specimens 87% faster and reduced cost 3-fold. Collectively, the elimination of costly equipment and courier fees, coupled with a shorter turnaround time, added value for both our satellite laboratories and for our patients.

Lipemia interferences in routine clinical biochemical tests

Lipemic specimens are a common and frequent, but yet unresolved problem in clinical chemistry, and may produce significant interferences in the analytical results of different biochemical parameters. The aim of this study was to examine the effect of lipid removal using ultracentrifugation of lipemic samples, on some routine biochemistry parameters. Among all the samples obtained daily in our laboratory, the ones which were visibly muddy were selected and underwent to a process of ultracentrifugation, being determined a variety of biochemical tests before and after ultracentrifugation. A total of 110 samples were studied. We found significant differences in all the parameters studied except for total bilirubin, glucose, gamma-glutamyl transferase (GGT) and aspartate aminotransferase (AST). The greatest differences in the parameters analyzed were found in the concentration of alanine aminotransferase (ALT) (7.36%) and the smallest ones in the concentration of glucose (0.014%). Clinically significant interferences were found for phosphorus, creatinine, total protein and calcium. Lipemia causes clinically significant interferences for phosphorus, creatinine, total protein and calcium measurement and those interferences could be effectively removed by ultracentrifugation.

Evaluation of an automated, homogeneous enzyme immunoassay for serum thyroxine measurement in dog and cat serum.

A homogenous enzyme immunoassay (EIA) for measurement of serum thyroxine (T4) concentration was evaluated for use with canine and feline serum. The EIA method was linear from 0 to 150 nmol T4/L for human serum, 0 to 94 nmol T4/L for feline serum and 10 to 60 nmol T4/L for canine serum. Intra- and interassay precision studies yielded coefficients of variation </= 8% using single point measurements. Method comparison studies gave close agreement between radioimmunoassay (RIA) and EIA results. Correlation coefficients (r values) were 0.88 and 0.97 for canine and feline samples respectively, after application of species-specific factors to correct the calibrator values assigned for human serum samples. The EIA showed no interference from hemolysis at hemoglobin concentrations </= 20 g/L or from moderate lipemia. Highly lipemic specimens could be tested after centrifugation to clarify the sample. The EIA showed less interference from autoantibodies to T4 than the RIA method. The EIA method allows automation of T4 testing in a veterinary hospital or laboratory, and can be integrated with the routine clinical chemistry panel.

Erroneous laboratory results from hemolyzed, icteric, and lipemic specimens

Erroneous laboratory results from hemolyzed, icteric, and lipemic specimens

Immunonephelometric Assay of Vitamin D-Binding Protein

An automated immunonephelometric assay was developed to measure vitamin D-binding protein (DBP) in serum. The assay is described for the Behring Nephelometer System, which uses rabbit anti-DBP antiserum and purified human DBP. The detection limit is 0.05 g/L (0.86 mumol/L), and the working range is less than or equal to 1.60 g/L (27.59 mumol/L). Intra- and interassay CVs of 2.0% and 2.8-3.8% compare favorably with alternative methods. When results were compared with those from a immunoradiometric assay, the correlation coefficient was 0.976 (P less than 0.001), and the regression equation [y = 0.866 +/- 0.085x + 0.05 (Syx = 0.042, n = 42)] identified a negative bias. Analysis indicated that both methods appeared to contribute equally to the bias. Although the assay was relatively free from analytical interference, falsely increased values were noted in severely lipemic specimens and in frozen specimens. Interference may be minimized by inclusion of Supplementary Precipitation reagent in a modified assay protocol. The range of concentrations expected in clinical samples was established from normal subjects [0.32-0.46 g/L (5.52-7.93 mumol/L), n = 28], pregnant subjects [0.51-0.70 g/L (8.79-12.07 mumol/L), n = 13], and subjects with liver diseases [0.12-0.33 g/L (2.07-5.69 mumol/L), n = 18].

Immunoturbidimetric method for routine determinations of apolipoproteins A-I and B

A simple immunoturbidimetric method for quantifying apolipoproteins (apo) A-I and B in serum or plasma is described. A special reagent formulation, including large amounts of suitable detergents, obviates the need for a sample blank even with grossly lipemic specimens. The assay is rapid, easily automated, and thus convenient for routine work. For both apo A-I and apo B, the assay range is about 0.2-3.5 g/L. The performance characteristics were assessed with discrete (Optimate and Olli CD) and centrifugal analyzers (Cobas Fara and IL Monarch 2000). Average analytical recovery was 101.5% for apo A-I and 99.4% for apo B. Dilution tests showed found/expected ratios of 101.2% (apo A-I) and 101.0% (apo B). Overall precision (CV) ranged from 1.4% to 3.3% for apo A-I and from 1.1% to 8.3% for apo B. Comparisons with commercially available rate nephelometry, radial immunodiffusion, and immunoturbidimetric methods gave good correlations (r greater than or equal to 0.938). Using the immunoturbidimetric method, we also established the relationships between apolipoproteins and lipids and determined the reference intervals. We conclude that the proposed method is suitable for routine use in clinical laboratories.

Four fluorescence polarization immunoassays for therapeutic drug monitoring evaluated.

We evaluated four fluorescence polarization immunoassays--those for phenytoin, procainamide, N-acetylprocainamide (NAPA), and quinidine--from Roche Diagnostic Systems, done in the Cobas Bio FP centrifugal analyzer. The assays for phenytoin, NAPA, and quinidine demonstrated a linear response over the expected ranges of concentrations, and analytical recovery of test drug added to drug-free sera was greater than 90%. However, recovery in the procainamide assay was poor (69-82%) for samples containing greater than 8 mg/L, owing to nonlinearity. Results of method-comparison studies of the four assays paralleled the recovery studies, although the quinidine assay demonstrated a bias (1.0 mg/L higher) when compared with EMIT (Syva). The precision of the phenytoin assay was acceptable at all concentrations tested (total CV less than 7.0%). Imprecision of the other assays was significant at certain concentrations: total CV greater than 10.0% at subtherapeutic values for NAPA and quinidine, and greater than 9.0% for low (2.4 mg/L) and moderate concentrations (9.6 mg/L) of procainamide. Interferences were not significant for hemolyzed, icteric, or lipemic specimens or for specimens with added drug metabolites. The calibration curves for all four assays had good stability (greater than 60 days).

Immunonephelometry of apolipoprotein B with a centrifugal analyzer.

We developed an automated immunonephelometric assay for the measurement of apolipoprotein B (apo B) with a light-scattering microcentrifugal analyzer. Pretreating specimens with a dilute solution of Tween 20 or triglyceride lipase decreased the nephelometric response of apo B. Polyethylene glycol is included in the reaction mixture, and the reaction is complete within 4 min. The method is precise (CV = 6.5%, mean = 0.68 g/L) and the standard curve is linear to an apo B concentration of 2.8 g/L. Lipemia does not interfere with the method if grossly lipemic specimens are centrifuged to remove chylomicrons.

Direct ultraviolet method for enzymatic determination of uric acid, with equilibrium and kinetic data-processing options.

We developed and evaluated a direct ultraviolet method for the enzymatic determination of uric acid in serum, plasma, or urine, without deproteinization of sera and plasma. Equilibrium and nonlinear curve-fitting kinetic options are evaluated and compared, and results of the proposed method are compared with those of a candidate Reference Method. All data-processing options yield a linear relation for absorbance and concentration of uric acid between 0.1 and 2 mmol/L; with the equilibrium option, results are linear to 5 mmol/L. For 100 plasma samples, results correlate well between the proposed method (y) and a reference method (x): y = 0.99x - 0.002 mmol/L. Between-run imprecision is about 2.3%, and interference by hemolyzed, icteric, or lipemic specimens or specimens containing high concentrations of xanthine or paraproteins is minimal. The kinetic option with a data-processing range of 100 s or longer yields results that correlate well with the equilibrium method: y = 1.006x + 0.002 mmol/L (n = 118). For 20 urine samples processed by the proposed (y) and a reference (x) methods, y = 1.04x + 0.038 mmol/L.

Calibration and Evaluation of an Enzymatic Procedure for Determination of Total Serum Cholesterol

A performance evaluation of the SPIN CHEM reagent for the enzymatic determination of cholesterol has been conducted with the Rotochem centrifugal analyzer. Special emphasis was directed toward the problem of calibration, the need for serum blank correction, and interferences associated with elevated lipids, bilirubin and hemoglobin. Accurate calibration requires a secondary serum standard with a cholesterol value established by a reference procedure. Serum blank correction is beneficial for clear specimens but results in overcorrection for lipemic specimens. Interferences resulting from bilirubin and hemoglobin are complex in nature but are minimized by omission of serum blank correction that would overcorrect for their respective spectral interferences. These principles should also be applicable to other similar enzymatic cholesterol procedures. Comparison of the enzymatic method, calibrated as described, with the method of Abell, et al. (J. Biol. Chem. 195:357, 1952) for analyses of clear and lipemic specimens yielded a linear regression equation of y = 0.99x + 13 mg/L, with a correlation coefficient (r) of 0.9982. Within-run precision (CV) was ≤1.2% and between-run precision was ≤1.6% at cholesterol levels of 1450 mg/L and 2770 mg/L.

Evaluation of the Beckman Creatinine Analyzer 2

The Beckman Creatinine Analyzer 2 was evaluated in our laboratory. This new instrument determines creatinine by a Jaffe rate method and utilizes an optical detection system which measures the rate of change in absorbance at 520 nm 25.6 seconds after sample introduction. The linearity of the instrument was verified up to creatinine concentrations of 24 mg/dl. Recovery averaged 92% from serum and 97% from urine. Comparison studies using serum specimens were made against a manual, non-kinetic Jaffe method and a Technicon SMA 12/60. Urine comparisons were made against the manual method. All comparisons indicate the Beckman analyzer yields lower creatinine values due to the minimization of interferences from non-creatinine chromogens. No significant change in these correlations was observed with elevated bilirubin concentrations (greater than 10 mg/dl) or with lipemic specimens (triglycerides up to 3390 mg/dl). Both within-day and day-to-day precision were shown to be within the stated manufacturer's specifications for both sera and urine specimens.