Analytical Quality Specifications Research Articles

Acceptable variability in external quality assessment programmes for basic semen analysis

External quality assessment is essential in modern andrology laboratories. To assess the proficiency of laboratories participating in an external quality assessment programme (EQAP), limits for acceptable variability must be determined. Limits currently specified largely depend on criteria set by the organizers of individual EQAP schemes. The objective of this study was to evaluate the different criteria described in ISO 13528: 2005 for calculating acceptable variability in EQAP when applied to basic semen analysis parameters. The data used in this study were the means and standard deviations obtained for independent samples from two EQAPs, one national (Spanish) and one international (European). The acceptable variability according to ISO 13528: 2005 was calculated using four types of criteria: (i) ± 3 standard deviations of the results of all participating laboratories; (ii) ± 3 standard deviations of the results of expert laboratories; (iii) quality specifications based on biological variability, state-of-the-art and clinicians' opinions and (iv) the same quality specifications adjusted for the uncertainty of the assigned value. The first two strategies resulted in very wide ranges of acceptable variability. Conversely, the strategy based only on quality specifications resulted in very narrow ranges. For the fourth strategy, acceptable ranges were intermediate between the results produced with the other strategies. The third and fourth strategies did not produce observable differences in acceptable ranges when the model used for calculating the specifications of analytical quality was changed. It is essential that EQAPs for semen parameters should determine the ranges for acceptable variability in results. Moreover, these ranges must be clinically useful, i.e. the variability should have a minimal negative impact on clinical decisions. The exact definition of 'expert laboratory' is more important than the model chosen for estimating analytical quality specifications in an EQAP for semen parameters in basic semen analysis.

Minimum analytical quality specifications of inter-laboratory comparisons: agreement among Spanish EQAP organizers

Four Spanish scientific societies organizing external quality assessment programs (EQAP) formed a working group to promote the use of common minimum quality specifications for clinical tests. Laboratories that do not meet the minimum specifications are encouraged to make immediate review of the analytical procedure affected and to implement corrective actions if necessary. The philosophy was to use the 95th percentile of results sent to EQAP (expressed in terms of percentage deviation from the target value) obtained for all results (except the outliers) during a cycle of 1 year. The target value for a number of analytes of the basic biochemistry program was established as the overall mean. However, because of the substantial discrepancies between routine methods for basic hematology, hormones, proteins, therapeutic drugs and tumor markers, the target in these cases was the peer group mean. The resulting specifications were quite similar to those established in the US (CLIA), and Germany (Richtlinie). The proposed specifications stand for the minimum level of quality to be attained for laboratories, to assure harmonized service performance. They have nothing to do with satisfying clinical requirements, which are the final level of quality to be reached, and that is strongly recommended in our organizations by means of documents, courses, symposiums and all types of educational activities.

European Specialist Porphyria Laboratories: Diagnostic Strategies, Analytical Quality, Clinical Interpretation, and Reporting As Assessed by an External Quality Assurance Program

The porphyrias are a group of rare metabolic disorders whose diagnosis depends on identification of specific patterns of porphyrin precursor and porphyrin accumulation in urine, blood, and feces. Diagnostic tests for porphyria are performed by specialized laboratories in many countries. Data regarding the analytical and diagnostic performance of these laboratories are scarce. We distributed 5 sets of multispecimen samples from different porphyria patients accompanied by clinical case histories to 18-21 European specialist porphyria laboratories/centers as part of a European Porphyria Network organized external analytical and postanalytical quality assessment (EQA) program. The laboratories stated which analyses they would normally have performed given the case histories and reported results of all porphyria-related analyses available, interpretative comments, and diagnoses. Reported diagnostic strategies initially showed considerable diversity, but the number of laboratories applying adequate diagnostic strategies increased during the study period. We found an average interlaboratory CV of 50% (range 12%-152%) for analytes in absolute concentrations. Result normalization by forming ratios to the upper reference limits did not reduce this variation. Sixty-five percent of reported results were within biological variation-based analytical quality specifications. Clinical interpretation of the obtained analytical results was accurate, and most laboratories established the correct diagnosis in all distributions. Based on a case-based EQA scheme, variations were apparent in analytical and diagnostic performance between European specialist porphyria laboratories. Our findings reinforce the use of EQA schemes as an essential tool to assess both analytical and diagnostic processes and thereby to improve patient care in rare diseases.

Aplicación de las especificaciones de calidad analíticas al cálculo de los límites admisibles de error en Programas de Evaluación Externa de la Calidad para concentración espermática

Introduction External quality assessment is an essential activity of the andrology laboratory. To evaluate the effectiveness of the laboratories participating in the Programme of External Quality Monitoring (PSEC) allowable limits of error are used, and the result of a laboratory is considered acceptable if it is within these limits. Currently allowable error limits depend to a large extent on the criteria of the organizers of the PSEC. Our goal is to compare the different criteria described in ISO 13 528:2005 for the calculation of the allowable limits of error discussed in the PSEC for sperm concentration, so that they can be used by all. Material and methods The data analysed in this study are the mean values and standard deviation (SD) obtained on independent control samples from two PSEC. Four different strategies for calculating the allowable limits of error according to ISO 13 528:2005 were set: 1) based on the results of participating laboratories, 2) based on the results of expert laboratories, 3) using quality specifications based on biological variability, state of the art and opinion of clinicians, and 4) using the quality specifications and adjusting for the uncertainty of the assigned value. Results Very large allowable limits of error were obtained with strategies 1 and 2, whereas strategy 3, based only on quality specifications, gave very narrow limits.. However, with strategy 4 we obtained values of intermediate acceptance intervals compared with the other strategies. There were no differences in the allowable limits of error in strategies 3 and 4 by varying the model for the calculation of analytical quality specifications. Discussion PSEC sperm concentration should use a strategy adjusted to the uncertainty of the value assigned to establish the allowable limits of error, since the acceptance intervals obtained are clinically useful, so that the values contained in them lead to clinically similar decisions. For expert laboratories to adequately define them is more important than the model chosen to estimate analytical quality specifications in a sperm concentratiońs PSEC.

Do new concepts for deriving permissible limits for analytical imprecision and bias have any advantages over existing consensus?

The Stockholm conference held in 1999 on "Strategies to set global analytical quality specifications (AQS) in laboratory medicine" reached a consensus and advocated the ubiquitous application of a hierarchical structure of approaches to setting AQS. This approach has been widely used over the last decade, although several issues remain unanswered. A number of new suggestions have been recently proposed for setting AQS. One of these recommendations is described by Haeckel and Wosniok in this issue of Clinical Chemistry and Laboratory Medicine. Their concept is to estimate the increase in false-positive results using conventional population-based reference intervals, the delta false-positive rate due to analytical imprecision and bias, and relate the results directly to the current analytical quality attained. Thus, the actual estimates in the laboratory for imprecision and bias are compared to the AQS. These values are classified in a ranking system according to the closeness to the AQS, and this combination is the new idea of the proposal. Other new ideas have been proposed recently. We wait, with great interest, as should others, to see if these newer approaches become widely used and worthy of incorporation into the hierarchy.

Closing the brain-to-brain loop in laboratory testing

Abstract The delivery of laboratory services has been described 40 years ago and defined with the foremost concept of "brain-to-brain turnaround time loop". This concept consists of several processes, including the final step which is the action undertaken on the patient based on laboratory information. Unfortunately, the need for systematic feedback to improve the value of laboratory services has been poorly understood and, even more risky, poorly applied in daily laboratory practice. Currently, major problems arise from the unavailability of consensually accepted quality specifications for the extra-analytical phase of laboratory testing. This, in turn, does not allow clinical laboratories to calculate a budget for the "patient-related total error". The definition and use of the term "total error" refers only to the analytical phase, and should be better defined as "total analytical error" to avoid any confusion and misinterpretation. According to the hierarchical approach to classify strategies to set analytical quality specifications, the "assessment of the effect of analytical performance on specific clinical decision-making" is comprehensively at the top and therefore should be applied as much as possible to address analytical efforts towards effective goals. In addition, an increasing number of laboratories worldwide are adopting risk management strategies such as FMEA, FRACAS, LEAN and Six Sigma since these techniques allow the identification of the most critical steps in the total testing process, and to reduce the patient-related risk of error. As a matter of fact, an increasing number of laboratory professionals recognize the importance of understanding and monitoring any step in the total testing process, including the appropriateness of the test request as well as the appropriate interpretation and utilization of test results.

Implementación de la variabilidad biológica como objetivo de la calidad en un laboratorio clínico

Introduction Various criteria have been developed to establish the quality that laboratories must achieve. At the Stockholm Conference a hierarchical model was established for analytical quality specifications. The aim of this study was to evaluate the biological variability in multiple biochemical parameters over one year. Materials and methods Every month, each parameter, was calculated for imprecision, systematic error (SE) and total error (TE), as well as calculating the relative variation coefficient (RVC). Results A total of 29 parameters were evaluated for routine and 31 for emergencies. For routine, the imprecision criteria, SE and TE were fulfilled in 80%, 81% and 97%, respectively; and for emergencies in 84%, 90% and 100% respectively. The sodium ion was the only parameter that did not fulfil any of the three criteria. The worst results were registered by those parameters with low biological variability. For the relative variation coefficient (RVC), only the sodium ion did not conform. There were no significant differences found between the results when comparing the quality specifications obtained between routine and emergency analyses. Discusion Even though the results obtained are acceptable, the analytical quality cannot be guaranteed for some parameters and should be reviewed. The best results were obtained using TE. Therefore, the Stockholm model for quality specifications should be implemented in both routine and emergencies laboratories.

Consenso sobre especificaciones mínimas de la calidad analítica para magnitudes hematológicas y de bioquímica especial

Introduction An Interdisciplinary Expert Committee was created by four Spanish Scientific Societies, organisers of external quality assurance programs, to provide laboratories a consensus of minimum analytical quality specifications with the aim of assuring similar analytical performance all over Spain. Material and method A total of 1,262,623 results was obtained from 3688 laboratories and 24 periods studied (2005 and 2006). For each analyte, the absolute difference between the result and the comparative value (peer group mean), expressed in percentage, was calculated and named “error”. From the error frequency distribution, the 95 percentile was considered as “candidate specification”. This value was compared with the specification used in Germany and USA and, in the case of being higher than one of them, the “candidate specification” value was increased iteratively, until 90% of participating laboratories had 75% of their results within the “candidate specification”. This value was considered to be the specification and the process ended. Results and conclusion Specifications for 37 analytes (3 immunology, 5 therapeutic drug monitoring, 15 hormones and tumour markers, 14 haematology and coagulation are proposed. They are considered to be the minimum level of quality that each laboratory had to reach, to assure common analytical performance. If the results in the external quality assurance program exceed these specifications, an immediate review should be made and, if necessary, corrective actions should be taken.

Strategies to set global analytical quality specifications in laboratory medicine: 10 years on from the Stockholm consensus conference

The setting of analytical quality specifications in laboratory medicine has attracted attention for many years. Over time, many strategies were advocated and all had advantages and disadvantages. In the final decade of the last millennium, considerable confusion existed on how to define analytical quality specifications correctly and how to apply them in everyday practice. This led to wide professional interest. In 1999, a consensus conference sponsored by IUPAC, IFCC and WHO was held in Stockholm on “Strategies to Set Global Analytical Quality Specifications in Laboratory Medicine”. The consensus set useful and well-documented strategies for the setting of analytical quality specifications into a hierarchy with the best strategy at the highest level, namely, (1) Evaluation of the effect of analytical performance on clinical outcomes in specific clinical situations, (2) Evaluation of the effect of analytical performance on clinical decisions in general, (3) Published professional recommendations, (4) Performance goals set by regulatory bodies and EQAS organisers, and (5) Goals based on the current state of the art. Much success has been achieved since the promulgation of the statement with the approach being adopted by many in laboratory medicine for a very wide variety of purposes, particularly in quality management. However, there is a requirement for additional investigation of, inter alia, quality specifications for examinations done on measurements performed on ordinal and nominal scales, pre-analytical factors and matrix effects, examinations done as POCT, target values of control materials, and ways in which analytical quality specifications can be used both to set what is the optimum performance and as a tool for assessment of everyday practice.

Oestradiol assays: fitness for purpose?

In this review we discuss the analytical inadequacies of oestradiol assays in relation to the clinical requirements for performing them, and make recommendations for their improvement. The measurement of oestradiol can be requested in a number of clinical scenarios (precocious puberty, infertility, assisted conception, hormone replacement therapy). The very wide dynamic range of oestradiol concentrations is a huge challenge for routine assays, which they are unlikely to meet on theoretical as well as practical grounds. The EQA performance of oestradiol assays in terms of trueness, comparability, recovery and analytical sensitivity leaves much to be desired and indicates that calibration is compromised by poor analytical specificity. To make oestradiol assays fit for purpose requires concerted action by all stakeholders to define analytical quality specifications for the various clinical scenarios involved, and then to encourage concerted action by the diagnostic industry to use the steroid reference measurement system to improve specificity, trueness and traceability.

Biological variation in inflammatory and hemostatic markers.

Concentrations of inflammatory and hemostatic variables are influenced by biological variation, which is the natural within-subject variation over time. The aim of this study was to determine fibrinogen, C-reactive protein (CRP), platelet aggregation, thrombin generation and prothrombin time (PT): (i) the number of repeated measurements needed to obtain the true habitual concentration of an individual; (ii) the recommended analytical imprecision for diagnosis and monitoring; (iii) the recommended analytical bias; (iv) the contribution of analytical imprecision to test result variability; (v) the index of individuality; (vi) the reference change value; and (vii) the seasonal variation. We collected 520 blood samples over a 1-year period from 40 healthy individuals, and determined the between-subject, within-subject and seasonal variation in fibrinogen, CRP, platelet aggregation, thrombin generation and PT. One or two repeated measurements were sufficient to establish the true habitual concentration, except for platelet aggregation and peak thrombin generation, where at least four and nine repeated measurements were needed, respectively. For diagnosis, the maximal recommended coefficient of analytical variation (CV) was 4%-27%, except for CRP (77.7%). For monitoring, these CVs were on average 3% lower. Recommended analytical bias varied between 1.7% and 33.2%. Finally, seasonal variation was observed in concentrations of fibrinogen and thrombin generation, which could explain approximately 11% of their total variation. This study provides insights into the biological variability of selected inflammatory and hemostatic markers, which can be used for sample size calculations and to determine the analytical quality specifications for their respective assays.

Influence of temperature and time before centrifugation of specimens for routine coagulation testing

The accurate standardization of the preanalytical phase is of pivotal importance for achieving reliable results of coagulation tests. Because information on the suitable storage conditions for coagulation testing is controversial, we aimed at investigating the sample stability with regard to the temperature and time before centrifugation. The activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen and D-dimer were assayed in specimens collected from 26 consecutive patients on antivitamin K therapy on the ACL TOP analyzer. Three primary 3.6-ml siliconized evacuated tubes containing 0.109 mol/l buffered trisodium citrate were sequentially collected from each patient. These three tubes were mixed, pooled and divided into seven identical aliquots. The first aliquot was immediately centrifuged according to the standard protocol [1500 g for 15 min at room temperature (RT)] and analyzed. The other aliquots were left for 3, 6 and 24 h, respectively, at RT or 4 degrees C, and then centrifuged and analyzed. Test results were compared with those obtained on the reference specimen. Statistically significant prolongations were observed for aPTT in all the samples. Such differences exceeded the analytical quality specifications for desirable bias in the samples stored for 24 h. A significant reduction, yet comprised within the desirable bias, was observed for PT and fibrinogen in uncentrifuged specimens stored at RT for 3 and 6 h. No significant biases could be recorded in D-dimer. In conclusion, a 6-h storage of uncentrifuged specimens at either RT or 4 degrees C may still be suitable to achieve results of routine coagulation testing comprised within the analytical quality specifications for desirable bias.

Biological variation in the plasma concentrations of polyunsaturated long chain fatty acids (PUFA) in older adults

Within‐ and between‐subject biological variation is often used to set analytical quality specifications and to evaluate the significance of changes in serial results. A placebo‐controlled PUFA intervention trial allowed estimation of intra‐ (CVi) and inter‐ (CVg) subject coefficients of variation of plasma concentrations of four n‐3 and six n‐6 PUFA in older adults (> 60 years old) all of whom were being supplemented with lutein and zeaxanthin for macular degeneration. Fasting blood samples were collected from 40 subjects at month 0 and from 20 subjects at months 1, 2, 3, 6, and 9 (placebo group). PUFA concentrations (umol/L) were quantitated using negative ion chemical ionization GC‐MS (Mol Gen Metab 2001;73: 38) with some modification. Analytical imprecision (CVa), calculated using inter‐assay CV for three quality control samples measured in duplicate over 20 assays, ranged from 12‐20%. CVg were calculated from the baseline blood draw (n=40). CVi were calculated using data from baseline and 5 subsequent blood draws (n=20). CVg was largest for eicosapentaenoic acid (72%) and smallest for linoleic acid (22%). CVi was largest for eicosapentaenoic acid (33%) and smallest for arachidonic acid (11%). To our knowledge there is little information available about the biological variation of absolute PUFA concentrations, independent of total fatty acid composition, either in older persons or the general population.

Quality specifications for seminal parameters based on clinicians' opinions

Background. The aim of this study was to identify analytical quality specifications for seminal parameters based on clinicians' opinions, and to compare with those based on biological variability and state of the art. Material and methods. Two questionnaires with case histories were sent to laboratories participating in the Spanish programme of external quality on semen analysis for distribution to as many specialist clinicians as possible. Our intention was to determine the critical difference (CD), defined as the difference needed between two consecutive results obtained from semen analysis to be 95 % confident that the two results actually are different. Subsequently, we calculated the specifications of analytical quality in accordance with the clinicians' opinions. Results. The CDs obtained from the median value of the differences between the initial value and that given in the clinicians' replies were similar in clinical situations of improvement or worsening in the infertile normozoospermic male, and also in worsening situations for male patients presenting a significant alteration in seminal parameters. For improvement in this latter case, the CD cited as necessary in the clinicians' opinion was much higher than that for the other clinical situations. At a desirable level of quality, for concentration and total motility the coefficients of variation in the clinicians' opinion were below those based on biological variability and the state of the art. However, for type “a+b” motility, type “a” motility, morphology and vitality the coefficients of variation based on the clinicians' opinions were higher than those based on biological variability and lower than those based on the state of the art. Conclusions. Quality specifications for seminal parameters based on clinicians' opinions depend to a large extent on the clinical situation and on the seminal parameter being analysed.

Non-homogeneous separation of triglycerides, γ-glutamyltransferase, C-reactive protein and lactate dehydrogenase after centrifugation of lithium-heparin tubes

Clinical chemistry testing is influenced by a variety of preanalytical variables, including sample preparation. The presence of a diluted plasma layer at the top of primary tubes containing plasma citrate has recently been reported. However, no indication is available so far on the potential non-homogeneous distribution of clinical chemistry analytes during centrifugation of primary tubes containing lithium-heparin as an additive. A total of 40 lithium-heparin plasma samples were collected from volunteers and immediately centrifuged. An aliquot was obtained from the upper 0.4 mL of plasma (upper aliquot), 1.0 mL of plasma was discarded, and a second aliquot (lower aliquot) was obtained from the remaining plasma. The concentrations of alanine aminotransferase, albumin, alkaline phosphatase, amylase, amylase pancreatic, aspartate aminotransferase, direct bilirubin, total bilirubin, blood urea nitrogen, calcium, chloride, cholesterol, C-reactive protein (CRP), creatinine, creatine kinase, gamma-glutamyltransferase (GGT), glucose, high-density lipoprotein-cholesterol, iron, lactate dehydrogenase (LDH), magnesium, phosphate, potassium, total protein, sodium, triglycerides and uric acid were assayed on a Roche/Hitachi Modular System P according to the manufacturer's specifications and using proprietary reagents. Sodium, chloride and potassium were measured on a Roche/Hitachi Modular System using indirect ion-selective electrode methods. We observed a statistically significant difference between the upper and lower aliquots for CRP (3.88+/-0.67 vs. 3.94+/-0.68 mg/L; p=0.025), GGT (32.1+/-8.0 vs. 31.8+/-8.0 U/L; p=0.013), LDH (395+/-19 vs. 386+/-20 U/L; p=0.010) and triglycerides (1.29+/-0.09 vs. 1.27+/-0.09 mmol/L; p=0.001); results for the other analytes were not significantly different. In no case did the mean percentage bias recorded between aliquots exceed the current analytical quality specifications for desirable bias. The results of our investigation show that plasma layer stratification might occur in primary lithium-heparin tubes for a limited number of routine clinical chemistry tests, introducing a statistically significant bias in the measurement of GGT, LDH, triglycerides and CRP in the upper vs. the bottom section. When delayed testing is necessary for these parameters, we suggest that plasma should be separated after centrifugation and appropriately mixed before delayed/repeated analysis or aliquoting.

Homocysteine measurement by Vitros® Microtip homocysteine assay

Elevated circulating total homocysteine is an independent vascular risk factor. Enzymatic homocysteine measurements represent an alternative to HPLC- or immunochemistry-based assays, suitable for automation. Here, we report on analytical performance of a commercial cystathionine beta-synthase-based assay, for use on Vitros automated analyzers. Linear range, limit of detection and analytical sensitivity were inferred from duplicate measurements of homocystine standard solutions (1-65 micromol/L). Imprecision was assessed using commercial controls according to NCCLS EP5-A2 and accuracy using NIST-SRM1955 reference material. Agreement with a clinically validated HPLC method was examined on 207 patient samples. The enzymatic assay was linear from 1 to 90 micromol/L homocysteine. Total (within-day) imprecision ranged from 4.5 (3.9)% to 2.8 (1.6)% at homocysteine 9.7-43.2 micromol/L. Accuracy was acceptable at 8.9 and 17.7 micromol/L homocysteine, with +6.4% and -1.2% bias, respectively, but showed substantial negative bias (-20.1%) at 4.0 micromol/L. High triglycerides (19.8 micromol/L) negatively interfered. The enzymatic method was slightly less sensitive than the HPLC method (limit of detection 0.7 and 0.2 micromol/L, respectively) but correlated well with the latter (r2=0.9997, slope=1.04, intercept=-0.66 micromol/L) and was more precise (p<0.05). The Vitros homocysteine assay met the CLIA Desirable Analytical Quality Specifications at homocysteine > or = 9 micromol/L. Its analytical performance and suitability for automation make the Vitros assay an analytically acceptable alternative to HPLC-based methods.

About the choice of acceptability limits in external quality assessment schemes

Quality control schemes are a practical tool used in clinical laboratories and an essential element for any quality assurance process. In France, external quality assessment schemes (EQAS) can be mandatory (as national quality control organized by AFSSAPS) or voluntary as those suggested by French associations (ProBioQual, CTCB or Asqualab). These EQAS usually evaluate participants according to their performances: this ranking depends on acceptability limits which are here compared. Various examples based on ProBioQual's background illustrate difficulties to plan out analytical quality specifications. A comment is given about the best criteria (state of the art or biological variation mainly) to be considered to delimit analytic goals. This discussion includes approaches suggested by French committee on accreditation (Cofrac). All criteria could be criticized but it is important to compare oneself laboratory to peers and also to take account of biological variation.

National Academy of Clinical Biochemistry and IFCC Committee for Standardization of Markers of Cardiac Damage Laboratory Medicine Practice Guidelines: Analytical Issues for Biomarkers of Heart Failure

### A. Background In 2005, the IFCC C-SMCD recommended analytical and pre-analytical quality specifications for natriuretic peptide and their related co-metabolites assays.1 The objectives developed were intended to guide manufacturers of commercial assays and clinical laboratories that utilize these assays. The overall goal was to establish uniform criteria so that the analytical qualities and clinical performance of assays natriuretic peptide and their related co-metabolites could be evaluated objectively. As B-type natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) become more heavily integrated into clinical practice as diagnostic and prognostic biomarkers, understanding the differences between individual assays becomes important. Further, the influence of clinical, analytical and preanalytical factors on the growing number of BNP and NT-proBNP assays commercially available begs for a better understanding of how to interpret findings of different studies predicated on BNP or NT-proBNP concentrations monitored by different assays. The Laboratory Medicine community must also work closely with the in vitro diagnostics companies to assist in defining all of the assay characteristics,1 a process that was poorly orchestrated during the developmental phase of cardiac troponin assays. When BNP or NT-proBNP assays are used as biomarkers for diagnosis, therapy decisions, and prognosis, or used in clinical trials or studies, they should be well characterized, as suggested by the list of recommendations that follow. We recommend that when designing studies that will use BNP or NT-proBNP assays, investigators …

Biological variation of asymmetric dimethylarginine and related arginine metabolites and analytical performance goals for their measurement in human plasma

Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide synthase which is believed to be a cause of endothelial dysfunction and has been shown to predict the occurrence of acute coronary events. Data regarding the biological variation of arginine and its methylated derivatives are conspicuously absent from the literature. Such data are important in setting analytical quality specifications, assessing the utility of population reference intervals and assessing the significance of changes in serial results from an individual. Arginine, homoarginine, ADMA and symmetric dimethylarginine (SDMA) are measured in plasma by high performance liquid chromatography. Twelve healthy volunteers underwent weekly blood sampling for 20 weeks in order to determine the intra- and inter-individual biological variation of these analytes, from which analytical quality specifications, indices of individuality (II) and reference change values (RCV) are derived. Plasma samples from 100 healthy individuals were obtained in order to determine population reference intervals. ADMA and symmetric dimethylarginine (SDMA) exhibit low intra-individual biological variation of 7.4% and 5.8%, respectively, imposing desirable imprecision goals (CV(A)) of < or = 3.7% and 2.9% for these analytes. The described methodology achieves these goals, with analytical CVs of < 3.5% for all analytes. Goals for bias and total error were 3.1-10.1% and 7.2-16.0%, respectively. Reference intervals for ADMA and SDMA were 0.29-0.63 micromol L(-1) and 0.24-0.55 micromol L(-1), but have IIs < 1. RCVs were at least 20% for all analytes studied. Dimethylarginine concentrations are tightly controlled in health, with the result that imprecision goals for laboratory methods require to be low. Relatively large differences are required between serial results to denote a significant change. Population reference intervals for dimethylarginines are likely to be of limited value in detecting 'abnormality' in an individual from a single result.

Preparation of a Quality Sample: Effect of Centrifugation Time on Stat Clinical Chemistry Testing

Background: Centrifugation time is a major bottleneck in laboratory specimen throughput. In most cases, 10 minutes is suggested to centrifuge samples on a swing-out rotor centrifuge, at room temperature, with a relative centrifugal force of 1,200±100 g; and 1,300 g or centrifugation times longer than 10 to 15 minutes may be advisable to get a better platelet clearance. Nevertheless, there is little evidence supporting the influence of different centrifuge times for primary lithium-heparin tubes with plasma separator on stat clinical chemistry testing. Methods: Five evacuated tubes collected from 10 consecutive subjects were centrifuged on an identical swing bucket centrifuge at 1,200 g for 1, 2, 5, 10, and 15 minutes respectively and tested for clinical chemistry stat analytes. Results: Statistically significant variations from the 15-minute centrifuge reference specimen were observed for ALT, calcium, glucose, potassium, urea nitrogen, and CK-MB (1 minute centrifugation), ALT, glucose, urea nitrogen, and CK-MB (2 minutes centrifugation), and glucose (5 and 10 minutes centrifugation). Meaningful biases according to the analytical quality specifi-cations for clinically-allowable variance were recorded for ALT, calcium, glucose, potassium (1 minute centrifugation), and for ALT, glucose, and potassium (2 minutes centrifugation).