Analytical Imprecision Research Articles

Simultaneous quantification of 20 synthetic cannabinoids and 21 metabolites, and semi-quantification of 12 alkyl hydroxy metabolites in human urine by liquid chromatography–tandem mass spectrometry

Clandestine laboratories constantly produce new synthetic cannabinoids to circumvent legislative efforts, complicating toxicological analysis. No extensive synthetic cannabinoid quantitative urinary methods are reported in the literature. We developed and validated a liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for simultaneously quantifying JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, JWH-398, RCS-4, AM-2201, MAM-2201, UR-144, CP 47,497-C7, CP 47,497-C8 and their metabolites, and JWH-203, AM-694, RCS-8, XLR-11 and HU-210 parent compounds in urine. Non-chromatographically resolved alkyl hydroxy metabolite isomers were considered semi-quantitative. β-Glucuronidase hydrolyzed urine was extracted with 1ml Biotage SLE+ columns. Specimens were reconstituted in 150μL mobile phase consisting of 50% A (0.01% formic acid in water) and 50% B (0.01% formic acid in 50:50 methanol:acetonitrile). 4 and 25μL injections were performed to acquire data in positive and negative ionization modes, respectively. The LC–MS/MS instrument consisted of a Shimadzu UFLCxr system and an ABSciex 5500 Qtrap mass spectrometer with an electrospray source. Gradient chromatographic separation was achieved utilizing a Restek Ultra Biphenyl column with a 0.5ml/min flow rate and an overall run time of 19.5 and 11.4min for positive and negative mode methods, respectively. Quantification was by multiple reaction monitoring with CP 47,497 compounds and HU-210 ionized via negative polarity; all other analytes were acquired in positive mode. Lower and upper limits of linearity were 0.1–1.0 and 50–100μg/l (r2>0.994). Validation parameters were evaluated at three concentrations spanning linear dynamic ranges. Inter-day analytical recovery (bias) and imprecision (N=20) were 88.3–112.2% and 4.3–13.5% coefficient of variation, respectively. Extraction efficiencies and matrix effect (N=10) were 44–110 and −73 to 52%, respectively. We present a novel LC–MS/MS method for simultaneously quantifying 20 synthetic cannabinoids and 21 metabolites, and semi-quantifying 12 alkyl hydroxy metabolites in urine.

Influence of analytical bias and imprecision on the number of false positive results using Guideline-Driven Medical Decision Limits

BackgroundDiagnostic decisions based on decision limits according to medical guidelines are different from the majority of clinical decisions due to the strict dichotomization of patients into diseased and non-diseased. Consequently, the influence of analytical performance is more critical than for other diagnostic decisions where much other information is included. The aim of this opinion paper is to investigate consequences of analytical quality and other circumstances for the outcome of “Guideline-Driven Medical Decision Limits”. TermsEffects of analytical bias and imprecision should be investigated separately and analytical quality specifications should be estimated accordingly. Biological variation and analytical performanceUse of sharp decision limits doesn't consider biological variation and effects of this variation are closely connected with the effects of analytical performance. Such relationships are investigated for the guidelines for HbA1c in diagnosis of diabetes and in risk of coronary heart disease based on serum cholesterol. The effects of a second sampling in diagnosis give dramatic reduction in the effects of analytical quality showing minimal influence of imprecision up to 3 to 5% for two independent samplings, whereas the reduction in bias is more moderate and a 2% increase in concentration doubles the percentage of false positive diagnoses, both for HbA1c and cholesterol. Frequency of follow-up laboratory testsAn alternative approach comes from the current application of guidelines for follow-up laboratory tests according to clinical procedure orders, e.g. frequency of parathyroid hormone requests as a function of serum calcium concentrations. Here, the specifications for bias can be evaluated from the functional increase in requests for increasing serum calcium concentrations. Probability function for diagnosesIn consequence of the difficulties with biological variation and the practical utilization of concentration dependence of frequency of follow-up laboratory tests already in use, a kind of probability function for diagnosis as function of the key-analyte is proposed.

The Effect of High-Calorie Meal Consumption on Oxidative Stress and Endothelial Dysfunction in Healthy Male Adults

Several authors have reported the association of postprandial hypertriglyceridemia with oxidative stress, systemic inflammation and endothelial dysfunction. Our aim was to investigate the effect of high-calorie meal on blood markers of oxidative stress and endothelial dysfunction and the association of APOA5 -1131T/C and -250G/A hepatic lipase (HL) polymorphisms with postprandial triglyceride response. This study included 102 healthy male volunteers. All participants consumed a high-calorie meal (823 calories, 50 g fat, 28 g protein, 60 g carbohydrates). Total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, hsCRP, TAS and ICAM-1 were measured at fasting state and postprandially. APOA5 -1131T/C and -250G/A HL polymorphisms were also determined. Postprandial triglycerides were significantly increased (1.4 (1.1-2.1) vs. 2.4 (1.9-3.3) mmol/l, P<0.001). Average triglyceride increase was 1.0+/-0.7 mmol/l (65 %). Concentration of triglycerides, HDL-cholesterol, LDL-cholesterol, TAS and ICAM-1 differed significantly between the fasting state and postprandial measurements (P<0.001). However, those differences were within the limits of analytical imprecision. Other parameters did not change 3 h after the meal. Triglycerides response did not differ respective to the APOA5 and HL polymorphisms. Family history of hypertension and acute myocardial infarction were associated with higher postprandial triglyceride concentrations. Postprandial hypertriglyceridemia is not associated with increased concentrations of hsCRP, TAS and ICAM-1. Furthermore, APOA5 -1131T/C and -250G/A HL polymorphisms are not associated with different postprandial triglyceride response.

Measurement of plasma viscosity by free oscillation rheometry: imprecision, sample stability and establishment of a new reference range

Plasma viscosity (PV) is used in the investigation of hyperviscosity syndrome. We have evaluated the performance of a new bench-top free oscillation rheometer, ReoRox from Medirox, and established a new reference range. Performance of the free oscillation rheometry (FOR) method was evaluated examining within-run and between-run imprecision, duration of measurement and operator simplicity. Furthermore, influence of storage on the PV measurements was assessed and a reference range based on plasma from 585 healthy men and women was established. Imprecision of the free oscillation rheometer was comparable to other viscometers and performance of the instrument was satisfying. The non-parametrical reference range established was 1.22-1.44 mPa s at 37℃ (2.5th and 97.5th percentiles). We have established a FOR PV reference range to support clinical decision-making. Furthermore, we found high pre-analytical stability of the samples and an analytical imprecision comparable to other PV methods.

Quantitative Mass Spectrometry Analysis of Intact Hemoglobin A2 by Precursor Ion Isolation and Detection

Precise and accurate quantification of proteins is essential in clinical laboratories. Here, we present a mass spectrometry (MS)-based method for the quantification of intact proteins in an ion trap mass spectrometer. The developed method is based on the isolation and detection of precursor ions for the quantification of the corresponding signals. The method was applied for the quantification of hemoglobin (Hb) A2, a marker used for the diagnosis of a β-thalassemia trait. The α and δ globin chains, corresponding to total Hb and HbA2, respectively, were isolated in the ion trap at specific charge states and ejected without activation. Areas of the corresponding isolated precursor ions were used to calculate the δ to α ratio. Three series of quantifications were performed on 7 different days. The standard curve fitted linearly (R(2) = 0.9982) and allowed quantification of HbA2 over a concentration range from 3% to 18% of total Hb. Analytical imprecision ranged from 3.5% to 5.3%, which is enough to determine if the HbA2 level is below 3.5% or above 3.7%. In conclusion, our method reaches precision requirements that would be acceptable for the quantitative measurement of diagnostic proteins, such as HbA2, in clinical laboratories.

Challenges of serial troponin testing: A symphony in need for harmony

In an earlier issue of this journal [1], we reported on early rule-out and rule-in of non-STEMI using 3 h and 6 h protocols of high sensitivity cardiac troponin T in an emergency department population. With interest, we now read a letter to the editor from Dr. Lippi [2] referring to a recent studybyCardinaels et al. [3]who found several cTnT degradation products in sera of patients admitted for STEMI and underwent primary PCI. Lippi now raised the possibility that cTnT degradation products might confound serial troponin testing that is endorsed by current ESC Guidelines on patients with non-ST segment elevationacute coronary syndromes [4]. The observationof Cardinaels et al. [3] is interesting but requires further validation of Western blot findings, as well as clinical evaluation of its clinical relevance. Lippi and Cervellin [2] suspect that antibodies of the current hsTnT assaymay fail to detect variousmolecular isoformsdue toheterogeneous immunoreactivity. For support, they refer to Cardinaels et al. [3] who demonstrated a consistentdegradationpatternusingantibodies directed against the C-terminal end of cTnTas compared to the pattern seenwith standard cTnT antibodies, but no fragments when the antibody was directed against the N-terminal end. Notably, the epitope of the cTnT assay is located centrally and thereby protected against epitope loss by protease cleavage [5,6]. In fact, this issue might be more relevant for some cTnI assays due to a higher susceptibility to posttranslational modifications, and heterogeneous epitope locations [5]. In addition to the central location of the epitope, cTnT is not prone to oxidation– reduction, phosphorylation or major modifications [5]. Nevertheless, it would be scientifically interesting to investigate the effect of degradation on measurable cTnT and cTnI concentrations over time. In clinical practice, the strength of hsTnT is earlier andmore accurate detection of NSTEMI. For STEMI, monitoring of reperfusion success is nowadays less important than in the era of fibrinolytic therapy, and estimation of infarct size using values beyond the first 24 h is not within the label. Therefore, the hsTnT assay was optimized at the low concentration range and truncated at the upper range as compared to the 4th generation cTnT assay [7]. Due to the excellent negative predictive value of hsTn assays in large clinical trials [8,9], current ESC guidelines on NSTE-ACS [4] endorse an early rule-out protocol with cTnTmeasurements on admission and after 3 h (optionally after 6 h). The finding that intact cTnT disappears 4 h after admission to the ED, and cTnT fragments start to become themajor fraction of cTnT does however not support the notion that cTnT fragments might disturb the diagnostic performance of hsTnT for early rule-out of NSTEMI. In addition, the mechanisms behind the formation of cTn degradation products remain unclear. Further work is needed to investigate the occurrenceof degradationproducts after small myocyte necrosis as compared to large STEMI with the subsequent release of large quantities of proteases, or the role of ischemia/ reperfusion injury after primary PCI. Although, the concerns of Dr. Lippi cannot be ruled out fully, his hypothesis is not supported by clinical experience where clinicians are rather confronted with issues of overdiagnosis of NSTEMI due to reduced clinical specificity rather than underdiagnosis. At least, at the moment, no data support an underestimation of MI due to epitope loss or modification, and the study by Cardinaels et al. [3] studied cTnT degradation products in a patient population that is not in the scope of troponin testing. In addition, as this is thefirst report,findings require confirmationusingmass spectroscopy followed by peptide sequencing to validate the identity of peptides. Furthermore, testing for fragments should be executed in heparin plasma, as well. Another issue raised by Dr. Lippi [2] is the analytical imprecision of hsTnT values below the LoQ which depends on the analytical platform used. Accordingly, Lippi [2] questioned the interpretation of small absolute concentration values for the diagnosis of NSTEMI. In our study [1], a diagnosis of NSTEMI was not only based on the presence of respective hsTnTchanges but required that the first or second hsTnT value exceeded the 99th percentile value of 14 ng/L. So far, the symphony is ready to be played but musicians still need to practice for harmony.

Short-term estimation and application of biological variation of small dense low-density lipoproteins in healthy individuals

A number of methods have been developed to measure small dense low-density lipoprotein cholesterol (sd-LDL-C) to evaluate atherogenic risk in the population. However, to our knowledge there are no reports about the biologic variability of these lipoproteins. Therefore, the aim of this work was to estimate sd-LDL-C biological variability, and with this information establish quality specifications, index of individuality (II) and reference change values (RCV). To estimate within- and between-subject biological variability, sd-LDL-C in serum was measured in 24 individuals (11 female and 13 male) for 5 consecutive days and then, at 2 and 3 weeks. Quality specifications, II, and RCVs were estimated according to procedures described. Total within- and between-subject biological variability, expressed as coefficient of variation, was 9.1% and 20%. Meanwhile, within- and between-biological variability in female and men was 10.9% and 6.7%, and 22% and 17%, respectively. Desirable quality specification to the sd-LDL-C method was 4.6% for analytical imprecision, bias 5.5% and total allowable error of 11.4%; the II was 0.46 and the RCV (calculated at 95% and 99% of significance) was 27.1% and 35.7%, for the total data. Short-term biological variability components were determined, and then used to estimate quality specifications, II and RCV for sd-LDL-C precipitation assay. To our knowledge, this is one of the first reports about sd-LDL-C biological variability, so that this information can be used as a starting point to develop long-term studies of biological variability for sd-LDL-C.

High-sensitivity Cardiac Troponin: From Theory to Clinical Practice

Since 2000, cardiac troponin (cTn) has been the recommended biomarker for the evaluation of patients with a possible diagnosis of acute myocardial infarction (AMI). Most of the cTn immunoassays currently in use lack the analytical sensitivity to accurately measure the upper reference limit (URL), ie, the 99th percentile reference value, which is the guideline-mandated cutoff value for myocardial injury. Consequently, current methods fail to detect some cTn values slightly higher than the 99th percentile, which can be present during the earlier phases of non-ST-segment elevation myocardial infarctions, and miss some smaller infarctions all together. This shortcoming has led to the development of the so called high-sensitivity cTn assays (hs-cTn), which can measure cTn concentrations 5 to 10 times lower than current assays and do so with improved analytical imprecision. With these assays, the diagnosis of AMI will remain specific, but will increase in frequency. In addition, it is likely that the percentage of elevations due to ischemic heart disease will diminish, because other more occult causes of cardiac injury will be revealed as being more common than previously appreciated. Since 2010, cardiac troponin T (cTnT) has been the only high-sensitivity assay (hscTnT) available. Recently, a high-sensitivity assay for troponin I (hs-cTnI) was released for use in Europe and Asia and other assays will become available in the near term. This Editorial will attempt to clarify some of the controversial issues intrinsic to this developing field and will discuss the usefulness of hs-cTn in different clinical scenarios. It will also alert readers to gaps in our understanding, some of which have recently been revealed in an important Spanish study.

Biological variation of thiopurine methyltransferase enzyme activity: when has a significant change taken place?

Thiopurine methyltransferase (TPMT) enzyme activity is measured before initiating thiopurine therapy to reduce the risk of severe drug-associated myelotoxicity in patients with low enzyme activity. TPMT activity may vary over time in relation to drug treatment and patient clinical condition. What constitutes a significant change in TPMT activity can be derived from biological variation and analytical imprecision. A large national laboratory database was used to identify patients with three or more TPMT activity measurements. Variance of TPMT activity was analysed by determining the total coefficient of variation (CVTOT) of repeated measurements and by correlation with parameters including gender and follow-up time. Between-run analytical imprecision (CVa) was determined by replicate analysis (n = 314). Of 7383 patients with TPMT measurements, 136 were identified as having three or more measurements over time (range 3-14). Median CVTOT for individual patient results was 14.5% (range 2.5-36.7%). Analytical imprecision (CVa) was 10.3%. A reference change value (or critical difference) with 95% probability was calculated as 42%. Therefore, a change in measured TPMT activity above 42% should lead to considering sources of variation other than biological variation and analytical imprecision. TPMT enzyme activity needs to change by at least 42% to determine that a true change has taken place beyond biological variation and analytical imprecision. A single measurement of TPMT activity is sufficient for most clinical purposes.

A systematic review of data on biological variation for alanine aminotransferase, aspartate aminotransferase and γ-glutamyl transferase

Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transferase (GGT) are enzymes measured in serum or plasma to investigate liver disease. The aim of this work is to assess the validity of published biological variation (BV) data currently available for these enzymes. Publications containing BV data for ALT, AST and GGT were identified by searching PubMed using the following keywords: biological varia*, RCV, CV(w), CV(i), CV(b), and CV(g). The 95% confidence intervals for the within- and between-subject coefficients of variation were calculated using the analytical imprecision, the number of subjects, samples and replicates. The searches identified 10 publications with ALT, 14 with AST and nine with GGT data. The protocols presented in those publications as used were varied. The ranges of within-subject variation reported were: ALT: 11.1%-58.1%, AST: 3.0%-32.3% and for GGT: 3.9%-14.5%. The median values (ALT: 18.0%, AST: 11.9% and GGT: 13.8%) were similar to those listed in a BV database commonly used as a reference source. Published BV data for ALT, AST and GGT demonstrate a wide range of values derived from inconsistent protocols. The quality of the presentations of the data is variable. These findings raise concerns around the utility of the data currently available and highlight the need for critical appraisal of such publications. The working group on BV of the European Federation of Clinical Chemistry and Laboratory Medicine is undertaking work to develop a critical appraisal checklist for the production and publication of reliable BV data.

Influence of temperature and storage duration on measurement of activated partial thromboplastin time, D‐dimers, fibrinogen, prothrombin time and thrombin time, in citrate‐anticoagulated whole blood specimens

We report the effect of temperature and storage duration on a range of haematological analytes: activated partial thromboplastin time (APTT), D-dimers, fibrinogen (Fbg), prothrombin time (PT) and thrombin time (TT). All analytes were measured using a Sysmex CA7000 System with SIEMENS reagents. One hundred and sixty patients were divided into two groups: samples from group A (80 patients) were used to assess the effect of storage at room temperature (RT) (0, 4, 8 and 24 h), and samples from group B (80 patients) were used to determine the effect of storage at 4 °C. Percentage changes compared with baseline results (T = 0 result) were calculated, and clinically relevant differences were defined as a percentage change of >10%. Changes in APTT, D-dimer, Fbg, PT and TT results following 4, 8 and 24 h storage were statistically significant at RT and 4 °C. For D-dimer, Fbg, PT and TT at both RT and 4 °C, the mean percentage changes after all storage periods were <10%, but for APTT, the mean percentage change following 24 h storage was >10% and exceeded the analytical within-batch imprecision. We demonstrate that a storage time interval up to 24 h for D-dimers, Fbg, PT, and TT, and 8 h for APTT at either RT or 4 °C is acceptable.

Preparation of a portable point-of-care in vitro diagnostic system, for quantification of canine C-reactive protein, based on a magnetic two-site immunoassay

In this study, characterization of the binding kinetics and optimization of a magnetic permeability based point-of-care (POC) immunoassay system for quantification of canine C-reactive protein (cCRP) is described. The reagent is based on a two-site heterogeneous immunoassay system utilizing conjugated superparamagnetic nanoparticles (SPION) and silica particles, both particles carrying covalently linked antibodies directed to the cCRP analyte. Detection is carried out using a magnetic permeability-based small instrument, adjusted in order to apply it in a POC setting near the patients. The kinetic parameters are characterized and applied in the final design of the assay system. In the cCRP system studied, 90% of the binding between immobilized solid-phase silica antibody and cCRP is complete after only 15 s, and 30 s for the binding between the antibody on the SPION and the bound cCRP on the silica particle. Additionally, the binding rate constants are determined to be 149 and 30 M(-1)s(-1), respectively. The analytical sensitivity, clinical sensitivity, and imprecision verifies the clinical usefulness of the system. Also, quantification of cCRP, using the system described, in dog clinical samples from mixed breeds shows a high correlation to a commercially available comparative cCRP ELISA system (y = 0.98 × +3.2, R(2) = 0.98, n = 47). The immunoassay system described can thus provide the veterinarian a valuable tool for rapid diagnosis and monitoring of inflammatory diseases in dogs in a setting near the patients.

Performance of Multiplex Cytokine Assays in Serum and Saliva among Community-Dwelling Postmenopausal Women

Multiplexing arrays increase the throughput and decrease sample requirements for studies employing multiple biomarkers. The goal of this project was to examine the performance of Multiplex arrays for measuring multiple protein biomarkers in saliva and serum. Specimens from the OsteoPerio ancillary study of the Women’s Health Initiative Observational Study were used. Participants required the presence of at least 6 teeth and were excluded based on active cancer and certain bone issues but were not selected on any specific condition. Quality control (QC) samples were created from pooled serum and saliva. Twenty protein markers were measured on five multiplexing array panels. Sample pretreatment conditions were optimized for each panel. Recovery, lower limit of quantification (LLOQ) and imprecision were determined for each analyte. Statistical adjustment at the plate level was used to reduce imprecision estimates and increase the number of usable observations. Sample pre-treatment improved recovery estimates for many analytes. The LLOQ for each analyte agreed with manufacturer specifications except for MMP-1 and MMP-2 which were significantly higher than reported. Following batch adjustment, 17 of 20 biomarkers in serum and 9 of 20 biomarkers in saliva demonstrated acceptable precision, defined as <20% coefficient of variation (<25% at LLOQ). The percentage of cohort samples having levels within the reportable range for each analyte varied from 10% to 100%. The ratio of levels in saliva to serum varied from 1∶100 to 28∶1. Correlations between saliva and serum were of moderate positive magnitude and significant for CRP, MMP-2, insulin, adiponectin, GM-CSF and IL-5. Multiplex arrays exhibit high levels of analytical imprecision, particularly at the batch level. Careful sample pre-treatment can enhance recovery and reduce imprecision. Following statistical adjustments to reduce batch effects, we identified biomarkers that are of acceptable quality in serum and to a lesser degree in saliva using Multiplex arrays.

A proposed reference change value for an IgA anti-tissue transglutaminase immunoassay to improve interpretation of serial results in celiac patients

BackgroundCeliac disease (CD) is an autoimmune disorder caused by an inappropriate immunological response to gluten ingestion in genetically susceptible individuals. IgA anti-tissue transglutaminase (tTG) antibodies have been widely employed as a specific biochemical marker for CD. Recent studies have also shown its usefulness in evaluating patient compliance with a gluten-free diet. MethodsA group of 28 subjects with CD was selected for the study. Each fulfilled the requirement of a gluten-free diet for more than one year. IgA anti-tTG determination was performed every two months for half a year. These data were used to estimate the biological variation (BV) of IgA anti-tTG in celiac patients and to calculate the reference change value (RCV). ResultsThe within-subject biological variation (CVI) and between-subject biological variation (CVG) were 19.2% and 75.6%, respectively, and the index of individuality was 0.25. The RCV calculated using these data together with our analytical imprecision (5.7%) was 55.5% for a 95% level of significance. ConclusionsWe have determined for the first time the BV and the RCV for IgA anti-tTG in a celiac population. This value and the probability curve generated from our data could be a valuable tool for monitoring patients' adherence to dietary treatment.

Reference values for urinary neutrophil gelatinase-associated lipocalin (NGAL) in pediatric age measured with a fully automated chemiluminescent platform

Neutrophil gelatinase-associated lipocalin (NGAL) has been suggested as the most promising biomarker of acute kidney injury. However, there are no reliable data on analytical sensitivity and reference limits of urinary NGAL (uNGAL) assay in pediatric age. The aim of the present study is to evaluate the analytical sensitivity and the reference range of uNGAL measured in urine specimens of pediatric age with the fully automated platform ARCHITECT i1000. A total of 333 urine samples were collected from 25 healthy newborns (16 males and 9 females; age 1-4 days) and 308 children (150 males and 147 females; mean age 80.7 months, range 0.63-248 months) and assayed for uNGAL by two different Italian centers (Department of Laboratory Medicine of the Fondazione Toscana G. Monasterio of Pisa and Massa and the Clinical Pathology Laboratory Unit of Istituto Giannina Gaslini of Genova). The calculated limits of blank (LOB) and detection (LOD) values were 0.5 ng/mL and 0.95 ng/mL, respectively. The distribution of uNGAL values approximated a log-normal distribution (median 5.2 ng/mL, interquartile range 2.5-12.8 ng/mL, 99th percentile 117.6 ng/mL). uNGAL values of the 25 neonates were significantly higher than those of 308 children (neonates: mean 44.2 ng/mL, median 30.3 ng/mL, range 5.2-137.4 ng/mL; children: mean 10.2 ng/mL, median 4.6 ng/mL, range 0.2-146.7 ng/mL; p<0.0001). uNGAL assay shows a good analytical sensitivity and imprecision, which allows the measurement of uNGAL values around the cut-off value (i.e., 117.6 ng/mL) with an imprecision <5 CV%. The distribution of uNGAL values in pediatric age approximates a log-normal distribution, with values which are higher in neonates compared to children.

Which point-of-care creatinine analyser for radiology: direct comparison of the i-Stat and StatStrip creatinine methods with different sample types

Availability of whole blood creatinine estimation for patients scheduled to undergo radiological contrast investigations can provide information to aid patient care by reducing adverse effects and improving departmental efficiencies. We performed imprecision studies, different patient sample type comparison in 40 participants, and a limited interference study with dopamine and dobutamine on the i-Stat and StatStrip point-of-care enzymatic analysers with the Beckman DxC800 Jaffe assay. Imprecision results showed that the i-Stat performed better. Patient comparison data indicated that the i-Stat provided better correlation than the StatStrip for all the different sample types with correlation coefficients (r(2)) being 0.995-0.996 and 0.918-0.995, respectively. The i-Stat results had a small positive bias of 6-9% for the three different sample types, which required different reference intervals. The StatStrip method showed greater scatter and overall small negative bias of -6% for the whole blood samples and a 10% positive bias with the plasma samples. Dopamine caused significant positive interference with the i-Stat only while dobutamine caused a small negative bias with the StatStrip method only. The findings indicated there are differences offered by the two systems. The StatStrip requires a very small finger prick capillary sample, calculates estimation of the glomerular filtration rate and has an adjustment option to improve correlation with the local method. The i-Stat offers better analytical imprecision and patient comparison with the laboratory method with the three sample types but showed significant interference from dopamine. A final consideration was the availability of middleware to capture patient results with the i-Stat. Based on all the study data, the i-Stat was recommended.

An improved candidate reference method for serum potassium measurement by inductively coupled plasma-mass spectrometry

BackgroundIn 2002, the National Institute of Standards and Technology (NIST) established a reference method for serum potassium based on inductively coupled plasma-mass spectrometry (ICP-MS). The aim of this study was to develop an inexpensive and improved candidate reference method for accurate and precise determination of serum potassium. MethodsSerum samples were diluted with 1% HNO3 supplemented with 59Cobalt as isotope internal standard, and potassium was measured by ICP-MS. The new method was evaluated with NIST standard reference materials (SRMs), according to the Clinical and Laboratory Standard Institute's evaluation protocols. ResultsAt 4.300 and 4.678mmol/l levels, the present method demonstrated analytical imprecision of 0.09% and 0.14%, and recoveries of 99.67% to 99.88%, respectively. The bias between the target values of SRMs were −0.02% to +0.28%, respectively. This method was linear between 0.0000 and 6.87mmol/l (R2=1.000). The method had an uncertainty (U95%) of 0.76%. ConclusionsThe proposed ICP-MS method to measure serum potassium is precise and accurate, with high sensitivity and specificity. It may be considered as a candidate reference method for the determination of serum potassium.

Repeating Tests: Different Roles in Research Studies and Clinical Medicine

Researchers often decide whether to average multiple results in order to produce more precise data, and clinicians often decide whether to repeat a laboratory test in order to confirm its validity or to follow a trend. Some of the major sources of variation in laboratory tests (analytical imprecision, within-subject biological variation and between-subject variation) and the effects of averaging multiple results from the same sample or from the same person over time are discussed quantitatively in this article. This analysis leads to the surprising conclusion that the strategy of averaging multiple results is only necessary and effective in a limited range of research studies. In clinical practice, it may be important to repeat a test in order to eliminate the possibility of a rare type of error that has nothing to do analytical imprecision or within-subject variation, and for this reason, paradoxically, it may be most important to repeat tests with the highest sensitivity and/or specificity (i.e., ones that are critical for clinical decision-making).

Confidence Intervals and Power Calculations for Within-Person Biological Variation: Effect of Analytical Imprecision, Number of Replicates, Number of Samples, and Number of Individuals

Reliable estimates of within-person biological variation and reference change value are of great importance when interpreting test results, monitoring patients, and setting quality specifications. Little information has been published regarding what experimental design is optimal to achieve the best estimates of within-person biological variation. Expected CIs were calculated for different balanced designs for a 2-level nested variance analysis model with varying analytical imprecision. We also simulated data sets based on the model to calculate the power of different study designs for detection of within-person biological variation. The reliability of an estimate for biological variation and a study's power is very much influenced by the study design and by the ratio between analytical imprecision and within-person biological variation. For a fixed number of measurements, it is preferable to have a high number of samples from each individual. Shortcomings in analytical imprecision can be controlled by increasing the number of replicates. The design of an experiment to estimate biological variation should take into account the analytical imprecision of the method and focus on obtaining the highest possible reliability. Estimates of biological variation should always be reported with CIs.

Robustness of ProsVue™ linear slope for prognostic identification of patients at reduced risk for prostate cancer recurrence: Simulation studies on effects of analytical imprecision and sampling time variation

ObjectiveThe ProsVue assay measures serum total prostate-specific antigen (PSA) over three time points post-radical prostatectomy and calculates rate of change expressed as linear slope. Slopes≤2.0pg/ml/month are associated with reduced risk for prostate cancer recurrence. However, an indicator based on measurement at multiple time points, calculation of slope, and relation of slope to a binary cutoff may be subject to effects of analytical imprecision and sampling time variation. We performed simulation studies to determine the presence and magnitude of such effects. Design and methodsUsing data from a two-site precision study and a multicenter clinical trial of 304 men, we performed simulation studies to assess whether analytical imprecision and sampling time variation can drive misclassifications or classification switching of patients with stable disease or recurrence. ResultsAnalytical imprecision related to expected PSA values in a stable disease population results in ≤1.2% misclassifications. For populations with recurrent disease, an analysis taking into account correlation between sampling time points demonstrates that classification switching across the 2.0pg/ml/month cutoff occurs at a rate ≤11%. In the narrow region of overlap between populations, classification switching maximizes at 12.3%. Lastly, sampling time variation across a wide range of scenarios results in 99.7% retention of proper classification for stable disease patients with linear slopes up to the 75th percentile of the distribution. ConclusionsThese results demonstrate the robustness of the ProsVue assay and the linear slope indicator. Further, these simulation studies provide a potential framework for evaluation of future assays that rely on the rate of change principle.