Total Imprecision Research Articles

The Roche Total Mycophenolic Acid® assay: An application protocol for the ABX Pentra 400 analyzer and comparison with LC–MS in children with idiopathic nephrotic syndrome

BackgroundFor TDM of mycophenolate acid (MPA), the Roche Total Mycophenolic Acid® assay based on the inhibition of recombinant inosine monophosphate dehydrogenase (IMPDH) has been shown to be a simple and reliable alternative to chromatographic methods. We have adapted this assay on the ABX Pentra 400 analyzer (HORIBA). ObjectiveTo investigate the analytical performances of the Roche Total Mycophenolic Acid® assay on the ABX Pentra 400 and to compare it to an LC-MS method using samples from children with nephrotic syndrome treated with mycophenolate mofetil (MMF). Material and methodsConfiguration of the open-channel on the ABX Pentra 400 was based on the Roche MPA assay package insert. Precision was determined as described in the CLSI protocol EP5-A2. Comparison with the LC-MS method was performed using 356 plasma samples from 42 children with nephrotic syndrome (8h pharmacokinetic profiles). ResultsThe enzymatic assay demonstrated high precision. The %CV for Within Run Imprecision ranged from 5.5% at 1.2mg/L to 1.5% at 14.1mg/L and Total Imprecision ranged from 9.3% to 2.5%. The method comparison with plasma samples from children yielded overall a good correlation and a good agreement between both methods. The Passing Bablok regression analysis showed the following results: [Roche MPA assay]=1.058 [MPA LC-MS] −0.06; rho=0.996. ConclusionThe Roche Total Mycophenolic Acid® assay is adaptable to the ABX Pentra 400 analyzer, and demonstrates accurate and precise measurement of MPA in plasma obtained from children with nephrotic syndrome.

Assessment of FUS-200 Performance: Comparison with Quantitative Urine Culture Shows that Identifying the Right Cutoff for Optimizing Analyzer Performance Is Challenging.

Urine sediment analysis is a frequently ordered test, since it permits screening for many clinical conditions. Here, the technical and bacteriuria diagnostic performance of FUS-200, a new sediment analyzer, was assessed. Carry-over, imprecision, and linearity were measured according to CLSI protocols. FUS-200 was compared to sediMAX™, our current laboratory analyzer, in terms of particle recognition/counting in 382 fresh urine samples, and bacteriuria diagnosis was based on white blood cell (WBC) and bacteria counts in a subgroup of the same samples. In the diagnostic study, quantitative bacterial cultures served to classify the samples as bacteria-positive or bacteria-negative. FUS-200 did not show carryover for the particles tested. Total imprecision for red blood cells (RBCs) and WBCs in positive controls was 3.6%-10.5% and complied with European guidelines. RBC and WBC recovery was linear. When FUS-200 particle counts were edited by a reviewer, concordance with sediMAX improved for epithelial cells, yeast, and crystals, and recognition of casts and crystals improved. FUS-200 concordance with sediMAX varied between 97% for yeast and 58% for bacteria and was satisfactory. FUS-200 detected bacteriuria better than sediMAX (P = 0.004). FUS-200 WBC and bacteria cutoff values based on the Youden index detected bacteriuria better than manufacturer cutoffs. The best sensitivity with which FUS-200 detected bacteriuria was 79%. Although casts and crystal recognition should be improved, the overall technical performance of FUS-200 was acceptable to good. FUS-200 exhibited good screening accuracy for bacteria and WBC. Editing only mildly influenced FUS-200 outcomes.

Practical LC-MS/MS Method for 5-Hydroxyindoleacetic Acid in Urine.

Serotonin, an endogenous biogenic amine found in enterochromaffin cells of the gastrointestinal tract, is produced in excess by carcinoid tumors. The primary urinary metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA), is used in the diagnosis and management of carcinoid disease. This study describes the development and validation of a dilute-and-shoot LC-MS/MS method for the measurement of 5-HIAA in urine. Samples were prepared by dilution in a 96-well format using an automated liquid handler. Chromatographic isolation of the analyte was achieved using a reversed-phase analytical column. A triple quadrupole mass spectrometer with electrospray ionization in positive mode was used for detection and quantification. Data were acquired by multiple reaction monitoring. Two transitions, quantifier (192.1/146.1) and qualifier (192.1/118.1), were monitored for the analyte and its stable isotope-labeled internal standard [5-hydroxyindole-4,6,7-d3-3-acetic-2,2-d2 acid (5-HIAA-d5)]. Chromatography was designed to elute the analyte outside of major suppression zones. Injection-to-injection time was 4 min. The method was validated for linearity, limit of quantification, accuracy, and imprecision. The analytical measurement range was 0.5-100 mg/L. Coefficients of variation for within-run, between-day, and total imprecision ranged from 0.8% to 5.4%. The method produced accurate 5-HIAA concentrations and correlated well (R = 0.9876) with a comparison HPLC method. Matrix effects were evaluated by post-column infusion of urine samples. An analytical specificity study of endogenous compounds, vitamins, medications, and drugs showed minimal interference. A simple, inexpensive LC-MS/MS method was developed for measurement of 5-HIAA in urine. Results from the assay can be used clinically to assess carcinoid tumors.

Analytical verification and quality assessment of the Tosoh HLC-723GX HbA1c analyzer

ObjectivesIon-exchange high-performance liquid chromatography (IE-HPLC) has long been used as a reproducible and versatile analytical tool for HbA1c measurement.In this study, we performed analytical verification and quality assessment of the recently introduced small IE-HPLC Tosoh HLC-723GX HbA1c analyzer, and a comparison of results to immunoassay (IA) and capillary electrophoresis (CE). Design and methodsThe total imprecision of Tosoh HLC-723GX was verified according to CLSI EP15-A2 protocol using commercial control materials (C-QC) and pooled human whole blood samples (HWB). The Sigma metric was used for the evaluation of quality targets. HbA1c results were compared to automated CE (MiniCap Flex Piercing, Sebia, France) and IA (Tina-quant HbA1c Gen 2, Cobas Integra 400+, Roche Diagnostics, USA) procedures. ResultsThe total imprecision of Tosoh HLC-723GX-HbA1c for IFCC(mmol/mol) and NGSP(%) units was: 1.91/1.25% (HbA1c=31mmol/mol/5.0%) and 0.51/0.63% (HbA1c=84mmol/mol/9.8%) for C-QC, and 0.39/0.2% (HbA1c=47mmol/mol/6.5%) and 0.77/0.46% (HbA1c=94mmol/mol/10.8%) in HWB samples, respectively. Bland-Altman analysis did not reveal any deviation of the results between Tosoh HLC-723GX and CE: mean difference 0.0% (95%CI: −0.02927 to 0.02653%), while the mean HbA1c difference against IA was −0.07% (95%CI: −0.1039 to −0.02765). At the selected HbA1c clinical decision level (48mmol/mol/6,5%), six sigma analysis gave σ value of 3.91, within a desirable classification of performance. ConclusionThe analytical performance of the Tosoh HLC-723GX complies with the rigorous quality criteria for clinical use of HbA1c, with the results comparable to the CE procedure. Tosoh HLC-723GX provides a plausible analytical choice for reliable HbA1c measurement in low-volume laboratories.

Development and implementation of an HPLC-ECD method for analysis of vitamin C in plasma using single column and automatic alternating dual column regeneration

ObjectivesVitamin C (l-ascorbic acid) is a water-soluble micronutrient necessary for human life. Inadequate intake can lead to the fatal disease scurvy. Measurement of vitamin C is used to assess nutritional status and to monitor supplementation. The goal of this study was to develop a chromatographic method for the quantitation of vitamin C in human plasma. Design and methodsSamples were prepared by protein precipitation, addition of internal standard, and reduction with dithiothreitol. Separation of ascorbic acid was accomplished by isocratic elution on a reverse-phase column; concentration was determined by coulometry. The method was validated through studies of assay linearity, sensitivity, imprecision, accuracy, analytical specificity, and carryover. ResultsThe new assay was developed using a single pump/single analytical column HPLC system. Results correlated well with our previously used spectrophotometric method. The analytical measurement range was 1.0–2500µmol/L. The injection-to-injection time was 13min. Subsequently, to increase method throughput and shorten turnaround time, a dual LC pump system with a 2-position/10-port switching valve capable of performing automatic alternating column regeneration was validated and implemented. The injection-to-injection time was reduced 2-fold to 6min. The method was linear to 5000µmol/L; limit of quantification was 1.9µmol/L. Total imprecision was less than 5%. ConclusionsWe have developed a robust method suitable for routine clinical measurement of vitamin C in plasma specimens. The method incorporates a simplified sample preparation and a stable, non-endogenous internal standard to specifically quantify vitamin C. Faster throughput was achieved by employing an automatic alternating column regeneration system.

Performance characteristics of the Access AMH assay for the quantitative determination of anti-Müllerian hormone (AMH) levels on the Access* family of automated immunoassay systems

ObjectivesAnti-Müllerian hormone (AMH) measurement is useful as an aid in the evaluation of ovarian reserve. In the past, its conventional use was restricted by the low-throughput and variability of existing manual AMH assays. We developed the automated Access AMH assay for the quantitative determination of AMH levels on the Access family of immunoassay systems. The analytical performance of this new assay was evaluated. Design and methodsSensitivity, dilution linearity, assay imprecision, AMH sample stability, lot-to-lot comparison and correlation with AMH Gen II assay (Beckman Coulter, Inc.) were evaluated. Reference intervals for Access AMH were established in healthy females, males, newborns (≤60days) and pediatric males classified by Tanner stages. ResultsThe limit of blank and limit of detection were below 0.0077 and 0.0098ng/mL, respectively. The limit of quantitation was 0.010ng/mL. The total imprecision ranged from 2.4 to 5.2%. Linearity was observed up to 24ng/mL. Sample storage at room temperature up to 48h, at 2–8°C up to 7days and at −20°C up to 15months had no impact on measured AMH. The correlation study gave a coefficient between 0.99 and 1 and a regression slope between 0.89 and 0.92. Excellent lot-to-lot comparability was observed on controls and patient samples with a maximum bias of 3.7% between 2.81 and 15.03ng/mL. ConclusionsThe fully automated Access AMH immunoassay demonstrates excellent analytical performance. As a consequence, the availability of this assay will represent a robust, fast and precise alternative to manual AMH assay testing.

Analytical evaluation of the novel Lumipulse G BRAHMS procalcitonin immunoassay

ObjectivesThis study was designed to evaluate the analytical performance of the novel Lumipulse G1200 BRAHMS procalcitonin (PCT) immunoassay. Design and methodsThis analytical evaluation encompassed the calculation of the limit of blank (LOB), limit of detection (LOD), functional sensitivity, intra- and inter-assay imprecision, confirmation of linearity and a comparison with the Vidas BRAHMS PCT assay. ResultsThe LOB, LOD and functional sensitivity were 0.0010ng/mL, 0.0016ng/mL and 0.008ng/mL, respectively. The total analytical imprecision was found to be 2.1% and the linearity was excellent (r=1.00) in the range of concentrations between 0.006–75.5ng/mL. The correlation coefficient with Vidas BRAHMS PCT was 0.995 and the equation of the Passing and Bablok regression analysis was [Lumipulse G BRAHMS PCT]=0.76×[Vidas BRAHMS PCT]+0.04. The mean overall bias of Lumipulse G BRAHMS PCT versus Vidas BRAHMS PCT was −3.03ng/mL (95% confidence interval [CI]: −4.32 to −1.74ng/mL), whereas the mean bias in samples with PCT concentration between 0–10ng/mL was −0.49ng/mL (95% CI: −0.77 to −0.24ng/mL). The diagnostic agreement was 100% at 0.5ng/mL, 97% at 2.0ng/mL and 95% at 10ng/mL, respectively. ConclusionsThese results attest that Lumipulse G BRAHMS PCT exhibits excellent analytical performance, among the best of the methods currently available on the diagnostic market. However, the significant bias compared to the Vidas BRAHMS PCT suggests that the methods cannot be used interchangeably.

Características operativas del método de electroquimioluminiscencia para la medida de la proteína epididimaria humana (HE4) en el analizador Cobas e411 (Roche Diagnostics®)

IntroductionHuman epididymis protein 4 (HE4) is an emerging tumor marker in diagnosis of ovarian cancer. Together with the cancer antigen CA 125 it can improve sensitivity and specificity by calculating the risk of ovarian malignancy algorithm (ROMA) score. Our purpose is to validate performance characteristics of the electrochemiluminiscent Cobas e411 assay for HE4, including the endogenous interference study. Material and methodsTwo different levels of commercial control were analyzed for imprecision and trueness study during 20 days. Limit of blank and limit of detection was assessed with 20 replicates of a sample lacking HE4. Linearity was assessed by regression analysis mixing specimens, with high HE4 concentration and a devoid analyte one. In addition carry-over assay and endogenous interference study were conducted. ResultsThe imprecision obtained for each level were 1.7 and 2.8% for within run, 2.59 and 1.28%, for between run and 4.67 and 4.47% for total imprecision. The relative biases were 6.11 and 1.54% for each level. The limit of blank was 1.25 pmol/L and the limit of detection 2.21 pmol/L. The linearity study yielded the following regression equation y=-18.22+0.98x, demonstrating a linear behavior. There was not significant carry-over. The method was not affected by hemolysis, bilirrubin, neither lipemia. ConclusionsElectrochemiluminiscence method for HE4 showed an excellent imprecision and trueness. The limit of detection resulted lower than the declared by the manufacturer. Linearity was verified within the limits studied. In addition the assay is devoid of carry-over and free of significant endogenous interferences.

Analytical evaluation of the BioPlex® 2200 25-OH vitamin D total assay

BackgroundTesting for 25-hydroxyvitamin D (25(OH)D) has increased dramatically over the past decade and several automated immunoassays exist to measure serum 25(OH)D. Here we assess the performance of the recently released automated Bio-Rad BioPlex® 2200 25-OH vitamin D immunoassay, claimed to equally detect 25(OH)D2 and 25(OH)D3, and compare its results against a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method and the well-established DiaSorin LIAISON® 25-OH vitamin D total immunoassay. MethodsImprecision was determined using third party controls over 20days. Linearity over the claimed measuring range was assessed using admixtures of a high and a low patient pool. Correlation between the BioPlex and LC–MS/MS (n=137) or the LIAISON (n=56) was assessed using patient samples with varying amounts of 25(OH)D3 and/or 25(OH)D2. ResultsThe total imprecision was 9.4%, 6.9% and 4.5% at concentrations of 39.4nmol/L, 70.6nmol/L and 242.8nmol/L, respectively. The assay was linear from 33.1–375.0nmol/L with a R2 of 0.993. Method comparison revealed a strong correlation between the BioPlex assay and LC–MS/MS for samples containing 25(OH)D2 alone (n=5; R2=0.999), 25(OH)D3 alone (n=119; R2=0.935) and both (n=13; R2=0.919). In samples tested by all three methods (n=56), the correlation between the BioPlex and the LIAISON (R2=0.853) was poorer than that of the BioPlex and LC–MS/MS (R2=0.942). ConclusionThe BioPlex assay is suitable for the measurement of total serum 25(OH)D. The strong correlation between the BioPlex assay and LC–MS/MS in detecting 25(OH)D2 and 25(OH)D3 provides evidence that the BioPlex assay is capable of the equivalent detection of both forms.

Analytical Evaluation of the DiaSys Albumin in Urine/CSF FS Kit for Urine Albumin Measurement Using a JEOL BioMajesty JCA-BM6010/C Analyzer

This article is available from http://www.labmedonline.org 2016, Laboratory Medicine Online This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Background: High albuminuria is defined as albumin excretion of >30 mg/24 hr or an albumin-to-creatinine ratio of 30 mg/g in a random urine sample. We assessed the analytical performance of the Albumin in Urine/CSF FS kit (DiaSys Inc., UK) using a BioMajesty JCA-6010/C analyzer (JEOL Inc., Japan). Methods: Urine albumin concentrations were measured by the Albumin in Urine/CSF FS kit using a BioMajesty JCA-BM6010/C analyzer. Imprecision, linearity, and carry-over were measured according to the Clinical Laboratory and Standards Institute documents EP10 and EP9. The assay was compared with the ALB-T TQ Gen.2 (Roche, Germany) assay on a Cobas8000 C702 (Roche, Germany), the Tina-Quant Albumin (Roche, Switzerland) assay on a Hitachi7600-210 (Hitachi, Japan), and an Abbott urine albumin assay (Abbott Laboratories, USA) on a TBA 200FR (Toshiba, Japan) using 50 random urine samples. Results: Within-run and total imprecision were 0.551-1.023% and 0.551-1.214%, respectively. Linearity ranged from 6.31 to 30.60 mg/dL, and functional sensitivity was 0.5 mg/dL. Results from the Albumin in Urine/CSF FS kit showed good correlation with the ALB-T TQ Gen.2 (r=0.987) and the Tina-Quant Albumin assays (r=0.991). However, the four assays categorized 18 of 50 urine samples into different albuminuria groups. Conclusions: Albumin in Urine/CSF FS testing on a BioMajesty JCA-BM6010/C analyzer showed good linearity, functional sensitivity, precision, and correlation with the ALB-T TQ Gen.2 and Tina-Quant Albumin assays. However, because some samples were categorized into different albuminuria groups by the different assays, further studies on the standardization of albuminuria assays are needed.

Quantitation of Aldosterone in Serum or Plasma Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Accurate determination of serum and plasma aldosterone is essential for screening, diagnosis, and subtype classification of primary aldosteronism (PA). Its measurement is also used in the investigation of adrenal incidentaloma, adrenal carcinoma, Addison's disease, congenital adrenal hyperplasia, renal artery stenosis, and renal tubular channelopathies. We describe a simple and robust method for the accurate and precise measurement of aldosterone in serum or plasma using liquid chromatography and tandem mass spectrometry (LC-MS/MS). After addition of internal standard, aldosterone is extracted from serum samples using supported liquid extraction (SLE) with methyl-t-butyl ether (MtBE). The MtBE is evaporated to dryness and sample is reconstituted with mobile phase before injection onto the LC-MS/MS and quantitation using an 8-point calibration curve. The assay calibration range is approximately 50-6500 pmol/L (0.16-234 ng/dL) with total imprecision between 6.8 and 4.1 % for concentrations between about 50 and 1000 pmol/L respectively.

LC-MS/MS Measurement of Parathyroid Hormone-Related Peptide.

Parathyroid hormone-related peptide (PTHrP) is involved in activating pathways, allowing tumor cells to form bone metastases. Measurement of PTHrP is used for the diagnosis and clinical management of patients suspected of hypercalcemia of malignancy. We developed an LC-MS/MS method for measuring PTHrP, established sex-specific reference intervals, and assessed the method's performance. PTHrP was enriched from plasma samples with rabbit polyclonal anti-PTHrP antibody conjugated to magnetic beads. Enriched PTHrP was digested with trypsin, and PTHrP-specific tryptic peptide was analyzed with 2-dimensional LC-MS/MS in multiple reaction monitoring mode. The lower limit of quantification was 0.6 pmol/L, and the upper limit of linearity was 600 pmol/L. Total imprecision was <10%. Very poor agreement was observed with the RIA (n = 207; Deming regression RIA = 0.059 × LC-MS/MS - 1.8, r = 0.483; Sy|x = 3.9). Evaluation of the clinical performance of the assay using samples from patients with and without hypercalcemia (n = 199) resulted in an area under the ROC curve of 0.874. In sets of consecutively analyzed routine samples of patients assessed for hypercalcemia, the PTHrP positivity rate by RIA (n = 1376) was 1.9%, and 26.6% by LC-MS/MS (n = 1705). Concentrations were below the lower limit of quantification in 95.6% of the samples by RIA and 2.0% by LC-MS/MS. PTHrP is a normal constituent in circulating blood and its concentrations are substantially underestimated by commercial RIAs, causing false-negative results in samples from patients suspected of hypercalcemia. Our observations suggest a link between increased concentrations of PTHrP in postmenopausal women with low body mass index and increased incidence of osteoporosis.

Measurement by a Novel LC-MS/MS Methodology Reveals Similar Serum Concentrations of Vitamin D-Binding Protein in Blacks and Whites.

Vitamin D deficiency is associated with poor bone health and other adverse health outcomes; however, the associations are greatly attenuated in black vs white individuals. One possible explanation for this attenuation is different concentrations of bioavailable vitamin D metabolites in plasma, which are estimated with equations that include the total concentration of vitamin D binding globulin (VDBG) and haplotype-specific dissociation constants. We developed a method to quantify VDBG with LC-MS/MS that could also identify the haplotypes/isoforms of VDBG present. We validated the method according to recent recommendations for publications of biomarker studies. We determined serum VDBG concentrations in samples from the Atherosclerosis Risk in Communities cohort and compared the results with a widely used monoclonal immunoassay. With 10 μL of serum or plasma, the lower limit of quantification for the assay (<20% CV) was 71 μg/mL. The assay was linear from 62 to 434 μg/mL, with total imprecision of 7.3-9.0% CV at approximately 250 μg/mL. Significant hemolysis interfered with quantification. The identification of isoforms was 97% concordant with genotyping (κ coefficient). Method comparison with immunoassay revealed significant isoform-specific effects in the immunoassay. Mean concentrations (SD) of VDBG by mass spectrometry were similar in whites and blacks [262 (25) vs 266 (35) μg/mL, respectively; P = 0.43]. Validated mass spectrometric methods for the quantification of proteins in human samples can provide additional information beyond immunoassay. Counter to prior observations by immunoassay, VDBG concentrations did not vary by race.

Evaluation of an Automated Chemiluminescent Immunoassay in Typing Detection of IgG Antibodies Against Herpes Simplex Virus.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are common infectious agents worldwide and the primary infection of HSV remains a major problem in the pregnant women in China nowadays. At present, typing detection of HSV is mainly based on ELISA in China. In this study, we evaluated the performance of a newly introduced chemiluminescent immunoassay assay (CLIA) for the determination of serum HSV-1 and HSV-2 immunoglobulin G (IgG) antibodies. The functional sensitivity of detecting HSV-1 and HSV-2 IgG were 0.7 Index and 0.6 Index, respectively. The repeatability and the total imprecision coefficient of variations were both below 10%, and the recoveries of these assays ranged from 90% to 110%. High concentration of hemoglobin, lipids, and bilirubin in samples did not affect the results. The infective rates of HSV-1 and HSV-2 were 919 (87.5%) and 169 (16.1%), respectively. HSV-1 seroprevalence was significantly higher than that of HSV-2 (P < 0.001). CLIA is an excellent method for HSV-1 and HSV-2 IgG measurement and can be used as a routine screening test. The infective rate of HSV was pretty high among women before pregnancy or in the period of pregnancy in Beijing.

Simultaneous quantification of cardiovascular disease related metabolic risk factors using liquid chromatography tandem mass spectrometry in human serum.

Recent observations from metabonomic studies have consistently found that branched-chain amino acids (BCAAs), aromatic amino acids (AAAs), glutamine (Gln), glutamic acid (Glu), Gln/Glu ratio, carnitine, and several species of acylcarnitines and lysophosphatidylcholines (LPCs) are possible risk factors for metabolic diseases such as diabetes mellitus (DM) and cardiovascular diseases (CVD). We described here a simple and reliable method for simultaneous quantification of these metabolic risk factors by liquid chromatography tandem mass spectrometry (LC-MS/MS). Serum samples were extracted with isopropanol, and the extracted metabolites were separated by hydrophilic interaction liquid chromatography (HILIC) and detected with electrospary ionization (ESI) inpositive ion mode with multiple reaction monitor (MRM) mode. All the metabolites were effectively separated within 5.5min. Analytical recoveries were in the range of 92.8-106.9%, with an average of 100.6%. The intra- run and total imprecisions for the measurement of these metabolites were 1.2-3.8% and 1.5-7.4%, respectively. Serum concentrations of the metabolites were analyzed in 123 apparently healthy volunteers. Significant associations between the metabolites and traditional CVD risk factors were observed. The newly developed LC-MS/MS method was simple, precise, and accurate and can be used as an efficient tool in CVD research and studies.

ARCHITECT STAT High Sensitive Troponin I Familiarization Study (FAM) in the Department of Laboratory Medicine, Collegium Medicum, Nicolaus Copernicus University in Bydgoszcz, Poland

Background. International guidelines recommend the use of cardiac troponin assays for early detection of acute myocardial infarction. New high-sensitivity assays along with improved precision and sensitivity, are now widely available, accelerating patient’s diagnosis, treatment and invasive therapy. In this study we evaluated analytical performance of the Abbott ARCHITECT STAT high-sensitive troponin-I immunoassay and its 99th percentile upper reference limit. Methods. We performed the analytical evaluation of the hs-cTnI assay using Abbott ARCHITECT i2000SR immunoanalyzers. Features of the assay including imprecision, detection limits, linearity of dilution, interferences and method comparisons were assessed, as well as the 99th percentile upper reference limits in a cohort of 427 presumably healthy individuals were established. Results. Total imprecision ranged from 3.1% to 4.7% and was lowest for the medium controls. The observed limit of blank, limit of detection and limit of quantitation assumed values of 0.1, 1.5 and 4.8 ng/L, respectively. Common interferences, sample dilution and carry over did not affect the hs-cTnI results. Hs-cTnI was detectable in 98% of presumably healthy individuals. The 99th percentile values were age and sex dependent in the presumably healthy, but not in the healthy individuals. Conclusion. The new high-sensitivity troponin-I assay has improved analytical features and may be a valuable diagnostic tool.

Relative contribution of biological variation and technical variables to zone diameter variations of disc diffusion susceptibility testing.

Disc diffusion is still largely based on manual procedures. Technical variations originate from inoculum preparation, variations in materials, individual operator plate streaking and reading accuracy. Resulting measurement imprecision contributes to categorization errors. Biological variation resembles the natural fluctuation of a measured parameter such as antibiotic susceptibility around a mean value. It is deemed to originate from factors such as genetic background or metabolic state. This study analysed the relative contribution of different technical and biological factors to total disc diffusion variation. For calculation of relative error factor contribution to disc diffusion variability, five experiments were designed keeping different combinations of error factors constant. A mathematical model was developed to analyse the individual error factor contribution to disc diffusion variation for each of the tested drug-species combinations. The contribution of biological variation to total diameter variance ranged from 10.4% to 98.8% for different drug-species combinations. Highest biological variation was found for Enterococcus faecalis WT and vancomycin (98.8%) and for penicillinase-producing Staphylococcus aureus and penicillin G (96.0%). Average imprecision of automated zone reading revealed that 1.4%-5.3% of total imprecision was due to technical variation, while materials, i.e. antibiotic discs and agar plates, contributed between 2.6% and 3.9%. Inoculum preparation and manual plate streaking contributed 6.8%-24.8% and 6.6%-24.3%, respectively, to total imprecision. This study illustrates the relative contributions of technical factors that account for a significant part of total variance in disc diffusion. The highest relative contribution originated from the operator, i.e. manual inoculum preparation and plate streaking. Further standardization of inoculum preparation and plate streaking by automation could potentially increase the precision of disc diffusion and improve the correlation of susceptibility reports with clinical outcome.

Head to head evaluation of the analytical performance of two commercial methotrexate immunoassays and comparison with liquid chromatography-mass spectrometry and the former fluorescence polarization immunoassay

Monitoring of plasma drug levels is mandatory in patients receiving high-dose methotrexate. This study evaluated the analytical performance of the novel Architect and the established ARK™ methotrexate immunoassay (running on the Roche Cobas© c502 analyzer) in comparison with liquid chromatography-mass spectrometry (LC-MS) and the TDx/TDxFLx Methotrexate II assay. Imprecision and linearity were verified for the Architect and ARK assay according to CLSI EP15-A3 and EP6-A guidelines, respectively. The reported limit of quantitation (0.04 μmol/L) was tested for both assays according to the CLSI EP17-A2 guideline. Correlation and agreement between the different assays were evaluated using residual plasma samples (n=153). Total imprecision was <6.3% and <9.5% for the Architect and ARK assay, respectively. The claimed linearity and limit of quantitation were confirmed for the Architect assay. For the ARK assay, imprecision at the limit of quantitation was <18% with a positive bias resulting in a high total error up to 58%, and hence the linearity could not be confirmed. Both assays showed strong correlations with the TDX assay and LC-MS but a positive bias of 12.2% and 20.5% in comparison to LC-MS for the Architect and ARK assay, respectively. For the ARK assay this bias increased dramatically for samples with concentrations towards the limit of quantitation. The Architect assay is suitable for monitoring plasma methotrexate, but the ARK assay showed unsatisfactory performance in the analysis of low concentrated samples. Unlike the TDX assay, both assays require manual dilution of samples at higher concentrations, which delays sample processing in clinical routine.

Simultaneous analysis of urinary metabolites for preliminary identification of primary hyperoxaluria.

The primary hyperoxalurias are inherited disorders of glyoxylate metabolism, which cause over-production of oxalate leading to urolithiasis and subsequent renal failure. Other metabolites may be produced in excess in the different forms of PH: glycolate in PH1, glycerate in PH2 and 4-hydroxy-2-oxoglutarate and 2,4-dihydroxyglutarate in PH3. The aim of this study was to set up and validate a method for the simultaneous analysis of these metabolites in urine and to evaluate its use for preliminary identification of primary hyperoxaluria prior to definitive diagnosis by genetic testing. Urine samples were derivitized by methoximation and silylation and extracted into organic solvent prior to analysis by gas chromatography mass spectrometry. Recovery of the analytes spiked into urine ranged from 91 to 103% and total analytical imprecision ranged from 3.0 to 13.6%. 4-Hydroxy-2-oxoglutarate was unstable in urine at room temperature, and preservation by acidification was required. Mean urinary glycolate, glycerate and 4-hydroxy-2-oxoglutarate or 2,4-dihydroxyglutarate (expressed as a ratio to creatinine) were significantly higher in patients with PH1, PH2 and PH3, respectively. Low 4-hydroxy-2-oxoglutarate was observed in some patients with PH3, probably due to the instability of this analyte, but all PH3 patients had elevated 2,4-dihydroxyglutarate. During five months of routine service, seven cases of PH were identified by this method and subsequently confirmed by gene sequencing including two with novel mutations in HOGA1. This study confirms that the method is useful in aiding the diagnosis of primary hyperoxaluria and can direct genetic testing.

Development and evaluation of analytical performance of a fully automated chemiluminescent immunoassay for protein induced by vitamin K absence or antagonist II

ObjectivesProtein induced by vitamin K absence or antagonist II (PIVKA-II), an abnormal form of prothrombin, has been used as an aid in the diagnosis of hepatocellular cancer (HCC) as a tumor marker. We developed a fully automated quantitative immunoassay for PIVKA-II on the ARCHITECT® i systems. The aim of this study was to evaluate the analytical performance of this assay. Design and MethodAssay imprecision, sensitivity, dilution linearity, high dose hook effect, sample type equivalency, assay interferences of potential interfering materials and correlation with Picolumi PIVKA-II (Eidia, Tokyo, Japan) were evaluated. ResultsThe percentage coefficient of variation (%CV) of total imprecision ranged from 2.8% to 5.4% with 10 levels of samples. The limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) were less than 0.63mAU/mL, 1.62mAU/mL, and 8.25mAU/mL, respectively. Linearity up to 30,000mAU/mL, no high dose hook effect, no difference among sample types and no interference of common drugs and endogenous substances were observed. Correlation study with the Picolumi PIVKA-II gave a correlation coefficient of 0.93 and a regression slope of 1.07. ConclusionsThe results demonstrate that the fully automated prototype ARCHITECT PIVKA-II assay is an accurate, highly sensitive and precise assay for the measurement of PIVKA-II levels in human sera and plasmas.