Intermediate Imprecision Research Articles

Assessment of the Androstenedione Assay on a Roche Analytical Platform and Comparison with Different Methods.

D4-androstenedione (D4ASD) is an intermediate hormone of androgen biosynthesis by the gonads and the adrenal glands. The interest in D4ASD concentration assessment resides in diagnostics of androgenic hyperproduction pathologies. Currently, many D4ASD quantification methods are available on the market including immunological methods that remain problematic due to the possible cross-reactivity with endogenous or exogenous steroids. Recently Roche® launched a new fully automated instrument for the measurement of D4ASD concentration. In this paper, the criteria of analytical performance (repeatability and intermediate precision) of the D4ASD Roche® assay were assessed and compared with 2 different methods including a radioimmunoassay (RIA) as well as a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Repeatability and intermediate precision of the D4ASD Roche® were acceptable according to the prede-fined RICOS standard (CV ≤ 7.9%) and the assay showed a good correlation with other assays considering the 95% CI obtained for the slope and the y-intercept. This method demonstrates acceptable criteria of analytical performance with an intermediate imprecision and a trueness within the fixed acceptance limits.

Evaluation of the Analytical Performances of the Biolabo SOLEA 100 Optical Coagulometer and Comparison with the Stago STA-R MAX Analyser in the Determination of PT, APTT, and Fibrinogen.

The Biolabo Solea 100 is a fully automated coagulation analyser using an optical system to detect coagulation designed to meet the needs of small- and medium-sized laboratories. This study aimed to evaluate the analytical performance in terms of bias, precision, and interference of the Biolabo Solea 100 coagulometer under routine laboratory conditions. In addition, a comparison was made with Stago STA-R MAX. Imprecision and bias were evaluated for activated partial thromboplastin time (APTT), fibrinogen (FIB), and prothrombin time (PT) at the medical decision levels. The results of 200, 181, and 206 plasma samples for APTT, FIB, and PT, respectively, were compared with those obtained by Stago STA-R MAX. In addition, the interference level of bilirubin, haemoglobin, triglycerides, and fractionated heparin was evaluated. Repeatability, intermediate imprecision, bias, and total error are overall below the defined limits of acceptability. Of interest is the high degree of agreement between Solea 100 and STA-R MAX with respect to PT (s), which fits perfectly with the theoretical line of identity (y = 0 + 1.00x). No interferences were found within the limits stated by the manufacturer, with some exceptions for APTT with heparin and APTT and PT for higher bilirubin concentrations. In conclusion, the performance of the Solea 100 optical analyser is satisfactory and adequate for the determination of routine coagulation tests. Moreover, they are perfectly comparable to mechanical systems, such as STA-R MAX and other upper-level analysers, even considering the low interference levels under routine conditions.

Development of an LC-MS/MS Proposed Candidate Reference Method for the Standardization of Analytical Methods to Measure Lipoprotein(a).

Use of lipoprotein(a) concentrations for identification of individuals at high risk of cardiovascular diseases is hampered by the size polymorphism of apolipoprotein(a), which strongly impacts immunochemical methods, resulting in discordant values. The availability of a reference method with accurate values expressed in SI units is essential for implementing a strategy for assay standardization. A targeted LC-MS/MS method for the quantification of apolipoprotein(a) was developed based on selected proteotypic peptides quantified by isotope dilution. To achieve accurate measurements, a reference material constituted of a human recombinant apolipoprotein(a) was used for calibration. Its concentration was assigned using an amino acid analysis reference method directly traceable to SI units through an unbroken traceability chain. Digestion time-course, repeatability, intermediate precision, parallelism, and comparability to the designated gold standard method for lipoprotein(a) quantification, a monoclonal antibody-based ELISA, were assessed. A digestion protocol providing comparable kinetics of digestion was established, robust quantification peptides were selected, and their stability was ascertained. Method intermediate imprecision was below 10% and linearity was validated in the 20-400 nmol/L range. Parallelism of responses and equivalency between the recombinant and endogenous apo(a) were established. Deming regression analysis comparing the results obtained by the LC-MS/MS method and those obtained by the gold standard ELISA yielded y = 0.98*ELISA +3.18 (n = 64). Our method for the absolute quantification of lipoprotein(a) in plasma has the required attributes to be proposed as a candidate reference method with the potential to be used for the standardization of lipoprotein(a) assays.

Development and Technical Validation of an Immunoassay for the Detection of APP669-711 (Aβ-3-40) in Biological Samples.

The ratio of amyloid precursor protein (APP)669–711 (Aβ−3–40)/Aβ1–42 in blood plasma was reported to represent a novel Alzheimer’s disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aβ−3–x and the development and “fit-for-purpose” technical validation of a sandwich immunoassay for the measurement of Aβ−3–40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aβ immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aβ−3–40 with no appreciable cross reactivity with Aβ1–40 or N-terminally truncated Aβ variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aβ−3–40 with a quantitative assay range of 22 pg/mL–7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aβ42/Aβ−3–40 and Aβ42/Aβ40 ratios were lower in patients with dementia of the Alzheimer’s type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aβ42/Aβ−3–40 ratio as a novel biomarker candidate for Alzheimer’s disease has been set.

Analytical performances of a chemiluminescence immunoassay for SARS-CoV-2 IgM/IgG and antibody kinetics.

Background Coronavirus disease 2019, abbreviated to COVID-19, represents an emerging health threat worldwide as, after initial reports in China, it has continued to spread rapidly. The clinical spectrum of the disease varies from mild to severe acute respiratory distress syndrome (ARDS). Moreover, many patients can be asymptomatic, thus increasing the uncertainty of the diagnostic work-up. Laboratory tests play a pivotal role in the diagnosis and management of COVID-19, the current gold standard being real-time reverse transcription polymerase chain reaction (rRT-PCR) on respiratory tract specimens. However, the diagnostic accuracy of rRT-PCR depends on many pre-analytical and analytical variables. The measurement of specific COVID-19 antibodies (both IgG and IgM) should serve as an additional, non-invasive tool for disease detection and management. Methods The imprecision of the MAGLUMI™ 2000 Plus 2019-nCov IgM and IgG assays (Snibe, Shenzhen, China) was assessed by adopting the Clinical and Laboratory Standards Institute (CLSI) EP15-A3 protocol. Linearity of dilution and recovery was evaluated by means of mixes of high-level pools and low-level pools of serum samples. Immunoglobulin time kinetics were evaluated using a series of serum samples, repeatedly collected from COVID-19-positive patients at different times, from <5 days up to 26-30 days. Results Findings at the analytical validation of the assay carried out according to the CLSI EP15-A3 guideline demonstrated that imprecision and repeatability were acceptable (repeatability was <4% and <6% for IgM and IgG, respectively, whilst intermediate imprecision was <6%). In addition, results of dilution and recovery studies were satisfactory. The kinetics of COVID-19 antibodies confirmed previously reported findings, showing a rapid increase of both IgM and IgG after 6-7 days from the symptom onset. IgG had 100% sensitivity on day 12, whilst 88% was the higher positive rate achieved for IgM after the same time interval. Conclusions The findings of this study demonstrate the validity of the MAGLUMI 2000 Plus CLIA assay for the measurement of specific IgM and IgG in sera of COVID-19 patients, and for obtaining valuable data on the kinetics of both (IgM and IgG) COVID-19 antibodies. These data represent a pre-requisite for the appropriate utilization of specific antibodies for the diagnosis and management of COVID-19 patients.

Comparison of the Quantum Blue® reader Point-of-Care system versus ELISA technique for therapeutic drug monitoring of Infliximab levels

BackgroundInfliximab (IFX) is a monoclonal antibody used to treat patients with inflammatory bowel disease (IBD). For IFX therapeutic drug monitoring (TDM), the most commonly used analysis is enzyme-linked immunosorbent assays (ELISA) which do not allow results to be provided in real-time. The aim of this study was to compare the in-house ELISA (Promonitor IFX) with the much faster assay Quantum Blue® IFX (QB) for quantification of serum IFX concentration among IBD patients in maintenance IFX therapy. MethodsWe studied 30 serum samples from outpatients in IFX maintenance therapy at Copenhagen University Hospital Hvidovre, Denmark. Samples were used to compare IFX measurements from Promonitor IFX with QB. Therapeutic intervals of <3 μg/mL, 3–7 μg/mL and >7 μg/mL were equally covered. Differences were evaluated using Bland-Altman plots and Student t-test. Correlation was evaluated using x,y-plot and Pearson’s correlation coefficient. The intermediate imprecision (CV%) of QB was measured at two levels (3 μg/mL and 7 μg/mL). For qualitative comparison, weighted kappa statistics (κ) were determined after stratification of results by therapeutic interval. ResultsPromonitor IFX and QB were strongly correlated (r = 0.92, p < 0.001). The mean difference between Promonitor IFX and QB was −0.57 μg/mL (p = 0.2). The CV% of QB was 16.3% at 3 μg/mL and 16.7% at 7 μg/mL. Classification of results according to therapeutic interval showed almost perfect agreement (κ = 0.81). ConclusionsQB is a suitable alternative to Promonitor IFX for TDM in patients treated with IFX for IBD. The results revealed a strong correlation between methods, in particular at lower IFX concentrations, representing the most interesting clinical range. When the samples were stratified according to the therapeutic interval, an almost perfect agreement between the methods was observed.

Multicenter Evaluation of a High-Sensitivity Troponin I Assay and Verification of an Early Rule-Out Algorithm

The objectives of this study were to independently evaluate the analytical performance of the STAT high-sensitivity troponin I (hs-cTnI) assay on a recently launched and CE-marked integrated chemistry and immunoassay system, confirm acceptable performance of the assay in line with The Third Global MI Task Force recommendations, and confirm suitability of the assay for continued use of an early rule-out algorithm for acute coronary syndrome at our Trust. A multicenter evaluation of the analytical performance characteristics of the hs-cTnI assay on the Abbott Alinity ci series was performed in 5 clinical laboratories across Europe. Comparison studies were performed vs the existing Abbott ARCHITECT hs-cTnI assay. Passing and Bablok regression analysis revealed a slope of 0.99 [95% confidence interval (CI), 0.98-1.00] and an intercept of -0.09 ng/L (95% CI, -0.21-0.11). Intermediate imprecision ranged from 3.7% to 5.4%, 2.6% to 4.5%, and 2.0% to 6.1% at concentrations of 19.1-21.1 ng/L, 196.6-205.5 ng/L, and 15229-16265 ng/L, respectively. There was good concordance between the 2 assays at the early rule-out cutoff. Comparable analytical performance of the hs-cTnI assay on new Abbott Alinity ci series supports the continued use of the early rule out algorithm for patients with suspected ACS at our Trust.

Development of a mass spectrometry method for 1,25-dihydroxy vitamin D3 using immunoextraction sample preparation.

Background 1,25-Dihydroxy vitamin D3 (DHVD) is the active metabolite of vitamin D, required to maintain blood calcium concentrations. Measurement has proved challenging as it circulates in picomolar concentrations and must be differentiated from other dihydroxyvitamin D species. Clinically, it is essential to be able to determine the cause of hypercalcaemia, which may be due to DHVD excess. Methods The liquid chromatography-mass spectrometry (LCMS) assay which has been developed uses immunoextraction of 0.5 mL serum followed by Amplifex™ derivatization of the dried eluent, with the analysis using the SCIEX 6500+ instrument taking a run time of 11 min. Results The limit of quantitation was determined (15 pmol/L) and the method is linear up to at least 600 pmol/L. Repeatability ranged from 6.1% at 23 pmol/L to 2.5% at 172 pmol/L and intermediate imprecision was 15.6% at 26 pmol/L to 8.3% at 173 pmol/L. The method is unaffected by icterus, haemolysis or lipaemia. Good performance was achieved with the samples from the vitamin D external quality assessment scheme, demonstrating a negative bias compared with the all lab trimmed mean (average –13.8%) and the specific method group (average –7.75%). A negative bias was observed across the concentration range found in 78 patient samples in comparison to a commercial radioimmunoassay (mean –47.8%). This was not unexpected and is likely due to better specificity of the mass spectrometry assay and the lack of a commutable standard reference calibrator. Conclusions We have developed a sensitive and robust LCMS method for the analysis of DHVD in serum, utilizing immunoextraction and derivatization to provide specificity.

Different top-down approaches to estimate measurement uncertainty of whole blood tacrolimus mass concentration values

BackgroundValues of mass concentration of tacrolimus in whole blood are commonly used by the clinicians for monitoring the status of a transplant patient and for checking whether the administered dose of tacrolimus is effective. So, clinical laboratories must provide results as accurately as possible. Measurement uncertainty can allow ensuring reliability of these results. The aim of this study was to estimate measurement uncertainty of whole blood mass concentration tacrolimus values obtained by UHPLC-MS/MS using two top-down approaches: the single laboratory validation approach and the proficiency testing approach. Material and methodsFor the single laboratory validation approach, we estimated the uncertainties associated to the intermediate imprecision (using long-term internal quality control data) and the bias (utilizing a certified reference material). Next, we combined them together with the uncertainties related to the calibrators-assigned values to obtain a combined uncertainty for, finally, to calculate the expanded uncertainty. For the proficiency testing approach, the uncertainty was estimated in a similar way that the single laboratory validation approach but considering data from internal and external quality control schemes to estimate the uncertainty related to the bias. ResultsThe estimated expanded uncertainty for single laboratory validation, proficiency testing using internal and external quality control schemes were 11.8%, 13.2%, and 13.0%, respectively. ConclusionsAfter performing the two top-down approaches, we observed that their uncertainty results were quite similar. This fact would confirm that either two approaches could be used to estimate the measurement uncertainty of whole blood mass concentration tacrolimus values in clinical laboratories.

Point of care testing of fecal calprotectin as a substitute for routine laboratory analysis

ObjectivesFecal calprotectin (FC) is widely used to monitor the activity of inflammatory bowel disease (IBD) and to tailor medical treatment to disease activity. Laboratory testing of fecal samples may have a turnaround time of 1–2 weeks, whereas FC home testing allows results within hours and thus enables a rapid response to clinical deterioration. Design and methodsFifty-five stool samples were analyzed by the IBDoc® Calprotectin Home Testing kit and the BÜHLMANN fCAL® turbo assay on a Roche Cobas 6000 c501. The correlation between the assays was assessed using Spearman's Rho correlation coefficient and the intermediate imprecision of both assays was calculated. ResultsWe found a strong correlation coefficient of 0.887 between FC measured on IBDoc® and the laboratory assay BÜHLMANN fCAL® turbo. The coefficients of variation (CVs) at three different FC levels were in the range 2.3–5.5% (BÜHLMANN fCAL® turbo) and in the range of 4.8–26.6% (IBDoc®). ConclusionsThis study suggests that IBDoc® is a suitable alternative for the assessment of disease activity in IBD patients.

Determination of serum calcium levels by42Ca isotope dilution inductively coupled plasma mass spectrometry

Serum calcium level is an important clinical index that reflects pathophysiological states. However, detection accuracy in laboratory tests is not ideal; as such, a high accuracy method is needed. We developed a reference method for measuring serum calcium levels by isotope dilution inductively coupled plasma mass spectrometry (ID ICP-MS), using 42Ca as the enriched isotope. Serum was digested with 69% ultrapure nitric acid and diluted to a suitable concentration. The 44Ca/42Ca ratio was detected in H2 mode; spike concentration was calibrated by reverse IDMS using standard reference material (SRM) 3109a, and sample concentration was measured by a bracketing procedure. We compared the performance of ID ICP-MS with those of three other reference methods in China using the same serum and aqueous samples. The relative expanded uncertainty of the sample concentration was 0.414% (k=2). The range of repeatability (within-run imprecision), intermediate imprecision (between-run imprecision), and intra-laboratory imprecision were 0.12%-0.19%, 0.07%-0.09%, and 0.16%-0.17%, respectively, for two of the serum samples. SRM909bI, SRM909bII, SRM909c, and GBW09152 were found to be within the certified value interval, with mean relative bias values of 0.29%, -0.02%, 0.10%, and -0.19%, respectively. The range of recovery was 99.87%-100.37%. Results obtained by ID ICP-MS showed a better accuracy than and were highly correlated with those of other reference methods. ID ICP-MS is a simple and accurate candidate reference method for serum calcium measurement and can be used to establish and improve serum calcium reference system in China.

Multicenter Analytical Evaluation of the Automated Electrochemiluminescence Immunoassay for Cyclosporine

Cyclosporine A (CsA) is used as a posttransplantation immunosuppressant drug, and careful monitoring of CsA concentration in whole blood is essential. A new automated electrochemiluminescence immunoassay (ECLIA) for CsA measurement has been assessed in a multicenter evaluation. Residual EDTA whole blood samples from patients undergoing CsA therapy after organ transplant were used in assay evaluation at 5 clinical laboratories in Europe. Experiments included imprecision according to CLSI EP5-A2 (within-run and intermediate), lower limit of quantification, linearity according to CLSI EP6-A, and recovery of commercial external quality control samples. In addition, comparisons to liquid chromatography-tandem mass spectrometry methods in routine use at each investigational site and to commercial chemiluminescent microparticle immunoassay and antibody-conjugated magnetic immunoassay methods were performed. Imprecision testing gave coefficients of variation of less than 9% in the 30-2000 mcg/L range for both within-run and intermediate imprecision. Lower limit of quantification of 6.8 mcg/L at one investigational site and 1.8 mcg/L at a second site at 20% coefficient of variation were observed. Linearity was measured over the concentration range 0-2000 mcg/L, yielding a deviation of less than ±12%. External quality control sample recovery by ECLIA was 93%-114% of LC-MS/MS sample recovery. Deming regression analysis of ECLIA method comparison to combined LC-MS/MS results yielded a slope of 1.04 [95% confidence interval (CI), 1.03-1.06] and intercept of 2.8 mcg/L (95% CI, 1.5-4.1 mcg/L). Comparison to chemiluminescent microparticle immunoassay yielded a slope of 0.87 (95% CI, 0.85-0.89) and intercept of 1.4 mcg/L (95% CI, -0.89 to 3.7 mcg/L); comparison to antibody-conjugated magnetic immunoassay yielded a slope of 0.96 (95% CI, 0.93-0.98) and intercept of -4.2 mcg/L (95% CI, -7.1 to -1.2 mcg/L). The data from this multicenter evaluation indicate that the new ECLIA-based cyclosporine assay is fit for its purpose, the therapeutic monitoring of CsA.

Multi-center analytical evaluation of a novel automated tacrolimus immunoassay

BackgroundTacrolimus (TAC) is a post-transplantation immunosuppressant drug used in patients for whom careful monitoring of TAC concentration is essential. A new semi-automated immunoassay for TAC measurement, the Elecsys Tacrolimus assay, is available and has been assessed in a multi-center evaluation. MethodsResidual whole blood samples from patients undergoing TAC therapy after organ transplant were used in assay evaluation at five clinical laboratories in Europe. Experiments included imprecision according to CLSI EP5-A2 (within-run and intermediate), functional sensitivity, linearity according to CLSI EP6-A, and recovery from external quality assessment scheme (EQAS) samples. The assay was compared to LC–MS/MS used routinely at each investigational site, and to the Abbott Architect immunoassay. ResultsLinearity from 0.5 to 40μg/L was observed and functional sensitivity of 0.3μg/L (CV≤20%) was determined. Within-run imprecision was ≤5.1% on cobas e 602 (5.1% at 1.5μg/L) and ≤8.9% (8.9% at 0.8μg/L) on cobas e 411. The intermediate imprecision for TAC concentrations ≥6.8μg/L was ≤6.5%. At lower therapeutic concentrations (to 1.5μg/L) it was consistently ≤10%. Deming regression analysis of method comparison to LC–MS/MS yielded slopes of 1.07 (95%CI: 1.05/1.10) for heart transplant samples, 1.13 (95%CI: 1.09/1.16) for kidney, and 1.05 (95%CI: 1.02/1.08) for lung transplant samples. ConclusionsThe Elecsys Tacrolimus assay has good linearity, functional sensitivity and intermediate imprecision and is comparable to LC–MS/MS methods. The over-all performance of ECLIA demonstrates a modern generation TAC assay that meets the demands of monitoring drug concentrations in current immunosuppressive regimens.

Simultaneous Determination of Antidementia Drugs in Human Plasma for Therapeutic Drug Monitoring

A simple liquid chromatography mass spectrometry method was developed and validated for the simultaneous determination of antidementia drugs, including donepezil, galantamine, rivastigmine and its major metabolite, NAP 226-90, and memantine. A solid phase extraction procedure with a mixed-mode sorbent was used to isolate the drugs from 0.5 mL human plasma. Reverse phase chromatographic separation of the compounds was obtained with a gradient elution of an ammonium acetate buffer at pH 9.3 and acetonitrile and the analytes were detected by mass spectrometry in the single ion monitoring mode. The method was validated according to the recommendations of the Food and Drug Administration, including assessment of trueness (-8.0% to +10.7%), imprecision (repeatability: 1.1-4.9%, intermediate imprecision: 2.1-8.5%), selectivity and matrix effects variability (less than 6%) as well as short- and long-term stability in plasma. The calibration ranges were from 1 ng/mL to 300 ng/mL (rivastigmine and memantine) and 2 ng/mL to 300 ng/mL (donepezil, galantamine, and NAP 226-90). The method was successfully applied to patients' samples and might contribute to evaluate whether a therapeutic drug monitoring-guided dose adjustment of antidementia drugs could contribute to minimize the risk of adverse reactions and to increase the probability of efficient therapeutic response.

Estimate of uncertainty of measurement from a single-laboratory validation study: application to the determination of lead in blood.

Lead is an environmental pollutant, and human exposure is assessed by monitoring lead concentrations in blood. Because the main source of environmental exposure has been the use of leaded gasoline, its phase-out has led to decreased lead concentrations in the general population. Therefore, validated analytical methods for the determination of lower lead concentrations in blood (<150 microg/L) are needed. In addition, new ISO standards require that laboratories determine and specify the uncertainty of their results. We validated a method to determine lead in blood at concentrations up to 150 microg/L by electrothermal atomic absorption spectrometry with Zeeman background correction according to EURACHEM guidelines. Blood samples were diluted (1:1 by volume) with 2 mL/L Triton X-100. NH4H2PO4 (5 g/L) and Mg(NO3)2 (0.5 g/L) were used as modifiers. Matrix-matched standards were used for calibration. We determined the limits of detection (3.1 microg/L) and quantification (9.4 microg/L). Repeatability and intermediate imprecision within the range 35-150 microg/L were <5.5% and <6.0%, respectively. We assessed trueness by use of certified reference materials, by recovery tests, and by comparison with target values of other reference materials (candidate external quality assessment samples). The expanded uncertainty ranged from 20% to 16% (with a confidence level of 95%) depending on concentration. This study provides a working example of the estimate of uncertainty from method performance data according to the EURACHEM/CITAC guidelines. The estimated uncertainty is compatible with quality specifications for the analysis of lead in blood adopted in the US and the European Union.