Reference limits of high-sensitive cardiac troponin T indirectly estimated by a new approach applying data mining. A special example for measurands with a relatively high percentage of values at or below the detection limit

A new model for the indirect estimation of reference limits (RLs) has been proposed recently and was coined TMC approach (truncated minimum chi-square estimation) which can be performed with R statistic. A spline function is applied to the RLs to get a continuous function if age is graphically presented vs. the RLs avoiding artificial “jumps” between different age groups. Most indirect models assume a power normal distribution and fail if this assumption is not fulfilled as e.g. if a relatively high percentage of measured values is below the detection limit and the data are distributed extremely skewed. This problem is handled by the TMC model. High-sensitive cardiac troponin T (hs cTnT) was chosen as an example. The hs cTnT concentration in serum or plasma is well accepted as a valuable marker in the diagnosis of acute myocardial infarction. Currently, the 99th percentile derived from a “healthy” subpopulation is the decision limit recommended by consensus groups. However, this decision limit is questioned by several authors for many reasons. In the present report, the 97.5th and the 99th percentile limits were reinvestigated by the TMC model with different subpopulations stratified according to age and sex and were finally compared to presently recommended decision limits. In summary, the generally recommended 99th percentile as a fixed decision limit should be reconsidered. It is suggested to apply more specific reference limits stratified for age and sex instead of a fixed decision limit.

Evaluating the performance of an updated high-sensitivity troponin T assay with increased tolerance to biotin

ObjectivesBiotin >20 ng/mL may interfere with the Elecsys® Troponin T-high sensitive assay (cTnT-hs; Roche Diagnostics International Ltd). We evaluated the performance of an updated assay, cTnT-hs*, which was designed to reduce biotin interference.MethodscTnT-hs* assay performance was assessed using up to two applications (18 min/9 min) on three analyzers (cobas e 411/cobas e 601/cobas e 801). Biotin interference was determined by measuring recovery in an 11-sample series dilution with biotin ranging from 0–3600 ng/mL. Repeatability/reproducibility were evaluated in five serum sample pools (n=75 each). Method comparisons tested: cTnT-hs* vs. cTnT-hs (18 min/cobas e 601); cTnT-hs* assay 18 vs. 9 min (cobas e 601); cTnT-hs* (18 min) on cobas e 601 vs. cobas e 411 and cobas e 601 vs. cobas e 801. Concordance at the 99th percentile decision limit between cTnT-hs* and cTnT-hs (9 min/cobas e 601) was calculated using 300 lithium-heparin plasma samples and a 14 ng/L assay cutoff.ResultscTnT-hs* assay (18 min/cobas e 601) recovery was ≥96% for biotin ≤1250 ng/mL. Across all applications/analyzers, coefficients of variation for repeatability/reproducibility with the cTnT-hs* assay were <5% in most serum sample pools (mean cardiac troponin T: 8.528–9484 ng/L). High correlation (Pearson’s r=1.000) was demonstrated for all method comparisons. Concordance at the 99th percentile decision limit was high between the cTnT-hs* and cTnT-hs assays.ConclusionsThe updated cTnT-hs* assay may provide greater tolerance to biotin interference, and shows good analytical and clinical agreement/concordance with the previous cTnT-hs assay.

Analytical and Clinical Validation for RT-qPCR detection of SARS-CoV-2 without RNA extraction

BackgroundThe recent COVID-19 pandemic has posed an unprecedented challenge to laboratory diagnosis, based on the amplification of SARS-CoV-2 RNA. With global contagion figures exceeding 4 million persons, the shortage of reagents for RNA extraction represents a bottleneck for testing globally. We present the validation results for a RT-qPCR protocol without prior RNA extraction. Because of its simplicity, this protocol is suitable for widespread application in resource-limited settings.MethodsOptimal protocol was selected by comparing RT-qPCR performance under a set of thermal (65°, 70°, and 95° for 5, 10, and 30 minutes) and amplification conditions (3 or 3,5 uL loading volume; 2 commercial RT-qPCR kits with limit of detection below 10 copies/sample) in nasopharyngeal swabs stored at 4°C in sterile Weise’s buffer pH 7.2. The selected protocol was evaluated for classification concordance with the standard protocol (automated RNA extraction) in 130 routine samples and in 50 historical samples with Cq values near to the clinical decision limit.ResultsOptimal selected conditions were: Thermal shock at 70° C for 10 minutes, loading 3.5 ul in the RT-qPCR. Prospective evaluation in 130 routine samples showed 100% classification concordance with the standard protocol. The evaluation in historical samples, selected because their Cqs were at the clinical decision limit, showed 94% concordance with our confirmatory-gold standard which includes manual RNA extraction.ConclusionsThese results validate the use of this direct RT-qPCR protocol as a safe alternative for SARS CoV-2 diagnosis in case of a shortage of reagents for RNA extraction, with minimal clinical impact.

Verification of Quantitative Measurement of Urinary Creatinine Enzymatic Method Kit by DIRUI CS-T240 Clinical Chemistry Analyser

Introduction: All urine samples sent for the diagnosis of inborn errors of metabolism (IEM) disorders testing will be first analysed for their creatinine levels. The urine creatinine analysis is applied either for calculation of urine amount needed in other tests or for quantitation of analytes per mol creatinine. Jaffe kinetic-end point method has been the preferred method. We aim to verify the performance of DIRUI enzymatic method reagent kit on DIRUI CS-T240 analyser and compare with Randox Jaffe kinetic-end point method reagent kit on same platform.Method: Precision study, linearity and method comparisons were carried out using three levels of internal quality control (IQC) samples and the measured results were compared against manufacturer’s claims, medical decision limit and biological variation (BV).Results: The within run coefficient variation (CV) were 3.57%, 2.61% and 1.35% for IQC Level 1, 2 and 3 respectively. The total precision and accuracy were 9.59% and 0.27% for Level 1, 3.17% and 0.57% for Level 2, while 2.03% and 3.63% for Level 3. Whereas, the verifica t ion value for CV based on analytical performance specifications (APS) BV desirable for CV and accuracy were 18.2% and 12.2% respectively. These values were compared, and the precision study was acceptable within desirable specification. Linearity met the manufacturer’s claim and range from 0.32 to 21.2mmol/L. The method comparison gave good correlation, r2 of 0.9975 and regression equation, y=0.9852x-0.022 with medical decision limit, 0.53mmol/L. The assessment of acceptability of this new method was acceptable as calculated total error (TEc) < total allowable error (TEa) (APS BV Desirable for TEa = 42.1%).Conclusion: The analytical verification study that we had carried out fulfilled the BV desirable in precision, accuracy and TEa. Therefore, the performances of enzymatic method kit on DIRUI CS-T240 analyser is verified to be fit-for-purpose and can replace the existing method.International Journal of Human and Health Sciences Supplementary Issue: 2019 Page: 53

Revisiting the sampling, sample preparation, and analytical variability associated with testing wheat for deoxynivalenol

Fifteen lots of wheat were sampled to characterise the total variance and distribution among sample test results associated with measuring deoxynivalenol (DON) in bulk wheat lots. An unbalanced nested experimental design based on past research was used to determine contributions to the total variance from sampling, sample preparation, and analysis. The wheat lots used in the study contained average DON concentrations that ranged from 0.17 to 24.5 mg/kg. Sampling was determined to be the largest contributor to the total variance of measuring DON at low mg/kg concentrations, which are relevant to existing maximum levels. With the experimental design parameters of 1 kg laboratory samples, sub-division of whole and ground grain using rotary sample division, sample comminution using a commercial-grade coffee grinder, extraction of 100 g test portions, and making one measurement of DON in the test portion by gas chromatography-mass spectrometry, the total variance of DON measurement at 2 mg/kg was 0.046 mg2/kg2 (coefficient of variation=10.7%). At this concentration, sampling contributed 67% to the total variance, followed by sample preparation (18%) and analysis (15%). The DON distribution among sample test results was accurately described by the normal distribution. The mathematical model of variance was used with the normal distribution of DON measurement results to construct operating characteristics curves to model the likelihood of mischaracterising a wheat lot as (non) compliant with a certain decision limit. With realistic laboratory sample and test portion sizes, as well as a practicable decision limit of 1.5 mg/kg, the estimated probability of mischaracterising a wheat lot containing 2 mg/kg DON as less than this concentration was reduced to 1%.

Procedure for the Screening of Eggs and Egg Products to Detect Oxolonic Acid, Ciprofloxacin, Enrofloxacin, and Sarafloxacin Using Micellar Liquid Chromatography

A method based on micellar liquid chromatography was developed to determine oxolinic acid, ciprofloxacin, enrofloxacin, and sarafloxacin in eggs and egg products. The antimicrobial drugs were obtained in a micellar solution which was directly injected. The analytes were resolved using a C18 column and a mobile phase of 0.05 M sodium dodecyl sulfate—7.5% 1-propanol—0.5% triethylamine, buffered at pH 3 with phosphate salt, running under the isocratic mode. The signal was monitored by fluorescence. Validation was successfully performed according to the EU Commission Decision 2002/657/EC in terms of specificity, calibration range (LOQ to 1 mg/kg), linearity (R2 > 0.9991), limit of detection and decision limit (0.01–0.05 mg/kg), limit of quantification (0.025–0.150 mg/kg), detection capability (<0.4 times decision limit), trueness (−14.2% to +9.8%), precision (<14.0%), robustness, and stability. The procedure was environmentally friendly, safe, easy-to-conduct, inexpensive, and had a high sample throughput, thus it is useful for routine analysis as a screening method in a laboratory for food residue control.

Determination of Sulfonamides in Feeds by High-Performance Liquid Chromatography after Fluorescamine Precolumn Derivatization

A new multi-residue method for the analysis of sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfaguanidine and sulfamethoxazole) in non-target feeds using high-performance liquid chromatography-fluorescence detection (HPLC-FLD) and precolumnderivatization was developed and validated. Sulfonamides (SAs) were extracted from feed with an ethyl acetate/methanol/acetonitrile mixture. Clean-up was performed on a Strata-SCX cartridge. The HPLC separation was performed on a Zorbax Eclipse XDB C18 column with a gradient mobile phase system of acetic acid, methanol, and acetonitrile. The method was validated according to EU requirements (Commission Decision 2002/657/EC). Linearity, decision limit, detection capability, detection and quantification limits, recovery, precision, and selectivity were determined, and adequate results were obtained. Using the HPLC-FLD method, recoveries were satisfactory (79.3–114.0%), with repeatability and reproducibility in the range of 2.7–9.1% to 5.9–14.9%, respectively. Decision limit (CCα) and detection capability (CCβ) were 197.7–274.6 and 263.2–337.9 µg/kg, respectively, and limit of detection (LOD) and limit of quantification (LOQ) were 34.5–79.5 and 41.3–89.9 µg/kg, respectively, depending on the analyte. Results showed that this analytical procedure is simple, rapid, sensitive, and suitable for the routine control of feeds.

Commutability of proficiency testing material containing tobramycin: a study within the framework of the Dutch Calibration 2.000 project

Background:Results from external quality assessment schemes (EQASs) can provide information about accuracy and comparability of different measurement methods, provided that the material used in these schemes behave identical to patient samples among the different methods, a characteristic also known as commutability. The aim of this study was to assess the commutability of different matrices for the material used in an EQAS for tobramycin.Methods:Proficiency testing material (PTM) and patient samples containing tobramycin were prepared, collected, pooled, and distributed to participating laboratories for analysis. Low, medium, and high tobramycin concentrations in liquid human, liquid bovine and lyophilized bovine serum were tested in this study. The patient serum results of every laboratory were plotted against each of the other laboratories, and the distances of the PTM results to the patient serum regression line were calculated. For comparison, these distances were divided by the average within-laboratory standard deviation (SDResults:With 10 laboratories participating in this study, 45 laboratory couples were formed. For human serum, only one relative residual for high concentrations of tobramycin was found outside the commutability decision limit. For liquid and lyophilized bovine sera, the number of relative residuals outside the decision limit was between 15 and 18 for low, medium, and high tobramycin concentrations.Conclusions:The PTM used for tobramycin is preferably prepared with human serum.