Journal of Applied Laboratory Medicine最新文献

Background: Validating a candidate method is commonly done by methods comparison (MC) using an accepted comparator to measure samples and to assess their agreement. The most powerful way of doing so is by parametric modeling: an "errors-in-variables," or Deming, regression. The measurement variance is rarely constant across the measuring interval, and so Deming regression requires suitable weights-the inverse of each measurement's variance-to achieve its theoretical good performance. Sometimes, independently of the data set, we have an explicit mathematical model, the precision profile, connecting each assay's variability to the analyte concentration. But many studies have no such external information, and the analysis must rely on the data set alone, with some assumptions about the form of the precision profile.

Methods: The mathematical theory melding mathematical precision profile models with errors-in-variables (Deming) regression is sketched.

Results: Weighted Deming regression is outlined, and R codes for implementing it in the known and unknown precision profile settings are discussed. The implementation includes a jackknife approach for standard errors, confidence intervals for the regression parameters, residuals for diagnostics on normality and linearity, and a methodology for identifying and testing outliers.

Conclusions: Existing weighted Deming regression publications assume either constant coefficient of variation or constant variance. Precision profile models fill this gap and allow for more general settings.

Background: Measurement of β-hydroxybutyrate (BOHB) is a valuable tool for the assessment of metabolic acidoses. In this study we evaluated hemolysis, icterus, and lipemia interference thresholds for a commonly used BOHB assay, the Stanbio β-Hydroxybutyrate LiquiColor reagent, on 3 different Roche cobas analyzers.

Methods: Remnant patient samples were combined to generate pools with target BOHB concentrations of approximately 0.20, 3.00, and 7.00 mmol/L into which increasing amounts of hemolysate, bilirubin, or Intralipid® were added. Samples were measured in triplicate for both interferences and BOHB concentration on Roche cobas c502, c501, and c311 analyzers.

Results: We determined that the Stanbio β-Hydroxybutyrate LiquiColor reagent will tolerate hemolysis to a hemolysis index of 400 (approximately 400 mg/dL hemoglobin) for BOHB concentrations ≤1.00 mmol/L, or 1010 for BOHB concentrations of >1.00 mmol/L. For icterus, the assay was unaffected by bilirubin to an icterus index of 25 [approximately 25 mg/dL bilirubin (427 μmol/L)] for all concentrations tested. Using the most conservative data from our study, the lipemia index limit was set at 800.

Conclusions: Our studies across 3 laboratories running 3 different cobas modules allowed our health system to better define permissible limits of sample quality and these updated values will reduce the number of unnecessary cancelations as compared to the limits defined by the manufacturer. Our data also demonstrated heterogeneity of interference between analyzer models, which is an important consideration for those seeking to adopt the limits determined on analyzers other than those in their own laboratories.

Background: Accurate and precise pipetting is critical for reliable laboratory results, especially when handling small volumes or sensitive analytical techniques. Despite the common use of piston-operated pipettes, operator-dependent variability remains a major source of error. This study aimed to evaluate pipetting performance among laboratory personnel with varying levels of experience.

Methods: The study included 108 participants: 26 laboratory school students (first to third year), 10 PhD students, 10 clinical chemists, and 62 laboratory technicians. Technicians were stratified into 3 groups based on the extent of sensitive manual pipetting in their work: minimal (Group A), moderate (Group B), and high (Group C). Pipetting performance was evaluated using gravimetric replicates at 10 µL and 100 µL volumes, and a dilution task involving serial dilutions of 1 M copper sulfate. Accuracy and precision metrics were compared across groups.

Results: The median coefficient of variation (CV%) was lower for 100 µL replicates (0.3% to 0.8%) than for 10 µL replicates (1.4% to 5.9%). Laboratory students showed the greatest variability and longest task completion times, although performance improved with advancing school year. Ten percent of participants exceeded ISO-defined accuracy limits for 10 µL pipetting, and 22% for 100 µL. Imprecision limits were surpassed by 36% and 20% of participants for 10 µL and 100 µL, respectively. Group C technicians achieved optimal dilution performance (R2 = 1.00), while students showed the highest deviation.

Conclusions: Pipetting performance was significantly influenced by experience level and task sensitivity. Even experienced personnel occasionally failed to meet ISO standards, underscoring the need for ongoing training and performance assessment.

Application of the results provided by medical laboratories plays an essential role in medical decision-making. This is not limited to diagnosis and monitoring of disease but also involves its use in other phases of the health continuum, e.g., predisposition, risk stratification, screening, staging, prognosis, and surveillance. With the growing importance of precision medicine, the importance of requirements related to clinical performance, and consequently analytical performance of laboratory tests, also grows. To allow the community of laboratory medicine to translate clinical need into a test arsenal with adequate performance, the application of metrology concepts is essential. This paper summarizes, for all steps in the examination process from test development to clinical interpretation, why and how metrological traceability is a fundamental requirement for adequate medical decision-making and is critical for correct use of test results in algorithms and artificial intelligence-led approaches. This includes the importance of metrology concepts and their correct implementation for obtaining equivalence of test results upon cross-facility result exchange for primary or secondary use in healthcare and research. This is not limited to biochemistry and hematology but is also of importance to other areas of laboratory medicine, including microbiology. This paper provides an overview of the purposes of the underappreciated science of metrology in modern laboratory medicine and its importance to patients and caregivers.

Background: Circuit-induced hemolysis is relatively common in extracorporeal membrane oxygenation (ECMO) patients. Intravascular release of cell-free hemoglobin can lead to complications and requires timely recognition. Validation of plasma free hemoglobin (PFH) measurement using a direct spectrophotometric method is presented.

Methodology: We evaluated a method modified from Kahn et al. (Ann Clin Lab Sci 1981;11:126-31) on a stand-alone spectrophotometer (Cary 60) and compared its performance to the semiquantitative H-index on an Abbott Alinity c, including precision, linearity, recovery, reference interval verification, interference, and stability. Method comparison was performed relative to the H-index and the same method on a different spectrophotometer (Beckman DU 720). Lipemia interference was performed on the Cary 60, Cary 3500, and Beckman DU 720. Surrogate biomarkers for hemolysis detection were also investigated in ECMO patients.

Results: The PFH method on the Cary 60 demonstrated imprecision ranging from 1% (96.0 mg/dL) to 4% (3.0 mg/dL), linearity to 100 mg/dL, and recovery >80% for values >2 mg/dL hemoglobin-spiked plasma. Dilution expanded the reportable range to the maximum dilution tested (1000 mg/dL). Lipemia interfered with PFH measurement by the direct method, but the same method on the Cary 3500 was resistant to lipemia. Bilirubin did not cause significant interference. Direct and H-index methods were comparable with a mean difference of 5.03 mg/dL (95% CI -1.38, 11.44). Lactate dehydrogenase was the most reliable surrogate biomarker for hemolysis. with AUC of 0.921 (0.894, 0.949) at >50 mg/dL.

Conclusion: PFH measurement by a direct spectrophotometric method is more precise and sensitive compared to the H-index; however, PFH measurement is susceptible to lipemia unless performed on a high-end spectrophotometer.

Background: Methotrexate (MTX), a synthetic antimetabolite and folic acid antagonist, has become a cornerstone in the management of malignant diseases. However, MTX toxicity can be severe and potentially life-threatening, especially with high-dose regimens used in oncology. Given its narrow therapeutic index, precise monitoring of MTX levels is critical to optimize efficacy while minimizing toxicity.

Methods: We validated the performance of the Roche ONLINE therapeutic drug monitoring (TDM) Methotrexate Assay on Cobas c502 and Cobas pro c503 platforms across 2 clinical sites, focusing on key analytical parameters such as linearity, accuracy, precision, dilution, carryover, and method comparison.

Results: The assay was linear across the analytical measurement interval (AMI) of 0.04-1.2 µmol/L. No significant carryover was observed (i.e., <5% bias). Automatic and manual dilutions demonstrated <10% bias, and the coefficients of variation (CVs) for precision and percentage bias for accuracy were <10%. A method comparison using 40 patient samples showed a strong correlation between the Roche and ARK MTX assays, with a Deming regression slope of 0.9634, an intercept of -0.0095, and Pearson correlation coefficient (r) = 0.9935, with a mean percentage difference (%Bias) of -12. Comparison with LC-MS/MS demonstrated high agreement for both assays. The ARK assay yielded a slope of 0.8873, an intercept of 0.0145, r = 0.9978, and %Bias of -6, while the Roche assay yielded a slope of 0.8541, an intercept of 0.0047, r = 0.9919, and %Bias of -18.

Conclusions: The Roche ONLINE TDM MTX assay is a robust and clinically adequate method for methotrexate quantification and therapeutic drug monitoring.

Background: The role of high-sensitivity cardiac troponin (cTn) assays for children and young adults is uncertain, and no guidance is available on diagnostic thresholds. This study evaluates the effect of applying pediatric compared to adult upper reference limits (URLs) for cTn.

Methods: We carried out a retrospective multicenter international cohort study of consecutive children and young adults (1 day to 18 years) undergoing cTn I or T testing at 4 tertiary care hospitals in Norway and Scotland, United Kingdom, from 2013 to 2023. Myocardial injury was classified using the adult sex-specific 99th percentile URL, a pediatric sex-specific 99th percentile, and a pediatric sex-specific 97.5th percentile. Diagnoses of myocarditis were obtained from the Norwegian Patient Register and the Scottish Morbidity Record.

Results: In total, 9833 (46.6% female) children and young adults underwent cTn testing. Applying the adult sex-specific 99th percentile, 1771 (18.0% [95% CI, 17.3%-18.8%]) had myocardial injury compared with 1762 (17.9% [95% CI, 17.2%-18.7%]) using a pediatric 99th percentile. In contrast, applying a pediatric sex-specific 97.5th percentile would identify 2261 (23.0% [95% CI, 22.2%-23.8%]) with myocardial injury (a 28% relative increase). Infants had a higher frequency of myocardial injury than those 1-18 years old (1035/1104; 93.8% [95% CI, 92.2%-95.1%] vs 1226/8729; 14.0% [95% CI, 13.3%-14.8%] using pediatric sex-specific 97.5th percentile, P < 0.001). Testing for cTn increased over the study period (τ = 0.42, P < 0.001).

Conclusions: The use of pediatric-specific 97.5th percentile URLs for cTn would increase classification of myocardial injury in children and young adults. The clinical implications of this are uncertain and require further study given cTn testing has increased over the last decade.

Background: Historically, 25-hydroxyvitamin D (25OHD) assays have under- or over-recovered 25-hydroxyvitamin D2 (25OHD2), but assay manufacturers have modified their reagents to address this problem. In this study, we compared the second- and third-generation Roche assays as well as two contemporary offerings from Diasorin and Beckman against liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Methods: We identified 50 remnant serum samples with 25OHD concentrations from across the analytical range of the second-generation Roche assay. To increase 25OHD2 representation, we identified 25 additional samples from individuals prescribed high-dose vitamin D2 supplements. We tested samples on Roche assays and circulated to laboratories performing Beckman and Diasorin 25OHD assays. We tested samples by LC-MS/MS to obtain concentrations for 25OHD2 and 25-hydroxyvitamin D3.

Results: Mean overall bias for each assay was 5.1 ng/mL or less against the LC-MS/MS measurement; mean proportional bias was 8.7% to 12.1%. Some individual specimens had much larger bias. 25OHD2 was under-recovered on average, but the bias for the third-generation Roche assay represents a significant improvement over the previous assay, and mean bias for current generation assays was no worse than -3.2 ng/mL. In most cases, clinical classification by automated assay values agreed with clinical classification by LC-MS/MS; where present, disagreements occurred near classification thresholds.

Conclusions: Automated 25OHD assays continue to improve, and 25OHD2 recovery no longer appears to be a significant concern for the assays evaluated here. All assays evaluated were adequate for clinical classification of vitamin D nutritional status and are suitable for routine use, including in patients prescribed high-dose vitamin D2.

Background: Measurement of serum free light chains (FLC) including kappa FLC, lambda FLC, and calculated kappa to lambda FLC ratio play a vital role in the diagnosis, prognosis, and monitoring of plasma cell disorders. Recent concerns regarding upward drift in The Binding Site's kappa FLC measurements have prompted a reformulation of the kappa FLC calibrator by the manufacturer. This study aims to assess the impact of the reformulated/new kappa FLC calibrator on both the kappa FLC reference interval (RI) and the FLC ratio diagnostic range.

Methods: Healthy volunteers (n = 124) from 3 tertiary care medical centers had FLCs measured using both the new and the prior lot of kappa FLC calibrators on an Optilite analyzer (The Binding Site). All samples were screened for monoclonal proteins using capillary electrophoresis. Creatinine was measured to confirm renal function.

Results: Comparison of kappa FLC values from the new vs prior calibrator demonstrated minimal differences. Neither calibrator was able to verify manufacturer-suggested RI in our study population. The proportion of samples that had FLC ratios within the manufacturer's claimed diagnostic range was equivalent between calibrators, with roughly 6% to 7% of study participants falling above the diagnostic range of 0.26 to 1.65.

Conclusion: The new reformulated kappa FLC calibrator from The Binding Site does not support verification of the manufacturer's claimed kappa FLC RI or improve the number of healthy patients falling within the FLC ratio diagnostic range. Both the kappa FLC RI and diagnostic range for FLC ratio should be reevaluated and updated in consensus guidelines.