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Evaluation of the clinical performance of anti-mutated citrullinated vimentin antibody and 14-3-3 eta testing in rheumatoid arthritis.

Early rheumatoid arthritis (RA) detection is crucial for improving patient prognosis. Anticyclic citrullinated peptide antibodies (anti-CCP) and rheumatoid factors (RF) support RA diagnosis but are undetectable in ∼20 % of cases. Recently, antibodies against mutated citrullinated vimentin (anti-MCV) and detection of 14-3-3 eta have emerged with implications for preclinical RA diagnosis and monitoring treatment. The objective of this study was to assess the clinical performance of anti-MCV antibodies and 14-3-3 eta in RA and to compare it to current RA criteria anti-CCP and RF markers, individually and in combination. A retrospective chart review of 326 subjects submitted for RA serology testing identified 134 RA positive and 192 RA negative disease control individuals. Fifty healthy controls specimens were also included. Performance of anti-MCV and 14-3-3 eta, alone and combined with CCP3.1 and RF, was assessed. Anti-MCV had a sensitivity of 71 % and a specificity of 92 %. 14-3-3 eta had a sensitivity of 43 % and a specificity of 90 %. In comparison, CCP3.1 and RF displayed a sensitivity of 79 % and 84 % and a specificity of 92 % and 61 %, respectively. ROC curve analysis demonstrated CCP3.1 and anti-MCV had superior diagnostic performance compared to RF and 14-3-3 eta. In our cohort, anti-MCV and 14-3-3 eta failed to identify seronegative RA patients. Different combinations of double antibody positivity increased specificity at the cost of lost sensitivity. Individually, 14-3-3 eta, anti-MCV and CCP3.1 assays had≥90 % specificity in diagnosed RA patients, with better sensitivities for anti-MCV and CCP3.1 than 14-3-3 eta. Overall diagnostic performance of anti-MCV was similar to CCP3.1 and RF, all of which outperformed 14-3-3 eta in our cohort.

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Evidence for stability of cardiac troponin T concentrations measured with a high sensitivity TnT test in serum and lithium heparin plasma after six-year storage at-80 °C and multiple freeze-thaw cycles.

As high-sensitivity cardiac troponin T (hs-cTnT) is making the transition from diagnostic to prognostic use, a long-term stability study of 5th generation hs-cTnT according to EFLM CRESS recommendations was set up for investigation of frozen clinical specimens (two matrices). Study samples collected in serum tubes and lithium heparin tubes with gel from patients admitted for suspected minor myocardial damage were measured directly after completion of the study (0 years), and after 3-year and 6-year storage at-80 °C, and recovery of hs-cTnT concentrations after long-term storage (%hs-cTnT concentration compared to 0-year) was calculated. Hs-cTnT changes were also compared to decisive delta changes, such as the ones proposed in the ESC NSTEMI 0 h/1 h algorithm (<3 or >5 ng/L for ruling out and ruling in suspected NSTEMI patients). Eighty-six patients were included in the study, whereof 28 both lithium heparin plasma and serum samples were collected simultaneously, in others only serum (n=30) or plasma (n=28). Multiple aliquots per patient were made, so that 479 serum and 473 plasma samples were available for analysis. Across the overall hs-cTnT measuring range, median recovery after 6 years was 105.4 % and 106.2 % for serum and plasma, respectively. Based on these decisive delta changes, serum showed consistent results upon long term storage (max 0.8 % of samples above delta threshold of >5 ng/L) as compared to heparin plasma (up to 19.2 % of samples above threshold). Over 6 years of storage at-80 °C, recovery of hs-cTnT in serum and heparin plasma was similar and within common lot-to-lot variation. Yet, when evaluating absolute delta increments around hs-cTnT clinical decision points, long-term stored sera displayed better clinical performance compared to heparin plasma samples.

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Evaluation of revised UK-NEQAS CSF-xanthochromia method for subarachnoid hemorrhage: outcome data provide evidence for clinical value.

Subarachnoid haemorrhage (SAH) has a high morbidity and mortality and requires prompt diagnosis. In patients with negative findings on computed-tomogram of the brain (CT-Brain) cerebrospinal fluid (CSF)-xanthochromia is considered the test of choice if performed 12 h or more after symptom onset. We audited the accuracy, usefulness and timing of CSF-xanthochromia testing and the interpretation of equivocal CSF-xanthochromia findings. We also investigated mortality outcomes for defined subsets of patients. A retrospective audit of CSF-xanthochromia tests over 8 years was performed. The service uses the revised UK-NEQAS (United Kingdom National External Quality Assessment Service) method. We analysed 543 cases (F=299, median age 44yrs) with 19 cases (3.5 %) having SAH. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of CSF-xanthochromia testing were 100 , 98.1, 65.9, 100 % respectively (equivocal results were counted as positives). 280 cases (F=153, median age 43yrs) had LP performed more than 24 h after the onset of headache (median time to LP=72 h). The sensitivity and specificity of the CSF-xanthochromia were 100 and 97.4 % in this group with NPV 100 % and PPV 66.6 %. 183 (65.4 %) CSF- xanthochromia negative cases in this subgroup had follow up data and survived without SAH occurring in the 12months follow up. In this study, supported by follow up outcome data, we show that CSF-xanthochromia testing using the revised UK-NEQAS method is fit-for-purpose for the use as a second line test to exclude SAH in patients with negative CT-brain including delayed presentation more than 24 h after headache onset.

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Comparison of a two-step Tempus600 hub solution single-tube vs. container-based, one-step pneumatic transport system.

Efficient and timely transportation of clinical samples is pivotal to ensure accurate diagnoses and effective patient care. During the transportation process, preservation of sample integrity is crucial to avoid pre-analytical aberrations on laboratory results. Here, we present a comparative analysis between a two-step Tempus600 hub solution single-tube and a one-step, container-based pneumatic transport system (PTS) from Airco, for the in-house transportation of blood samples. Ten blood samples from healthy volunteers were split in 10 mL collection tubes filled at full or half capacity for transportation with the two PTS (about 250 m). To compare the impact of transportation, markers of hemolysis such as lactate dehydrogenase (LDH), potassium (K+), and the hemolysis index (HI), were determined. Additionally, differences in HI in routine samples and repeated transportation was investigated. To assess and compare the mechanistic impact profiles, we recorded the acceleration profiles of the two PTS using a shock data logger. Transportation using the Tempus600 hub solution resulted in 49 and 46 % higher HI with samples filled to total or half capacity, respectively. Routine samples transported with the Tempus600 hub solution showed a higher median HI by 23 and 33 %. Additionally, shock logger analysis showed an elevated amount of shocks (6.5 fold) and shock intensities (1.8 fold). The Tempus600 hub solution caused an increased number of unreportable LDH or K+ results based on the hemolysis index. However, it was only statistically significant for LDH (p<0.01 and p<0.08)- while the comparisons for K+ were not statistically significant (p<0.28 and p<0.56).

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Stimulating thyrotropin receptor antibodies in early pregnancy.

Thyrotropin-receptor antibodies (TRAb) are used to diagnose Graves' hyperthyroidism in pregnant women. Bioassays provide a measure of thyrotropin-receptor stimulatory antibodies (TSI) specifically. The objective was to measure TSI in pregnant women for establishment of a pregnancy-specific cut-off and comparison with immunoassay measurements of TRAb. The retrospective Danish study was performed within the North Denmark Region Pregnancy Cohort (2011-2015) that includes stored biobank samples from early pregnancy (median week 10) with immunoassay measurements of thyroid function parameters and TRAb. TSI were measured in the same samples using the Turbo TSI bioassay (Quidel/Ortho-Clinical Diagnostics) with a recommended cut-off of 0.0241 IU/L in non-pregnant adults. A pregnancy-specific TSI cut-off (95-percentile) was established using Regression on Order Statistics. The established TSI cut-off was 0.0418 IU/L (95 % CI: 0.0417-0.0419). Among women with early pregnancy hyperthyroidism (n=438), 43 women (9.8 %) were TSI positive using the established cut-off, and these women had lower TSH (median 0.008 mIU/L) compared to women with TSI levels below 0.0241 (median TSH 0.040 mIU/L) or in the range from 0.0241 to 0.0418 (median TSH 0.033 mIU/L). Among the 438 women with early pregnancy hyperthyroidism, 22 women were positive for TSI and TRAb, 388 were negative for both, and 28 women were positive for either TSI or TRAb. This is the first study on TSI measurements in a large cohort of early pregnant women. A pregnancy-specific cut-off for TSI was established and agreement in the classification with immunoassay measurements of TRAb was seen in 94 % of cases.

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Using Bland-Altman plot-based harmonization algorithm to optimize the harmonization for immunoassays.

Harmonization has been recommended by theInternational Organization for Standard (ISO) to achieve equivalent results across invitro diagnostic measurement devices (IVD-MDs). We aim to evaluate the effectiveness of Bland-Altman plot-based harmonization algorithm (BA-BHA) created in this study and compare it with weighted Deming regression-based harmonization algorithm (WD-BHA) proposed in ISO 21151:2020. Eighty patient sera were used as the harmonization reference material (HRM) to develop IVD-MD-specific harmonization algorithms. Another panel of 40 patient sera was used to validate the effectiveness of harmonization algorithms. We compared regression slopes, intercepts, Bland-Altman plot layouts, percent differences, limits of agreement (LoAs), between-method coefficients of variation (CV) before and after harmonization. After harmonization by WD-BHA, acceptable slopes and intercepts between measured values and HRM targets were observed in weighted Deming regression, but not in Passing-Bablok analysis. Mean differences were-5.5 to 5.0 % and differences at specific levels were-33.9 to 23.9 %. LoAs were-64.6 to 74.6 %. Between-method CV was 22.9 % (±12.9 %). However, after harmonization by BA-BHA, both weighted Deming and Passing-Bablok regressions equations presented harmonized results. Mean differences were-0.3 to 0.2 % and differences at specific levels were-1.1 to 1.6 %. LoAs were-23.3 to 23.2 %. Between-method CV was 8.4 % (±4.0 %). The data points were evenly distributed at both sides of the mean in Bland-Altman plots. The inequivalence of test results between different methods can be improved but unacceptable analytical differences at specific levels may be hidden in terms of an acceptable slope and intercept on WD-BHA. The new protocol BA-BHA may be a viable alternative to optimize the harmonization for immunoassays.

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Thyroglobulin measurement is the most powerful outcome predictor in differentiated thyroid cancer: a decision tree analysis in a European multicenter series.

An accurate prognostic assessment is pivotal to adequately inform and individualize follow-up and management of patients with differentiated thyroid cancer (DTC). We aimed to develop a predictive model for recurrent disease in DTC patients treated by surgery and 131I by adopting a decision tree model. Age, sex, histology, T stage, N stage, risk classes, remnant estimation, thyroid-stimulating hormone (TSH), thyroglobulin (Tg), administered 131I activities and post-therapy whole body scintigraphy (PT-WBS) were identified as potential predictors and put into regression algorithm (conditional inference tree, c-tree) to develop a risk stratification model for predicting persistent/recurrent disease over time. The PT-WBS pattern identified a partition of the population into two subgroups (PT-WBS positive or negative for distant metastases). Patients with distant metastases exhibited lower disease-free survival (either structural, DFS-SD, and biochemical, DFS-BD, disease) compared to those without metastases. Meanwhile, the latter were further stratified into three risk subgroups based on their Tg values. Notably, Tg values >63.1 ng/mL predicted a shorter survival time, with increased DFS-SD for Tg values <63.1 and <8.9 ng/mL, respectively. A comparable model was generated for biochemical disease (BD), albeit different DFS were predicted by slightly different Tg cutoff values (41.2 and 8.8 ng/mL) compared to DFS-SD. We developed a simple, accurate and reproducible decision tree model able to provide reliable information on the probability of structurally and/or biochemically persistent/relapsed DTC after a TTA. In turn, the provided information is highly relevant to refine the initial risk stratification, identify patients at higher risk of reduced structural and biochemical DFS, and modulate additional therapies and the relative follow-up.

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QC Constellation: a cutting-edge solution for risk and patient-based quality control in clinical laboratories.

Clinical laboratories face limitations in implementing advanced quality control (QC) methods with existing systems. This study aimed to develop a web-based application to addresses this gap, and improve QC practices. QC Constellation, a web application built using Python 3.11, integrates various statistical QC modules. These include Levey-Jennings charts with Westgard rules, sigma-metric calculations, exponentially weighted moving average (EWMA) and cumulative sum (CUSUM) charts, and method decision charts. Additionally, it offers a risk-based QC section and a patient-based QC module aligning with modern QC practices. The codes and the web application links for QC Constellation were shared at https://github.com/hikmetc/QC_Constellation, and http://qcconstellation.com, respectively. Using synthetic data, QC Constellation demonstrated effective implementation of Levey-Jennings charts with user-friendly features like checkboxes for Westgard rules and customizable moving averages graphs. Sigma-metric calculations for hypothetical performance values of serum total cholesterol were successfully performed using allowable total error and maximum allowable measurement uncertainty goals, and displayed on method decision charts. The utility of the risk-based QC module was exemplified by assessing QC plans for serum total cholesterol, showcasing the application's capability in calculating risk-based QC parameters including maximum unreliable final patient results, risk management index, and maximum run size andoffering risk-based QC recommendations. Similarly, the patient-based QC and optimization modules were demonstrated using simulated sodium results. In conclusion, QC Constellation emerges as a pivotal tool for laboratory professionals, streamlining the management of quality control and analytical performance monitoring, while enhancing patient safety through optimized QC processes.

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Strategies to verify equimolar peptide release in mass spectrometry-based protein quantification exemplified for apolipoprotein(a).

Quantitative protein mass spectrometry (MS) is ideally suited for precision diagnostics and for reference standardization of protein analytes. At the Leiden Apolipoprotein Reference Laboratory we apply MS strategies to obtain detailed insight into the protein-to-peptide conversion in order to verify that quantifier peptides are not partly concealed in miscleaved protein backbone. Apolipoprotein(a) (apo(a)) was digested in a non-optimal manner to enhance the number of miscleaved peptides that were identified by high resolution liquid chromatography tandem-MS measurements. The protein-to-peptide conversion was carefully mapped with specific attention for miscleaved peptides that contain an apo(a) quantifier peptide. Four different isotopologues of each apo(a)-quantifier peptide were applied to evaluate linearity of internal peptide standards during measurement of specific real-life samples. Two apo(a) quantifier peptides that were concealed in two different miscleaved peptides were included into a multiple reaction monitoring list in our targeted MS-based apo(a) quantifications to alert for potential protein digestion discrepancies. The presence of miscleaved peptides could be ruled out when applying our candidate reference measurement procedure (RMP) for apo(a) quantification. These data further corroborate the validity of our apo(a) candidate RMP as higher order method for certification of commercial Lp(a) tests that is endorsed by theInternational Federation of Clinical Chemistry and Laboratory Medicine. MS-based molecular detection and quantification of heterogeneous apo(a) proteoforms will allow manufacturers' transitioning from confounded lipoprotein(a) [Lp(a)] mass levels into accurate molar apo(a) levels.

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