Imprecision CVs Research Articles

B-093 Analytical performances of a novel fluorescent immunoassay of anti-Müllerian hormone and establishment of the reference intervals in Chinese children

Abstract Background Anti-Müllerian Hormone (AMH) plays a important role in children growth and the concentrations change with age as well as gender. This study aimed to evaluate the analytical performances of a novel AMH assay based on the cycle enhanced fluorescence immunoassay (CEFIA) and establish the pediatric reference intervals of AMH Methods The experiments on imprecision, sensitivity, linearity, linear range and comparison were carried out to evaluate the analytical performances. Furthermore, the concentrations of serum AMH in 238 females and 346 males aged 0-18 years were measured by the Pylon AMH assays and the reference ranges for ages group were calculated using robust methods. Results The repeatability and the within-laboratory imprecision CVs of the assay were 3.7% and 6.4% at at 2.25 ng/mL, and 4.6% and 6.4% at 15.49 ng/mL, respectively. The sensitivity of the assay (LoB =0.05 ng/mL, LoD= 0.1 ng/mL and LoQ= 0.3 ng/mL) was verified. The AMH Pylon assays showed good linearity within the detection range (between 0.1 and 24 ng/ml) and can report up to 480 ng/mL with 20x pre-dilution of samples. The AMH concentrations of serum samples measured by the Pylon assays correlated to those measured by the Elecsys assays. In the males under 18 years old, the AMH levels were high at birth (0-1 month: median 95.10 ng/mL) and continued to increase to a peak between 7 months to one year old (median 158.80 ng/mL) before they started to decrease with age to the lowest level between 15 and 18 years old (median 6.31 ng/mL). On the other side, the serum AMH concentrations in females were low at birth (0-1 month: median 0.20 ng/mL) and increased with age (15-18 years old: median 3.03 ng/mL). The reference intervals were provided for each gender and age groups,respectively. Conclusions The Pylon AMH assays showed good analytical performances, and the AMH reference intervals in chinese children determined may provide a basis for the use in clinical diagnosis and treatment of related diseases.

A Multicentric Verification of Hereditary and Acquired Thrombophilia Coagulation Assays

Aims: With an ever-growing number of patients being admitted to our University Hospital Sveti Duh (Zagreb, Croatia), there was a need to positively respond to clinicians' demands about hereditary and acquired thrombophilia testing.
 Study Design: An extensive and multicentric verification of coagulation assays included in thrombophilia testing was performed on the BCS XP coagulation analyzer (Siemens Healthineers, Marburg, Germany): antithrombin activity (AT) (Innovance Antithrombin), protein C activity (PC) (Berichrom Protein C), protein S activity (PS) (Protein S Ac), free protein S antigen (free PS:Ag) (Innovance Free PS Ag), activated protein C resistance (APCR) (ProC Ac R and ProC Global + Coagulation Factor V Deficient Plasma), lupus anticoagulant (LA) screening (LA1 and activated partial thromboplastin time by using Dade Actin FSL as reagent) and confirmation test (LA2), factor VIII activity (FVIII) (Dade Actin FS and coagulation FVIII Deficient Plasma).
 Place and Duration of Study: Department of Medical Laboratory Diagnostics, University Hospital Sveti Duh, Zagreb, Croatia, between January 2020 and December 2021.
 Methodology: Verification protocol included: the precision study (CLSI EP15-A3 protocol), trueness estimation by comparison of seventy remnant plasma samples with two large Croatian hospital laboratories with established thrombophilia testing, and verification of reference intervals and cut-off values (CLSI EP28-A3C and CLSI HA-60 guidelines).
 Results: All of the obtained imprecision CVs were within the manufacturer's claims (<5/10/15%). While the observed bias for PC (+1.9%) was within the EFLM performance specifications (6.7%), the average bias for AT was higher than acceptance criteria (+10.8% vs 3.2% allowed). P&B regression revealed a significant positive proportional difference (slope=1.14). Comparison of PS activity yielded a high negative bias (-33.4%) that exceeded the acceptance criteria of 8.1%. Regarding LA testing, the diagnostic accuracy was 99% when compared to Sestre milosrdnice UHC (N=70) and 95% when compared to University Hospital Centre Zagreb (N=19). All manufacturer's reference intervals and cut-offs were verified.
 Conclusion: The verification study confirmed all manufacturer's claims, except for PS which has been replaced with free PS:Ag determinations.

Analytical performances of a new rapid assay of soluble ST2 for cardiac and inflammatory diseases and establishment of the reference intervals for children and adolescence in China

BackgroundsST2 has emerged as a potential disease biomarker of cardiac and inflammatory diseases in pediatrics. This study aimed to evaluate the performance of the new Pylon sST2 assay and establish the reference intervals of sST2 in children and adolescence in China. MethodsThe experiments on precision, linearity, effects of interferents and sample stability were carried out to evaluate the analytical performances. A total of 240 healthy participants, aged from 2 to 17 years were enrolled. The nonparametric method was used to calculate the age- and sex-specified reference intervals. sST2 levels were measured in children with different diseases to evaluate the assay's diagnostic performance. ResultsThe repeatability and within-laboratory imprecision CVs of the assay were 6.0% and 7.6% at 19.5 ng/ml, and 3.1% and 5.9% at 289.8 ng/ml, respectively. The method showed linearity between 2.5 and 918.5 ng/ml. It was also noteworthy that the sST2 level was not affected in the presence of hemoglobin (2 mg/ml), triglyceride (30 mg/ml), bilirubin (0.3 mg/ml) and cholesterol (5 mg/ml). sST2 was found stable for 5 days at 4 °C in serum sample. The reference interval was determined as 2.1–21.0 ng/ml in general. No significant variation was observed by sex. However, sST2 increased constantly with age, especially in male. Increased sST2 was found in patients of systemic lupus erythematosus, sepsis, Crohn's diseases, respiratory failure and post cardiac surgery. ConclusionsThe Pylon sST2 assay showed good analytical performances. The reference intervals were established in children and adolescence and sST2 showed potential clinical values in several diseases in pediatrics.

Absolute Quantification of Total Hemoglobin in Whole Blood by High-Performance Liquid Chromatography Isotope Dilution Inductively Coupled Plasma-Mass Spectrometry.

Accurate and traceable measurement of hemoglobin (HGB) is of great importance in clinical testing. Although the HiCN method is the internationally accepted conventional reference method for this biomarker and frequently used in clinical routine diagnostics, the HiCN method cannot be traceable to the International System of Units (SI) and thus does not meet highest metrological demands. In this study, an absolute quantitative approach for total HGB in a whole blood sample is proposed based on the determination of natural Fe and S present in the heme-group of HGB by HPLC isotope dilution ICP-MS. IRMM/IFCC-467 is used for method validation, and then clinical blood samples are measured by the established strategy and HiCN method. The measurable ranges of total HGB were 10.0-240.0 g L-1. Limits of detection via Fe and S were 0.01 and 0.07 g L-1, respectively. The intra-assay imprecision CVs via Fe and S were 0.89-1.35 and 0.99-1.56%, and the interassay CVs were 1.19-2.15 and 1.55-2.55%, respectively. Good agreement was achieved in the method validation. In the comparison with HiCN experiments, the ID-ICP-MS assays via Fe and S showed correlations of r2 = 0.991 and 0.970 against HiCN methods. Moreover, the concentration of transferrin (Tf) was also simultaneously measured. This strategy has potential to serve as a reference measurement procedure for total HGB in whole blood, which could be traceable to SI and does not require toxic derivation reagent.

Analytical and clinical evaluation of DiaSorin Liaison® Calprotectin fecal assay adapted for serum samples.

BackgroundCalprotectin is a calcium‐binding protein that can be measured in serum, plasma, and feces. Increased serum and plasma calprotectin concentrations have been found in chronic inflammatory rheumatic disorders. An analytical and clinical evaluation of the DiaSorin Liaison® fecal Calprotectin assay using LIAISON® XL was performed.MethodsThe protocol included an analytical and clinical evaluation in which imprecision, the linearity of dilution, differences between serum and plasma samples and method comparison with CalproLab™ ELISA kit were assessed. Serum calprotectin concentrations in active (n = 26) and remission (n = 23) rheumatoid arthritis (RA) patients were compared.ResultsThe intra‐day and inter‐day analytical imprecision CVs ranged from 2.9% to 4.0% and 2.7% to 10.4%, respectively. Correlation between measured and expected values was high (R > 0.99), indicating good linearity. The Wilcoxon signed‐rank test showed that serum and plasma matched samples presented statistically significant differences (p < 0.001) being the highest concentrations of calprotectin observed in serum samples. Deming regression equation was as follows: Diasorin calprotectin (μg/ml) = −0.32 (95% CI: −0.65 ‐ −0.05) +1.58 (95% CI: 1.42–1.79).* Calprolab calprotectin (μg/ml). Significantly higher serum calprotectin levels were found in RA patients with active disease when compared to patients with low disease activity or in clinical remission (mean ± SD) [(3.35 μg/ml ± 1.55) vs. (1.63 μg/ml ± 0.52), p < 0.001] and these levels correlated well with all disease activity indices.ConclusionsThe DiaSorin Liaison® fecal Calprotectin assay adapted for serum samples showed adequate technical performances and the clinical performances were similar to other assays.

Evaluation of the Analytical Performance of Lifotronic H9 Glycated Hemoglobin Analyzer and its Effect on Clinical Decision Making

We aimed to analyze the analytical performance of Lifotronic H9 glycated hemoglobin analyzer (Lifotronic H9) with Arkray Adams HA-8160 glycated hemoglobin analyzer (Arkray HA8160), which is currently used for measurement of HbA1c test in our laboratory. The verification studies of the Lifotronic H9 were performed by comparison with the Arkray HA8160, which has been verified and is already used in our laboratory, and in accordance with the CLSI EP5-A3 and CLSI 15-A3 guidelines. The method comparison study was performed with 316 subjects. Both instruments demonstrated perfect repeatability (within-run) CVs (0.8% - 1.6%), between-run CVs (0.2% - 1.0%), within-day CVs (0.8% - 1.7%), between-day CVs (0.3% - 0.6%), and within laboratory (total im-precision) CVs (0.8% - 1.8%) with two levels of quality control materials. Very good linearity was observed in the method-comparison studies based on Passing-Bablok regression analysis (y = -0.03 + 1.02 x, CUSUM test p = 0.07). Intraclass correlation coefficient (ICC = 0.995) for results and Kappa value (κ = 0.86) for classification of results according to the American Diabetes Association recommendations showed almost perfect agreement. The Lifotronic H9 analyzer shows good precision with lower than recommended total CV 2% and linearity with Arkray HA8160. The Lifotronic H9 is a suitable analyzer for the diagnosis of diabetes and monitoring diabetic control.

Analytical evaluation of the ADAMS™ A1c HA 8180 thalassemia mode high-pressure liquid chromatography analyser for the measurement of HbA2 and HbF.

ADAMS™ A1cHA-8180T is a HPLC system; within 3.5min, it quantifies HbF, HbA2 , and HbA0 and flags abnormal peaks. We evaluate its analytical performance for routine estimation of HbA2 and HbF, and critical tests were performed for identifying β-thalassemia carriers. Trueness imprecision, carry over, linearity, and effect of anemia were evaluated according to ICLH, ICLS, or manufacture's guidelines. Comparison (ADAMS™ A1c HA-8160T) was performed by running 400 samples from healthy subjects, 30 alpha and 80 beta carriers (range: 1.9-5.7 %). Trueness - HbA2 2.7 %, bias 0.81 %; HbA2 5.8 %, bias 0.38 %. HbA2 4.0% is not affected by Hb in the range 221-40g/L. Carry over was negligible. Within run: normal control - CV 1.5 %, high control - CV 0.9 %.Within laboratory: normal control - total CV% 1.59%; high control - 0.92 %. Linearity - y=1.034x-0.17, R2 =0.998 (range: 2.8-4.8%).Method comparison - y=0.93x+0.22, R2 =0.997. HbF imprecision CVs between 0.66 and 1.24% and trueness between 0 and 2.8%. Linearity - y=1.088x-0.27, R2 =0.999 (0.1-5.7%). ADAMS™ A1c HA-8180T provides a rapid and reliable separation of HbA2 . The measurement is accurate and reproducible, which is needed because of the slight difference between normal and pathological values. The gap in HbA2 values between normal subjects and β-thalassemia carriers makes this an appropriate method for rapid screening for carriers.

Performance evaluation of an automated electrochemiluminescent calcitonin (CT) immunoassay in diagnosis of medullary thyroid carcinoma

ObjectivesThe aim was to evaluate the Elecsys® hCT electrochemiluminescence immunoassay on the immunoanalyser Cobas e411. Design and methodsWithin-run, between-run imprecision, linearity, recovery after dilution, the high dose hook effect, as well as the stability of calcitonin were examined. A method comparison to an established immunoradiometric assay was executed in 135 routine plasma samples. ResultsWithin- and between-run imprecision CVs were ≤1.4% and ≤2.5%, respectively. The Elecsys® hCT assay was linear over the measuring range of 0.5–2000ng/L. Recoveries after dilution of a high sample in Diluent MultiAssay were within the range of 95% to 97%. The high dose hook point occurred at an hCT concentration of 56,000ng/L. Calcitonin was found to be stable up to 5h at RT, up to 24h at 2–8°C and up to one month at −20° and −80°C. Passing Bablok regression analysis showed no significant deviation from linearity (p=0.10) with a slope of 1.00 (95% CI 0.95–1.06) and an intercept of −0.81 (95%CI −1.01 to −0.46). ConclusionsThe Elecsys® hCT immunoassay provides precision and reliability in combination with reduced turnaround time as compared to the hCT IRMA.

Development and validation of 14 human serum protein assays on the Roche cobas® c 501

Many laboratories rely on dedicated nephelometers and turbidimeters for the measurement of serum proteins. There are, however, a number of chemistry analyzers that offer open channel configurations for end-user applications. We developed and validated 14 human serum protein assays (α(1)-antitrypsin, α(2)-macroglobulin, albumin, apolipoproteins AI and B, complement components 3 and 4, haptoglobin, immunoglobulins A, G, and M, orosomucoid, transferrin, and transthyretin) on the Roche cobas(®) c 501. We obtained excellent precision at low, normal, and high physiologic concentrations of each protein (within-run imprecision CVs ≤2.5%, total imprecision CVs ≤3.6%). Linearity for each method was within 5% of the expected value throughout the calibration range, and method comparison studies to commercial assays from Roche or Siemens were in good agreement (r>0.975). We observed no significant interference from bilirubin (up to 414 mg/l), hemoglobin (up to 8.9 g/l), triglyceride (up to 28 g/l), or rheumatoid factor (up to 3,930 IU/ml). Calibration was stable for at least 14 days. The instrument's small reaction cell allowed us to conserve nearly 60% of our specimen and reagent volume compared with our previous system. These newly developed assays provide precise and accurate results with high throughput, but without the associated cost of a dedicated instrument.

Quantification of Galactose-1-Phosphate Uridyltransferase Enzyme Activity by Liquid Chromatography–Tandem Mass Spectrometry

The diagnosis of galactosemia usually involves the measurement of galactose-1-phosphate uridyltransferase (GALT) activity. Traditional radioactive and fluorescent GALT assays are nonspecific, laborious, and/or lack sufficient analytical sensitivity. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay for GALT enzyme activity measurement. Our assay used stable isotope-labeled alpha- galactose-1-phosphate ([(13)C(6)]-Gal-1-P) as an enzyme substrate. Sample cleanup and separation were achieved by reversed-phase ion-pair chromatography, and the enzymatic product, isotope-labeled uridine diphosphate galactose ([(13)C(6)]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [(13)C(6)]-Glu-1-P (265 > 79) as an internal standard. The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) mumol x (g Hgb)(-1) x h(-1) in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 micromol x (g Hgb)(-1) x h(-1) (0.2% of normal control value). Intraassay imprecision was determined at 4 different levels (100%, 25%, 5%, and 0.2% of the normal control values), and the CVs were calculated to be 2.1%, 2.5%, 4.6%, and 9.7%, respectively (n = 3). Interassay imprecision CVs were 4.5%, 6.7%, 8.2%, and 13.2% (n = 5), respectively. The assay recoveries at the 4 levels were higher than 90%. The apparent K(m) of the 2 substrates, Gal-1-P and UDPGlc, were determined to be 0.38 mmol/L and 0.071 mmol/L, respectively. The assay in erythrocytes of 33 patients with classical galactosemia revealed no detectable activity. This LC-MS/MS-based assay for GALT enzyme activity will be useful for the diagnosis and study of biochemically heterogeneous patients with galactosemia, especially those with uncommon genotypes and detectable but low residual activities.

Multicenter evaluation of the first automated Elecsys sFlt-1 and PlGF assays in normal pregnancies and preeclampsia

ObjectivesPerformance evaluation of Elecsys® sFlt-1 and PlGF assays. Design and methodsWithin-, between-run, total imprecision, functional sensitivity, inter-laboratory comparison, method comparison and lot-to-lot reproducibility were evaluated. ResultsWithin- and between-run CVs were below 4% for sFlt-1 >60 and PlGF >20pg/mL. Total imprecision CVs were below 4.3%. Functional sensitivity was <5pg/mL. Inter-laboratory CVs were <5%. Elecsys correlated well with Quantikine VEGF-R1 (r=0.960) and PlGF (r=0.968). Lot-to-lot comparisons yielded highly correlated results (r>0.999).In healthy pregnancies, the median levels of sFlt-1 remained constant in first (1107pg/mL) and second trimesters (1437pg/mL) but increased in the third trimester (2395pg/mL), while median PlGF levels increased in the first (30pg/mL) and second trimesters (279pg/mL) and peaked at 29 to 32weeks (626pg/mL) and decreased thereafter (340pg/mL). The sFlt-1/PlGF ratio is highest in the first trimester (median: 28) but remained constant in the second (median: 4.7) and third trimesters (median: 5.1).In PE/HELPP samples matched for gestational age the sFlt-1 levels were significantly higher (6894–34,624pg/mL), whereas PlGF levels were lower (9.2–80pg/mL) and the median sFlt-1/PlGF ratio is much higher (461; range: 121–2614) than in apparently healthy pregnancies (3.6; range: 0.3–105). ConclusionThe new Roche Elecsys sFlt-1 and PlGF immunoassay showed excellent precision and reliability. There was a clear difference in the Elecsys sFlt-1/PlGF ratio between samples obtained from women with apparently normal pregnancy at the time of blood collection and those diagnosed with PE/HELLP at the same age of gestation.

Quantification of Urinary Albumin by Using Protein Cleavage and LC-MS/MS

Urinary albumin excretion is a sensitive diagnostic and prognostic marker for renal disease. Therefore, measurement of urinary albumin must be accurate and precise. We have developed a method to quantify intact urinary albumin with a low limit of quantification (LOQ). We constructed an external calibration curve using purified human serum albumin (HSA) added to a charcoal-stripped urine matrix. We then added an internal standard, (15)N-labeled recombinant HSA ((15)NrHSA), to the calibrators, controls, and patient urine samples. The samples were reduced, alkylated, and digested with trypsin. The concentration of albumin in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and linear regression analysis, in which the relative abundance area ratio of the tryptic peptides (42)LVNEVTEFAK(51) and (526)QTALVELVK(534) from albumin and (15)NrHSA were referenced to the calibration curve. The lower limit of quantification was 3.13 mg/L, and the linear dynamic range was 3.13-200 mg/L. Replicate digests from low, medium, and high controls (n = 5) gave intraassay imprecision CVs of 2.8%-11.0% for the peptide (42)LVNEVTEFAK(51), and 1.9%-12.3% for the (526)QTALVELVK(534) peptide. Interassay imprecision of the controls for a period of 10 consecutive days (n = 10) yielded CVs of 1.5%-14.8% for the (42)LVNEVTEFAK(51) peptide, and 6.4%-14.1% for the (526)QTALVELVK(534) peptide. For the (42)LVNEVTEFAK(51) peptide, a method comparison between LC-MS/MS and an immunoturbidometric method for 138 patient samples gave an R(2) value of 0.97 and a regression line of y = 0.99x + 23.16. Urinary albumin can be quantified by a protein cleavage LC-MS/MS method using a (15)NrHSA internal standard. This method provides improved analytical performance in the clinically relevant range compared to a commercially available immunoturbidometric assay.

Evaluation of a 1,25-Dihydroxyvitamin D Enzyme Immunoassay

Radioactive reagents are used in most assays for measurement of 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. We evaluated a 1,25(OH)(2)D enzyme immunoassay (EIA) from IDS Ltd. that uses solid-phase immunoextraction and colorimetric detection and compared results to those of the thymus radioreceptor assay (RRA) for 1,25(OH)(2)D. We collected serum samples (n = 145) representing an even distribution (0-200 pmol/L) of 1,25(OH)(2)D concentrations and Vitamin D External Quality Assessment Scheme (DEQAS) proficiency survey samples from 2004 surveys (n = 15) and stored them at -20 degrees C. We analyzed all samples with both EIA and RRA methods. We calculated imprecision using 5 QC samples in quadruplicate in each run (n = 6), including both pooled patient material used for QC with the RRA and QC material included in the EIA reagent set. We evaluated calibration stability by analyzing calibrators from different lots on the same plate and determining if calculated sample values drifted significantly. Deming linear regression between IDS EIA and RRA methods yielded slope 1.25 (95% CI 1.13-1.37), y-intercept -3 (95% CI -18 to 12), R(2) = 0.74. DEQAS proficiency survey samples for 2004 were all within 30% of the all-methods-trimmed mean. Imprecision CVs were 12%-16% within-run and 15%-20% between-run. We find no evidence of inferiority to the classic calf-thymus receptor assay for 1,25(OH)(2)D and no disadvantage in the results generated by the IDS EIA using samples from the major proficiency survey for 1,25(OH)(2)D. According to the product insert, however, the IDS EIA underestimates 1,25(OH)(2)D(2) compared with the D(3) form.

Measurement of free IGF-I saliva levels: Perspectives in the detection of GH/IGF axis in athletes

Objectives: To examine an immunoassay for measuring free IGF-I in a saliva specimen (free sIGF-I) and to study the levels in relation to the training conditions comparing young athletes and sedentary females. Design and methods: The analysis was carried out by modifying a commercial kit for plasma matrix to measure the free sIGF-I. The plasma free and total IGF-I fractions, hGH and salivary total proteins were also measured. Saliva and blood specimens were collected from 15 well-trained young female volleyball athletes and from a control group of 14 young sedentary females. Results: The calibration curve to assay free sIGF-I covered the range 0.05–5.00 μg/L. The detection limit was 0.07 μg/L. The within-run and between-run imprecision CVs were 10% and 13% respectively. The average recovery was 88%. Free sIGF-I, measured in 15 athletes and in 14 young sedentary females, was 0.10 ± 0.03 and 0.20 ± 0.05 μg/L respectively ( p < 0.001). Conclusions: There were decreased levels of free sIGF-I in well-trained athletes, compared with sedentary females. This decrease could be related to a greater tissue requirement by the active muscle subjected to intense exercise for several days.