Anne-Catherine Bekaert

Neutrophil gelatinase-associated lipocalin (NGAL) determined in urine with the Abbott Architect or in plasma with the Biosite Triage? The laboratory's point of view.

Neutrophil gelatinase-associated lipocalin (NGAL) has emerged as a potential marker for the early detection of acute kidney injury (AKI) (1–3). However, most studies used cumbersome techniques (ELISA) that are particularly difficult to implement in routine practice (3, 4). Recently, two commercially available kits for determination of NGAL have appeared on the market. The first one, from Abbott Laboratories (Abbott Park, IL, USA), is an automated immunoassay that allows determination of urinary NGAL with the Architect platform. The second, the Triage NGAL Test (Biosite-Inverness Medical, Waltham, MA, USA) is a point-ofcare immunoassay for the quantitative determination of NGAL in EDTA anticoagulated whole blood or plasma. The aim of this study was to perform an analytical validation and a comparison of imprecision of these new tests. The Architect NGAL assay is a non-competitive two-site sandwich immunoassay that utilizes two mouse antibodies recognizing distinct NGAL epitopes. The Triage NGAL is a rapid fluorescence immunoassay that is used with the Triage Meters. All tests were performed by a trained laboratory technician according to the manufacturer’s instructions. Both tests have been correlated against an established and validated ELISA that uses mouse monoclonal antibody raised against human NGAL ( HYB211-05; AntibodyShop, Gentofte, Denmark) (5, 6). We used e-noval (Arlenda, Liège, Belgium) software for the statistical evaluation of results.

Analytical evaluation of the new Abbott Architect 25-OH vitamin D assay

OBJECTIVES Validation of the Architect 25-OH vitamin D assay. DESIGN AND METHODS Determination of repeatability, reproducibility, accuracy profile and 25(OH)-vitamin D2 recovery on native samples. Comparison with DiaSorin Liaison and RIA. RESULTS AND CONCLUSION Coefficients of variation: <6% (13.6 ng/mL) and 2.2% (78.1 ng/mL). Functional sensitivity: 5 ng/mL. Accuracy profile shows that the method is validated between 13.6 and 78.1 ng/mL. Recovery of 25(OH)D2: 75,8%( 95% CI: 61.9-89.7%). Good correlation with DiaSorin RIA and Liaison <50 ng/mL; above this threshold a systematic positive bias was observed.

IDS iSYS automated intact procollagen-1-N-terminus pro-peptide assay: method evaluation and reference intervals in adults and children.

Abstract Background: We carried out a technical evaluation of the Immunodiagnostic Systems (IDS) automated intact procollagen-I N-terminus propeptide (PINP) assay on the iSYS platform, and established reference intervals for PINP in both adults and children. Methods: Assay imprecision, recovery and interference were studied. Serum and plasma values were compared, and PINP stability was assessed. Using 828 specimens, IDS iSYS intact PINP and Roche E170 total PINP values were compared. Specimens from 597 adults and 485 children and adolescents were used to establish reference intervals for intact PINP. Results: The method demonstrated good recovery and acceptable imprecision. The assay was unaffected by icterus and lipaemia, but haemolysis decreased measured PINP. Serum and plasma values were comparable. There was a non-linear relation between IDS intact and Roche total PINP values. Pre- and post-menopausal women had comparable PINP values, but there was a difference between women of different age groups. Serum PINP in men showed a decline in young age up to 45 years, but remained steady thereafter. Separate reference intervals were established for four age groups in women and for two age groups in men. Data for children were partitioned into four-year age groups, and these showed PINP to be high with no major gender differences until 12 years of age. Thereafter, values in females decreased in 13–16 years age groups and further in 17–20 years age groups, whereas PINP increased in boys of 13–16 years of age with a subsequent decline at 17–20 years. Conclusions: The IDS iSYS PINP intact assay appears to be reliable. We have established gender- and age-related reference intervals for children and adults based on a relatively large healthy North European population.

Analytical validation of serum bone alkaline phosphatase (BAP OSTASE) on Liaison

Abstract Background: The goal of this study was to validate the DiaSorin Liaison BAP OSTASE, a new method for measurement of bone alkaline phosphatase (BAP), and to compare this method with the Beckman-Coulter Access Ostase. We also wanted to establish the reference range for BAP in adults and children. Methods: We determined the precision, functional sensitivity, recovery, linearity and measurement uncertainty, accuracy profile and β-expectation limits. We defined an adult reference interval using individuals with 25-OH vitamin D >80 nmol/L, parathormone <58 ng/L, and normal calcium, phosphorous and estimated glomerular filtration rate. Each adult subclass (men/non-menopausal women/menopause women) contained 120 individuals. We also determined the 2.5th and 97.5th percentiles from a population of 450 children, stratified according to age and gender. Results: The results of the validation showed: precision <6%, functional sensitivity <0.74 μg/L, mean recovery 98.8±4.2% and good linearity. Relative uncertainty ranged from 9.0% to 12.9%, and the risk of one result falling out of the ±15% acceptance limits was <5% for concentrations between 7 and 94 μg/L. The Bland-Altman plot showed no systematic bias between the two methods. In adults, we did not find any statistical difference between the different subclasses. The upper limit of normality observed in the entire population (n=360) was 21.3 μg/L (90% CI: 18.3–24.2 μg/L). Conclusions: The Liaison BAP OSTASE is a robust method, and is completely validated between 7 and 93 μg/L: in this range, 95% of the values obtained will be within ±15% of the true value. Clin Chem Lab Med 2010;48:67–72.

Analytical and clinical evaluation of the new Fujirebio Lumipulse G non-competitive assay for 25(OH)-vitamin D and three immunoassays for 25(OH)D in healthy subjects, osteoporotic patients, third trimester pregnant women, healthy African subjects, hemodialyzed and intensive care patients

Abstract Background: In this study, we provide a short analytical evaluation of the new Fujirebio Lumipulse®G non-competitive immunoassay for 25(OH)D. Clinical performance was compared with three commercial competitive automated immunoassays against a Vitamin D Standardization Program (VDSP)-traceable liquid chromatography-tandem mass spectrometry (LC-MS/MS) in six different clinically relevant populations. Methods: Lumipulse®G 25(OH)D precision, measurement uncertainty, recovery, limit of quantification were assessed, as well as 25(OH)D2 and C3-epimer recovery. For method comparison, 250 serum samples obtained in healthy Caucasians and Africans, osteoporotic, hemodialyzed and intensive care patients and 3rd trimester pregnant women were analyzed by all methods. Correlation was studied using Passing-Bablok and Bland-Altman analysis. Concordance correlation coefficient (CCC) was calculated to evaluate agreement between immunoassays and the LC-MS/MS. Results: The Lumipulse®G 25(OH)D assay presented interesting analytical features and showed excellent correlation to the LC-MS/MS results (y=1.00×–1.35 ng/mL), as obtained in healthy Caucasian individuals. In the other special populations, Lumipulse®G presented a concordance with LC-MS/MS which was generally higher than competitors, even if all methods significantly under-recovered 25(OH)D in hemodialyzed patients. Intra-assay CV ranged from 12.1% at 9.6 ng/mL to 2.1% at 103.7 ng/mL and inter-assay CV ranged from 16.2 to 3.7% at the same concentrations, respectively. Measurement uncertainty, with a probability of 95%, were respectively 33.1 and 7.6% at these concentrations. LOQ was found to be at 4.6 ng/mL. Mean (95% CI) 25(OH)D2 revovery was 77% (74–81) and no cross-reactivity was observed with C3-epimer. Conclusions: Fujirebio Lumipulse®G 25-OH Vitamin D Total assay is therefore considered suitable for assessment of vitamin D status in clinical routine.

Analytical and clinical validation of the new Abbot Architect 25(OH)D assay: fit for purpose?

Abstract Background: We provide a clinical and analytical evaluation of the reformulated version of the Abbott Architect 25-hydroxyvitamin D assay. We compared this assay with three commercial automated immunoassays and against a VDSP-traceable liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in six different populations. We also supplemented 40 healthy volunteers with either 600,000 IU of vitamin D2 or 100,000 of vitamin D3 to evaluate the performance of the immunoassays vs. the LC-MS/MS. Methods: Precision and limit of quantification were assessed, 25(OH)D2 and C3-epimer recovery were calculated. Two hundred and forty samples obtained in healthy Caucasians and Africans, osteoporotic, hemodialyzed and intensive care patients and 3rd trimester pregnant women were analyzed by all methods. Correlation was studied using Passing-Bablok and Bland-Altman analysis. Concordance correlation coefficient (CCC) was calculated to evaluate agreement between immunoassays and LC-MS/MS. We verified if patients were homogeneously classified with the immunoassays when they took vitamin D2 or vitamin D3 after 1, 7 and 28 days. Results: We observed excellent analytical features and showed a very good correlation to the LC-MS/MS results in the overall population. Compared to the other immunoassays, concordance of the new Abbott assay with the LC-MS/MS was at least similar, and often better in diseased populations. Althought the cross-reactivity with 25(OH)D2 was not of 100%, there was no significant difference in the classifications of the patients, either supplemented with D2 or D3 or after 7 or 28 days. Conclusions: This modified version of the Abbott Architect assay is clearly improved compared to the previous one and presents a better agreement with the LC-MS/MS.

Analytical validation of the Liaison Calcitonin_II-Gen (DiaSorin)

Abstract Background: We validated the DiaSorin Liaison Calcitonin_II-Gen, an improved method for calcitonin (CT) measurements, compared this method with the Cisbio_h-CT kit and established the reference range of CT in a normal adult population. Methods: We determined the precision, functional sensitivity, traceability to the 2nd IS 89/620, linearity and measurement uncertainty, accuracy profile and β-expectation limits. We evaluated the specificity, the susceptibility to human anti-animal antibodies (HAMA), hook-effect and carry over. To establish a reference range, we selected 267 adults without renal insufficiency presenting with normal thyroid stimulating hormone (TSH), free thyroxin (T4) and calcium concentrations and without anti-thyroglobulin antibodies as our “reference” healthy population. We compared the method with Cisbio on 250 consecutive and 45 samples from a post-pentagastrin stimulation test. Results: Precision (expressed as CV) was <10% for the measurement range, functional sensitivity: 5.3 ng/L and the method was found linear until to a 1/10 dilution. Uncertainty ranged from 25% to 7.2%, and the risk that one result falls out of the ±20% acceptance limits was <5% between 2.9 and 1513 ng/L. The Bland and Altman plot showed no systematic bias between the two methods. The test is still prone to HAMA influence, does not present any hook-effect, although carry over was observed. Ninety-five percent of our adult reference population showed CT concentrations <7.4 ng/L, with an important gender difference: 95% of the men showed CT values <9.8 ng/L, whereas 95% of women were <4.0 ng/L. Conclusions: The Liaison Calcitonin_II-Gen is an analytically robust method. The important difference in gender observed in our population might lead to re-evaluation of the generally used “10 ng/L” cut-off in a multicentre prospective study.