Agnès Carlisi

Evaluation of automated immunoassays for 25(OH)-vitamin D determination in different critical populations before and after standardization of the assays

INTRODUCTION Standardization of immunoassays for 25(OH)-vitamin D determination is a major problem in clinical practice. A worldwide standardization program has started to address this and will reduce the bias observed between immunoassays. We aimed to calibrate 5 immunoassays on a LC-MS/MS traceable to the SRM 2972 and the ID-LC-MS/MS 25(OH)D Reference Method Procedure to see if the re-standardization would be efficient in a population of 3rd trimester pregnant women (PW), hemodialysis (HD) and osteoporosis (OP) patient. MATERIAL AND METHODS 184 serum samples (25(OH)D: 8.4-87 ng/ml) were selected to calibrate the immunoassays (Abbott-Architect, Roche-Elecsys, DiaSorin-Liaison, Siemens-Centaur and IDS-iSYS). Chromsystems MassChrom method was used as the referenced. Serum obtained in 34 PW, 25 HD and 34 OP patients were used as comparatives. RESULTS After adjusting to LC-MS/MS, immunoassays had regression slopes nearly identical to 1.0 with intercepts <0.5 ng/ml. However, in special populations, a systematic bias was still observed, except for iSYS. CONCLUSIONS Re-standardization of 25(OH)D immunoassay will globally improve the differences. However, patients with a different serum matrix will still present significantly different results when they will be run with different methods. For those patients, the LC-MS/MS method seems to be the method of choice, even if some immunoassays are less influenced than others.

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.

Cross-reactivity of 25-hydroxy vitamin D2 from different commercial immunoassays for 25-hydroxy vitamin D: an evaluation without spiked samples

Etienne Cavalier*, A. Michael Wallace, Agnès Carlisi, Jean-Paul Chapelle, Pierre Delanaye and Jean-Claude Souberbielle 1 Department of Clinical Chemistry, University of Liege, CHU Liege, Liege, Belgium 2 Department of Clinical Biochemistry, Glasgow Royal Infirmary, Glasgow, Scotland, UK 3 Department of Nephrology and Dialysis, University of Liege, CHU Liege, Liege, Belgium 4 Laboratoire d’Explorations Fonctionnelles, Hôpital Necker-Enfants Malades, Paris, France

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.

Analytical Quality of Calcitonin Determination and Its Effect on the Adequacy of Screening for Medullary Carcinoma of the Thyroid

Calcitonin, a 32–amino acid calcium-lowering peptide secreted by the C cells (parafollicular cells) of the thyroid, is used as a marker for medullary carcinoma of the thyroid (MCT). However, calcitonin is not specific for MCT, because it is also secreted by other neoplasms, including breast cancer and small cell lung cancer. Secretion of calcitonin is regulated primarily by the concentration of extracellular calcium, but can also be stimulated by gastrin. Although calcitonin concentrations are higher in men than in women and tend to decline with age, many laboratories use a cutoff value of 10 ng/L instead of a population-based reference interval. Prior studies have shown that basal calcitonin concentrations are below this threshold in a normal population(1)(2) and in 90% of …

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.

Human anti-mouse antibodies interferences in Elecsys PTH assay after OKT3 treatment.

We report the case of a 61-year-old white man with end-stage renal disease secondary to an autosomal dominant polycystic kidney disease. Dialysis therapy was commenced in 2003, and he received a renal transplant from a deceased donor in 2004. His circulating parathormone (PTH) concentration before transplantation was 287 pg/mL as measured by the Roche Elecsys immunoassay analyser (Mannheim, Germany), in the target of the K/DOQI recommendations (1). The transplanted kidney functioned until 2007, and then failed because of acute cellular rejection (type II B according to Banff classification). He received standard antirejection medication (intravenously steroids and OKT3) but did not respond, lost function of his renal graft and returned to hemodialysis in February 2007. Four months later, the renal graft was removed because of systemic manifestations of rejection. From March to December 2007, plasma PTH concentrations were measured every 3 months again by Roche Elecsys. The results showed a rise of PTH after 7 months on hemodialysis, and this was even more dramatic after 10 months (Table 1). No parathyroid mass was identified by TcSestamibi or ultrasound scans. An interference in the PTH assay was thus suspected, and the sample was sent to the reference laboratory where extra-investigations were performed, as already published (2). The treatment of the sample with heterophilic blocking tubes (Scantibodies, Shantee, CA), which removes human anti-animal antibodies, resulted in an important decrease of PTH, from 3748 to 552 pg/mL. Treatment with antirheumatoid factor (IBL, Hamburg, Germany) did not result in a significant change in PTH. PTH was then measured by a different second-generation chemiluminescent immunoassay (Liaison, Diasorin, Saluggia, Italy) that uses polyclonal anti-goat antibodies, one directed against the N-terminal (aa 1–34) region, and the other one directed against the C-terminal (aa 39 – 84) part, which showed a result at 605 pg/mL, not altered by inclusion of human anti-animal antibodies or rheumatoid factor (RF). By contrast the suspect Roche Elecys intact PTH assay uses two murine monoclonal antibodies each of which bind to different epitopes on the PTH molecule; the Nterminal (aa 26 –32) portion (3), and the C-terminal fragment (aa 38 – 84). Thus these two assays use antibodies derived from different animal species which bind to different epitopes on the PTH molecule. This led us to conclude that there was an analytical interference in the PTH determination by Elecsys, and that this interference was because of a human anti-mouse IgM. Treatment with OKT3, a murine monoclonal antibody directed against the CD23 of human T-cell antibodies, is well known to induce the production of human anti-mouse antibodies (4). These antibodies can reduce the efficiency of the drug by blocking the interaction with the target cells, but can also interfere with the immunoassays that use murine antibodies (5). Such interferences are not always obvious to detect, particularly in the case of hemodialyzed or transplanted patients, where high circulating concentrations of PTH can be expected. In conclusion, physicians should always keep in mind that OKT3 treatment can result in the production of human anti-mouse antibodies. These antibodies can interfere with any immunoassay and give spurious results, leading to unnecessary cost-effective extra-investigations.

Standardization of DiaSorin and Roche automated third generation PTH assays with an international standard: impact on clinical populations

Abstract Background: Standardization of parathyroid hormone (PTH) assays is a major issue, especially in hemodialyzed (HD) patients. Two automated third generation PTH assays (Roche Elecsys and DiaSorin Liaison) are now available. These assays are specific for the (1-84) PTH and do not cross-react with the (7-84) fragment, contrary to second generation (intact) assays. We aimed to calibrate the two methods against the WHO International PTH Standard (IS) 95/646 to see if the two assays could provide comparable results in a population of healthy subjects, HD patients and patients suffering from primary hyperparathyroidism (PHP). Methods: We selected 79 healthy subjects and two populations of patients presenting PTH disorders: 56 HD and 27 PHP patients. We reconstituted the IS in a pool of human serum containing undetectable levels of 1-84 PTH and prepared 13 serum standards ranging from 0 to 2000 pg/mL. The standards were run on the two instruments to calibrate the assays on the IS. The different populations were run before and after restandardization. Results: As these kits were differently calibrated, the results obtained after restandarization were significantly different. Restandardization process improved concordance between assays and, taking the analytical variability of the two kits into account, the results could be considered to be similar. Conclusions: Restandardization of automated third generation PTH assays with the WHO 1-84 PTH Standard significantly reduces inter-method variability. Reference ranges and raw values are totally transposable from one method to the other in healthy subjects, but also in diseased patients, e.g., with HD or those suffering from PHP.

Analytical validation of the new version of the Liaison N-Tact PTH assay

Abstract We performed analytical validation of the new version of the Liaison N-Tact PTH (parathormone) assay according to NCCLS guidelines and compared this new generation of reagent with the Roche Elecsys PTH assay. We showed that this new version is a sensitive and precise method with good recovery. Significant carryover was observed, but with limited clinical implications. We demonstrated that the new version of the Liaison PTH is in reasonably good agreement with the Roche Elecsys PTH assay, and as we observed no differences in a hemodialyzed population, moving from one method to the other should not affect the daily follow-up of patients. However, one should be cautious with the highest values (>500 pg/mL). We established reference intervals of 12–54 pg/mL for the Liaison and 14–52 pg/mL for the Elecsys assay. Clin Chem Lab Med 2007;45:105–7.