Comparison of two chromogranin A assays and investigation of nonlinear specimens.

Background: As a marker for functional and non-functional neuroendocrine tumors, serum chromogranin A (CgA) concentrations have shown value for detecting and monitoring disease. Here we describe a comparison between an established micro-titer plate assay (Cisbio CgA ELISA) and an analyzer-based assay (B·R·A·H·M·S CgA II KRYPTOR). Reference limits were established along with a performance evaluation of the KRYPTOR assay. Nonlinearity observed in approximately 0.03% of patients was also investigated. Methods: Samples were tested according to kit manufacturer's protocols. Reference limits were established for both assays testing the same cohort of healthy volunteers. Potential causes of nonlinearity investigated were HAMA, macromolecule effects and elevated serum creatinine. Results: KRYPTOR vs. Cisbio: slope=0.692, y-intercept=-40.0 (r2=0.967, n=186). Upper reference limits were 160 and 103 ng/mL for the Cisbio and KRYPTOR assays, respectively. Linearity: slope=1.012 (r2=0.998) with 95.0-105.5% recoveries. Precision: repeatability ≤2.4%, within-laboratory ≤3.1% (79 and 738 ng/mL). Limit of detection: 8 ng/mL. Strong nonlinear specimens (n=6) retested for HAMA interference generated differences (block-no block) ranging -3.2-4.2%. Polyethylene glycol precipitation recoveries ranged from 157 to >5714% for affected specimens versus 71-79% for normal specimens. Eight of 14 nonlinear specimens (57%) had elevated serum creatinine results (>1.20 mg/dL). Conclusions: The CgA II KRYPTOR assay performs acceptably for quantifying CgA in human serum. While adequate correlation is observed against the Cisbio ELISA, there is significant disagreement overall. Efforts to identify a cause of the nonlinearity observed in a small percentage of patients were inconclusive, but neither HAMA interference, macromolecule effects nor renal failure appear as major factors.

Decision Threshold for Kryptor sFlt-1/PlGF Ratio in Women With Suspected Preeclampsia: Retrospective Study in a Routine Clinical Setting.

Background The objective was to evaluate predictive performance and optimal decision threshold of the Kryptor soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) ratio when implemented for routine management of women presenting with symptoms of preeclampsia. Methods and Results Observational retrospective study of a cohort of 501 women with suspected preeclampsia after 20 weeks of gestation. Women referred to maternity ward for observation of preeclampsia had an sFlt-1/PlGF ratio test included in routine diagnostic workup. Maternal and offspring characteristic data included maternal risk factors, outcomes, delivery mode, and indication for suspected preeclampsia. Biochemical measurements to determine sFlt-1/PlGF ratio were performed using the BRAHMS/Kryptor sFlt-1/PlGF ratio immunoassays. Results were analyzed by area under receiver-operating characteristic curve. Preeclampsia occurred in 150 of 501 (30%) of symptomatic women with an sFlt-1/PlGF ratio determined before the time of diagnosis. Area under receiver-operating characteristic curve for diagnosis of early-onset preeclampsia within 1 and 4 weeks was 0.98 (95% CI, 0.96-1.00) and 0.95 (95% CI, 0.92-0.98), respectively. For late-onset preeclampsia, predictive performance within 1 and 4 weeks was lower: 0.90 (95% CI, 0.85-0.94) and 0.85 (95% CI, 0.80-0.90), respectively. The optimal single sFlt-1/PlGF ratio threshold for all preeclampsia and late-onset preeclampsia within 1 and 4 weeks was 66. The negative and positive predictive values for ruling out and ruling in developing preeclampsia within 1 week were 96% and 70%, respectively. Conclusions The Kryptor sFlt-1/PlGF ratio is a useful clinical tool ruling out and in preeclampsia within 1 week. Prediction within 4 weeks is superior for early-onset preeclampsia. A single decision threshold of 66 is indicated for use in clinical routine.

Concordance of Three Automated Procalcitonin Immunoassays at Medical Decision Points.

Procalcitonin (PCT) is a useful bacterial infection biomarker with the potential for guiding antibiotic therapy. We evaluated the concordance of three automated PCT immunoassays: Kryptor (BRAHMS GmbH, Hennigsdorf, Germany), Atellica IM 1600 (Siemens Healthcare Diagnostics, Munich, Germany), and Cobas e801 (Roche Diagnostics, Mannheim, Germany). In 119 serum samples with a PCT concentration <5.00 µg/L, Kryptor (reference assay) was compared with the other two immunoassays by Spearman's rank correlation, regression analysis, and concordance at two antibiotic stewardship medical decision points: 0.25 and 0.50 µg/L. The Atellica IM 1600 and Cobas e801 results showed high correlations with those of Kryptor, with correlation coefficient (ρ) values of 0.97 and 0.99, respectively. However, negative biases were observed in both immunoassays (slope/y-intercept: 0.75/-0.00 for Atellica IM 1600; 0.88/-0.01 for Cobas e801). Atellica IM 1600 and Cobas e801 demonstrated excellent concordance with Kryptor at both medical decision points, with linearly weighted κ values of 0.90 and 0.92, respectively, despite discrepancies, which were more prominent at the 0.25 µg/L medical decision point. Based on these biases and discrepancies, the alternate use of different PCT immunoassays in repeat examinations is inadvisable. Standardization is required before comparing the results of different PCT immunoassays.

Analytical performance evaluation of thyroid-stimulating hormone receptor antibody (TRAb) immunoassays.

BACKGROUND: Thyroid-stimulating hormone receptor (TSHR)-activating autoantibodies stimulate thyroid growth and hormone synthesis/secretion, causing hyperthyroidism of Graves' disease (GD). TRAb measurement helps diagnose GD and is an important first test in evaluating hyperthyroidism according to the recent American Thyroid Association guidelines. We compared the performance of the BRAHMS TRAK Kryptor (Thermo Scientific) and Roche cobas TRAb immunoassays for use in GD. METHOD: Method comparison (n = 40) and clinical agreement were assessed between the Kryptor, cobas e411, and cobas e601. The analytical performance of Kryptor and cobas e411 were assessed for within- and between-day imprecision across 20 days, linearity, functional assay sensitivity (FAS), dilution recovery, and cut-off verification. RESULTS: The Kryptor, e411, and e601 TRAb immunoassays correlated well (r > 0.95, overall percent agreement = 0.95, Cohen's kappa = 0.90). With a total allowable error of 20%, percent bias was within 13%, which was minimally negative at <20 IU/L, but highly positive (33%-34%) >20 IU/L. The Kryptor, but not e411, was linear across the claimed analytical measuring range (AMR). The claimed functional assay sensitivity (FAS), which was close to the clinical GD cut-off 1.8 IU/L, was verified for Kryptor and e411. CONCLUSION: Overall, our evaluation demonstrates acceptable comparability between TRAb immunoassays with in-house imprecision up to 13% and 10% on Kryptor and e411, respectively. While Roche has preferable calibration frequency and on-board reagent stability, both platforms demonstrate acceptable imprecision using patient samples at their claimed FAS, which is important for GD diagnosis. Diluted results (using a negative patient pool as diluent) exhibits proportional positive bias on the Kryptor relative to the Roche methods.

Serum Chromogranin A as a Complementary Marker for the Prediction of Prostate Cancer-Specific Survival.

Better prognostication of clinically localized prostate cancer (PCA) is urgently needed. Former studies using different study end-points provided controversial results regarding the prognostic value of serum chromogranin A (CGA) in clinically localized PCA. However, serum CGA was not tested for correlation with the most significant study end-point of long-term disease-specific survival (DSS). CGA and matrix metalloproteinase-7 (MMP7) levels were measured by the BRAHMS KRYPTOR in two independent patient groups with 127 serum and 110 plasma samples. CGA and MMP7 concentrations were correlated with clinicopathological and survival data. In addition, we tested the combinations of CGA with PSA and with a currently identified prognostic factor, MMP7, for their prognostic value. CGA concentrations were significantly elevated in advanced compared to clinically localized cases both in serum and plasma samples (45 vs. 23 ng/ml, p < 0.001 and; 41 vs. 22 ng/ml; p = 0.002 respectively). In accordance, high CGA levels were correlated with poor DSS. In clinically localized cases, CGA levels alone were not prognostic, but its dichotomized combinations with PSA or MMP7 were independently associated with DSS (HR: 4.88, 95% CI: 1.35-17.71, p = 0.016, HR: 7.46, 1.65-33.63, p = 0.009, respectively). Elevated serum CGA levels in progressed PCA and its prognostic value suggest a potential for CGA in disease monitoring. Our results revealed no independent prognostic value for CGA as a single serum marker in clinically localized cases. However, when combining with PSA or MMP7, CGA may improve both marker's performance in distinguishing between clinically significant and indolent PCAs.

Development of medians generators for the calculation of MoM for the first-trimester Down syndrome maternal serum markers.

The Down syndrome risk calculation software, Fast Screen pre I plus, from Thermo Fisher Scientific, converts the First-trimester maternal serum markers concentrations (PAPP-A and hCGß) into degrees of extreme (DoE). To meet the requirements of the French legislation as well as those of the certified biologists for Down syndrome screening, a conversion tool for DoE into multiple of the median (MoM), intermediate reference value for the screening, was developed. In absence of any median polynomial allowing to obtain MoM easily, the calculations were made first on the basis of the polynomials centiles from the software. The subsequent reviews of the risk algorithms showed the fragility of this approach based on the Gaussian hypothesis. Medians generators, calculated from the data of the Kryptor club's biologists, were established.

KRYPTOR-automated angiogenic factor assays and risk of preeclampsia-related adverse outcomes.

OBJECTIVE: To evaluate KRYPTOR assays for circulating soluble fms-like tyrosine kinase-1 (sFlt1) and placental growth factor (PlGF) in risk assessment of adverse outcomes in women with suspected preeclampsia. METHODS: We studied 412 women carrying a singleton pregnancy from a previous study cohort who were evaluated for suspected preeclampsia. Another 434 nonpreeclamptic patients with plasma samples drawn throughout pregnancy were used to derive normative data. Plasma sFlt1 and PlGF levels were measured on the automated KRYPTOR platform and evaluated for prediction of adverse maternal and perinatal outcomes within 2 weeks. Normative values were used to create a ratio of markers and these values were reported as multiples of median (MoM) for women with and without adverse outcomes. The KRYPTOR assay results were also compared with previously reported measurements obtained using the automated Elecsys platform. RESULTS: Among participants presenting at <34 weeks (N = 110), patients with subsequent adverse outcome had higher sFlt1, lower PlGF, and higher sFlt1/PlGF ratio compared with women without adverse outcomes: the median (25th, 75th centile) sFlt1 (pg/ml), 9030 (3197, 12,140) versus 1976 (1248, 2937); PlGF (pg/ml), 36 (16, 111) versus 318 (108, 629); and ratio, 285.6 (32.2, 758.5) versus 6.1 (2.3, 20.3) (all p < 0.0001). Higher sFlt1/PlGF ratio correlated negatively with timing of delivery (r = -0.60, p < 0.001) and the risk of adverse outcomes was markedly elevated among women in highest tertile compared with lower tertile (odds ratio, 14.77; 95% confidence interval (CI), 4.28-51.00). The addition of sFlt1/PlGF ratio (≥85) to hypertension and proteinuria significantly improved the prediction for subsequent adverse outcomes (AUC 0.89 (95% CI): 0.82, 0.95) for hypertension, proteinuria, and sFlt1/PlGF (AUC = 0.75 (0.65, 0.85)) for hypertension alone (p = 0.002). Compared with normative controls, women who were evaluated for preeclampsia without adverse outcomes had higher MoM for sFlt1/PlGF ratio; these values were further elevated in women with adverse outcomes. sFlt1/PlGF ratios measured on the KRYPTOR platform were highly correlated with measurements obtained using Elecys platform (r = 0.97, p < 0.001). CONCLUSIONS: In women with suspected preeclampsia presenting prior to 34 weeks of gestation, KRYPTOR assays for circulating sFlt1 and PlGF when used in conjunction with standard clinical evaluation performs well in the prediction of adverse maternal and perinatal outcomes occurring within 2 weeks of presentation.

Validation of the diagnostic and prognostic relevance of serum MMP-7 levels in renal cell cancer by using a novel automated fluorescent immunoassay method.

PURPOSE: Despite encouraging results in other cancers, in renal cell cancer, no consensus exists regarding the diagnostic and prognostic relevance of MMP-7. The aim of this study was to assess the diagnostic and prognostic potential of serum MMP-7 levels in renal cell cancer. Furthermore, parallel to the widely used ELISA method, we tested a new, fluid-phase, fluorescent immunoassay (B.R.A.H.M.S KRYPTOR®) for the quantitation of MMP-7. METHODS: We analyzed the serum samples of 174 individuals (77 patients and 97 age-matched healthy controls) by a commercially available sandwich ELISA and by a novel, automated, fluid-phase immunofluorescent assay (B.R.A.H.M.S KRYPTOR®). Results were correlated with the clinicopathological and follow-up data. RESULTS: MMP-7 concentrations showed a high concordance level (R (2) = 0.979) between the two methods (p < 0.001). Serum MMP-7 concentrations were significantly higher in patients compared to controls. At a cutoff value of 3.15 ng/ml, a specificity and a sensitivity of 70 and 82 % for the detection of RCC was found. Patients with metastasis had significantly higher MMP-7 levels as those without metastasis (p = 0.038 by KRYPTOR, p = 0.011 by ELISA). High MMP-7 levels proved to be independently associated with shorter overall, disease-specific and metastasis-free survival, regardless of the analytical method. CONCLUSIONS: Based on these results, serum MMP-7 levels have both diagnostic and prognostic potential. The KRYPTOR method provided comparable results to the standard ELISA analysis, with a high concordance level and can therefore be considered as a surrogate method. Its flexibility and automated operation make the KRYPTOR MMP-7 assay suitable for routine laboratory use in the daily practice.

Automated two-site immunofluorescent assay for the measurement of serum chromogranin A.

OBJECTIVES: Chromogranin A (CgA) is the best-characterized biological marker common to neuroendocrine tumours and is therefore recommended for their diagnosis. The measurement of serum CgA is of great importance for reaching an early diagnosis and thus reducing the delay before treatment is instigated. The Kryptor CgA assay is the first fully automated assay available. The aim of this study was to evaluate its analytical performance. DESIGN AND METHODS: The imprecision and linearity of the Kryptor CgA assay were evaluated. This assay was compared with the Cis Bio CgA RIA assay in 78 serum samples. Its clinical utility was assessed in serum from 229 patients. RESULTS: The study performed on imprecision of Kryptor measurements showed intra- and inter-run CVs ≤ 5%. The study of linearity showed a satisfactory recovery rate for CgA concentrations up to 1200 µg/L. The Kryptor and RIA assays agreed well on the basis of the cut-off values provided by the two manufacturers. The Bland and Altman plot of the values obtained (range: 20-5560 µg/L) provided a mean difference of -10.1 µg/L (SD: 116). The clinical sensitivities of Kryptor CgA for diagnosis of pheochromocytoma and paraganglioma (n 20) and gastroenteropancreatic NETs (n 17) were respectively 100 and 94%. CONCLUSIONS: The Kryptor assay for CgA shows reliable analytical and clinical characteristics and allows a fast delivery of results.

[Prenatal risk calculation: comparison between Fast Screen pre I plus software and ViewPoint software. Evaluation of the risk calculation algorithms]. / Calcul du risque de trisomie 21 fœtale : comparaison du logiciel Fast Screen pre I plus avec le logiciel ViewPoint. Évaluation des algorithmes de calcul.

The Fetal medicine foundation (FMF) has developed a new algorithm called Prenatal Risk Calculation (PRC) to evaluate Down syndrome screening based on free hCGß, PAPP-A and nuchal translucency. The peculiarity of this algorithm is to use the degree of extremeness (DoE) instead of the multiple of the median (MoM). The biologists measuring maternal seric markers on Kryptor™ machines (Thermo Fisher Scientific) use Fast Screen pre I plus software for the prenatal risk calculation. This software integrates the PRC algorithm. Our study evaluates the data of 2.092 patient files of which 19 show a fœtal abnormality. These files have been first evaluated with the ViewPoint software based on MoM. The link between DoE and MoM has been analyzed and the different calculated risks compared. The study shows that Fast Screen pre I plus software gives the same risk results as ViewPoint software, but yields significantly fewer false positive results.