Assessing the commutability of reference material formats for the harmonization of amyloid-ß measurements.

BACKGROUND: The cerebrospinal fluid (CSF) amyloid-ß (Aß42) peptide is an important biomarker for Alzheimer's disease (AD). Variability in measured Aß42 concentrations at different laboratories may be overcome by standardization and establishing traceability to a reference system. Candidate certified reference materials (CRMs) are validated herein for this purpose. METHODS: Commutability of 16 candidate CRM formats was assessed across five CSF Aß42 immunoassays and one mass spectrometry (MS) method in a set of 48 individual clinical CSF samples. Promising candidate CRM formats (neat CSF and CSF spiked with Aß42) were identified and subjected to validation across eight (Elecsys, EUROIMMUN, IBL, INNO-BIA AlzBio3, INNOTEST, MSD, Simoa, and Saladax) immunoassays and the MS method in 32 individual CSF samples. Commutability was evaluated by Passing-Bablok regression and the candidate CRM termed commutable when found within the prediction interval (PI). The relative distance to the regression line was assessed. RESULTS: The neat CSF candidate CRM format was commutable for almost all method comparisons, except for the Simoa/MSD, Simoa/MS and MS/IBL where it was found just outside the 95% PI. However, the neat CSF was found within 5% relative distance to the regression line for MS/IBL, between 5% and 10% for Simoa/MS and between 10% and 15% for Simoa/MSD comparisons. CONCLUSIONS: The neat CSF candidate CRM format was commutable for 33 of 36 method comparisons, only one comparison more than expected given the 95% PI acceptance limit. We conclude that the neat CSF candidate CRM can be used for value assignment of the kit calibrators for the different Aß42 methods.

Technical performance of a novel, fully automated electrochemiluminescence immunoassay for the quantitation of ß-amyloid (1-42) in human cerebrospinal fluid.

INTRODUCTION: Available assays for quantitation of the Alzheimer's disease (AD) biomarker amyloid-beta 1-42 (Aß [1-42]) in cerebrospinal fluid demonstrate significant variability and lack of standardization to reference measurement procedures (RMPs). We report analytical performance data for the novel Elecsys ß-amyloid (1-42) assay (Roche Diagnostics). METHODS: Lot-to-lot comparability was tested using method comparison. Performance parameters were measured according to Clinical & Laboratory Standards Institute (CLSI) guidelines. The assay was standardized to a Joint Committee for Traceability in Laboratory Medicine (JCTLM) approved RMP. RESULTS: Limit of quantitation was <11.28 pg/mL, and the assay was linear throughout the measuring range (200-1700 pg/mL). Excellent lot-to-lot comparability was observed (correlation coefficients [Pearson's r] >0.995; bias in medical decision area <2%). Repeatability coefficients of variation (CVs) were 1.0%-1.6%, intermediate CVs were 1.9%-4.0%, and intermodule CVs were 1.1%-3.9%. Estimated total reproducibility was 2.0%-5.1%. Correlation with the RMP was good (Pearson's r, 0.93). DISCUSSION: The Elecsys ß-amyloid (1-42) assay has high analytical performance that may improve biomarker-based AD diagnosis.

Development and clinical validation of a novel algorithmic score (GAAD) for detecting HCC in prospective cohort studies.

BACKGROUND: Alpha-fetoprotein (AFP) and des-gamma carboxyprothrombin (DCP), also known as protein induced by vitamin K absence-II (PIVKA-II [DCP]) are biomarkers for HCC with limited diagnostic value when used in isolation. The novel GAAD algorithm is an in vitro diagnostic combining PIVKA-II (DCP) and AFP measurements, age, and gender (biological sex) to generate a semi-quantitative result. We conducted prospective studies to develop, implement, and clinically validate the GAAD algorithm for differentiating HCC (early and all-stage) and benign chronic liver disease (CLD), across disease stages and etiologies. METHODS: Patients aged ≥18 years with HCC or CLD were prospectively enrolled internationally into algorithm development [n = 1084; 309 HCC cases (40.7% early-stage) and 736 controls] and clinical validation studies [n = 877; 366 HCC cases (47.6% early-stage) and 303 controls]. Serum samples were analyzed on a cobas® e 601 analyzer. Performance was assessed using receiver operating characteristic curve analyses to calculate AUC. RESULTS: For algorithm development, AUC for differentiation between early-stage HCC and CLD was 90.7%, 84.4%, and 77.2% for GAAD, AFP, and PIVKA-II, respectively. The sensitivity of GAAD for the detection of early-stage HCC was 71.8% with 90.0% specificity. Similar results were shown in the clinical validation study; AUC for differentiation between early-stage HCC and CLD was 91.4% with 70.1% sensitivity and 93.7% specificity. GAAD also showed strong specificity, with a lower rate of false positives regardless of disease stage, etiology, or region. CONCLUSIONS: The GAAD algorithm significantly improves early-stage HCC detection for patients with CLD undergoing HCC surveillance. Further phase III and IV studies are warranted to assess the utility of incorporating the algorithm into clinical practice.

Performance evaluation of the Elecsys PIVKA-II and Elecsys AFP assays for hepatocellular carcinoma diagnosis.

Background and Aims: Prothrombin induced by vitamin K absence-II (PIVKA-II) is a serum biomarker linked to hepatocellular carcinoma (HCC), showing superiority to alpha-fetoprotein (AFP) for early disease detection. We aimed to assess the clinical and analytical performance of the Elecsys® PIVKA-II immunoassay in diagnosing HCC and evaluate PIVKA-II's technical performance. Methods: Serum samples from adult cases (i.e. patients with a first-time HCC diagnosis; n = 168) and disease controls (i.e. patients without HCC with an at-risk condition; n = 208) were assessed. An AFP cut-off of 20 ng/mL was used to differentiate between HCC cases and disease controls. Clinical performance of the Elecsys PIVKA-II assay was compared with that of comparator assays (Lumipulse G PIVKA-II, µTASWako DCP, ARCHITECT PIVKA-II) using receiver operating characteristic curve analysis to determine the area under the curve (AUC) values. Results: The Elecsys PIVKA-II assay compared favorably with comparator assays. Using a 28.4 ng/mL cut-off, the Elecsys PIVKA-II assay detected HCC with 86.9% sensitivity and 83.7% specificity. Clinical performance of the Elecsys PIVKA-II assay (AUC: 90.8%) was equivalent to that of comparator assays (AUC: 88.3-89.6%). Relatively high PIVKA-II concentrations were observed for cholangiocarcinoma and pancreatic cancer with the Elecsys assay in specificity panel analyses, indicating that high PIVKA-II concentrations should not be used alone in the absence of other clinical data. Conclusions: The Elecsys PIVKA-II assay showed good analytical performance under routine laboratory conditions, comparing favorably with comparator assays. These findings support the suitability of the Elecsys PIVKA-II assay as an aid in HCC diagnosis.

Formation of circular polyribosomes in wheat germ cell-free protein synthesis system.

We report a morphological study of functioning ribosomes in a efficient and robust cell-free protein synthesis system prepared from wheat embryos. Sucrose density gradient analysis of translated mixtures programmed with luciferase mRNAs having different 5' and 3' untranslated regions showed formation of large polysomes. Electron microscopic examination of translation mixtures programmed with those of capped and polyadenylated mRNA revealed that ribosomes assemble into a circular-type polysome in vitro. Furthermore, a series of experiments using mRNAs lacking either cap, poly(A) tail or both also resulted in the formation of circular polysomes, which are indistinguishable from those with the original mRNA. The wheat germ cell-free system may provide a good experimental system for understanding functional ribosomes at the molecular level.

Rapid translation system: a novel cell-free way from gene to protein.

Proteome research has recently been stimulated by important technological advances in the field of recombinant protein expression. One major breakthrough was the development of a new generation of cell-free transcription/translation systems. The open and flexible character of these systems allows direct control over expression conditions via the addition of supplements to the expression reaction. The possibility of working with linear expression templates instead of cloned plasmids and the ease of downstream processing, circumventing the need for cell-lysis, makes them ideally suited for high-throughput screening applications. Among these novel cell-free systems, the Rapid Translation System (RTS) developed by Roche is the first one that is scalable from micrograms to milligrams of protein. This review describes the basic principles of RTS which differentiate it from traditional in vitro expression technologies, starting from template generation to high-end applications like labeling for structural biology research. Recent results obtained by RTS users from different institutions are presented to illustrate each step of a novel cell-free protein expression workflow and its benefits compared to traditional cell-based expression.