New Generation Of Assays Research Articles

Companion diagnostics: the key to personalized medicine

This special focus issue of Expert Review of Molecular Diagnostics on in vitro companion diagnostics aims to provide the reader with up-to-date knowledge on this fast-evolving area of medical research. Companion diagnostics takes up a central role in the development of targeted drugs and to a large extent, the success of this type of therapy depends on their performance. Companion diagnostic assays have a single patient as a point of reference and they will be decisive for the move toward a more precise and individualized pharmacotherapy. The ‘first generation’ of companion diagnostic assays relies on single biomarker detection but with our increasing understanding of disease pathophysiology a new generation of assays is under development, which will be based on patient profiling and multiplex platforms.

A national survey of troponin testing and recommendations for improved practice.

The introduction of troponin assays with higher analytical sensitivity and enhanced performance has produced new challenges for both laboratory and clinician in the optimal investigation of patients with cardiovascular disease. After some years of collective experience with this new generation of assays, this survey aimed to assess the level of consensus that exists regarding their application. A questionnaire was designed, based on a review of published evidence and current opinion, to obtain information on a number of key areas relating to troponin analysis and reporting and was circulated to lead laboratory consultants across the UK and Ireland. Completed questionnaires were received from 94 laboratory contacts. Sixty per cent of those who responded had implemented a high-sensitivity troponin assay, with the Roche Cobas troponin T high sensitivity assay the most widely used. It is evident that some confusion remains regarding the definition of high-sensitivity assays and there was considerable variation in practice, even among those using the same manufacturer's assay. There is a need for greater consensus in the approach to the clinical utilization of troponin assays with improved sensitivity and it is important that laboratories are fully aware of the capabilities of their assay and provide useful guidance to users. On the basis of survey findings and the existing evidence base, a number of recommendations have been proposed to improve current practice and enhance patient safety.

Development and validation of a quantitative real-time polymerase chain reaction assay specific for the detection of Rickettsia felis and not Rickettsia felis-like organisms.

Human infections with Rickettsia felis have been reported worldwide. Recent studies have revealed the presence of many closely related but unique rickettsiae, referred to as Rickettsia felis-like organisms (RFLO), identified in various arthropods. Due to the recent discovery of the lack of specificity of earlier R. felis-specific assays, there has become a need to develop a new generation of R. felis-specific molecular assays that will differentiate R. felis not only from other rickettsiae but more importantly from other members of the R. felis genogroup that may not be pathogenic to humans. This new generation of assays is essential for determining the true risk for flea-borne spotted fever (FBSF) by surveying arthropod vectors/hosts. Because of the lack of specificity of previous assays developed to detect R. felis infections, prior surveys may have overestimated the prevalence of R. felis in arthropod vectors and thus the perceived risk of FBSF. We have developed a specific quantitative real-time polymerase chain reaction (qPCR) assay to detect R. felis (RfelB). Specificity of the assay was determined by testing it with a panel of 17 related Rickettsia species and 12 nonrickettsial bacterial DNA preparations. The RfelB qPCR assay was positive for R. felis DNA and negative for all of the 17 related Rickettsia species and 12 nonrickettsia bacterial DNA preparations. The limit of detection of the RfelB qPCR assay was determined to be two copies (two genoequivalents) per microliter of R. felis target ompB fragment-containing plasmid. Validation of the RfelB qPCR assay was accomplished by testing 83 previously sequence-confirmed R. felis and RFLOs containing DNA preparations from human and flea samples collected from different geographical locations around the world. This assay will be useful for rapid detection, identification, and enumeration of R. felis, an emerging human pathogen of worldwide importance, from both clinical and environmental samples.

Seralite© Rapid Point-Of-Care Detection Of Free Light Chain Escape, Non-Secretory Relapse and Light Chain Only Relapse In Multiple Myeloma

Free light chain (FLC) testing in serum is a more sensitive method of monitoring FLC secretion from myeloma cells than FLC testing in urine because FLC do not appear in urine until their secretion exceeds the renal tubule threshold for FLC reabsorption. Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) are insensitive methods of detecting FLC in serum. In 2002 quantitative detection of serum FLC by turbidimetric and nephelometric assays using polyclonal sheep anti-sera specific for FLC (Freelite®) became available; this has become the internationally recommended method of measuring FLC levels in serum. Two thirds of non-secretory (NS) patients who are IFE negative in serum and urine have abnormal FLC levels in serum and might be better classified as oligosecretory light chain only (LCO) patients. Serum FLC testing is particularly useful in diagnosis and monitoring of these and conventional LCO patients in whom FLC are detectable in urine when their disease is very active. In 5% IgG and 10% IgA M-protein myeloma patients, relapse is characterised by an increase in abnormal serum FLC without a corresponding increase in whole M-protein, termed ‘free light chain escape' and their relapse is detected earliest by serum FLC testing.Despite the clinical advantages of serum FLC testing, existing quantitative serum FLC assays are restricted to centralised laboratories. The time between venesection and availability of FLC results, for most clinicians, varies from days to weeks. This delays diagnosis and delays changes in clinical management when assessing response or monitoring for relapse. We have developed a second generation of serum FLC tests that utilise monoclonal rather than polyclonal anti-FLC antibodies. One result of this new generation of assays has been the development of a new rapid serum FLC test (Seralite®, Serascience Ltd, Oxford UK) that provides point of care measurement of serum FLC levels in just ten minutes.We assessed FLC levels at diagnosis, remission and relapse in 31 patients diagnosed with intact immunoglobulin myeloma who relapsed with abnormal serum FLC but without an increase in M-protein level (termed ‘light chain escape'). In all evaluable samples, Seralite® detected an abnormal serum FLC level at diagnosis, followed by a normalisation of the serum FLC ratio during remission, and a subsequent abnormal FLC level at relapse. Seralite® FLC results correlated diagnostically with the existing laboratory serum FLC assay for all samples.Additionally, we analysed FLC levels at diagnosis, remission and relapse in 166 myeloma patients characterised as having no intact immunoglobulin M-protein by serum IFE at diagnosis. 16% had a negative urine IFE and were identified only by the presence of an abnormal FLC ratio in their serum (NS or oligosecretory LCO). The remaining 84% had FLC detected in urine by IFE at diagnosis (conventional LCO). All diagnosis samples had an imbalanced FLC ratio detected by Seralite®. With a median follow-up of over 5 years, 83% of these patients relapsed with abnormal serum FLC levels. Half of the conventional LCO patients were IFE negative in the urine at relapse. Seralite® FLC results correlated diagnostically with the existing laboratory FLC assay for all samples.In summary, Seralite® detected abnormal serum FLC levels in all myeloma patients relapsing with light chain escape, and patients relapsing with non-secretory (oligosecretory LCO) and conventional LCO myeloma. There were no false negatives and no false positives supporting the use of Seralite® for the monitoring of these distinct myeloma patient groups. Disclosures:Campbell:Serascience Ltd: Equity Ownership. Stride:Serascience Ltd: Employment. Goodall:Serascience Ltd: Equity Ownership. Drayson:Serascience Ltd: Equity Ownership.

Troponina en el diagnóstico de infarto al miocardio: Consideraciones desde el laboratorio clínico

Among the many biomarkers studied to evaluate myocardial damage, troponin is considered the most sensitive and specific. However, current methodologies present pre-analytical, analytical and post-analytical problems, of which the more significant are the lack of standardization and a high uncertainty in the level of decision or cut-off. It is hoped that a new generation of assays called "ultra sensitive" will improve analytical performance characteristics of the technique and achieve compliance with the internationally recommended quality specifications.

Troponin in the diagnosis of myocardial infarction: An approach from the Clinical Laboratory

Among the many biomarkers studied to evaluate myocardial damage, troponin is considered the most sensitive and specific. However, current methodologies present pre-analytical, analytical and post-analytical problems, of which the more significant are the lack of standardization and a high uncertainty in the level of decision or cut-off. It is hoped that a new generation of assays called "ultra sensitive" will improve analytical performance characteristics of the technique and achieve compliance with the internationally recommended quality specifications.

NucliSENS® EasyQ® HPV v1 test – Testing for oncogenic activity of human papillomaviruses

Analytical sensitivity of DNA-based assays to detect infection with human papillomaviruses is very high, but clinical specificity for cervical cancer strongly depends on the age of the patient and case classification. To solve the dilemma between sensitivity and specificity, a new generation of assays focuses on the pathogenic factors that underlie the development of HPV-associated tumors: the expression of the viral oncogenes E6 and E7. Demonstration of persistent expression of these mRNAs or expression in the context of relevant clinical symptoms has a strong positive predictive value for the development of HPV-associated carcinomas and strongly warrants further diagnostic action. The NucliSENS EasyQ HPV v1 test was designed to test cervical scrapes for the expression of the oncogenic E6/E7 mRNA from the five most common carcinogenic HPV types (16, 18, 31, 33 and 45). The test can be used for confirmation and risk stratification of individuals with proven infection with high risk papillomaviruses. In order to establish performance of the assay, sensitivity, specificity, repeatability, and reproducibility were determined with artificial and clinical specimens. Further, a total of 420 cervical scrapes were tested and the results directly compared to the CE-market device PreTect HPV-Proofer assay (NorChip, Klokkarstua, Norway). For arbitration of discordant clinical results, the specimens were rated according to Pap-classification and the presence of HPV DNA was determined. The limit of detection for the five HPV types 16, 18, 31, 33, and 45 ranged from 2.3x10(2) to 3.0x10(4) copies/mL on a background of 5x10(3) HPV-negative HS67 cells. No cross-reactivity for other viral, bacterial, or fungal agents known to be potentially present in cervical fluids was detected. Repeatability and reproducibility were shown by testing panels of HPV-spiked artificial and clinical samples. A comparative analysis of 420 cervical scrapes demonstrated an overall concordance of >90% between the NucliSENS EasyQ HPV test and the technologically related PreTect HPV-Proofer assay.

A Novel Diagnostic Target in the Hepatitis C Virus Genome

BackgroundDetection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5′-noncoding region (5′-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.Methods and FindingsIn this study we determined by de novo sequencing that the 3′-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1–6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3–24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10−9 IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1–6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5′-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.ConclusionThis study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.

Monospecific and bispecific antibodies against E. coli O157 for diagnostics

Escherichia coli O157:H7 is a serious human pathogen that causes hemorrhagic colitis, and occasionally hemolytic uremic syndrome. Identification of the O157 antigen is an essential part of the detection and management of E . coli O157:H7. A quadroma P126 secreting a bispecific hybrid MAb (bsMAb), which recognizes both E. coli O157 and horseradish peroxidase in one molecule was produced by somatic hybridization of hybridomas specific for E. coli O157 and HRPO molecule. A bridge ELISA was used to select the quadromas obtained for bispecific monoclonal antibody purification and characterization. Benzhydroxamic-acid agarose (BHA) affinity co-chromatography was used as a convenient one-step method for purifying the HRPO–bsMAb complex for ultrasensitive diagnostic applications. Sandwich ELISA for detecting E. coli O157:H7 with HRPO–bsMAb allows quick one step detection of spiked E. coli O157:H7. The detection sensitivities were 100 CFU, 750 CFU and 500 CFU per 1 ml of tap water, lake water and apple juice respectively by microtiter assay. E. coli O157:H7 detection with immunofilter ELISA and immunomagnetic ELISA formats was approximately 1 CFU/ml and 10 CFU/ml respectively. BsMAbs avoid enzyme conjugation, has highest specific activity and molecular uniformity without aggregates and contribute to good signal to noise ratios. This new bispecific antibody can be generated and purified from quadroma cultures by affinity co-chromatography in one step and can be used to develop a new generation of assays for public health applications in water, food and human sample testing.

Bone and bone related biochemical examinations. Bone and collagen related metabolites. Osteocalcin (OC)

Osteocalcin is produced by mature osteoblasts and primarily deposited in the extracellular matrix of skeletal tissue. It has been shown that serum osteocalcin is a marker of osteoblastic activity, and the levels reflect the rate of bone formation. The first osteocalcin assays were competitive radioimmunoassays using bovine osteocalcin as a standard, and a new generation of assays was developed to measure the major circulating forms of the protein, which is the intact and the large N-terminal fragment. Since osteocalcin binds to hydroxyapatite crystal after gamma-carboxylation at three residues, increased amounts of immature undercarboxylated osteocalcin might reflect risks for fractures. However, at present, measurement of osteocalcin does not substitute for bone mass measurement and only provide limited clinical values on evaluation of patients with osteoporosis or metabolic bone diseases.

Early detection of hepatitis B virus infection: comparative sensitivity of HBV DNA detection by PCR and HBSAG detection by a new generation of assays: Robin Biswas, Chu-Chieh Hsia, FDA, Bethesda, MD; Michael Busch, Megan Laycock, Dale Hirschkorn, UCSF, San Francisco, CA; David Wright, Westat, San Luis Obispo, CA; Jay Epstein, Edward Tabor, FDA, Rockville, MD

Early detection of hepatitis B virus infection: comparative sensitivity of HBV DNA detection by PCR and HBSAG detection by a new generation of assays: Robin Biswas, Chu-Chieh Hsia, FDA, Bethesda, MD; Michael Busch, Megan Laycock, Dale Hirschkorn, UCSF, San Francisco, CA; David Wright, Westat, San Luis Obispo, CA; Jay Epstein, Edward Tabor, FDA, Rockville, MD

Evaluation of Roche Diagnostics ONLINE(R) DAT II, a New Generation of Assays for the Detection of Drugs of Abuse

A new generation of ONLINE assays has been developed that offers improved performance and enhanced ease of use. This family of assays is being applied to both the COBAS INTEGRA and Roche/Hitachi line of analyzers. The four ONLINE DAT II assays that were evaluated included cocaine (benzoylecgonine) (BE), methadone (MDN), opiates (OP), and tetrahydrocannabinol (THC). The BE assay has a dual cutoff (150/300 ng/mL) with a dynamic range from 0 to 5000 ng/mL. The MDN assay has a cutoff of 300 ng/mL with a dynamic range from 0 to 2000 ng/mL. The opiates assay has a 300 ng/mL cutoff with a 0 to 2000 ng/mL range and a 2000 ng/mL cutoff with a 0 to 8000 ng/mL range. The THC assay has 20, 50, and 100 ng/mL cutoffs with 0 to 100, 0 to 300, and 0 to 300 ng/mL dynamic ranges, respectively. The ranges of the intra-assay precision (coefficients of variation for n = 20) run in the semiquantitative mode are 2.3-7.5% for BE, 2.0-3.8% for MDN, 1.9-4.2% for OP, and 3.9-5.2% for THC. The intra-assay qualitative precision for all of the assays as calculated from absorbance values is generally higher than that of the intra-assay semiquantitative precision at the cutoff. The qualitative precision ranges between 0.4% and 3.1%. The standard curve stability defined for the COBAS INTEGRA systems for these reagents ranges from 35 to 68 days. The clinical sensitivity and specificity were compared to the OnLine generation I and CEDIA immunoassays, as well as gas chromatography-mass spectrometry (GC-MS). The results indicate that for each assay, the sensitivity and specificity were the same or greater when compared to the other two immunoassay technologies. The results of each assay also correlated very well (> 99%) when compared with GC-MS.

Use of bispecific antibodies in molecular velcro assays whose specificity approaches the theoretical limit of immunodetection for Bordetella pertussis.

A bispecific monoclonal antibody (bsMAb) that detects Bordetella pertussis, the causative agent of whooping cough, and horseradish peroxidase (HRPO) has been developed by use of the quadroma technology. A quadroma, P123, was produced by fusing two well-characterized hybridomas against the bacterium and the enzyme and was subcloned to obtain a stable bsMAb-secreting cell line. The quadroma was theoretically expected to produce up to 10 different molecular species of immunoglobulins, so secreted bispecific antibody was complexed with excess HRPO and the HRPO-bsMAb complex was purified in one step by benzhydroxamic acid-agarose affinity cochromatography. An ultrasensitive homosandwich molecular "velcro" enzyme-linked immunosorbent assay for the detection of B. pertussis whole bacteria with HRPO-bsMAb was established in both microplate and nasopharyngeal swab formats. This assay demonstrates a high sensitivity that approaches the theoretical limit of detection of one bacterium. This new nanoprobe can be used to develop a new generation of assays that are simple, inexpensive alternatives to quantitative PCR and that can be used by clinical laboratories. This strategy of homosandwich assays with solid-phase monospecific antibodies and solution-phase bsMAb with specificity for the same repeating surface determinants can be applied to generate ultrasensitive immunodiagnostic assays for viruses and bacteria.

Molecular and diagnostic clinical virology in real time.

During the last decade, the application of both qualitative and quantitative nucleic acid detection techniques has had a major impact on diagnostics in clinical virology. Both signal and target amplification‐based systems are currently used routinely in most if not all virology laboratories. However, commercial assays are only available for a very limited number of targets, and this has resulted in the development and introduction of assays developed in‐house for most viral targets. With improved and automated nucleic acid sample isolation techniques, as well as real‐time detection methods, a new generation of assays for most clinically important viruses is being developed. These technological improvements also make it possible to generate results with a very short turnaround time. As an example of a more individual‐patient disease‐management concept, we have introduced in our clinical setting the quantitative detection of Epstein–Barr virus (EBV) in T‐cell‐depleted allogeneic stem cell transplant patients. This has enabled us to develop models for pre‐emptive anti‐B‐cell immunotherapy for EBV reactivation, and for reducing not only the incidence of EBV lymphoproliferative disease (EBV‐LPD), but the virus‐related mortality. It is now also feasible to introduce molecular testing for those viruses that can easily be detected using classical virological methods, such as culture techniques or antigen detection. Prospective studies are needed to evaluate the clinical importance of the additional positive samples detected. It should, however, be clear that a complete exchange of technology is unlikely to occur, and that complementary methods should stay operational, making possible the discovery of new viruses. Furthermore, the ability to characterise viruses more easily by sequencing opens new possibilities for epidemiological studies. There is also an urgent need, with regard to molecular diagnostic methods, for the introduction and use of standardised materials and participation in international quality control programmes. Finally, with the introduction of a universal internal control throughout the whole procedure, the accuracy of the results generated is warranted.

Novel bispecific immunoprobe for rapid and sensitive detection of prostate-specific antigen

Prostate-specific antigen (PSA) is one of the most useful tumor markers for the screening and follow-up of prostate cancer. Bispecific monoclonal antibodies (bsMAbs) are unique immunoprobes that incorporate two different binding sites in the same antibody molecule. This antibody designing can bring important advantages in the development of new immunoassays. We have developed a new hybrid hybridoma that secretes bsMAb anti-PSA x anti-horseradish peroxidase. This bsMAb has shown rapid kinetics and an excellent detection limit in a sandwich single-step assay with a total incubation time of 15 min and a 5-min substrate development. This assay in a manual format has a detection limit of 0.028 microgram/L. Comparison with the Hybritech Tandem-E PSA assay yielded a regression equation with slope = 0.433 [95% confidence interval (CI) = 0.415-0.451], intercept = 0.88 (CI = 0.45-1.31), and Sy/x = 1.83 micrograms/L (r = 0.98). This new immunoprobe can be used to develop a new generation of assays for clinical laboratories and can be adapted to screening devices for physicians' offices and even home diagnostics.

Measurement of Parathyroid Hormone

Interpretation of serum immunoreactive PTH measurements requires an understanding of the secretion, metabolism, and heterogeneity of circulating immunoreactive PTH. Both intact hormone and biologically inactive carboxyl fragments containing the middle and C-terminal regions are secreted by the parathyroid glands. Inactive fragments also are produced peripherally by metabolism of intact hormone by liver and kidney. Inactive fragments represent 75 to 95% of the total immunoreactivity in serum, a consequence of their long half-life in vivo as compared with intact hormone. Immunoassays for PTH can be divided into those measuring intact hormone (N-terminal, intact) and those measuring both inactive fragments and intact hormone (mid-region, C-terminal, polyvalent). The latter principally measures inactive fragments because of their greater concentration as compared with intact hormone in peripheral serum. The clinical utility of PTH assays varies considerably because of differences in their specificity and sensitivity. Serum PTH levels have been more often observed to be elevated in individuals with primary hyperparathyroidism with the use of research quality radioimmunoassays that recognize both inactive fragments and intact hormone than with conventional N-terminal or intact assays. Homologous mid-region assays have provided exceptional clinical sensitivity in confirming primary hyperparathyroidism. Comparison of a sensitive mid-region radioimmunoassay with a recently developed two-site, noncompetitive chemiluminescent immunoassay for intact PTH indicated that both methods were highly useful in the differential diagnosis of hypercalcemia. The mid-region assay provided the best diagnostic sensitivity in primary hyperparathyroidism with more elevated levels of PTH. The sensitivity of the intact assay was good, a significant improvement over conventional N-terminal and intact assays. The specificity of the intact assay was clearly superior, with measured PTH levels found to be suppressed to below normal in most subjects with hypercalcemia associated with malignancy. In contrast, measured levels were primarily normal with the mid-region assay. The higher levels of immunoreactive PTH observed in nonparathyroid hypercalcemia with the mid-region assay are in agreement with the measurement of biologically inactive carboxyl fragments, which continue to be secreted in hypercalcemia.