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Background and aims Creatinine-based MDRD and CKD-EPI equations include a race correction factor, which results in higher eGFR in Black patients. We evaluated the impact on our patient population upon adoption of the CKD-EPI equation and the removal of the race correction factor from the equation. Materials and methods Retrospective analysis of blood creatinine results and respective eGFR values calculated by the MDRD or CKD-EPI equation without the race correction factor (CKD-EPINoRace) in a large academic medical system over a 20.5-month period. Results In our population, when changing from MDRD to CKD-EPINoRace, we observed that 3.5% of all patients were reclassified to categorically have worse kidney function. However, we also observed fewer patients overall with eGFR below 60 mL/min/1.73 m2. Around 60 and 20 mL/min/1.73 m2, 2.96% and 0.16% of all patients > 65 years of age were reclassified, as were 4.29% and 0.03% of all Black patients, respectively. When calculated with CKD-EPINoRace, median eGFR was not meaningfully different between Black and non-Black patients (p = 0.02). Conclusions Changing from MDRD to CKD-EPINoRace could lead to a lower referral rate to nephrology. The distributions of creatinine and eGFR calculated with CKD-EPINoRace were not meaningfully different in Black and non-Black patients.

Background Conventional HER2-targeting therapies improve outcomes for patients with HER2-positive breast cancer (BC), defined as tumors showing HER2 protein overexpression by immunohistochemistry and/or ERBB2 gene amplification determined by in situ hybridization (ISH). Emerging HER2-targeting compounds show benefit in some patients with neither HER2 protein overexpression nor ERBB2 gene amplification, creating a need for new assays to select HER2-low tumors for treatment with these compounds. We evaluated the analytical performance of a targeted mass spectrometry-based assay for quantifying HER2 protein in formalin-fixed paraffin-embedded (FFPE) and frozen BC biopsies. Methods We used immunoaffinity-enrichment coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) to quantify HER2 protein (as peptide GLQSLPTHDPSPLQR) in 96 frozen and 119 FFPE BC biopsies. We characterized linearity, lower limit of quantification (LLOQ), and intra- and inter-day variation of the assay in frozen and FFPE tissue matrices. We determined concordance between HER2 immuno-MRM-MS and predicate immunohistochemistry and ISH assays and examined the benefit of multiplexing the assay to include proteins expressed in tumor subcompartments (e.g., stroma, adipose, lymphocytes, epithelium) to account for tissue heterogeneity. Results HER2 immuno-MRM-MS assay linearity was ≥103, assay coefficient of variation was 7.8% (FFPE) and 5.9% (frozen) for spiked-in analyte, and 7.7% (FFPE) and 7.9% (frozen) for endogenous measurements. Immuno-MRM-MS-based HER2 measurements strongly correlated with predicate assay HER2 determinations, and concordance was improved by normalizing to glyceraldehyde-3-phosphate dehydrogenase. HER2 was quantified above the LLOQ in all tumors. Conclusions Immuno-MRM-MS can be used to quantify HER2 in FFPE and frozen BC biopsies, even at low HER2 expression levels.

Supplemental Digital Content is Available in the Text., Background: The opioid crisis has had a substantial financial impact on the health care system in the United States. This study evaluates how health plans have been affected financially and shows how a laboratory benefit management (LBM) program can be used to address related drug testing in an outpatient setting. Methods: Monthly claims data from private health plans were collected from June 1, 2016 to February 29, 2020. The total number of claims (units) for definitive and presumptive drug testing were calculated and the number of paid claims recorded. Claims distribution by laboratory type and medical code billed, the paid rate and compound annual growth rate, and the test distribution and paid rate of rendering providers who had submitted a minimum of 1000 claims were determined. Results: In total, 2,004,230 drug testing claims were submitted. After the LBM program was implemented, the percentage of paid claims for definitive drug testing (Healthcare Common Procedure Coding System code G0483) decreased and the paid rate for the low-cost tests (HCPCS code G0480) in physician office and independent laboratory settings increased. The compound annual growth rate for G0483 claims submitted indicated a 70.5% and 31.9% decrease in payments to physician offices and independent laboratories, respectively, for the period ending February 2020. Conclusions: An LBM program can positively address policy enforcement while reducing unnecessarily complex tests and limiting potential fraud, waste, and abuse by directing testing toward laboratories amenable to cost-efficient contractual savings. Moreover, for definitive drug testing, the enforcement of the use of Healthcare Common Procedure Coding System codes and a move toward more cost-efficient tests (G0480), when clinically applicable, supported by clinical practice guidelines, or evidence-based medicine, is an approach to providing medical benefits while maintaining health costs.

Background Fentanyl was developed in the 1960s as an alternative to morphine, but quickly became a drug of abuse due to its potency, inexpensiveness, and ease of synthesis. One source of exposure is mixing fentanyl into other drugs of abuse (e.g., heroin), but users also actively seek out this potent opioid. While monitoring for pain medication compliance and office-based opioid treatment, we noticed increasing fentanyl use. We sought to investigate this increase in the local population, and see if this reflected the regional health, morbidity, and mortality statistics. Methods This data review was determined not to involve “human subjects” as defined by federal regulations by the University of Washington (UW) Human Subjects Division (STUDY00014988). Local data were extracted from the laboratory information system and analyzed. Data from the King County Medical Examiner’s Office derives from cases sent to the Washington State Toxicology Laboratory. The Addictions, Drug, and Alcohol Institute (ADAI) at the UW compiled data from the Washington State Department of Health, the Forensic Laboratory Services Bureau, Washington State Patrol, and the state Office of Financial Management. Results We found a significant increase in fentanyl positivity in clinical LC-MS/MS assays, an increase in deaths due to fentanyl, and an increase in the fentanyl usage documented by the public health laboratory. Conclusions Clinical data from community toxicology testing performed at academic medical centers can reflect trends in society at large, and as such, there may be a compelling reason to publish and use these data to inform public health approaches.

Precision tissue diagnostics rely on high quality input specimens so that assay results are not affected by artifact, but advances in collection and processing of tissue specimens have lagged behind innovations in diagnostic assay development. Therefore, we have designed and evaluated a novel surgical tissue collection device that maintains and monitors sample temperature and motion throughout transport so that the major preanalytical variable of tissue temperature can be controlled and measured. This device, in combination with an improved cold–hot tissue fixation protocol affords optimal biomarker preservation in less overall time, thereby simultaneously improving diagnostic quality and turnaround time. We collected 50 primary and metastatic liver tumors using a novel transport device. Tissue was fixed using a rapid cold–hot fixation protocol and immunohistochemical assays were used to assess the performance of the device, in comparison to control tissue preserved using standard clinical fixation protocol. Two pathologists evaluated the IHC studies in a blinded fashion to determine the immunophenotype of each tumor. The observed IHC staining intensities and the clinical impressions of the immunophenotypes did not differ between tissue collected with the novel device and control tissue, while improvements in processing time were achieved. The novel cold transport device and rapid fixation protocol can be successfully and safely combined and used to monitor specimen conditions, thus preserving the diagnostic utility of specimens and improving the overall turn-around time of the diagnostic process. Electronic supplementary material The online version of this article (10.1007/s10561-019-09800-8) contains supplementary material, which is available to authorized users.

The widespread use of opioid drugs has contributed to escalating rates of addiction, overdoses, and drug-related deaths. Targeted urine drug screening plays an important role in supporting the care of patients with chronic pain or addiction. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can provide excellent sensitivity and specificity, and, as a result, remains the definitive choice for confirmatory urine drug testing. However, the complexities of LC-MS/MS operation present major challenges to the clinical laboratory. In this study, we leveraged upgraded instrumentation to develop and validate a simplified "dilute-and-shoot" LC-MS/MS opioid assay. By modifying the chromatographic gradient, isobaric interferences were well-resolved and eliminated. Analytical ranges were expanded by utilizing alternative mass transitions, and updated quality assurance parameters were established. Results from 204 clinical samples correlated well between the new method and a previous version. The upgraded systems provided better sensitivity, greater dynamic ranges, and the new method reduced carryover, which enabled us to eliminate extra injections and chromatogram reviews. The new method also reduced turnaround time and doubled testing capacity. These improvements could serve as a model for other laboratories approaching a similar transition in mass spectrometric instrumentation.

The development of precision testing for disease diagnosis has advanced medicine by specifically matching patients with drugs to treat specific diseases. High-quality diagnostics start with high-quality tissue specimens. The development and optimization of tissue handling and processing have lagged behind bioassay development. Ultrasound time-of-flight (TOF) technology has been successfully used to monitor the critical processing step of tissue fixation with formalin. In this study, we expand the use of this technology to monitor tissue dehydration and clearing by analyzing TOF signals from 270 different specimens, representing 13 different tissue types obtained through surgical resections. We determined the time constant τ90 for each tissue type for the following tissue processing solvents: 70% ethanol, 90% ethanol, 100% ethanol, and xylene. The TOF signals were correlated with tissue morphology to ensure that high-quality tissue was produced. Tissues can be grouped into those exhibiting fast and slow reagent diffusion. We monitored incomplete dehydration of tissue by skipping a key processing step, dehydration in absolute ethanol, and then correlated the τ90 with poor histomorphology, demonstrating that the technique can detect significant processing errors. Ultrasound TOF technology can therefore be used to monitor all phases of tissue processing cycle and yields an important preanalytical quality metric.

Objective We evaluated the early impact of a new hospital-based health technology assessment (HB-HTA) program, called Smart Innovation, at the University of Washington Medical Center (UWMC). Methods We compared the UWMC's utilization trends for two surgical procedures to control hospitals by evaluating the difference before and after adoption decisions: (i) a new filter for transcatheter aortic valve replacement (TAVR) procedures that treat aortic valve stenosis and (ii) microwave ablation (MWA) for treating hepatocellular carcinoma. We used descriptive statistics to assess the difference between the UWMC and controls for TAVR and MWA procedures and multivariate difference-in-differences (DID) analyses to test for statistical significance. Results The UWMC experienced a 10 percent reduction in TAVR procedures compared with controls following the implementation of the TAVR Sentinel filter. The DID regression model indicated a 1.5 reduction in the number of TAVR procedures per quarter at the UWMC between the pre- and post period, which was not statistically significant (p-value: .87). The UWMC experienced a 51 percent reduction in utilization when compared with controls for MWA procedures in the pre- and post periods. The DID model for MWA indicated an 18.8 decrease in utilization per quarter during the study period for the UWMC, which was statistically significant (p-value: .0007). For MWA procedures, the UWMC experienced a $647,658 dollar reduction in total costs in the post period compared with controls. Conclusions When the UWMC used HB-HTA methods for technology adoption, there was a reduction in utilization and total costs when compared with controls; however, when the UWMC adopted a new technology without using HB-HTA methods, there was no difference in utilization.

To ensure that clinical laboratories produce results that are both accurate and of clinical utility it is essential that only samples of adequate quality are analysed. Although various studies and databases assessing the stability of analytes in different settings do exist, guidance on how to perform and report stability studies is lacking. This results in studies that often do not report essential information, thus compromising transferability of the data. The aim of this manuscript is to describe the C hecklist for R eporting S tability S tudies (CRESS) against which future studies should be reported to ensure standardisation of reporting and easy assessment of transferability of studies to other healthcare settings. The EFLM WG-PRE (European Federation of Clinical Chemistry and Laboratory Medicine Working Group for the Preanalytical Phase) produced the CRESS checklist following a detailed literature review and extensive discussions resulting in consensus agreement. The checklist consists of 20 items covering all the aspects that should be considered when producing a report on a stability study including details of what should be included for each item and a rationale as to why. Adherence to the CRESS checklist will ensure that studies are reported in a transparent and replicable way. This will allow other laboratories to assess whether published data meet the stability criteria required in their own particular healthcare scenario. The EFLM WG-PRE encourage researchers and authors to use the CRESS checklist as a guide to planning stability studies and to produce standardised reporting of future stability studies.

We describe the methods and decision from a health technology assessment of a new molecular test for bladder cancer (Cxbladder), which was proposed for adoption to our send-out test menu by urology providers. The Cxbladder health technology assessment report contained mixed evidence; predominant concerns were related to the test's low specificity and high cost. The low specificity indicated a high false-positive rate, which our laboratory formulary committee concluded would result in unnecessary confirmatory testing and follow-up. Our committee voted unanimously to not adopt the test system-wide for use for the initial diagnosis of bladder cancer but supported a pilot study for bladder cancer recurrence surveillance. The pilot study used real-world data from patient management in the scenario in which a patient is evaluated for possible recurrent bladder cancer after a finding of atypical cytopathology in the urine. We evaluated the type and number of follow-up tests conducted including urine cytopathology, imaging studies, repeat cystoscopy evaluation, biopsy, and repeat Cxbladder and their test results. The pilot identified ordering challenges and suggested potential use cases in which the results of Cxbladder affected a change in management. Our health technology assessment provided an objective process to efficiently review test performance and guide new test adoption. Based on our pilot, there were real-world data indicating improved clinician decision-making among select patients who underwent Cxbladder testing.

A series of unfortunate events The history of how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread around the planet has been far from clear. Several narratives have been propagated by social media and, in some cases, national policies were forged in response. Now that many thousands of virus sequences are available, two studies analyzed some of the key early events in the spread of SARS-CoV-2. Bedford et al. found that the virus arrived in Washington state in late January or early February. The viral genome from the first case detected had mutations similar to those found in Chinese samples and rapidly spread and dominated subsequent undetected community transmission. The other viruses detected had origins in Europe. Worobey et al. found that early introductions into Germany and the west coast of the United States were extinguished by vigorous public health efforts, but these successes were largely unrecognized. Unfortunately, several major travel events occurred in February, including repatriations from China, with lax public health follow-up. Serial, independent introductions triggered the major outbreaks in the United States and Europe that still hold us in the grip of control measures. Science, this issue p. 571, p. 564, The first case of SARS-CoV-2 in Washington state occurred at the end of January and, by going undetected, sparked community transmission., After its emergence in Wuhan, China, in late November or early December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus rapidly spread globally. Genome sequencing of SARS-CoV-2 allows the reconstruction of its transmission history, although this is contingent on sampling. We analyzed 453 SARS-CoV-2 genomes collected between 20 February and 15 March 2020 from infected patients in Washington state in the United States. We find that most SARS-CoV-2 infections sampled during this time derive from a single introduction in late January or early February 2020, which subsequently spread locally before active community surveillance was implemented.

ObjectivesWe designed, developed, and implemented a new hospital-based health technology assessment (HB-HTA) program called Smart Innovation. Smart Innovation is a decision framework that reviews and makes technology adoption decisions. Smart Innovation was meant to replace the fragmented and complex process of procurement and adoption decisions at our institution. Because use of new medical technologies accounts for approximately 50 percent of the growth in healthcare spending, hospitals and integrated delivery systems are working to develop better processes and methods to sharpen their approach to adoption and management of high cost medical innovations.MethodsThe program has streamlined the decision-making process and added a robust evidence review for new medical technologies, aiming to balance efficiency with rigorous evidence standards. To promote system-wide adoption, the program engaged a broad representation of leaders, physicians, and administrators to gain support.ResultsTo date, Smart Innovation has conducted eleven HB-HTAs and made clinician-led adoption decisions that have resulted in over $5 million dollars in cost avoidance. These are comprised of five laboratory tests, three software-assisted systems, two surgical devices, and one capital purchase.ConclusionsSmart Innovation has achieved cost savings, avoided uncertain or low-value technologies, and assisted in the implementation of new technologies that have strong evidence. The keys to its success have been the program's collaborative and efficient decision-making systems, partnerships with clinicians, executive support, and proactive role with vendors.

Hemolysis is conventionally defined as membrane disruption of red blood cells and other blood cells that is accompanied by subsequent release of intracellular components into the serum or plasma. It accounts for over 60% of blood sample rejections in the laboratory and is the most common preanalytical error in laboratory medicine. Hemolysis can occur both in vivo and in vitro. Intravascular hemolysis (in vivo) is always associated with an underlying pathological condition or disease, and thus careful steps should always be taken by the laboratory to exclude in vivo hemolysis with confidence. In vitro hemolysis, on the other hand, is highly preventable. It may occur at all stages of the preanalytical phase (i.e. sample collection, transport, handling and storage), and may lead to clinically relevant, yet spurious, changes in patient results by interfering with laboratory measurements. Hemolysis interference is exerted through several mechanisms: (1) spectrophotometric interference, (2) release of intracellular components, (3) sample dilution and (4) chemical interference. The degree of interference observed depends on the level of hemolysis and also on the assay methodology. Recent evidence shows that preanalytical practices related to detection and management of hemolyzed samples are highly heterogeneous and need to be standardized. The Working Group for Preanalytical Phase (WG-PRE) of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) has published many recommendations for facilitating standardization and improvement of this important preanalytical issue. Some key EFLM WG-PRE publications related to hemolysis involve: (i) a call for more transparency and some practical recommendations for improving the harmonization of the automatic assessment of serum indices and their clinical usefulness, specifically the hemolysis index (H- index), (ii) recommendations on how to manage local quality assurance of serum or plasma hemolysis/icterus/lipemia-indices (HIL-indices) and (iii) recommendations on how to detect and manage hemolyzed samples in clinical chemistry testing. In this review we provide a comprehensive overview of hemolysis, including its causes and effects on clinical laboratory assays. Furthermore, we list and discuss the most recent recommendations aimed at managing hemolyzed samples in everyday practice. Given the high prevalence of hemolyzed blood samples, the associated costs, the great heterogeneity in how hemolysis is handled across healthcare settings, countries and continents, and increasing patient cross-border mobility, standardization and quality improvement processes aimed at combatting this important preanalytical problem are clearly warranted.

The first automobile accident in the US occurred in 1891, only 6 years after Karl Benz developed the first gasoline-powered production automobile. Less poorly documented is the interval of time that elapsed between Yalow's invention of the immunoassay, Mullis' development of the polymerase chain reaction, or Fenn's demonstration of electrospray mass spectrometry and the first orders for clinical laboratory tests based on these technologies that were placed on the wrong patients, for the wrong reasons, at the wrong times. Our lack of documentary evidence of the timeline of laboratory test malutilization notwithstanding, we in the laboratory industry know full well that each step of progress we make towards technical and biomedical innovation in clinical laboratory testing is accompanied by small but significant steps backwards, as medical practitioners misorder the tests we provide owing to confusion, poorly implemented electronic ordering, a culture of medical practice that encourages daily testing, and a panoply of other reasons. The available data converge on an estimate that on average 30% of laboratory tests are ordered inappropriately or are unneeded, and, equally as worrisome, an unknown but sizeable fraction of laboratory tests are underutilized, meaning that they should be ordered but are not. Enter the concept of laboratory test utilization management, or “stewardship,” or “demand management,” or any one of a host of names that describe the role laboratorians can and should play in addressing the problems in our medical systems that conspire to prevent the performance of laboratory tests on the right patients, at the right times, for the right reasons. Although interventions that work (and don't work) to optimize laboratory test utilization have been reported and reviewed extensively in the literature, this Q&A focuses on current topics in the field, with some as-yet unanswered questions posed to leaders in this field. Laboratory test utilization …

Background Unrecognized pseudohyperkalemia (PHK), defined as an artificial increase in measured potassium concentration, due to thrombocytosis and leukocytosis can lead to inappropriate patient treatment. Understanding the laboratory and patient characteristics that increase risk of PHK is key to preventing diagnostic errors. Methods Serum/plasma potassium results collected at 2 laboratories over 4 years were selected based on blood cell counts collected within 24 h and whole blood potassium concentrations determined within 2 h of the serum/plasma sample. Differences between whole blood and serum or plasma potassium were compared as functions of platelet or leukocyte count, fit to linear models, and stratified based on leukemia diagnosis codes. Patients having a serum/plasma potassium concentration that was at least 1 mEq/mL higher than the whole blood concentration were defined as having PHK. Based on this analysis, high-risk patients were prospectively identified and PHK risk was communicated to providers. Medication administration records were queried to compare rates of kayexalate use pre- and post-intervention. Results Approximately 14% of serum samples with platelet counts > 500 × 109/L had a > 1 mEq/L increase relative to whole blood potassium. > 25% of serum and plasma samples showed a > 1 mEq/L increase relative to whole blood potassium when leukocyte counts were > 50 × 109/L. Patients with chronic lymphocytic leukemia and high WBC count demonstrated the highest rates of PHK. The rate of kayexalate administration prior to confirmatory testing decreased from 37% to 16% after the laboratory started verbally communicating the possibility of PHK to treating providers. Conclusions According to our data, a leukocyte count threshold for plasma samples of 50 × 109/L is appropriate for indicating a high risk of PHK. Direct communication by the laboratory to the care team reduces inappropriate potassium lowering treatment in populations at high risk.

Supplemental Digital Content is available in the text., Personalized medicine promises diagnosis and treatment of disease at the individual level and relies heavily on clinical specimen integrity and diagnostic assay quality. Preanalytics, the collection and handling steps of a clinical specimen before immunohistochemistry or other clinical assay, are critically important to enable the correct diagnosis of disease. However, the effects of preanalytics are often overlooked due to a lack of standardization and limited assessment tools to quantify their variation. Here, we report a novel real-time ultrasound time-of-flight instrument that is capable of monitoring and imaging the critical step in formalin fixation, diffusion of the fixative into tissue, which provides a quantifiable quality metric for tissue fixation in the clinical laboratory ensuring consistent downstream molecular assay results. We analyzed hundreds of tissue specimens from 34 distinct human tissue types and 12 clinically relevant diseased tissues for diffusion and fixation metrics. Our measurements can be converted into tissue diffusivity constants that correlate with the apparent diffusion constant calculated using magnetic resonance imaging (R2=0.83), despite the differences in the approaches, indicating that our approach is biophysically plausible. Using data collected from time-of-flight analysis of many tissues, we have therefore developed a novel rapid fixation program that could ensure high-quality downstream assay results for a broad range of human tissue types.

Phosphoproteins are the key indicators of signaling network pathway activation. Many disease treatment therapies are designed to inhibit these pathways and effective diagnostics are required to evaluate the efficacy of these treatments. Phosphoprotein IHC have been impractical for diagnostics due to inconsistent results occurring from technical limitations. We have designed and tested a novel cold transport device and rapid cold plus warm formalin fixation protocol using phosphoproteins IHC. We collected 50 liver tumors that were split into two experimental conditions: 2 + 2 rapid fixation (2 hours cold then 2 hour warm formalin) or 4 hour room-temperature formalin. We analyzed primary hepatocellular carcinoma (n = 10) and metastatic gastrointestinal tumors (n = 28) for phosphoprotein IHC markers pAKT, pERK, pSRC, pSTAT3, and pSMAD2 and compared them to slides obtained from the clinical blocks. Expression of pERK and pSRC, present in the metastatic colorectal carcinoma, were better preserved with the rapid processing protocol while pSTAT3 expression was detected in hepatocellular carcinoma. Differences in pSMAD2 expression were difficult to detect due to the ubiquitous nature of protein expression. There were only 3 cases expressing pAKT and all exhibited a dramatic loss of signal for the standard clinical workflow. The rapid cold preservation shows improvement in phosphoprotein preservation.

Study objective Several methods are available to predict unbound (free) phenytoin concentrations in patients with hypoalbuminemia; however, predictive methods have not been evaluated in patients with concurrent hypoalbuminemia and kidney dysfunction or in patients with mild to moderate (estimated glomerular filtration rate [eGFR] 30-90 ml/min/1.73 m2 ) kidney dysfunction alone. Thus the objective was to evaluate the accuracy and precision of predictive methods to estimate free phenytoin concentrations in patients with varying albumin concentrations and/or kidney dysfunction. Design Retrospective chart review. Setting Large academic medical center. Patients A total of 344 patients with free and total phenytoin, albumin, and serum creatinine concentrations obtained between November 2012 and May 2017. Measurements and main results Free phenytoin concentrations were estimated in patients without kidney dysfunction using the Winter-Tozer, Anderson, Kane, and Cheng equations. For the analysis in patients with eGFR lower than 90 ml/min/1.73 m2 , free phenytoin concentrations were estimated using the Shiner-Tozer derivation with adjusted affinity coefficients (C = 0.15, 0.20, 0.25, and 0.30). For both analyses, accuracy of predictive methods was evaluated by P20, the proportion of estimations within 20% of the measured free phenytoin concentration. In 158 patients with normal kidney function/normal albumin concentrations, 73 with normal kidney function/hypoalbuminemia, or 47 with mild kidney dysfunction/normal albumin concentrations, the Anderson method had the highest accuracy (86%, 82%, and 92%, respectively) and highest precision compared with the other methods. In 47 patients with normal albumin concentrations and mild kidney dysfunction or 13 with moderate kidney dysfunction, the free fraction was unchanged, and total phenytoin concentrations accurately reflected free concentrations. In 17 patients with hypoalbuminemia and mild or 17 with moderate kidney dysfunction, the Winter-Tozer (67% and 50%, respectively) and the Anderson (56% and 67%, respectively) methods had the highest accuracy compared with other methods with significantly lower accuracy compared with patients with normal kidney function. In the 14 patients with severe kidney dysfunction and hypoalbuminemia, none of the coefficients had a P20 accuracy greater than 45%. Conclusion In patients with normal albumin concentrations, with or without mild or moderate kidney dysfunction and not receiving a protein-binding displacer, the free fraction of phenytoin is unchanged, and it is not necessary to measure a free phenytoin concentration. Free phenytoin concentrations should be measured directly in patients with hypoalbuminemia and kidney dysfunction.

The contrasts could not be starker. One is a world-renowned scientist with graduate degrees from Johns Hopkins and Caltech, an august record of scientific and academic achievement recognized by countless international accolades and awards, and who, importantly, invented DNA and protein synthesizers and sequencers that started the genomic and proteomic revolutions. The other is a college dropout with a scant history of any meaningful scientific contributions and who has been indicted for hoodwinking investors and patients into believing that the next-generation, Apple-inspired, point-of-care laboratory instruments she claimed to have invented were anything more than repurposed and unreliable glue dispensers. Despite these contrasts, though, both of these individuals seem to have gravitated towards the same push to convince the public and the medical establishment that the road to health is paved with laboratory test results. Specifically, not just a few laboratory test results, but LOTS of laboratory test results, the more the better, and ideally performed on people when they are healthy, in an effort to preserve wellness and fend off one's future demise. The 2 individuals described above, of course, are Lee Hood and Elizabeth Holmes; Lee Hood is the one who is the scientist. Hood's inventions of the first sequencers and synthesizers precedes Holmes' birth, and his conception of the term …

Cancer genomics research aims to advance personalized oncology by finding and targeting specific genetic alterations associated with cancers. In genome-driven oncology, treatments are selected for individual patients on the basis of the findings of tumour genome sequencing. This personalized approach has prolonged the survival of subsets of patients with cancer. However, many patients do not respond to the predicted therapies based on the genomic profiles of their tumours. Furthermore, studies pairing genomic and proteomic analyses of samples from the same tumours have shown that the proteome contains novel information that cannot be discerned through genomic analysis alone. This observation has led to the concept of proteogenomics, in which both types of data are leveraged for a more complete view of tumour biology that might enable patients to be more successfully matched to effective treatments than they would using genomics alone. In this Perspective, we discuss the added value of proteogenomics over the current genome-driven approach to the clinical characterization of cancers and summarize current efforts to incorporate targeted proteomic measurements based on selected/multiple reaction monitoring (SRM/MRM) mass spectrometry into the clinical laboratory to facilitate clinical proteogenomics.

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R(2) = 0.94) and immuno-MRM (R(2) = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens.

BACKGROUND The CDC states that laboratory testing for persons under investigation for Ebola virus disease can be safely performed using automated laboratory instruments by adhering to bloodborne pathogen practices. We therefore sought to investigate the levels of viral contamination of a total laboratory automation (TLA) system to guide risk mitigation strategies for handling infectious agents. METHODS Environmental swabs followed by PCR for hepatitis B (HBV) and hepatitis C (HCV) viruses were taken from a chemistry TLA system during routine clinical use and after running a small number of high-titer HCV samples. Control experiments were performed to ensure the recovery of DNA and RNA viruses by swabs from a representative nonporous surface. RESULTS Of 79 baseline swabs for nucleic acids performed on the TLA system, 10 were positive for HBV and 8 for HCV. Viral nucleic acid was consistently detected from swabs taken from the distal inside surface of the decapper discharge chute, with areas adjacent to the decapper instrument and the centrifuge rotor also positive for HBV or HCV nucleic acid. Contamination was occasionally detected on exposed surfaces in areas without protective barriers between samples and personnel. After running known HCV-positive samples, at least one additional site of contamination was detected on an exposed area of the line. CONCLUSIONS A low level of viral contamination of automated clinical laboratory equipment occurs in clinical use. Given the risks associated with highly infectious agents, there is a need for risk-mitigation procedures when handling all samples.

Background Beckman Coulter recently introduced a new hCG assay manufactured for the Access 2 and DxI platforms. This assay is the first to use the 5th International Standard (5th IS) as its primary calibration material. Clinical laboratories are required to validate the method performance before testing and reporting patient results. Methods Beckman Coulter Access 2 instruments (n = 41) across Kaiser Permanente Northern California were evaluated for their performance characteristics using the hCG5 reagent. Precision, linearity, dilution verification, and patient sample comparisons were performed on each instrument. Results The assay was linear up to 1350 IU/L. Intra-day and inter-day precision ranged from 1.0%–3.3% and 1.8–7.3%, respectively, for the low QC material (mean concentration 4.6 IU/L). Percent bias between the previous assay (hCG2) and the hCG5 assay was 3.2 to 22.7% for hCG concentrations 1000 IU/L. On board and manual dilutions agreed within 15% following proper adjustment of the instrument dilution factor. Conclusions Achieving Access 2 inter-instrument agreement on specimens needing dilutions (hCG > 1350 IU/L) requires validation of the on board dilution factor. Laboratories should use QC material above the linear range to monitor instrument dilution accuracy and precision.

Flibanserin is a medication recently approved by the FDA for treatment of generalized hypoactive sexual desire disorder (HSDD) in premenopausal women. Its mechanism of action is not fully understood but is thought to modulate serotonin receptors and increase levels of norepinephrine and dopamine. While much is known about toxicity of other drugs which affect these systems, there is little information about toxicity of flibanserin at this time.We present a case of a 2-year-old boy who ingested an estimated 600 mg of his mother's flibanserin. Following ingestion, the child developed facial twitching and unresponsiveness to pain, concerning for seizure-like activity. In the emergency department (ED) he was found to have hypertension, mydriasis, slurred speech, and normal labs. He responded well to supportive care including administration of benzodiazepines. Shortly after admission to the hospital, his temperature increased to 38.4 °C. Toxicology testing revealed the presence of 1-(3-trifluoromethylphenyl)-piperazine (TFMPP), a flibanserin metabolite. TFMPP is a recreational drug used as an alternative to 3,4-methylenedioxymethamphetamine (more commonly known as "MDMA" or "ecstasy").This case highlights potential toxicity associated with ingestion of flibanserin.

Background Prescription opioid use and opioid related deaths continue to increase nationwide. Several states have adopted legislation allowing for recreational use of cannabis. Little is known about how recreational cannabis laws impact compliance in chronic pain patients who have been prescribed opioid therapy. The goals of this study were to (1) retrospectively assess the effect of cannabis use on compliance with opioid therapy in a high-risk patient population and (2) determine the impact of legalization of recreational cannabis on patients prescribed therapeutic opioids. Methods We conducted a retrospective cohort study on results from a “high-risk” urine drug testing panel. Results from 1 year before and 1 year after initiation of recreational cannabis legislation were analyzed. This testing panel included qualitative assays for cannabinoids and 9 other common drugs of abuse in addition to a quantitative LC-MS/MS assay for 23 different opioids and metabolites. Opioid compliance was assigned by reviewing pathologists’ interpretations. Results In the pre-legalization period, 1776 panels were performed, and in the post-legalization, 1648 panels were performed. An increase (6%) in the rate of positive cannabinoids screening results was observed after legalization of recreational cannabis; however, the overall compliance rate was consistent. Conclusions The results of this study suggest that legalization of recreational cannabis does not affect compliance rate in patients treated with opioid therapy for chronic pain.

The United States Choosing Wisely initiative was started in 2012 by the American Board of Internal Medicine Foundation and focused on reducing medical resource overutilization. Since its inception in the US, similar efforts have arisen in at least a dozen countries. Strongly patient-focused, and in fact started in collaboration with the consumer magazine Consumer Reports, the effort has resulted in a collection of greater than 500 recommendations from over 80 US professional societies intended to inform both patients and doctors about medical practices whose necessity should be questioned or discussed. Targets of recommendations include practices that lack a basis in scientific evidence, practices that may be duplicative of other care already received, practices that may be harmful and practices that are simply unnecessary. While critiques have been levied against the Choosing Wisely initiative over its intent, methods and efficacy, it is clear that many of its recommendations have been adopted by large medical practices, and several positive outcomes, i.e. reductions in perceived waste, have been reported in relation to many of the recommendations, including those specifically targeting laboratory tests. The future success of Choosing Wisely will likely hinge on whether or not significantly positive and durable outcomes can be demonstrated, especially at a time where there is increasing pressure to drive down costs in medical care while concomitantly increasing quality.

Objectives Naturopathy is defined as the practice of medicine for the treatment of human diseases with natural agents. It also emphasizes prevention and promotion of health through the self-healing process of the body. Even without any illness or symptoms, naturopathic doctors (NDs) often advocate for and use unique lab tests to make clinical judgments. Tests that are performed outside of the ordering institution, send-out tests, are at increased risk of ordering the wrong or unnecessary test and misinterpreting test results. Here we evaluate the laboratory send-out tests that were ordered by NDs and general practitioners (GPs) at a tertiary pediatric care center to identify opportunities for improved laboratory stewardship. Methods We performed a retrospective analysis from Seattle Children’s laboratory send-out tests, which were ordered between January 1, 2018, and December 31, 2018. We compared the tests ordered by NDs with general practitioners in our practice, by grouping the following provider specialties: adolescent medicine, family medicine, or pediatrics. All the requests were reviewed and categorized by test type: heavy metal, allergen, infectious disease, hormone and vitamin, hematology and immunology, toxicology, oncology, genetics, and others. Ordering frequency and abnormal rate from each category were analyzed. The abnormal rate was defined as percentage of abnormal results divided by total number of ordered tests. Results During the 12-month period, there were 20,312 send-out tests; 1,028 (5.1%) of them were ordered by 83 NDs for 329 patients, and 3,862 (19.0%) of them were ordered by 462 GPs for 2,139 patients. The most frequently ordered tests by NDs were heavy metal screens (506 tests, 49.2% of total), allergen panels (31.7%), and infectious disease tests (7.0%). The corresponding abnormal rates were 8.5%, 0.9%, and 8.3%, respectively, with an overall abnormal rate of 6.6%. For GPs, the most frequently ordered tests were hormone and vitamin analyses (1,346 tests, 34.9% of total), hematology and immunology tests (22.5%), and heavy metal screens (10.8%). The corresponding abnormal rates were 18.1%, 35.9%, and 18.8% and overall abnormal rate was 17.8%. Conclusion We observed different ordering patterns between GPs and NDs, with rates of abnormal findings significantly higher from the tests ordered by GPs. These data suggest some of the tests ordered by NDs have low positive predictive value or clinical significance and could be potentially mis- or overutilized. Understanding the patterns and the variety of testing from different providers can help target interventions to improve laboratory stewardship in this area.

Background Idiopathic normal pressure hydrocephalus (INPH) is a neurologic disease that affects 65 years, but is difficult to distinguish from other diseases that present in this age group, such as Alzheimer’s disease. Large volume lumbar puncture and an external lumbar drain trial (ELD) are used to make a clinical diagnosis of INPH, but the accuracy of ELD is suspected. Objective To investigate proteomic cerebrospinal fluid (CSF) biomarker patterns in patients with INPH undergoing ELD to develop a quantitative diagnostic. Methods Twenty patients with suspected INPH underwent an ELD trial and the CSF biomarkers AB1-42, total tau, and tau phosphorylated at amino acid 181 (p-tau) were quantified with immunoassays in specimens taken prior to ELD placement, after the ELD trial, and from ventricular samples collected at the time of permanent ventriculoperitoneal shunt placement. Results CSF total tau was elevated, on average, in pre- and post-ELD samples from patients who failed to improve clinically during the ELD trial, but the findings were marginally significant after correction for multiple comparisons. AB1-42 and p-tau concentrations were not significantly different in patients who either did or did not clinically improve after the ELD. Conclusions CSF total tau is a potential novel biomarker for suspected INPH patients who will clinically improve, or have clinically improved, after an ELD trial. The small sample size of this study, which was due to the relative rarity of this condition, indicates that larger studies are needed to confirm the utility of this approach.

It is now undeniable that laboratory testing is vital for the diagnosis, prognostication and therapeutic monitoring of human disease. Despite the many advances made for achieving a high degree of quality and safety in the analytical part of diagnostic testing, many hurdles in the total testing process remain, especially in the preanalytical phase ranging from test ordering to obtaining and managing the biological specimens. The Working Group for the Preanalytical Phase (WG-PRE) of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) has planned many activities aimed at mitigating the vulnerability of the preanalytical phase, including the organization of three European meetings in the past 7 years. Hence, this collective article follows the previous three opinion papers that were published by the EFLM WGPRE on the same topic, and brings together the summaries of the presentations that will be given at the 4th EFLM-BD meeting "Improving quality in the preanalytical phase through innovation" in Amsterdam, 24-25 March, 2017. ispartof: Clinical Chemistry and Laboratory Medicine vol:55 issue:4 pages:489-500 ispartof: location:Germany status: published

Efficiently managing laboratory test utilization requires both ensuring adequate utilization of needed tests in some patients and discouraging superfluous tests in other patients. After the difficult clinical decision is made to define the patients that do and do not need a test, a wealth of interventions are available to the clinician and laboratorian to help guide appropriate utilization. These interventions are collectively referred to here as the utilization management toolbox. Experience has shown that some tools in the toolbox are weak and other are strong, and that tools are most effective when many are used simultaneously. While the outcomes of utilization management studies are not always as concrete as may be desired, what data is available in the literature indicate that strong utilization management interventions are safe and effective measures to improve patient health and reduce waste in an era of increasing financial pressure.

Patients suffering from trauma, burns, and sepsis are frequently in a hypermetabolic state, and as the majority of urinary nitrogen comes from amino acid metabolism, the determination of total urinary nitrogen (TUN) provides an accurate measurement of all nitrogen excreted in urine and can be used as an indicator of catabolic stress. To evaluate whether time of collection influences the performance of TUN test, 12-hour urine specimens were collected from ICU trauma patients and the 12-hour results were correlated with results from 24-hour collections. Two consecutive 12-hour urine samples were collected (06:00-18:00 hours and 18:00-06:00 hours) from surgical ICU patients. Total urinary nitrogen levels were measured in each 12-hour sample, as well as in a pooled sample (24 hours). Pyrochemiluminescence was used to determine TUN level on an Antek 9000N elemental analyzer. In this approach, the urine sample is completely oxidized at high temperature in a quartz pyrotube. Nitrogen is converted to nitric oxide (·NO), then mixed with O3 (ozone) to form nitrogen dioxide (NO2). Light is emitted and specific wavelengths between 650 and 900 nm were measured in a photomultiplier tube. The measured chemiluminescent emission is specific and proportional to the amount of nitrogen in the sample. Fifty 12-hour urine samples from 16 patients were collected. One patient was excluded from the study due to acute renal injury during the collection period. There was no significant observed circadian effect on measured TUN. The 12-hour TUN (g/total volume) was multiplied by 2 to compare to 24-hour TUN for statistical analysis. There was strong correlation between either day or night 12-hour TUN and corresponding pooled 24-hour TUN, with correlation coefficients ranging from 0.93 to 0.98 and regression slopes ranging from 0.98 to 1.01. No statistically significant difference was found between the 12-hour TUN and 24-hour TUN approaches. A 12-hour TUN collection was overall highly predictive of 24-hour TUN collection and has the advantage of convenience of sample collection and improved clinical efficiency. Serial 12-hour urine collection is therefore preferred for monitoring nitrogen balance and adjusting protein intake for critically ill ICU patients.

Elizabeth Holmes, Theranos' CEO, delivered a special session at the 2016 AACC Annual Scientific Meeting & Clinical Lab Expo titled “Theranos Science and Technology: The Miniaturization of Lab Testing.” AACC's decision to host the event, intended to describe the company's laboratory testing framework for the first time, was controversial and stimulated much discussion among AACC membership. The following pieces present the personal, thoughts, feelings, and reflections of 5 session attendees who share their perspectives before and after the Holmes session. I will begin with a bias: disruptive technology is a good thing and should be actively encouraged. Our profession has a long history of embracing such disruption, and avoiding new technology to maintain an existing business model (even a laboratory or academic model) is likely to fail. When I first heard claims that Theranos could perform large numbers of tests on a drop of blood, my reaction was a desire to find out more and get on the bandwagon. It quickly became clear, however, that their model of operation focused on secrecy and vertical integration in a way that effectively prevented independent verification, scientific scrutiny, and an open understanding of the limitations of their technology. Disruptive technology was, and is, coming; however, it was difficult to tell if Theranos would be the …

BACKGROUNDThe current gold standard for diagnostic classification of many solid-tissue neoplasms is immunohistochemistry (IHC) performed on formalin-fixed, paraffin-embedded (FFPE) tissue. Although IHC is commonly used, there remain important issues related to preanalytic variability, nonstandard methods, and operator bias that may contribute to clinically significant error. To increase the quantitative accuracy and reliability of FFPE tissue–based diagnosis, we sought to develop a clinical proteomic method to characterize protein expression in pathologic tissue samples rapidly and quantitatively.METHODSWe subclassified FFPE tissue from 136 clinical pituitary adenoma samples according to hormone translation with IHC and then extracted tissue proteins and quantified pituitary hormones with multiplex bead-based immunoassays. Hormone concentrations were normalized and compared across diagnostic groups. We developed a quantitative classification scheme for pituitary adenomas on archived samples and validated it on prospectively collected clinical samples.RESULTSThe most abundant relative hormone concentrations differentiated sensitively and specifically between IHC-classified hormone-expressing adenoma types, correctly predicting IHC-positive diagnoses in 85% of cases overall, with discrepancies found only in cases of clinically nonfunctioning adenomas. Several adenomas with clinically relevant hormone-expressing phenotypes were identified with this assay yet called “null” by IHC, suggesting that multiplex immunoassays may be more sensitive than IHC for detecting clinically meaningful protein expression.CONCLUSIONSMultiplex immunoassays performed on FFPE tissue extracts can provide diagnostically relevant information and may exceed the performance of IHC in classifying some pituitary neoplasms. This technique is simple, largely amenable to automation, and likely applicable to other diagnostic problems in molecular pathology.

An important precondition for the successful development of diagnostic assays of cerebrospinal fluid (CSF) biomarkers of age-related neurodegenerative diseases is an understanding of the dynamic nature of the CSF proteome during the normal aging process. In this study, a novel proteomic technology was used to quantify hundreds of proteins simultaneously in the CSF from 90 cognitively normal adults 21 to 85 years of age. SomaLogic's highly multiplexed proteomic platform can measure more than 800 proteins simultaneously from small volumes of biological fluids using novel slow off-rate modified aptamer (SOMAmer) protein affinity reagents with sensitivity, specificity, and dynamic ranges that meet or exceed those of enzyme-linked immunosorbent assays. In the first application of this technology to CSF, we detected 248 proteins that possessed signals greater than twofold over background. Several novel correlations between detected protein concentrations and age were discovered that indicate that both inflammation and response to injury in the central nervous system may increase with age. Applying this powerful proteomic approach to CSF provides potential new insight into the aging of the human central nervous system that may have utility in discovering new disease-related changes in the CSF proteome.

The recent Circulation article, “Aptamer-Based Proteomic Profiling Reveals Novel Candidate Biomarkers and Pathways in Cardiovascular Disease,” reports the use of an established oligonucleotide aptamer–based proteomic analysis platform in identifying early biomarkers of cardiovascular disease (1). The core of this technology is a set of 1129 aptamers comprising modified-base single-stranded DNA, each targeting a plasma protein. Readout of the assay involves binding fluorescent aptamers to biomarker proteins in the sample, with several biochemical manipulations to account for the fact that only single aptamers, rather than “sandwich” pairs, are used to bind analytes, and final readout on an Agilent array facilitated by sequence-specific binding of the fluorescent aptamers to their cognate immobilized sequences. Several such studies have now been published (2), but of particular interest to the readership of Clinical Chemistry , the authors of the Circulation article followed up with experiments technically validating the biomarker “hits” using aptamers as affinity pulldown reagents before targeted mass spectrometry–based proteomic analysis. Indeed, the results from the mass spectrometric analysis for a specific panel of proteins (n = 29) closely mirrored the results from the aptamer platform. From the perspective of a clinical chemist, a novel platform to reliably and simultaneously quantify more than 1000 proteins in human serum would be welcomed. There are a few different technologies that have been developed to quantify proteins in a multiplexed fashion including bead-based immunoassays analyzed using flow cells and LC-MS/MS. In many cases, multiplexed immunoassays suffer from a lack of specificity due to cross-reactivity …

Use of a coagulation panel [prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT) and fibrinogen], intended for evaluation of bleeding, tripled over 6 years, out of proportion to admissions, surgery, or transfusions. To determine whether the panels were ordered appropriately, we classified 28,737 sets of panel results into groups followed by chart reviews to determine typical patient histories. In 39% of panels, PT/PTT was normal. Prolonged PT occurred in 33% of results, due to liver failure (8%), warfarin (23%), and presumed vitamin K deficiency (69%). Prolonged PTT occurred in 34% of results and was primarily associated with long PT or lupus inhibitors. Prolonged PTT and TT (15% of panels) indicated heparin therapy. Fibrinogen was normal in 98% and low in 1.4%. Critical fibrinogen (below 100 mg/dl, 0.6% of panels) was associated with bleeding in 90% of patients. Only 8% of panel orders were clinically indicated based on patient history. Clinician interviews indicated many were unaware the panel included fibrinogen and TT. Interventions included an education program and an order form change. The education program had no effect on overall order volume or test selection. A later order form change made TT and fibrinogen a separate order. This reduced TT and fibrinogen testing by 90% without complaints or changes in blood transfusion statistics. We conclude that many coagulation test panel orders were not clinically indicated, that PT more often diagnosed vitamin K deficiency than bleeding risk, and that order-based restriction of testing was more effective than educational programs at introducing change in clinical test utilization.

Background Despite their widespread use, the performance of delta check rules is rarely evaluated because errors are rare and lack a gold standard for detection. In this study we used a simulation-based approach to compare strategies for empirically defining criteria for univariate delta checks, and assessed the performance of these rules for detecting mislabeled specimens in 2 inpatient populations. Methods We performed simulations using historical laboratory test results by randomly sampling pairs of specimens successively drawn from the same patient or two different patients. We evaluated the performance of delta check rules using a variety of thresholds, including those currently in use in our laboratory. Result Mean corpuscular volume had the highest positive predictive value for specimen mislabeling, and produced the fewest false positives. Conversely, rules using other laboratory tests had considerably poorer performance. Several of the “best guess” thresholds historically used in our laboratory, notably those for potassium and anion gap, were predicted to have extremely low yields. In addition, rule performance was not consistent between the two patient populations. Conclusions The low yield of delta checks based on any single analyte should prompt careful evaluation of their practical utility. Furthermore, our results indicate that it may not be possible to generalize delta rules across institutions.

Immunohistochemistry is used in both research and clinical settings to identify proteins in tissue samples. Despite the power and versatility of immunohistochemistry, limitations are imposed by the slow diffusion of antibodies through tissue and the need for secondary staining or signal amplification. Aptamers can circumvent these limitations, but their application has been hindered by nonspecific binding to cellular components, particularly in the nucleus. Here we describe unique slow off-rate modified aptamers that facilitate rapid and selective binding to target proteins in tissue. Specifically, we have developed a fluorescent aptamer that binds to the human epidermal growth factor receptor 2 (HER2) in breast carcinomas quickly and specifically, and we have shown that the slow off-rate of the aptamer from the HER2 protein contributes to its selectivity. These findings open the door to aptamer histochemistry applications in both research and clinical settings, including intraoperative diagnostics in which speed and accuracy are paramount.

In the inaugural issue of The Journal of Applied Laboratory Medicine , Deng et al. (1) reported the utility of antinuclear antibody screening by various methodologies. …

The enzyme horseradish peroxidase is routinely used in immunohistochemistry to facilitate the chromogenic oxidation of 3,3'-diaminobenzidine, producing an insoluble brown precipitate marking the location and quantity of a tissue protein. In an effort to develop non-protein reagents for tissue diagnostics, we have developed a peroxidase DNAzyme construct that can be used as a chromogenic functional group in immunohistochemistry assays. The DNAzyme is based on a reported 18-mer G-quadruplex-forming DNA sequence, PS2.M, and has been covalently linked to its catalytically active moiety, hemin, to avoid the high background signal associated with the use of free hemin in histochemical studies. The activity of the covalent construct is maintained under conditions where G-quadruplex formation is unfavored and where the noncovalent DNAzyme-hemin complex has no activity.

Biomarkers are urgently needed for the diagnosis and monitoring of disease progression in Parkinson's disease. Both DJ-1 and alpha-synuclein, two proteins critically involved in Parkinson's disease pathogenesis, have been tested as disease biomarkers in several recent studies with inconsistent results. These have been largely due to variation in the protein species detected by different antibodies, limited numbers of patients in some studies, or inadequate control of several important variables. In this study, the nature of DJ-1 and alpha-synuclein in human cerebrospinal fluid was studied by a combination of western blotting, gel filtration and mass spectrometry. Sensitive and quantitative Luminex assays detecting most, if not all, species of DJ-1 and alpha-synuclein in human cerebrospinal fluid were established. Cerebrospinal fluid concentrations of DJ-1 and alpha-synuclein from 117 patients with Parkinson's disease, 132 healthy individuals and 50 patients with Alzheimer's disease were analysed using newly developed, highly sensitive Luminex technology while controlling for several major confounders. A total of 299 individuals and 389 samples were analysed. The results showed that cerebrospinal fluid DJ-1 and alpha-synuclein levels were dependent on age and influenced by the extent of blood contamination in cerebrospinal fluid. Both DJ-1 and alpha-synuclein levels were decreased in Parkinson's patients versus controls or Alzheimer's patients when blood contamination was controlled for. In the population agedor = 65 years, when cut-off values of 40 and 0.5 ng/ml were chosen for DJ-1 and alpha-synuclein, respectively, the sensitivity and specificity for patients with Parkinson's disease versus controls were 90 and 70% for DJ-1, and 92 and 58% for alpha-synuclein. A combination of the two markers did not enhance the test performance. There was no association between DJ-1 or alpha-synuclein and the severity of Parkinson's disease. Taken together, this represents the largest scale study for DJ-1 or alpha-synuclein in human cerebrospinal fluid so far, while using newly established sensitive Luminex assays, with controls for multiple variables. We have demonstrated that total DJ-1 and alpha-synuclein in human cerebrospinal fluid are helpful diagnostic markers for Parkinson's disease, if variables such as blood contamination and age are taken into consideration.

Background: Ionized calcium (iCa) is measured frequently in hospitalized patients, and hypocalcemia is frequently found, seemingly supporting the practice. Methods: We retrieved the results of 58 040 iCa tests and records of intravenous (IV) and oral calcium supplementation from laboratory and hospital information systems and evaluated them for frequency of testing, frequency of hypocalcemia, and effects of calcium supplementation. Results: Serial and daily iCa testing was common and responsible for a substantial fraction of all iCa tests ordered. Half of all patients tested had iCa values below the reference interval. IV, but not oral, calcium therapy increased mean iCa concentrations, but the effect of calcium administration was small compared with the spontaneous increase in iCa that occurred in similar patients who received no calcium treatment. A retrospective analysis suggested that a low total calcium (tCa) concentration ( Conclusions: Testing for iCa and IV calcium supplementation were significantly reduced with a reflexive calcium testing strategy that provided iCa testing only to patients with low tCa. Adverse clinical outcomes possibly associated with hypocalcemia did not increase.