High-throughput tagging of endogenous loci for rapid characterization of protein function.

To facilitate the interrogation of protein function at scale, we have developed high-throughput insertion of tags across the genome (HITAG). HITAG enables users to rapidly produce libraries of cells, each with a different protein of interest C-terminally tagged. HITAG is based on a modified strategy for performing Cas9-based targeted insertions, coupled with an improved approach for selecting properly tagged lines. Analysis of the resulting clones generated by HITAG reveals high tagging specificity, with most successful tagging events being indel free. Using HITAG, we fuse mCherry to a set of 167 stress granule-associated proteins and elucidate the features that drive a subset of proteins to strongly accumulate within these transient RNA-protein granules.

Flexible-Interval High-Sensitivity Troponin Velocity for the Detection of Acute Coronary Syndromes.

Many algorithms for emergency department (ED) evaluation of acute coronary syndrome (ACS) using high-sensitivity troponin assays rely on the detection of a "delta," the difference in concentration over a predetermined interval, but collecting specimens at specific times can be difficult in the ED. We evaluate the use of troponin "velocity," the rate of change of troponin concentration over a flexible short interval for the prediction of major adverse cardiac events (MACEs) at 30 days. We conducted a prospective, observational study on a convenience sample of 821 patients who underwent ACS evaluation at a high-volume, urban ED. We determined the diagnostic performance of a novel velocity-based algorithm and compared the performance of 1- and 2-hour algorithms adapted from the European Society of Cardiology (ESC) using delta versus velocity. A total of 7 of 332 patients (2.1%) classified as low risk by the velocity-based algorithm experienced a MACE by 30 days compared with 35 of 221 (13.8%) of patients classified as greater than low risk, yielding a sensitivity of 83.3% (95% confidence interval [CI] 68.6% to 93.0%) and negative predictive value (NPV) of 97.9% (95% CI 95.9% to 98.9%). The ESC-derived algorithms using delta or velocity had NPVs ranging from 98.4% (95% CI 96.4% to 99.3%) to 99.6% (95% CI 97.0% to 99.9%) for 30-day MACEs. The NPV of the novel velocity-based algorithm for MACE at 30 days was borderline, but the substitution of troponin velocity for delta in the framework of the ESC algorithms performed well. In conclusion, specimen collection within strict time intervals may not be necessary for rapid evaluation of ACS with high-sensitivity troponin.

Functional map of SARS-CoV-2 3CL protease reveals tolerant and immutable sites.

The SARS-CoV-2 3CL protease (3CLpro) is an attractive therapeutic target, as it is essential to the virus and highly conserved among coronaviruses. However, our current understanding of its tolerance to mutations is limited. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the 3CLpro and validate a subset of our results within authentic viruses. We reveal that the 3CLpro is highly malleable and is capable of tolerating mutations throughout the protein. Yet, we also identify specific residues that appear immutable, suggesting that these may be targets for future 3CLpro inhibitors. Finally, we utilize our screening as a basis to identify E166V as a resistance-conferring mutation against the clinically used 3CLpro inhibitor, nirmatrelvir. Collectively, the functional map presented herein may serve as a guide to better understand the biological properties of the 3CLpro and for drug development against coronaviruses.

The Functional Landscape of SARS-CoV-2 3CL Protease.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) as the etiologic agent of COVID-19 (coronavirus disease 2019) has drastically altered life globally. Numerous efforts have been placed on the development of therapeutics to treat SARS-CoV-2 infection. One particular target is the 3CL protease (3CL pro ), which holds promise as it is essential to the virus and highly conserved among coronaviruses, suggesting that it may be possible to find broad inhibitors that treat not just SARS-CoV-2 but other coronavirus infections as well. While the 3CL protease has been studied by many groups for SARS-CoV-2 and other coronaviruses, our understanding of its tolerance to mutations is limited, knowledge which is particularly important as 3CL protease inhibitors become utilized clinically. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the SARS-CoV-2 3CL pro , and validate our results both in yeast and in authentic viruses. We reveal that the 3CL pro is highly malleable and is capable of tolerating mutations throughout the protein, including within the substrate binding pocket. Yet, we also identify specific residues that appear immutable for function of the protease, suggesting that these interactions may be novel targets for the design of future 3CL pro inhibitors. Finally, we utilize our screening results as a basis to identify E166V as a resistance-conferring mutation against the therapeutic 3CL pro inhibitor, nirmatrelvir, in clinical use. Collectively, the functional map presented herein may serve as a guide for further understanding of the biological properties of the 3CL protease and for drug development for current and future coronavirus pandemics.

Impact of a rapid high-sensitivity troponin pathway on patient flow in an urban emergency department.

Study Objective: To evaluate whether the introduction of a 1-hour high-sensitivity cardiac troponin-T (hs-TnT) pathway for patients who present to the emergency department (ED) with suspected acute coronary syndrome (ACS) improves ED patient flow without changing the rate of "missed" major adverse cardiac events (MACE), compared to use of conventional cardiac troponin with an associated 3-hour pathway. Methods: This was a prospective, uncontrolled observational study conducted before and after implementation of a 1-hour hs-TnT pathway at a high-volume urban ED. Patients undergoing evaluation for ACS in the ED were enrolled during their initial visit and clinical outcomes were assessed at 30 and 90 days. Throughput markers were extracted from the electronic medical record and compared. The primary outcome was provider-to-disposition decision time. Results: A total of 1892 patients were enrolled, 1071 patients while using conventional troponin and 821 after introduction of hs-TnT. With the new assay and pathway, median interval between troponin tests decreased from 4.7 hours (interquartile range [IQR] 3.9-5.7 hours) to 2.3 hours (IQR 1.5-3.4 hours) (P < 0.001). However, there was no difference in median provider-to-disposition decision time, which measured 4.7 hours (IQR 2.9-7.2) and 4.8 hours (IQR 3.1-7.1) (P = 0.428) respectively. Total 30-day MACE rate in discharged patients was low in both groups, occurring in only 4/472 (0.85%) encounters in the first cohort and 4/381 (1.0%) encounters in the second. Conclusion: Introduction of a 1-hour hs-TnT ACS evaluation pathway reduced the troponin collection interval but did not reduce provider to disposition time. There was no difference in rate of 30-day MACE in patients discharged from the ED.

Impact of pre-emptive rapid testing for glucose-6-phosphate dehydrogenase deficiency prior to rasburicase administration at a tertiary care centre: A retrospective study.

AIMS: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymopathy in humans, can cause acute haemolysis resulting from exposure to certain medications, chemicals, infections and fava beans. Rasburicase, used to manage elevated uric acid levels in the oncologic emergency of tumour lysis syndrome, is one such drug. The US Food and Drug Administration (FDA) recommends testing of G6PD status prior to rasburicase administration for patients at higher risk for G6PD deficiency. METHODS: We performed a retrospective chart review of all oncology patients for whom a semi-quantitative biochemical test for detecting G6PD deficiency was performed prior to rasburicase administration over a 2.5-year period, in a large academic metropolitan hospital. RESULTS: We identified 16 out of 260 tested individuals as G6PD-deficient (6.1%), including six females. On average, test results were electronically available to health care providers within 4 hours of sample collection, with most results available within 2-3 hours. Four G6PD-deficient patients developed elevated uric acid levels. Two of the G6PD-deficient patients were treated with rasburicase, and subsequently developed haemolysis, which was appropriately managed. CONCLUSION: In summary, by providing information about G6PD status with a rapid turnaround time, we have taken a significant step towards personalized medicine in our institution. In spite of the test implementation, two out of four G6PD-deficient patients, who were no longer candidates for rasburicase use, still received the drug, highlighting the need for improved provider education.

An artificial intelligence-assisted diagnostic platform for rapid near-patient hematology.

Hematology analyzers capable of performing complete blood count (CBC) have lagged in their prevalence at the point-of-care. Sight OLO (Sight Diagnostics, Israel) is a novel hematological platform which provides a 19-parameter, five-part differential CBC, and is designed to address the limitations in current point-of-care hematology analyzers using recent advances in artificial intelligence (AI) and computer vision. Accuracy, repeatability, and flagging capabilities of OLO were compared with the Sysmex XN-Series System (Sysmex, Japan). Matrix studies compared performance using venous, capillary and direct-from-fingerprick blood samples. Regression analysis shows strong concordance between OLO and the Sysmex XN, demonstrating that OLO performs with high accuracy for all CBC parameters. High repeatability and reproducibility were demonstrated for most of the testing parameters. The analytical performance of the OLO hematology analyzer was validated in a multicenter clinical laboratory setting, demonstrating its accuracy and comparability to clinical laboratory-based hematology analyzers. Furthermore, the study demonstrated the validity of CBC analysis of samples collected directly from fingerpricks.

Center-related Bias in MELD Scores Within a Liver Transplant UNOS Region: A Call for Standardization.

BACKGROUND: Model for End-Stage Liver Disease (MELD) score-based liver transplant allocation was implemented as a fair and objective measure to prioritize patients based upon disease severity. Accuracy and reproducibility of MELD is an essential assumption to ensure fairness in organ access. We hypothesized that variability in laboratory methodology between centers could impact allocation scores for individuals on the transplant waiting list. METHODS: Aliquots of 30 patient serum samples were analyzed for creatinine, bilirubin, and sodium in all transplant centers within United Network for Organ Sharing (UNOS) region 9. Descriptive statistics, intraclass correlation coefficients (ICCs), and linear mixed-effects regression were used to determine the relationship between center, bilirubin, and calculated MELD-sodium (MELD-Na) score. RESULTS: The mean MELD-Na score per sample ranged from 14 to 38. The mean range in MELD-Na per sample was 3 points, but 30% of samples had a range of 4-6 points. Correlation plots and intraclass correlation coefficient analysis confirmed bilirubin interfered with creatinine, with worsening agreement in creatinine at high bilirubin levels. Center and bilirubin were independently associated with creatinine reported in mixed-effects models. Unbiased hierarchical clustering suggested that samples from specific centers have consistently higher creatinine and MELD-Na values. CONCLUSIONS: Despite implementation of creatinine standardization, centers within a single UNOS region report clinically significant differences in MELD-Na on an identical sample, with differences of up to 6 points in high MELD-Na patients. The bias in MELD-Na scores based upon center choice within a region should be addressed in the current efforts to eliminate disparities in liver transplant access.

Digital morphology analyzers in hematology: ICSH review and recommendations.

INTRODUCTION: Morphological assessment of the blood smear has been performed by conventional manual microscopy for many decades. Recently, rapid progress in digital imaging and information technology has led to the development of automated methods of digital morphological analysis of blood smears. METHODS: A panel of experts in laboratory hematology reviewed the literature on the use of digital imaging and other strategies for the morphological analysis of blood smears. The strengths and weaknesses of digital imaging were determined, and recommendations on improvement were proposed. RESULTS: By preclassifying cells using artificial intelligence algorithms, digital image analysis automates the blood smear review process and enables faster slide reviews. Digital image analyzers also allow remote networked laboratories to transfer images rapidly to a central laboratory for review, and facilitate a variety of essential work functions in laboratory hematology such as consultations, digital image archival, libraries, quality assurance, competency assessment, education, and training. Different instruments from several manufacturers are available, but there is a lack of standardization of staining methods, optical magnifications, color and display characteristics, hardware, software, and file formats. CONCLUSION: In order to realize the full potential of Digital Morphology Hematology Analyzers, pre-analytic, analytic, and postanalytic parameters should be standardized. Manufacturers of new instruments should focus on improving the accuracy of cell preclassifications, and the automated recognition and classification of pathological cell types. Cutoffs for grading morphological abnormalities should depend on clinical significance. With all current devices, a skilled morphologist remains essential for cell reclassification and diagnostic interpretation of the blood smear.

A Human Organotypic Microfluidic Tumor Model Permits Investigation of the Interplay between Patient-Derived Fibroblasts and Breast Cancer Cells.

Tumor-stroma interactions significantly influence cancer cell metastasis and disease progression. These interactions are partly comprised of the cross-talk between tumor and stromal fibroblasts, but the key molecular mechanisms within the cross-talk that govern cancer invasion are still unclear. Here, we adapted our previously developed microfluidic device as a 3D in vitro organotypic model to mechanistically study tumor-stroma interactions by mimicking the spatial organization of the tumor microenvironment on a chip. We cocultured breast cancer and patient-derived fibroblast cells in 3D tumor and stroma regions, respectively, and combined functional assessments, including cancer cell migration, with transcriptome profiling to unveil the molecular influence of tumor-stroma cross-talk on invasion. This led to the observation that cancer-associated fibroblasts (CAF) enhanced invasion in 3D by inducing expression of a novel gene of interest, glycoprotein nonmetastatic B (GPNMB), in breast cancer cells, resulting in increased migration speed. Importantly, knockdown of GPNMB blunted the influence of CAF on enhanced cancer invasion. Overall, these results demonstrate the ability of our model to recapitulate patient-specific tumor microenvironments to investigate the cellular and molecular consequences of tumor-stroma interactions. SIGNIFICANCE: An organotypic model of tumor-stroma interactions on a microfluidic chip reveals that CAFs promote invasion by enhancing expression of GPNMB in breast cancer cells.

Electronic reporting of all reference laboratory results: An important step toward a truly all-encompassing, integrated health record.

Results from reference laboratories are often not easily available in electronic health records. This article describes a multi-pronged, long-term approach that includes bringing send-out tests in-house, upgrading the laboratory information system, interfacing more send-out tests and more reference laboratories, utilizing the "miscellaneous assay" option offered by some reference laboratories, and scanning all remaining paper reports from reference laboratories for display in the electronic health record. This allowed all laboratory results obtained in association with a patient visit, whether performed in-house or at a reference laboratory, to be available in the integrated electronic health record. This was achieved without manual data entry of reference laboratory results, thereby avoiding the risk of transcription errors. A fully integrated electronic health record that contains all laboratory results can be achieved by maximizing the number of interfaced reference laboratory assays and making all non-interfaced results available as scanned documents.

Downtime procedures for the 21st century: using a fully integrated health record for uninterrupted electronic reporting of laboratory results during laboratory information system downtimes.

OBJECTIVES: Downtimes of the laboratory information system (LIS) or its interface to the electronic medical record (EMR) disrupt the reporting of laboratory results. Traditionally, laboratories have relied on paper-based or phone-based reporting methods during these events. METHODS: We developed a novel downtime procedure that combines advance placement of orders by clinicians for planned downtimes, the printing of laboratory results from instruments, and scanning of the instrument printouts into our EMR. RESULTS: The new procedure allows the analysis of samples from planned phlebotomies with no delays, even during LIS downtimes. It also enables the electronic reporting of all clinically urgent results during downtimes, including intensive care and emergency department samples, thereby largely avoiding paper- and phone-based communication of laboratory results. CONCLUSIONS: With the capabilities of EMRs and LISs rapidly evolving, information technology (IT) teams, laboratories, and clinicians need to collaborate closely, review their systems' capabilities, and design innovative ways to apply all available IT functions to optimize patient care during downtimes.

Analytical and clinical performance of the epoc blood analysis system: experience at a large tertiary academic medical center.

OBJECTIVES: To describe validation and performance of epoc, a blood gas analysis point-of-care system, in a live clinical setting. METHODS: Data were collected for 156 epoc systems over 12 months. Preimplementation precision and correlation studies and postimplementation quality assurance data were collected, including test card, reader, and personal data assistant (PDA) failure rates. RESULTS: The coefficient of variation was clinically acceptable for all analytes. Correlation studies yielded an R(2) from 0.901 (for sodium) to 0.994 (for potassium) with the Nova analyzer and from 0.961 (sodium) to 0.991 (glucose) with the i-STAT. Average test card failure rate was 13%. Of the PDA/reader units, 55% needed repair within 1 year. CONCLUSIONS: The analytical performance showed high precision and good correlation with the Nova and i-STAT platforms. Test card and instrument failure rates were higher than that of the i-STAT system.

Determination of turnaround time in the clinical laboratory: "accessioning-to-result" time does not always accurately reflect laboratory performance.

Laboratories often focus on computer-generated components of turnaround time (TAT) reports that capture time from sample accessioning to reporting of results, and use this indicator to determine performance. This study assesses limitations of "accessioning-to-results" times and details ways in which to evaluate laboratory-controlled TAT. Samples were sent via pneumatic tube and times from arrival to accessioning were determined. Staffing was increased and the delay between sample arrival and accessioning was measured again. Significant delays were seen between specimen arrival and accessioning, which were not captured with computer-generated TAT reports. When TAT was calculated to include these delays, the TAT goal was not achieved. Increasing the number of employees significantly decreased delays. Laboratories must ensure that TAT reports encompass all laboratory-controlled parts of the testing processes. Analysis of causes for discrepancies between computer reports and clinician perceptions, combined with targeted measurements and well-designed interventions, can decrease TAT and improve service.

An application for monitoring order set usage in a commercial electronic health record.

Organizations that use electronic health records (EHRs) often maintain a considerable amount of clinical content in the form of order sets, documentation templates, and decision support rules. EHR vendors seldom provide analytic tools for customers to maintain such content and monitor its usage. We developed an application for tracking order sets, documentation templates and clinical alerts in a commercial electronic health record. Using the application, we compared trends in order set creation and usage at two academic medical centers over a three-year period. In January 2012, one medical center had 873 order sets available to clinicians; the other had 787. Approximately 50-75 new order sets were added each year at each medical center. We found that 46% of order sets at the first medical center and 39% at the second medical center were unused over the three-year period.

Altered DNA methylation in leukocytes with trisomy 21.

The primary abnormality in Down syndrome (DS), trisomy 21, is well known; but how this chromosomal gain produces the complex DS phenotype, including immune system defects, is not well understood. We profiled DNA methylation in total peripheral blood leukocytes (PBL) and T-lymphocytes from adults with DS and normal controls and found gene-specific abnormalities of CpG methylation in DS, with many of the differentially methylated genes having known or predicted roles in lymphocyte development and function. Validation of the microarray data by bisulfite sequencing and methylation-sensitive Pyrosequencing (MS-Pyroseq) confirmed strong differences in methylation (p<0.0001) for each of 8 genes tested: TMEM131, TCF7, CD3Z/CD247, SH3BP2, EIF4E, PLD6, SUMO3, and CPT1B, in DS versus control PBL. In addition, we validated differential methylation of NOD2/CARD15 by bisulfite sequencing in DS versus control T-cells. The differentially methylated genes were found on various autosomes, with no enrichment on chromosome 21. Differences in methylation were generally stable in a given individual, remained significant after adjusting for age, and were not due to altered cell counts. Some but not all of the differentially methylated genes showed different mean mRNA expression in DS versus control PBL; and the altered expression of 5 of these genes, TMEM131, TCF7, CD3Z, NOD2, and NPDC1, was recapitulated by exposing normal lymphocytes to the demethylating drug 5-aza-2'deoxycytidine (5aza-dC) plus mitogens. We conclude that altered gene-specific DNA methylation is a recurrent and functionally relevant downstream response to trisomy 21 in human cells.

A method for optimizing and validating institution-specific flagging criteria for automated cell counters.

CONTEXT: Automated cell counters use alerts (flags) to indicate which differential white blood cell counts can be released directly from the instrument and which samples require labor-intensive slide reviews. The thresholds at which many of these flags are triggered can be adjusted by individual laboratories. Many users, however, use factory-default settings or adjust the thresholds through a process of trial and error. OBJECTIVE: To develop a systematic method, combining statistical analysis and clinical judgment, to optimize the flagging thresholds on automated cell counters. DESIGN: Data from 502 samples flagged by Sysmex XE-2100/5000 (Sysmex, Kobe, Japan) instruments, with at least 1 of 5 user-adjustable, white blood cell count flags, were used to change the flagging thresholds for maximal diagnostic effectiveness by optimizing the Youden index for each flag (the optimization set). The optimized thresholds were then validated with a second set of 378 samples (the validation set). RESULTS: Use of the new thresholds reduced the review rate caused by the 5 flags from 6.5% to 2.9% and improved the positive predictive value of the flagging system for any abnormality from 27% to 37%. CONCLUSIONS: This method can be used to optimize thresholds for flag alerts on automated cell counters of any type and to improve the overall positive predictive value of the flagging system at the expense of a reduction in the negative predictive value. A reduced manual review rate helps to focus resources on differential white blood cell counts that are of clinical significance and may improve turnaround time.

Validation and implementation of an algorithm for reporting the automated absolute neutrophil count from selected flagged specimens.

Samples that are flagged by automated cell counters and, therefore, require a time-consuming microscopic review cause unacceptable wait times for patients in hematology/oncology clinics. We used a set of 518 samples to validate that 5 flags on Sysmex XE/XT instruments (Sysmex, Kobe, Japan) could safely be ignored when the absolute neutrophil count (ANC) was the primary clinical question. The R(2) between automated and manual ANCs was 96.9% for samples triggering non-ANC flags, with 1 clinically significant discrepant sample out of 296 (0.3%). A new test code allowing clinicians to specify "ANC-only" was implemented, and these non-ANC specific flags were disregarded. The new order set was used in 16.3% of patients. Automated reporting of the ANC in selected flagged samples reduced the review rate by 60% and decreased the turnaround time by 100 minutes. This approach to automatically report the ANC in selected flagged specimens in which the ANC is the primary clinical interest safely decreases the turnaround time for many ANC samples.