Returning actionable genomic results in a research biobank: Analytic validity, clinical implementation, and resource utilization.

Over 100 million research participants around the world have had research array-based genotyping (GT) or genome sequencing (GS), but only a small fraction of these have been offered return of actionable genomic findings (gRoR). Between 2017 and 2021, we analyzed genomic results from 36,417 participants in the Mass General Brigham Biobank and offered to confirm and return pathogenic and likely pathogenic variants (PLPVs) in 59 genes. Variant verification prior to participant recontact revealed that GT falsely identified PLPVs in 44.9% of samples, and GT failed to identify 72.0% of PLPVs detected in a subset of samples that were also sequenced. GT and GS detected verified PLPVs in 1% and 2.5% of the cohort, respectively. Of 256 participants who were alerted that they carried actionable PLPVs, 37.5% actively or passively declined further disclosure. 76.3% of those carrying PLPVs were unaware that they were carrying the variant, and over half of those met published professional criteria for genetic testing but had never been tested. This gRoR protocol cost approximately $129,000 USD per year in laboratory testing and research staff support, representing $14 per participant whose DNA was analyzed or $3,224 per participant in whom a PLPV was confirmed and disclosed. These data provide logistical details around gRoR that could help other investigators planning to return genomic results.

Integrated Carbon Dioxide Capture and Conversion to Methanol Utilizing Tertiary Amines over a Heterogenous Cu/ZnO/Al2O3 Catalyst.

Increasing carbon dioxide emissions has sparked a growing interest in capturing these emissions at the source of their release. For such processes, amines can be used as carbon dioxide capture agents. Herein, CO2 was captured under ambient conditions using solutions of amines and polyamines in ethylene glycol. The captured solutions were then successfully hydrogenated to methanol under hydrogen pressure with a heterogeneous Cu/ZnO/Al2O3 industrial catalyst. An extensive amine scope found that tetramethyl-1,6-hexanediamine, with two tertiary amine sites, provided the highest methanol productivity. This reaction was then optimized to achieve up to 89% methanol yield under relatively mild conditions of 250 °C and 80 bar H2 pressure. The catalyst was shown to be recyclable over five reaction cycles.

Reanalysis of eMERGE phase III sequence variants in 10,500 participants and infrastructure to support the automated return of knowledge updates.

PURPOSE: The clinical genomics knowledgebase is dynamic with variant classifications changing as newly identified cases, additional population data, and other evidence become available. This is a challenge for the clinical laboratory because of limited resource availability for variant reassessment. METHODS: Throughout the Electronic Medical Records and Genomics phase III program, clinical sites associated with the Mass General Brigham/Broad sequencing center received automated, real-time notifications when reported variants were reclassified. In this study, we summarized the nature of these reclassifications and described the proactive reassessment framework we used for the Electronic Medical Records and Genomics program data set to identify variants most likely to undergo reclassification. RESULTS: Reanalysis of 1855 variants led to the reclassification of 2% (n = 45) of variants, affecting 0.6% (n = 67) of participants. Of these reclassifications, 78% (n = 35) were high-impact changes affecting reportability, with 8 variants downgraded from likely pathogenic/pathogenic to variants of uncertain significance (VUS) and 27 variants upgraded from VUS to likely pathogenic/pathogenic. Most upgraded variants (67%) were initially classified as VUS-Favor Pathogenic, highlighting the benefit of VUS subcategorization. The most common reason for reclassification was new published case data and/or functional evidence. CONCLUSION: Our results highlight the importance of periodic sequence variant reevaluation and the need for automated approaches to advance routine implementation of variant reevaluations in clinical practice.

Automated typing of red blood cell and platelet antigens from whole exome sequences.

BACKGROUND: Genotyping has expanded the number red blood cell (RBC) and platelet (PLT) antigens that can readily be typed, but often represents an additional testing cost. The analysis of existing genomic data offers a cost-effective approach. We recently developed automated software (bloodTyper) for determination of RBC and PLT antigens from whole genome sequencing. Here we extend the algorithm to whole exome sequencing (WES). STUDY DESIGN AND METHODS: Whole exome sequencing was performed on samples from 75 individuals. WES-based bloodTyper RBC and PLT typing was compared to conventional polymerase chain reaction (PCR) RHD zygosity testing and serologic and single-nucleotide polymorphism (SNP) typing for 38 RBC antigens in 12 systems (17 serologic and 35 SNPs) and 22 PLT antigens (22 SNPs). Samples from the first 20 individuals were used to modify bloodTyper to interpret WES followed by blinded typing of 55 samples. RESULTS: Over the first 20 samples, discordances were noted for C, M, and N antigens, which were due to WES-specific biases. After modification, bloodTyper was 100% accurate on blinded evaluation of the last 55 samples and outperformed both serologic (99.67% accurate) and SNP typing (99.97% accurate) reflected by two Fyb and one N serologic typing errors and one undetected SNP encoding a Jknull phenotype. RHD zygosity testing by bloodTyper was 100% concordant with a combination of hybrid Rhesus box PCR and PCR-restriction fragment length polymorphism for all samples. CONCLUSION: The automated bloodTyper software was modified for WES biases to allow for accurate RBC and PLT antigen typing. Such analysis could become a routing part of future WES efforts.

Precision and reliability of 5 platelet function tests in healthy volunteers and donors on daily antiplatelet agent therapy.

BACKGROUND: Anticoagulation protocols used during mechanical circulatory support call for titration of antiplatelet agents. We compared the precision and reliability of 5 platelet function tests in healthy volunteers and donors on daily antiplatelet therapy to distinguish their efficacy for titrating antiplatelet therapy. METHODS: We assessed arachidonic acid-induced platelet function by light transmission aggregometry (LTA), Multiplate impedance aggregometry, VerifyNow, and platelet mapping by thromboelastography (TEG PM). We assessed ADP-induced platelet function by the same methods and flow cytometry. Forty healthy volunteers and 10-13 volunteers on daily aspirin and/or clopidogrel therapy were evaluated. We compared tests for intraassay precision, interassay precision (samples from 2 separate blood draws), and reliability coefficient. RESULTS: For arachidonic acid-induced platelet aggregation in healthy volunteers, intra- and interassay CVs were ≤ 10% for all methods. Intra- and interassay precision among donors on daily aspirin was ≤ 30% for all methods except LTA (38% interassay CV) and TEG PM (95% intraassay and 104% interassay CV). For ADP-induced platelet function, intra- and interassay precision was ≤ 10% and ≤ 30% for all methods. Only Multiplate demonstrated moderate or greater (R > 0.40) reliability coefficients for arachidonic acid-induced platelet function among all subjects. All methods of ADP-induced platelet function, except TEG PM, demonstrated substantial or greater (R > 0.60) reliability among all subjects. CONCLUSIONS: TEG PM is least suited to monitor effects of antiplatelet agents. Multiplate impedance aggregometry was the only method to demonstrate an acceptable reliability coefficient among healthy volunteers and donors on both aspirin and clopidogrel therapy.

Local verification and assignment of mean normal prothrombin time and International Sensitivity Index values across various instruments: recent experience and outcome from North America.

Warfarin dosing relies on accurate measurements of international normalized ratio (INR), which is calculated from the prothrombin time (PT), International Sensitivity Index international sensitivity index (ISI) of the thromboplastin, and the geometric mean of normal PT (MNPT). However, ISI assignments of certain reagent/instrument combinations are frequently unavailable, especially when the reagent and instrument are not from the same manufacturer. The effort to be in compliance with widely endorsed Clinical and Laboratory Standards Institute (CLSI) guidelines by locally verifying or assigning an ISI to an unsupported reagent/instrument combination is further hindered by the lack of US Food and Drug Administration (FDA)-approved certified plasmas designated for a particular reagent/instrument combination. The objectives of the study include development of a process to verify/assign ISI and MNPT of a single thromboplastin reagent from one manufacturer across multiple instruments including several from another manufacturer and across several campuses of a single institution, the Mayo Clinic. In this study, RecombiPlasTin 2G (R2G), was evaluated on the ACL TOP 700 (IL), STA-R Evolution, STA Compact, and STA Satellite. Random normal donor samples (n = 25) were used to verify/assign MNPT. A subset of the normal donors (n = 8) and 13 warfarin pools (INR range: 1.3-3.9), created from stable warfarin patient plasma, were used for ISI verification/assignment. The manufacturer's assigned ISI was first verified on the ACL TOP 700 (reference method), then assigned on three unsupported instruments using orthogonal regression analysis. The MNPT and manufacturer assigned ISI (11.0, 0.95) were verified on the ACL TOP 700 and subsequently assigned on the STA-R Evolution (11.6, 1.04); STA Compact (11.5, 1.02); and STA Satellite (10.9, 0.99). Linear correlations of the INR results from all the four instruments demonstrated an r2 > 0.99. This process provides a reproducible approach to assigning ISIs on unsupported reagent/instrument combinations. Our data also confirm that ISIs of the same PT reagent differ significantly on different instruments, thus confirming the requirement for evaluations and validation of ISIs for different reagent/instrument combinations.

Addition of Imidazolium-Based Ionic Liquid to Improve Methanol Production in Polyamine-Assisted CO2 Capture and Conversion Systems Using Pincer Catalysts.

Ionic liquids have been studied as CO2 capture agents. However, they are rarely used in combined CO2 capture and conversion processes. Utilizing imidazolium-based ionic liquids, the conversion of CO2 to methanol was greatly improved in polyamine assisted systems catalyzed by homogeneous pincer catalysts with Ru and Mn metal centers. Among the ionic liquids tested, [BMIM]OAc was found to perform the best under the given reaction conditions. Among the polyamine tested, pentaethylenehexamine (PEHA) led to the highest conversion rates. Ru-Macho and Ru-Macho-BH were the most active catalysts. Direct air capture utilizing PEHA as the capture material was also demonstrated and produced an 86 % conversion of the captured CO2 to methanol in the presence of [BMIM]OAc.

Bridging the gap: The critical role of laboratory developed tests in clinical toxicology.

•Toxicology testing provides valuable information for patient management.•Current in vitro diagnostics (IVDs) are unable to meet all clinical needs.•Lab-developed tests (LDTs) in toxicology can be used to close clinical care gaps.•LDTs in clinical toxicology are almost exclusively mass spectrometry-based methods.

Resolving icteric interference by a novel photoisomerization device: Bilibox.

Icterus, a phenomenon caused by bilirubin elevation in the blood, is a common endogenous interference in chemistry testing, occurring either spectrally or through chemical reactivity with assay reagents. Often, laboratories have few options other than to dilute or reject samples exceeding icteric thresholds. However, recent studies have optimized in vitro photoisomerization of bilirubin to a 17-minute bilirubin half-life using 500 nm light at 37 °C. Using an enzymatic creatinine assay as a model, due to its prevalence in routine laboratory testing and susceptibility to icteric interference, our study explores the usage of in vitro photoisomerization by replicating these conditions in a device, the Bilibox, as means of resolving icterus in laboratory testing. Left-over icteric and non-icteric clinical samples, collected by lithium heparin vacutainer (n = 10), were analyzed for baseline creatinine, diluted creatinine (1:4 0.9 % NaCl), total bilirubin, direct bilirubin, and hemolysis, icterus and lipemia (HIL) indices. Samples were then placed in the Bilibox in two intervals of 30 min with repeat measurements of the aforementioned analytes. On average, icteric-index, total bilirubin (TBIL), and direct bilirubin (DBIL) decreased by 33.5, 39.1 and 39.9 % respectively following 30 min of Bilibox treatment; and by 47.6 %, 63.7 % and 59.8 % following 60 min. The average percent difference between the pre-exposure diluted and undiluted creatinine was 5.8 %, demonstrating the icterus interference. Following Bilibox treatment, the difference between undiluted (post-exposure) and diluted (pre-exposure) creatinine decreased to 0.02 % (p = 0.0232) and 2.2 % (p = 0.0021) at 30 and 60 min of treatment respectively, demonstrating resolution of interference. Consequently, photoisomerization can be utilized as an additional and reasonably quick method for resolving icterus when dilutional methods cannot be applied.

Appearance of aseptic vascular grafts after endovascular aortic repair on [(18)F]fluorodeoxyglucose positron emission tomography/computed tomography.

BACKGROUND: Diagnosis of prosthetic vascular graft infection with [(18)F]fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) allows for early detection of functional changes associated with infection, based on increased glucose utilization by activated macrophages and granulocytes. Aseptic vascular grafts, like all foreign bodies, can stimulate an inflammatory response, which can present as increased activity on 18F-FDG PET/CT. Consequently, distinguishing aseptic inflammation from graft infection, though important, can be difficult. In the case of endovascular aneurysm repair (EVAR), a minimally invasive procedure involving the transfemoral insertion of an endoprosthetic stent graft, the normal postoperative appearance of these grafts on 18F-FDG PET/CT can vary over time, potentially confounding study interpretation. AIM: To investigate the visual, semiquantitative, and temporal characteristics of aseptic vascular grafts in patients status post EVAR. METHODS: In this observational retrospective cohort study, patients with history of EVAR who underwent 18F-FDG PET/CT for indications other than infection were identified retrospectively. All patients were asymptomatic for graft infection - no abdominal pain, fever of unknown origin, sepsis, or leukocytosis - at the time of imaging and for ≥ 2 mo after each PET/CT. Imaging studies such as CT for each patient were also reviewed, and any patients with suspected or confirmed vascular graft infection were excluded. One hundred two scans performed on 43 patients (34 males; 9 females; age = 77 ± 8 years at the time of the final PET/CT) were retrospectively reviewed. All 43 patients had an abdominal aortic (AA) vascular graft, 40 patients had a right iliac (RI) limb graft, and 41 patients had a left iliac (LI) limb graft. Twenty-two patients had 1 PET/CT and 21 patients had from 2 to 9 PET/CTs. Grafts were imaged between 2 mo to 168 mo (about 14 years) post placement. Eight grafts were imaged within 6 mo of placement, including three that were imaged within three months of placement. The mean interval between graft placement and PET/CT for all 102 scans was 51 ± 39 mo. PET/CT data was reconstructed with region-of-interest analysis of proximal, mid and distal portions of the grafts and background ascending aorta. Maximum standardized uptake value (SUVmax) was recorded for each region. SUVmax-to-background uptake ratios (URs) were calculated. Visual assessment was performed using a 2-pattern grading scale: Diffuse (homogeneous uptake less than liver uptake) and focal (one or more areas of focal uptake in any part of the graft). Statistical analysis was performed. RESULTS: In total, there were 306 AA grafts, 285 LI grafts, 282 RI grafts, and 306 ascending aorta background SUVmax measurements. For all 102 scans, mean SUVmax values for AA grafts were 2.8-3.0 along proximal, mid, and distal segments. Mean SUVmax values for LI grafts and RI grafts were 2.7-2.8. Mean SUVmax values for background were 2.5 ± 0.5. Mean URs were 1.1-1.2. Visual analysis of the scans reflected results of quantitative analysis. On visual inspection, 98% revealed diffuse, homogeneous 18F-FDG uptake less than liver. Graft URs and visual pattern categories were significantly associated for AA graft URs (F-ratio = 21.5, P < 0.001), LI graft URs (F-ratio = 20.4, P < 0.001), and RI graft URs (F-ratio = 30.4, P < 0.001). Thus, visual patterns of 18F-FDG uptake corresponded statistically significantly to semiquantitative URs. The age of grafts showing focal patterns was greater than grafts showing diffuse patterns, 87 ± 89 vs 50 ± 37 mo, respectively (P = 0.02). URs were significantly associated with graft age for AA grafts (r = 0.19, P = 0.001). URs were also significantly associated with graft age for LI grafts (r = 0.25, P < 0.0001), and RI grafts (r = 0.31, P < 0.001). Quartiles of similar numbers of graft (n = 25-27) grouped by graft age indicated that URs were significantly higher for 4th quartile vs 2nd quartile URs (F-ratio = 19.5, P < 0.001). When evaluating URs, graft SUVmax values within 10%-20% of the ascending aorta SUVmax is evident in aseptic grafts, except for grafts in the oldest quartiles. In this study, grafts in the oldest quartiles (> 7 years post EVAR) showed SUVmax up to 30% higher than the ascending aorta SUVmax. CONCLUSION: Characteristics of an aseptic vascular stent graft in the aorta and iliac vessels on 18F-FDG PET/CT include graft SUVmax values within 10%-20% of the ascending aorta background SUVmax. The SUVmax of older aseptic grafts can be as much as 30% above background. The visual uptake pattern of diffuse, homogeneous uptake less than liver was seen in 98% of aseptic vascular grafts, making this pattern particularly reassuring for clinicians.

Rapid serum clot tubes reduce haemolysis due to pneumatic tube transport.

AIMS: Pneumatic tube systems (PTSs) are critical for modern hospital operations, allowing for rapid sample transport. Despite widespread use, PTSs can compromise specimen integrity and affect laboratory values. Our objective was to prove that rapid serum clot tubes (RST) provide protective benefits over plasma during PTS transport and can be a practical solution for certain PTS routes. METHODS: In this study, we compared the effects of PTS transport on cell lysis indicators: h-index, lactate dehydrogenase (LDH) and potassium (K+), in RST versus lithium heparin gel separator tubes using 10 volunteers. RESULTS: In comparison with plasma, RST showed a median reduction in PTS-induced haemolysis of 80.4% (p=0.0049), with a reduction in post-PTS median LDH concentration (49.7%, p=0.04) and K+ concentration (50.0%, p=0.0273). CONCLUSION: This study demonstrates RST tubes can significantly reduce PTS-induced haemolysis and can be recommended for poor PTS routes.

The ALT upper reference interval debate: Blame it on the alcohol.

BACKGROUND: In 2017, the American College of Gastroenterology (ACG) and the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition published clinical guidelines recommending the use of alanine aminotransferase (ALT) upper reference limits (URL) of 33, 25, 26, and 22 U/l for men, women, boys, and girls, respectively. This was opposed by laboratory experts who advocated for the use of higher URL of 59, 41, 33, and 24 U/l instead. We suspected that the variable inclusion of individuals who consumed alcohol to be a major contributing source of URL variability and debate. METHODS: Outpatient ALT data (n = 7379) were collected from unique individuals ≥13 y with BMIs of ≥19 and ≤25. A total of 222 (3%) were excluded due to suspected liver disease. Patients were split into a pediatric group (age 13-17 y), an alcohol-restricted adult group (age 18-20 y), and adults with access to alcohol by decade (i.e., age 21-29, 30-39, 40-49, 50-59, 60-69, 70-79, and ≥ 80 y). All ALT values were measured on Roche Cobas 8000 with pyridoxal phosphate and traceable to the IFCC-reference measurement procedure. RESULTS: We derived URL similar to CALIPER for our pediatric population, but closer to ACG-proposed URL in our alcohol-restricted adult group. The URL increased significantly in men and women for all other age groups. CONCLUSIONS: The discrepancy in ALT URL in clinical laboratories may be attributable in part due to the variable inclusion of individuals who recently consumed alcohol in local population derivation studies.

gem-Halofluorocyclopropanes via [2 + 1] Cycloadditions of In Situ Generated CFX Carbene with Alkenes.

An efficient and operationally simple synthesis of gem-bromofluorocyclopropanes under mild conditions has been developed. The method employs ethyl dibromofluoroacetate (EDBFA) as an accessible and inexpensive source of the bromofluorocarbene (:CFBr) intermediate. The protocol provides the bromofluorocyclopropane products in excellent yields, including examples synthesized in multigram scales. The chlorinated ester, ethyl dichlorofluoroacetate (EDCFA), is also utilized to make the analogous gem-chlorofluorocyclopropanes.

Automated Pharmacogenomic Reports for Clinical Genome Sequencing.

Clinical laboratories offering genome sequencing have the opportunity to return pharmacogenomic findings to patients, providing the added benefit of preemptive testing that could help inform medication selection or dosing throughout the lifespan. Implementation of pharmacogenomic reporting must address several challenges, including inherent limitations in short-read genome sequencing methods, gene and variant selection, standardization of genotype and phenotype nomenclature, and choice of guidelines and drugs to report. An automated pipeline, lmPGX, was developed as an end-to-end solution that produces two versions of a pharmacogenomic report, presenting either Clinical Pharmacogenetics Implementation Consortium or US Food and Drug Administration guidelines for 12 genes. The pipeline was validated for performance using reference samples and pharmacogenetic data from the Genetic Testing Reference Materials Coordination Program. To determine performance and limitations, lmPGX was compared with three additional publicly available pharmacogenomic pipelines. The lmPGX pipeline offers clinical laboratories an opportunity for seamless integration of pharmacogenomic results with genome reporting.

Development of a clinical polygenic risk score assay and reporting workflow.

Implementation of polygenic risk scores (PRS) may improve disease prevention and management but poses several challenges: the construction of clinically valid assays, interpretation for individual patients, and the development of clinical workflows and resources to support their use in patient care. For the ongoing Veterans Affairs Genomic Medicine at Veterans Affairs (GenoVA) Study we developed a clinical genotype array-based assay for six published PRS. We used data from 36,423 Mass General Brigham Biobank participants and adjustment for population structure to replicate known PRS-disease associations and published PRS thresholds for a disease odds ratio (OR) of 2 (ranging from 1.75 (95% CI: 1.57-1.95) for type 2 diabetes to 2.38 (95% CI: 2.07-2.73) for breast cancer). After confirming the high performance and robustness of the pipeline for use as a clinical assay for individual patients, we analyzed the first 227 prospective samples from the GenoVA Study and found that the frequency of PRS corresponding to published OR > 2 ranged from 13/227 (5.7%) for colorectal cancer to 23/150 (15.3%) for prostate cancer. In addition to the PRS laboratory report, we developed physician- and patient-oriented informational materials to support decision-making about PRS results. Our work illustrates the generalizable development of a clinical PRS assay for multiple conditions and the technical, reporting and clinical workflow challenges for implementing PRS information in the clinic.

Determination of 97.5th and 99th percentile upper reference limits for heart-type fatty acid-binding protein (H-FABP) in a US population.

BACKGROUND: Heart-type fatty acid binding protein (H-FABP), a low molecular weight protein found primarily in myocardial tissue, has been identified as a potential biomarker in the detection of acute coronary syndrome and acute kidney injury. To further investigate clinical utility, we sought to establish an upper reference limit (URL) of H-FABP within a healthy U.S. METHODS: Serum samples of healthy donors were acquired from the AACC Universal Sample Bank. We analyzed 355 samples for H-FABP concentration using the Randox Laboratories immunoturbidimetric assay on the Roche Cobas 8000 series analyzer. RESULTS: The final sample population consisted of individuals aged 18-74 y, with 170 males and 185 females. The distribution of the population exhibited a strong positive skew, affecting outlier analysis and URL determination. The 97.5th-percentile URL was found to be 7.4 ng/ml (95% CI: 6.3-9.2), while the 99th-percentile URL was 12.1 ng/ml (8.6-14.9). CONCLUSION: As the URL for H-FABP is highly affected by population distribution and outlier removal, final determination for an assay cutoff should be made in the context of clinical utility, either as a standalone assay or in conjunction with other biomarkers, and the desired clinical sensitivity and specificity.