Discovery of small molecule inhibitors for the C. elegans caspase CED-3 by high-throughput screening.

C. elegans has been widely used as a model organism for programmed cell death and apoptosis. Although the CED-3 caspase is the primary effector of cell death in C. elegans, no selective inhibitors have been identified. Utilizing high-throughput screening with recombinant C. elegans CED-3 protein, we have discovered and confirmed 21 novel small molecule inhibitors. Six compounds had IC50 values < 10 µM. From these, four distinct chemotypes were identified. The inhibitor scaffolds described here could lead to the development of selective molecular probes to facilitate our understanding of programmed cell death in this model organism.

Method-dependent Discrepancies in Fetal Hemoglobin Quantification in Patients With Hemoglobin S.

Hemoglobin F (HbF) concentration is used in the diagnosis of certain hemoglobinopathies and accurate quantification is central to treatment of patients with sickle cell disease. The 2 most commonly used methods to quantify HbF are high performance liquid chromatography and capillary zone electrophoresis. This study reports discrepancies in HbF quantification between these methods when hemoglobin S is present in the sample. Clinicians and investigators should be mindful of the method used for HbF quantification when evaluating and treating patients who produce hemoglobin S.

Development and validation of an LC-MSMS method to quantify creatinine from dried blood spots.

Introduction: Screening for chronic kidney disease relies on accurate and precise creatinine measurements. Traditionally, creatinine is measured in serum or plasma using high-throughput chemistry analyzers. However, dried blood spots (DBS) can also be utilized to improve testing access. Methods: Samples were obtained from a 6 mm DBS punch, which was reconstituted in water before undergoing an acetonitrile crash. The resulting supernatant was diluted using an 80:20 acetonitrile: water before injection. Creatinine was identified using an isocratic gradient, and detected using an API 4000 triple quadrupole mass analyzer. Quantification relied on matrix-matched calibrators, with values harmonized to the Roche Cobas enzymatic assay. Validation studies assessing method performance included precision, linearity, accuracy, method comparison, stability, interference, and matrix effects. Results: The LC-MSMS assay was linear from 0.3 to 20 mg/dL (y = 1.02x-0.11; R2 = 0.996). Precision ranged from 5.2 to 8.1 % using matrix-matched controls (n = 4) that spanned the analytical measurement range. LC-MSMS results corresponded to the enzymatic assay (Roche) with a fitted line equation of y = 0.956x-0.07 (R2 = 0.995; n = 173). The Siemens and Roche enzymatic assays demonstrated higher accuracy in correlating to the DBS creatinine concentration (n = 40 paired venous/DBS collections) compared to the Beckman Jaffe assay (-2.5 % and -0.8 % versus -6.3 % and -4.1 %, respectively) or the iSTAT (-28.4 % and -27.1 %, respectively). Accuracy was unaffected by hematocrit, blood spot volume, excess IgG or IgA, or hypertriglyceridemia. No matrix effects were observed, and both extraction and processing efficiency were robust.Ambient stability extended to at least 10 days, and exposure to extreme temperature did not affect the creatinine results. Conclusion: We successfully developed an accurate and precise LC-MSMS method for quantifying creatinine in DBS.

A Review of Current Practices and Future Trends in Body Fluid Testing.

BACKGROUND: Body fluid testing in the clinical chemistry laboratory is a cornerstone in the diagnostic workup of pathological effusions. Laboratorians may not be aware of the preanalytical workflows used in the collection of body fluids though the value is evident whenever processes change or issues arise. The analytical validation requirements can vary depending on the regulations dictated by the laboratories' jurisdiction and accreditor requirements. Much of analytical validation hinges on how useful testing is to clinical care. Usefulness of testing varies with how well established and incorporated the tests and interpretation are in practice guidelines. CONTENT: Body fluid collections are depicted and described so clinical laboratorians have a basic appreciation of what specimens are submitted to the laboratory for testing. A review of validation requirements by major laboratory accreditation entities is presented. A review of the usefulness and proposed decision limits for common body fluid chemistry analytes is presented. Body fluid tests that show promise and those that are losing (or lost long ago) value are also reviewed. SUMMARY: The total testing process from collection to result interpretation can be complicated and easily overlooked by the clinical laboratory. This review aims to improve the understanding and awareness of collections, validation, result interpretation, and provide an update on recent trends.

Comparison of Five Common Analyzers in the Measurement of Chemistry Analytes in an Authentic Cohort of Body Fluid Specimens.

OBJECTIVES: Interpretation of body fluid (BF) results is based on published studies and clinical guidelines. The aim of this study is to determine whether the assays from five common commercial vendors produce similar results in BFs for 12 analytes in a BF cohort. METHODS: BFs (n = 25) and serum (n = 5) were analyzed on five instruments (Roche cobas c501, Ortho 5600, Beckman AU5800 and DXI800, Siemens Vista 1500, and Abbott Architect c8000) to measure albumin, amylase, total bilirubin, cholesterol, creatinine, glucose, lactate dehydrogenase (LDH), lipase, total protein, triglycerides, urea nitrogen, and carcinoembryonic antigen. Deming regression and Bland-Altman analysis were used for method comparison to Roche. RESULTS: Results were significantly different from Roche for LDH and lipase on Ortho and lipase on Siemens but similar for both BFs and serum. BF differences were larger than serum differences when measuring creatinine, glucose, and urea nitrogen on Ortho and glucose on Siemens. CONCLUSIONS: Five instruments used to perform BF testing produce results that are not significantly different except for lipase and LDH measurements. Bias of similar magnitude observed in both BF and serum should not affect interpretation. Further investigations into Ortho and Siemens measuring glucose and Ortho measuring creatinine and urea nitrogen are warranted.

The Lines That Held Us: Assessing Racial and Socioeconomic Disparities in SARS-CoV-2 Testing.

BACKGROUND: Racial disparities in SARS-CoV-2 prevalence are apparent. Race is a sociocultural construct, necessitating investigation into how sociocultural factors contribute. METHODS: This cross-sectional study linked laboratory data of adult patients between February 29 and May 15, 2020 with socio-demographics variables from the 2018 American Community Survey (ACS). Medical sites included healthcare organizations in Michigan, New York, North Carolina, California, Florida, Pennsylvania, and Washington. Race was treated as a proxy for racism and not biological essentialism. Laboratory data included patient age, sex, race, ethnicity, test result, test location, and residential ZIP code. ACS data included economic and educational variables contributing to an SES Index, population density, proportion Medicaid, and racial composition for corresponding ZIP code. Associations between race/socioeconomic variables and test results were examined using odds ratios (OR). RESULTS: Of 126 452 patients [mean (SD) age 51.9 (18.4) years; 52 747 (41.7%) men; 68 856 (54.5%) White and 27 805 (22.0%) Black], 18 905 (15.0%) tested positive. Of positive tests, 5238 (SD 27.7%) were White and 7223 (SD 38.2%) were Black. Black race increased the odds of a positive test; this finding was consistent across sites [OR 2.11 (95% CI 1.95-2.29)]. When subset by race, higher SES increased the odds of a positive test for White patients [OR 1.10 (95% CI 1.05-1.16)] but decreased the odds for Black patients [OR 0.92 (95% CI 0.86-0.99)]. Black patients, but not White patients, who tested positive overwhelmingly resided in more densely populated areas. CONCLUSIONS: Black race was associated with SARS-CoV-2 positivity and the relationship between SES and test positivity differed by race, suggesting the impact of socioeconomic status on test positivity is race-specific.

The 2018 AACC/SYCL PhD Clinical Chemist Compensation Survey.

BACKGROUND: Doctoral level board-certified clinical chemists play an invaluable role in many facets of laboratory medicine and healthcare. However, information concerning their total compensation is sparse. CONTENT: A confidential self-reported compensation survey was conducted by the American Association for Clinical Chemistry's Society for Young Clinical Laboratorians (AACC SYCL) Core Committee from April 1 to April 17, 2018. Respondents provided information on geographic location, employment sector, gender, and years of experience to account for the influence of these variables on compensation. There were 199 respondents in total from the United States and Canada, however, only respondents employed in the United States with an earned doctoral degree and certification by the American Board of Clinical Chemistry (n = 133), were included in the full analysis. In comparison to compensation reported in AACC SYCL salary surveys conducted in 2010 and 2013, early career median salaries are trending upwards after correction for inflation. SUMMARY: This survey is the first to collect the gender of respondents, and identify a pay gap for some geographic groups. However, this gap could be due in part to a difference in the years of experience, since males were highly represented in the group with >20 years of experience (25 out of 35, 71%). Future studies on compensation trends within clinical chemistry that do not rely on self-report are needed to ensure accuracy and completeness of the dataset.

An Introduction to Drug Testing: The Expanding Role of Mass Spectrometry.

Measurement of drugs and their metabolites in biological fluids is the foundation of both therapeutic drug monitoring (TDM) and toxicology. The introduction of methods based on mass spectrometry (MS), coupled with gas or liquid chromatography, has revolutionized these areas. This chapter will introduce the reader to the application of MS to TDM and toxicology, the steps that should be considered during implementation and the processes that should be implemented to assure continued quality. Points of emphasis include advances and recent trends since the publication of the first edition of this book, such as high-resolution mass spectrometry and increased interest in alternate matrices.

Ca(2+)/Calmodulin-binding proteins from the C. elegans proteome.

Calmodulin (CaM) is the primary Ca(2+)-sensor that regulates a wide variety of cellular processes in eukaryotes. Although many Ca(2+)/CaM-binding proteins have been identified, very few such proteins could be found from the genome-wide protein-protein interaction maps of Caenorhabditis elegans constructed by yeast two-hybrid screening. Using a genotype-phenotype conjugation method called mRNA-display, we performed a selection for Ca(2+)/CaM-binding proteins from a proteome library of C. elegans. The method allowed the identification of 9 known and 47 previously uncharacterized Ca(2+)-dependent CaM-binding proteins from the adult worm proteome. The Ca(2+)/CaM-binding properties of these proteins were characterized and their binding motifs were identified. The availability of such information could facilitate our understanding of the signaling pathways mediated by Ca(2+)/CaM in C. elegans. Due to its simplicity and efficiency, the method could be readily applied to examine the Ca(2+)-dependent binding partners of numerous other Ca(2+)-binding proteins, which may play important roles in many signaling pathways in C. elegans.

Modulation of nucleobindin-1 and nucleobindin-2 by caspases.

Nucleobindin-1 (NUCB1) and nucleobindin-2 (NUCB2) are multifunctional proteins that interact with Ca(2+), nucleic acids and various regulatory proteins in different signaling pathways. So far, our understanding of the regulation of the biological functions of nucleobindins remains limited. In our proteome-wide selection for downstream caspase substrates, both NUCB1 and NUCB2 are found to be the downstream substrates of caspases. We report here the detailed analyses of the cleavage of nucleobindins by caspases. Significantly, the caspase cleavage sites are located exactly at one of the Ca(2+)-binding EF-hand motifs. Our results suggest that the functions of nucleobindins could be modulated by caspase-mediated cleavage in apoptosis.

Reference intervals and diagnostic ranges for serum free κ and free λ immunoglobulin light chains vary by instrument platform: Implications for classification of patient results in a multi-center study.

BACKGROUND: Serum free light chain (FLC) immunoglobulins are key biomarkers that aid in the diagnosis, prognosis and assessment of treatment response in patients with plasma cell disorders (PCD). Here we investigated the transference of manufacturer's reported κFLC, λFLC and κ to λ FLC reference intervals (RI) and established de novo FLC RI and diagnostic ranges on four instruments at three academic medical centers. In addition, we also compared the classification of patient FLC results using manufacturer's versus established RIs and diagnostic ranges. METHODS: CLSI EP28-A3C protocol was applied to investigate transference and establishment of FLC reference intervals on the cobas (Roche), Immage (Beckman), Optilite and SPA Plus (Binding Site). Serum κ FLC and λ FLC were measured in reference sera (N = 126) with estimation of central 95% RIs and FLC ratio diagnostic range (total range). Frequencies (%) in patient FLC results (N > 380 per institution) classified above, below or within manufacturer's versus established FLC RI were compared. RESULTS: Three of four instrument platforms did not exhibit acceptable transference of manufacturer's reported κ FLC RI. The manufacturer's reported FLC total diagnostic range did not encompass all values observed in reference sera for any of the four platforms evaluated. Established FLC ratio diagnostic ranges reduced the frequency of patient results classified above range for three of four platforms evaluated. CONCLUSIONS: Transference of manufacturer's reported FLC RIs may be inappropriate for select instrument platforms. De novo establishment of FLC RIs specific to instrument platform is highly recommended in order to assure correct patient result classification.

Method Performance of Bicarbonate and Electrolytes by Use of Duodenal Fluid for Assessment of Exocrine Pancreatic Function.

BACKGROUND: Evaluation of exocrine pancreatic insufficiency is challenging for both clinicians and laboratories. Indirect pancreatic function tests such as serum trypsinogen, fecal elastase, and fecal fat measurements are moderately sensitive for diagnosis of advanced chronic pancreatitis but show reduced sensitivity and specificity for diagnosis of early disease. An alternative is the endoscopic pancreatic function test, which uses duodenal secretions after administration of IV secretin. Samples are collected at various times via the endoscopic tube and then analyzed for bicarbonate, sodium, potassium, and chloride. METHODS: Precision, linearity, method comparison, and stability studies were performed on the Beckman Coulter AU5822 chemistry analyzer with duodenal fluid. Comparison with the Vitros 4600 dry slide chemistry instrument was used to interrogate differences between methods. RESULTS: All assays produced a CV <2% without any measurable effects from the endoscopy fluid matrix and showed acceptable imprecision near the limit of detection (CV < 5%). All analytes showed linear dilution across the analytical measuring range. All the calculated error biases from dilutions were within 50% of the CLIA-allowable error for serum for each of the respective analytes. The calculated slopes ranged from 0.841 to 1.274 when compared to the Vitros 4600. Stability studies demonstrated that sodium, potassium, chloride, and bicarbonate remained stable after storage at -20 °C and after multiple freeze-thaw cycles. The percent change for all analytes was <5% mmol/L. CONCLUSIONS: The AU5800 series demonstrated adequate performance for the analysis of bicarbonate in duodenal fluid and therefore can be used for assessment of exocrine pancreatic function. However, notable discrepancies were observed for sodium, potassium, and chloride between the AU5800 series and the Vitros 4600.

Cleavage of BNIP-2 and BNIP-XL by caspases.

BNIP-2 and BNIP-XL are BCH domain-containing proteins that are implicated in programmed cell death. It has been reported that overexpression of BNIP-2 in neuroblastoma cell lines resulted in massive cell death, whereas BNIP-XL was upregulated during NGF-depletion-induced apoptosis in neuroblastoma and was involved in the regulation of differentiation, survival, and aggressiveness of tumor cells. Despite their importance in apoptosis, our understanding of BNIP-2 containing proteins is limited. In this communication, we demonstrate that both BNIP-2 and BNIP-XL are cleaved by caspases during apoptosis. Significantly, the caspase cleavage sites on BNIP-2 are located on its N-terminal EF-hand motif, while that on BNIP-XL is located upstream of the C-terminal BCH domain. Our results suggest that the caspase-mediated cleavage of BNIP-2 and BNIP-XL could result in the release of the BCH domain or smaller fragments that are crucial for their proapoptotic activities.

Method performance and clinical workflow outcomes associated with meconium and umbilical cord toxicology testing.

OBJECTIVE: Neonatal abstinence syndrome (NAS) is a rising concern with unknown long-term effects. It is apparent that higher cost of care, impact on the community and reduced quality of life are associated with similar etiologies (e.g., fetal alcohol syndrome). Detection of drug exposure in utero allows for earlier intervention to potentially reduce undesired outcomes. Umbilical cord tissue (UCT) has been documented as a readily accessible specimen for detection of drug exposure and has emerged as an alternative specimen to meconium. METHODS: The analytical and clinical impact of umbilical cord tissue relative to meconium was evaluated for assessment of in utero drug exposure. Quality metrics relating to turnaround-time and diagnosis of NAS were investigated after switching from meconium to UCT. RESULTS: Umbilical cord tissue showed higher clinical sensitivity but lower specificity for prediction of NAS diagnosis. Birth to result time decreased with adoption of UCT. CONCLUSIONS: Birth to result time decreased by the switching to UTC as well as the number of missed collections. The clinical sensitivity and negative predictive value for NAS increased with UCT; however both meconium and UTC samples were negative for opiates for a significant percentage of newborns with a diagnosis of NAS.