bluuebunny/crossref_metadata_2025 huggingface dataset:
Practical LC-MS/MS Method for 5-Hydroxyindoleacetic Acid in Urine
Authors: Clark Zlatuse D , Cutler Jeaneah M , Frank Elizabeth L
Published: July 2018

Background

Serotonin, an endogenous biogenic amine found in enterochromaffin cells of the gastrointestinal tract, is produced in excess by carcinoid tumors. The primary urinary metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA), is used in the diagnosis and management of carcinoid disease. This study describes the development and validation of a dilute-and-shoot LC-MS/MS method for the measurement of 5-HIAA in urine.

Methods

Samples were prepared by dilution in a 96-well format using an automated liquid handler. Chromatographic isolation of the analyte was achieved using a reversed-phase analytical column. A triple quadrupole mass spectrometer with electrospray ionization in positive mode was used for detection and quantification. Data were acquired by multiple reaction monitoring. Two transitions, quantifier (192.1/146.1) and qualifier (192.1/118.1), were monitored for the analyte and its stable isotope–labeled internal standard [5-hydroxyindole-4,6,7-d3-3-acetic-2,2-d2 acid (5-HIAA-d5)]. Chromatography was designed to elute the analyte outside of major suppression zones.

Results

Injection-to-injection time was 4 min. The method was validated for linearity, limit of quantification, accuracy, and imprecision. The analytical measurement range was 0.5–100 mg/L. Coefficients of variation for within-run, between-day, and total imprecision ranged from 0.8% to 5.4%. The method produced accurate 5-HIAA concentrations and correlated well (R = 0.9876) with a comparison HPLC method. Matrix effects were evaluated by post-column infusion of urine samples. An analytical specificity study of endogenous compounds, vitamins, medications, and drugs showed minimal interference.

Conclusions

A simple, inexpensive LC-MS/MS method was developed for measurement of 5-HIAA in urine. Results from the assay can be used clinically to assess carcinoid tumors.
Routine Supercritical Fluid Chromatography Tandem Mass Spectrometry Method for Determination of Vitamin K1 Extracted from Serum with a 96-Well Solid-Phase Extraction Method
Authors: Sandvik Trude Athammer , Husa Asgeir , Buchmann Marie , Lundanes Elsa
Published: July 2018

Background

The concentration of vitamin K1 in serum or plasma is the most common index for assessing vitamin K status. The aim of this study was to develop and validate a rapid and reliable routine method for quantifying vitamin K1 above 0.1 ng/mL. Semi-automation of a simple sample preparation with fast analysis by supercritical fluid chromatography–tandem mass spectrometry (SFC-MS/MS) was exploited.

Methods

Vitamin K1 was extracted from 250-μL serum samples by the use of protein precipitation and reversed-phase solid-phase extraction (SPE) in 96-well plates and quantified by SFC on a 2.1 × 100 mm Torus 1-Aminoanthracene (1-AA) column in 3.8 min with electrospray ionization—tandem mass spectrometry (MS/MS) detection.

Results

This method shows good linearity in the concentration range of 0.1–50 ng/mL with a correlation coefficient of R  2 >0.999. Imprecision was satisfactory, with repeatability and reproducibility <10% CV. The lower limit of the measuring interval was 0.1 ng/mL, and no systematic bias was observed for the method, which used vitamin K1-d7 as internal standard. Recovery of vitamin K1 in external quality controls was satisfactory compared to other laboratories participating in the external quality assurance scheme. The method is currently in routine use for analysis of serum samples.

Conclusions

The method allows high-throughput reliable determination of vitamin K1 in serum in the range 0.1–50 ng/mL.
Validation of an Extensive CYP2D6 Assay Panel Based on Invader and TaqMan Copy Number Assays
Authors: Leung Edward Ki Yun , Agolini Emanuele , Pei Xun , Melis Roberta , McMillin Gwendolyn A , Friedman Paula N , Peterson Patrick , Danahey Keith , O'Donnell Peter H , Yeo Kiang-Teck J
Published: July 2018

Background

CYP2D6 is involved in the oxidative metabolism of approximately 20% of all clinically used medications. Genotyping cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6), is a challenge because of the high complexity of the locus.

Methods

Twenty-nine CYP2D6 sequence variants were genotyped in 50 deidentified patient samples and 29 Coriell DNAs by Invader assay, and results were compared with Infiniti assay and Sanger sequencing. To determine CYP2D6 copy number, 3 TaqMan real-time hydrolysis probes were used and results were compared with long-range PCR. Discrimination of the duplicated alleles was done on 17 DNA samples with 3 copies of CYP2D6 by long-range PCR followed by Invader genotyping and single nucleotide extension for the comparison.

Results

Complete concordance was observed for all samples between platforms except for 2 samples due to the lack of the *45 allele in the Infiniti panel. Reproducibility with the Invader assay and TaqMan copy number was 100%. Analytical sensitivity using DNA with 2 copies was determined to be 10 ng DNA for the Invader assay and 1 ng/μL DNA for the TaqMan assay, respectively. Complete concordance and reproducibility were observed for duplicated allele discrimination with the exception of 1 sample, determined to be *29/*43X2 by the Invader test and *1X2/*29 by the Infiniti method, which did not test for *43.

Conclusions

This validation study showed that Invader and TaqMan assay combined panel provides an attractive, valid, highly accurate, and reproducible approach for CYP2D6 genotyping for clinical implementation.
An Ultraperformance LC-MS/MS Method for the Quantification of the Antimalarial Atovaquone in Plasma
Authors: Chambliss Allison B , Parsons Teresa L , Marzinke Mark A
Published: July 2018

Background

A primary modality in the treatment and prevention of malaria is the administration of antimalarial agents. Atovaquone (ATQ) has been used in single-drug and multidrug antimalarial applications; however, studies have demonstrated high interindividual drug variability. With the scarcity of analytical methodologies available in the literature, we have developed and optimized a rapid, ultraperformance (UP) LC-MS/MS method for the quantification of ATQ in human plasma.

Methods

ATQ was extracted from 25 μL K2-EDTA human plasma via protein precipitation with acetonitrile. Sample solutions were separated on a Synergi 2.5-μm Polar-RP 100A (100 × 2 mm) column. ATQ and its internal standard were detected over 1.3 min on an API 4000 mass analyzer using an electrospray ionization source operated in negative ionization and selected reaction monitoring modes. The method was validated in accordance with the Food and Drug Administration (FDA) Guidance for Industry: Bioanalytical Method Validation recommendations.

Results

Owing to pharmacokinetic parameters associated with ATQ, 2 calibration curves were generated to quantify the drug across a dynamic concentration range. Two standard curves were established ranging from 250 to 5000 ng/mL and 5000 to 50000 ng/mL, respectively. QC levels for both lower and higher concentration ranges prepared at low (750 ng/mL, 12000 ng/mL), mid (2000 ng/mL, 22500 ng/mL), and high (4250 ng/mL, 42500 ng/mL) concentrations yielded interassay precision ≤9.1% and accuracy ≤±9.4%. Dilutional, stability, and matrix effects studies were also performed, and results were within acceptability limits.

Conclusions

This work describes the development and analytical evaluation of a UPLC-MS/MS method for ATQ quantification in plasma. The described method is sufficiently sensitive for ATQ quantification in plasma to support preclinical and clinical trials.
An Automated Assay for Growth Differentiation Factor 15
Authors: Wollert Kai C , Kempf Tibor , Giannitsis Evangelos , Bertsch Thomas , Braun Siegmund L , Maier Harald , Reim Manfred , Christenson Robert H
Published: July 2018

Background

Growth differentiation factor 15 (GDF-15) can serve as a biomarker for cardiovascular disease burden and risk. We evaluated a new, fully automated electrochemiluminescence immunoassay for measuring GDF-15.

Methods

Six laboratories independently characterized the Elecsys® GDF-15 assay (Roche Diagnostics) under routine conditions. Within-run precision (repeatability), within-laboratory precision (intermediate precision), and between-laboratory precision (reproducibility) were assessed. Plasma-serum sample correlation, reagent lot-to-lot reproducibility, and instrument comparisons were performed. The Elecsys assay was compared to a research immunoradiometric assay (IRMA) and a commercially available ELISA. GDF-15 concentrations were measured with the Elecsys assay in 739 apparently healthy individuals.

Results

CVs for within-run and within-laboratory precision ranged from 0.7% to 7.7% and 1.7% to 8.6%, respectively, for samples containing 670–16039 ng/L. CVs for between-laboratory precision ranged from 7.1% to 8.9% (766–14289 ng/L). Recovery of GDF-15 was comparable for serum, Li-heparin plasma, K2- and K3-EDTA plasma, and citrated plasma, between 2 reagent lots, and on the cobas e 411 and cobas e 601 analyzers (Roche Diagnostics). GDF-15 concentrations in the clinically relevant range (400–3000 ng/L) measured with the Elecsys assay showed a good correlation and agreement with those measured by IRMA or ELISA. GDF-15 concentrations in apparently healthy individuals increased with age but did not vary by sex.

Conclusions

The Elecsys GDF-15 assay demonstrates a robust analytic performance under routine conditions and provides an automated method for measuring GDF-15 concentrations in serum and plasma.
Creation of a Universal Sample Bank for Determining the 99th Percentile for Cardiac Troponin Assays
Authors: Wu Alan H B , Apple Fred , Love Sara A , Koch David , Myers Gary L , Christenson Robert H ,   on behalf of the AACC's Biomarkers of Acute Cardiovascular Disease Division
Published: July 2018

Background

International guidelines authored and endorsed by professional societies of cardiology, emergency medicine, and laboratory medicine are unanimous that the cutoff concentration for establishing a diagnosis of acute myocardial infarction be set at the 99th percentile of a healthy population. The establishment of the actual 99th percentile value is assay- and sample-dependent and is influenced by the population of individuals selected for testing. We created a sample bank that will enable manufacturers of troponin assays a consistent comparison of the 99th percentile.

Methods

Participants were recruited from those attending the 2015 Annual Meeting of the AACC for the creation of a universal sample bank of apparently healthy individuals (free from uncontrolled diabetes, renal insufficiency, and heart disease). For those who met eligibility criteria and signed a written consent to participate, 60 mL blood was collected into 6 10-mL tubes each (2 serum, 3 heparin plasma, and 1 EDTA). Whole blood was tested for hemoglobin (Hb) A1c, and serum was tested for N-terminal pro–B-type natriuretic peptide (NT-proBNP) and creatinine.

Results

There were a total of 764 individuals who consented during the AACC Annual Meeting. After this initial enrollment, it was determined that there was an insufficient number of male participants recruited. Under the same protocol and consent, blood from 131 additional males was collected at the University of Maryland. Samples were centrifuged, and 240 μL aliquots of the 2 serum, 3 heparin plasma, and 1 EDTA tubes were frozen at 70 °C within 2 h of collection. The labeled samples were divided into boxes containing 1 aliquot from each individual. Sets of these samples were made available for purchase to manufacturers of cardiac troponin assays. Eighty-eight samples were excluded from the database for having a high NT-proBNP (>300 ng/L), low estimated glomerular filtration rate (eGFR) (<60 mL/min/1.73 m2), high Hb A1c (≥6.5%), or preanalytical sample issues and consenting/data issues. The final total was 808 individuals (402 females and 406 males; 60% Caucasian, 26% African or African American, 11% Asian or Pacific Islander, and 3% other).

Conclusions

The creation of a bank of samples from healthy individuals enables a consistent comparison of the 99th percentile results from manufacturers of cardiac troponin assays.
Quantification of Relaxin-2 Connecting Peptide (Pro-RLX2) in Human Blood Samples
Authors: Rehfeldt Miriam , Sparwasser Andrea , Funk Eugenia , Köhrle Josef , Bergmann Andreas
Published: July 2018

Background

The peptide hormone relaxin-2 is implicated in diverse physiological and pathophysiological processes. Several assays are available for quantification of human relaxin-2, but because stability of the mature peptide in serum is limited, measurement of the more stable connecting peptide (pro-RLX2) might be beneficial.

Methods

Pro-RLX2 was measured in a sandwich immunoluminometric assay using 2 monoclonal antibodies. The concentration of pro-RLX2 was detected in healthy pregnant (n = 100) and healthy male and nonpregnant female (n = 81) subjects and compared with the concentration of mature relaxin-2 in a subset of samples.

Results

The pro-RLX2 immunoassay has an analytical and functional assay sensitivity (FAS) of 1.59 pmol/L and 1.7 pmol/L, respectively. The analyte is stable in EDTA plasma samples for 8 days at room temperature, dilutes in a linear fashion, and recovery was 103%. The assay system is not biased by common interfering substances. Measurement of 80% of plasma samples from healthy males and females is below the FAS {median 1.49 pmol/L [interquartile range (IQR) of 0.925–2.14 pmol/L]}, and no concentration difference between male and nonpregnant female plasma samples was observed. The median plasma concentration in healthy pregnant women is increased up to 562 pmol/L (IQR 341–789 pmol/L). During pregnancy, pro-RLX2 concentrations decrease with increasing gestation. The correlation coefficient with the R&D assay for mature relaxin-2 was 0.96 (P < 0.0001).

Conclusion

Pro-RLX2 is stable in plasma of healthy individuals. Although samples of pregnant women are reliably measurable, most samples from healthy nonpregnant women and men are below the detection limit. Determination of pro-RLX2 concentrations might indicate rate of synthesis of relaxin-2 during pregnancy and therapeutic application of recombinant relaxin (Serelaxin).
Validation of a Circulating Tumor-Derived DNA Blood Test for Detection of Methylated BCAT1 and IKZF1 DNA
Authors: Murray David H , Baker Rohan T , Gaur Snigdha , Young Graeme P , Pedersen Susanne K
Published: July 2018

Background

Colvera™ is a test that detects circulating tumor-derived DNA in patients with colorectal cancer by assaying for the presence of methylated BCAT1 and IKZF1 in blood. This study describes the analytical and clinical performance characteristics of the test.

Methods

Validation was performed in accordance with ISO15189 and National Pathology Accreditation Advisory Council requirements. Spiked samples including 264 plasma and 120 buffer samples were randomized, divided into 8 batches of 48 samples, and processed over 8 days using 2 equipment lines (each line consisting of a QIAsymphony SP/AS, QIACube HT, and LC480); 2 reagent batches; and 2 operators to determine limit of detection, selectivity/specificity, precision, reproducibility, ruggedness, and susceptibility to commonly known interfering substances. Clinical performance was validated by assaying 222 archived plasma samples from subjects (n = 26 with cancer) enrolled in a previous prospective trial.

Results

The limit of detection for Colvera was 12.6 pg/mL (95% CI, 8.6–23.9 pg/mL), which equates to 2 diploid genome copies per milliliter plasma. No statistically significant difference was determined between testing days (n = 8), instrumentation, operators, or reagent batches in precision studies for the methylation-specific assays. The assay performance was unaffected by 9 commonly known interference substances, variations in bisulfite conversion, or quantitative PCR settings (cycling temperatures, incubation times, and oligonucleotide concentrations). For this clinical cohort, sensitivity and specificity estimates for Colvera were 73.1% (19 of 26; 95% CI, 52.2–88.4) and 89.3% (175 of 196; 95% CI, 84.1–93.2), respectively.

Conclusion

Colvera is a robust test and suitable for detection of circulating tumor-derived DNA by measuring levels of methylated BCAT1 and IKZF1 in human blood plasma.
Pharmacogenetics of Opioid Use and Implications for Pain Management
Authors: Nerenz Robert D , Tsongalis Gregory J
Published: July 2018

Background

Opioid analgesics are frequently prescribed to manage acute and chronic pain, but individual differences in opioid response make effective pain control in all patients an elusive goal. Furthermore, the risk of addiction following opioid consumption varies among individual patients. Although many psychosocial factors contribute to an individual's opioid response and risk for addiction, a strong genetic component has also been demonstrated.

Content

Opioids undergo substantial enzymatic modification that can generate metabolites with either increased or decreased opioid activity relative to the parent compound. To elicit their analgesic effect, parent compounds and active metabolites must be transported into the central nervous system where they bind to opioid receptors and inhibit neurotransmission. Inherited genetic variants that alter the function of proteins involved in these processes have been associated with differences in opioid response and risk for addiction. Detection of these variants can help guide opioid selection, inform dosing decisions, or encourage use of a nonopioid analgesic.

Whereas some genetic variants are clearly associated with differences in opioid response and have been included in consensus clinical practice guidelines, the impact of other variants on opioid response remains unclear. Studies performed to date have generated promising results, but inconsistent findings, reimbursement challenges, and the lack of robust decision support tools have hampered widespread adoption of pharmacogenetic testing to guide pain management treatment decisions. Future work involving the simultaneous evaluation of large numbers of variants and demonstration of a clear clinical benefit provided by pharmacogenetic testing will be required to overcome these obstacles.
Planning Risk-Based Statistical Quality Control Strategies: Graphical Tools to Support the New Clinical and Laboratory Standards Institute C24-Ed4 Guidance
Authors: Bayat Hassan , Westgard Sten A , Westgard James O
Published: July 2018

Background

Clinical and Laboratory Standards Institute (CLSI)'s new guideline for statistical quality control (SQC; C24-Ed4) (CLSI C24-Ed4, 2016; Parvin CA, 2017) recommends the implementation of risk-based SQC strategies. Important changes from earlier editions include alignment of principles and concepts with the general patient risk model in CLSI EP23A (CLSI EP23A, 2011) and a recommendation for optimizing the frequency of SQC (number of patients included in a run, or run size) on the basis of the expected number of unreliable final patient results. The guideline outlines a planning process for risk-based SQC strategies and describes 2 applications for examination procedures that provide 9-σ and 4-σ quality. A serious limitation is that there are no practical tools to help laboratories verify the results of these examples or perform their own applications.

Methods

Power curves that characterize the rejection characteristics of SQC procedures were used to predict the risk of erroneous patient results based on Parvin's MaxE(Nuf) parameter (Clin Chem 2008;54:2049–54). Run size was calculated from MaxE(Nuf) and related to the probability of error detection for the critical systematic error (Pedc).

Results

A plot of run size vs Pedc was prepared to provide a simple nomogram for estimating run size for common single-rule and multirule SQC procedures with Ns of 2 and 4.

Conclusions

The “traditional” SQC selection process that uses power function graphs to select control rules and the number of control measurements can be extended to determine SQC frequency by use of a run size nomogram. Such practical tools are needed for planning risk-based SQC strategies.
Proenkephalin (PENK) as a Novel Biomarker for Kidney Function
Authors: Beunders Remi , Struck Joachim , Wu Alan H B , Zarbock Alexander , Di Somma Salvatore , Mehta Ravindra L , Koyner Jay L , Nadim Mitra K , Maisel Alan S , Murray Patrick T , Neath Sean-Xavier , Jaffe Allan , Pickkers Peter
Published: July 2018

Background

The assessment of kidney function and detection of acute kidney injury (AKI) remain cumbersome. On the one hand, because of limited accuracy of established tests: The most widely used methods are creatinine based, which lack in sensitivity, as creatinine is not purely filtrated by the kidney and rises relatively late after onset of AKI. On the other hand, because of labor-intensiveness: Gold standard inulin clearance and comparable methods involve intravenous compound infusion, blood sampling at several time points, and have error-sensitive determination methods. In recent years, several biomarkers have been put forward (e.g., NGAL, KIM-1, TIMP-2*IGFBP-7), but clinical implementation is limited up to now.

Content

Proenkephalin (PENK) represents a new candidate to determine kidney function. This peptide is cleaved from the precursor peptide preproenkephalin A alongside enkephalins (endogenous opioids) and is filtrated in the glomerulus. PENK plasma concentration appears to accurately represent glomerular filtration rate in patients diagnosed with sepsis or cardiac diseases. Moreover, increased PENK concentration is found to be associated with longer-term outcome concerning AKI and cardiac diseases. Lastly, the predominant receptor of enkephalins, the δ-opioid receptor, is expressed with the highest density in the kidney, suggesting that enkephalins could also exert a direct effect on kidney function.

In this review, we present an overview of enkephalins and the assessment of kidney function using this possible new functional biomarker PENK and compare it with established and novel biomarkers.
Transforming Laboratory Utilization Review into Laboratory Stewardship: Guidelines by the PLUGS National Committee for Laboratory Stewardship
Authors: Dickerson Jane A , Fletcher Andrew H , Procop Gary , Keren David F , Singh Ila R , Garcia Joaquin J , Carpenter Robert B , Miles Joe , Jackson Brian , Astion Michael L
Published: July 2018

Appropriate utilization of clinical laboratory services is important for patient care and requires institutional stewardship. Clinical laboratory stewardship programs are dedicated to improving the ordering, retrieval, and interpretation of appropriate laboratory tests. In addition, these programs focus on developing, maintaining, and improving systems to provide proper financial coverage for medically necessary testing. Overall, clinical laboratory stewardship programs help clinicians improve the quality of patient care while reducing costs to patients, hospitals, and health systems. This document, which was created by a new multiinstitutional committee interested in promoting and formalizing laboratory stewardship, summarizes core elements of successful hospital-based clinical laboratory stewardship programs. The core elements will also be helpful for independent commercial clinical laboratories.
Interindividual Variability and Normal Ranges of Whole Blood and Plasma Thrombin Generation
Authors: Bloemen Saartje , Huskens Dana , Konings Joke , Kremers Romy M , Miszta Adam , de Laat Bas , Kelchtermans Hilde
Published: July 2018

Background

Assays measuring thrombin generation (TG) in plasma increasingly gained attention in the field of thrombosis and hemostasis. Adaptation of the method enabled the measurement of TG in whole blood (WB). Despite their potential, TG assays did not reach the stage of universal clinical application, partly because of the absence of normal ranges. Our study aimed to accurately determine normal ranges and interindividual variability of TG and correlate results with coagulation factor levels, sex, and oral contraceptive usage.

Methods

The study protocol was evaluated by the local medical ethical board. In total, 129 healthy volunteers gave full informed consent. Normal ranges of TG in platelet-poor plasma (PPP), platelet-rich plasma (PRP), and WB were determined according to CLSI guidelines.

Results

Our study is the first to measure normal ranges of TG in PPP, PRP, and WB in a large healthy cohort. Significant correlations were found between TG in plasma and WB. Interindividual variability of TG in WB was comparable to that of plasma. Oral contraceptive use increased TG in PPP, PRP, and WB. The inhibitory effect of thrombomodulin on TG was significantly lower in females than in males. This effect was more pronounced upon oral contraceptive use. Primary clotting factor determinants for TG parameters depended on the tissue factor concentration, but were similar in WB and plasma.

Conclusions

Establishing normal ranges for TG brings us 1 step closer to clinical use. Good correlations between plasma and WB (including clotting factor determinants for TG) suggest that WB TG can be reliably used in clinic.
Sandwich Immunoassay for Bioactive Plasma Adrenomedullin
Authors: Weber Julia , Sachse Janine , Bergmann Sarah , Sparwaßer Andrea , Struck Joachim , Bergmann Andreas
Published: July 2018

Background

Adrenomedullin (ADM) is a circulating peptide known to regulate vasodilation and vascular integrity. Increased plasma ADM concentrations have been described for several life-threatening conditions, including cardiovascular diseases and septic shock. Reliable methods for the simple quantification of bioactive ADM (bio-ADM) are lacking.

Methods

Monoclonal antibodies against the amidated C-terminus and middle portion of bio-ADM were generated and used for the development of a 1-step immunometric assay for the specific quantification of bio-ADM in plasma. The assay was developed in a microtiter plate/chemiluminescence label format with a significantly reduced incubation time. Precision, linearity, specimen stability, and distribution of results in healthy subjects were evaluated.

Results

The use of monoclonal antibodies against predetermined epitopes of bio-ADM enabled the development of an assay for the determination of bio-ADM directly in EDTA plasma. Plasma samples were stable for up to 24 h at ambient temperature and over multiple freeze–thaw cycles without loss of immunoreactivity. The assay had a limit of detection of 3 pg/mL and a limit of quantification of 11 pg/mL. The assay exhibited acceptable linearity characteristics and was not influenced by complement factor H, a putative ADM-binding protein. In healthy subjects, bio-ADM concentrations were all above the limit of detection, and approximately half of them were above the limit of quantification.

Conclusions

By using monoclonal antibodies with defined epitope specificities, we have developed a simple, rapid, accurate, and sensitive sandwich immunoassay for bio-ADM. The assay is a potentially novel tool to support patient management, particularly in acute care in the field of sepsis and other indications, which are currently being investigated, such as acute heart failure.
Expanding the Utility of High-Sensitivity Dried Blood Spot Immunoassay Testing with Single Molecule Counting
Authors: Mukherjee Ali , Dang Tam , Morrell Heather , Yerramilli Rama , Bishop Jeffrey J
Published: July 2018

Background

Dried blood spot (DBS) testing has been used for years in newborn screening and for other applications when obtaining blood by venipuncture is impractical or expensive. However, several technical challenges have restricted the use of DBS testing to qualitative assays or to analytes that are present in relatively high concentrations. The application of high-sensitivity detection using single molecule counting (SMC™) technology can potentially overcome the limitations of DBS as specimen source.

Methods

A method was developed for reproducibly collecting, storing, and subsequently reconstituting DBS samples to be used with assays based on the SMC technology. Before extraction, DBS samples were scanned, and the blood spot area was calculated to normalize for sample volume and spot variability. DBS sample extraction was done using an efficient high-salt extraction buffer. DBS samples were tested using SMC-based cardiac troponin I (cTnI), prostate-specific antigen (PSA), and C-reactive protein (CRP) assays.

Results

The SMC-DBS assays showed reproducible sensitivity, precision, and the stability required for quantifying low-abundance biomarkers. These assays were not significantly impacted by normal variations in hematocrit or sample collection technique. Correlation coefficients obtained from method comparisons between SMC-DBS and laboratory-developed tests or Food and Drug Administration-cleared tests using traditional sample types were 1.08, 1.04, and 0.99 for cTnI, PSA, and high-sensitivity CRP, respectively.

Conclusions

Combining DBS finger-stick blood collection with next-generation immunoassay technology will aid the expansion of DBS testing to protein biomarkers that are in low abundance or to low-volume samples, and will enable the development and adoption of DBS testing to far-reaching applications.
Variability in Microplate Surface Properties and Its Impact on ELISA
Authors: Lilyanna Shera , Ng Enoch Ming Wei , Moriguchi Shiho , Chan Siew Pang , Kokawa Ryohei , Huynh So Hung , Chong P C Jenny , Ng Yan Xia , Richards A Mark , Ng Tuck Wah , Liew Oi Wah
Published: July 2018

Background

Microplate-based immunoassays are widely used in clinical and research settings to measure a broad range of biomarkers present in complex matrices. Assay variability within and between microplates can give rise to false-negative and false-positive results leading to incorrect conclusions. To date, the contribution of microplates to this variability remains poorly characterized and described. This study provides new insights into variability in immunoassays attributable to surface characteristics of commercial microplates.

Methods

Well-to-well assay variation in γ-treated and nontreated 96-well opaque microplates suitable for chemiluminescence assays was determined by use of a validated sandwich ELISA. Microplate surface characteristics were assessed by sessile drop contact angle measurements, scanning electron microscopy, energy dispersive x-ray spectroscopy, and atomic force microscopy.

Results

All microplate types tested exhibited vendor-specific assay response profiles; and “rogue” plates with very high intraassay variation and deviant mean assay responses were found. Within-plate, location-dependent bias in assay responses and variability in well contact angle were also observed. We demonstrate substantial differences in well-surface properties with putative effects on protein-coating reproducibility and hence consistency in immunoassay responses. A surface “cleaning” effect on manufacturing residues was attributed to γ-irradiation, and treated microplates manifest increased polar functionalities, surface roughness, and assay responses.

Conclusions

Our data suggest that tighter control of variability in surface roughness, wettability, chemistry, and level of residual contaminants during microplate preparation is warranted to improve consistency of ELISA assay read out.
Opioids: Clinical Practice, Monitoring, and Medicolegal Considerations
Authors: Eldrige Jason S , Rho Richard H , Martin Joanne L , Murphy Joshua B , Hooten W Michael
Published: July 2018

Background

In the past decade, opioid use in the general population of the US has increased dramatically. Concomitantly, opioid-related morbidity and mortality have also risen steeply. The causes of opioid over-prescribing are multifactorial, with pressure from medical agencies, patients, aggressive patient-directed marketing, and use of patient surveys as an evaluation tool to unduly influence medical providers' decision-making. Providers also have significant concerns regarding scrutiny from their medical board and exposure to medicolegal liability.

Content

To provide the basis for best practice recommendations, several aspects of modern opioid prescribing practice require review. The practicing clinician should be aware of: (a) the pervasiveness of current opioid prescribing within the US; (b) the availability and applicability of clinical tools used to assess patient risk; (c) the new guidelines designed to facilitate appropriate and safe medical use of opioids; (d) the incorporation of clinical tools and best documentation practices to reduce medicolegal exposure.

On completion of this review, the reader should be able to recognize the magnitude of the problem of opioid over-prescribing in the US and identify several patient risk factors for opioid misuse, abuse, and/or diversion. Specifically, better familiarity with the recently published CDC guidelines for opioid prescribing will help ensure the adoption of best-use practices that maximize therapeutic efficacy while attenuating the possibility of harm to patients. A thorough review of the legal principles and risk-mitigation strategies relevant to opioid prescribing will also lessen the clinician's exposure to medicolegal liability.
Mass Spectrometric Identification of Cardiac Troponin T in Urine of Patients Suffering from Acute Myocardial Infarction
Authors: Streng Alexander S , van der Linden Noreen , Kocken Jordy M M , Bekers Otto , Bouwman Freek G , Mariman Edwin C M , Meex Steven J R , Wodzig Will K W H , de Boer Douwe
Published: July 2018

Background

Because of its high cardiospecificity, cardiac troponin T (cTnT) is one of the first-choice biomarkers to diagnose acute myocardial infarction (AMI). cTnT is extensively fragmented in serum of patients suffering from AMI. However, it is currently unknown whether all cTnT is completely degraded in the body or whether some cTnT fragments can leave the body via urine. The aim of the present study is to develop a method for the detection of cTnT in urine and to examine whether cTnT is detectable in patient urine.

Methods

Proteins in urine samples of 20 patients were precipitated using a cTnT-specific immunoprecipitation technique and a nonspecific acetonitrile protein precipitation. After in-solution digestion of the precipitated proteins, the resulting peptides were separated and analyzed using HPLC and mass spectrometry with a targeted selected ion monitoring assay with data-dependent tandem mass spectrometry (t-SIM/dd-MS2).

Results

The t-SIM/dd-MS2 assay was validated using a synthetic peptide standard containing 10 specific cTnT peptides of interest and with purified human intact cTnT spiked in urine from healthy individuals. Using this assay, 6 different cTnT-specific peptides were identified in urine samples from 3 different patients, all suffering from AMI.

Conclusions

We show here for the first time that cTnT can be present in the urine of AMI patients using a targeted LC-MS/MS assay. Whether the presence of cTnT in urine reflects a physiological or pathophysiological process still needs to be elucidated.
Multicenter Laboratory Comparison of Iohexol Measurement
Authors: Schwartz George J , Wang Hongyue , Erway Brian , Nordin Gunnar , Seegmiller Jesse , Lieske John C , Bäck Sten-Erik , Miller W Greg , Eckfeldt John H
Published: July 2018

Background

Iohexol is used for measurement of kidney glomerular filtration rate (GFR). Until recently, there have not been available proficiency standards to assist in calibrating a laboratory's results. In view of a shift in calibration at the University of Rochester Medical Center (URMC) laboratory, serving as Central Biochemistry Laboratory for the CKiD study, we performed a multicentered laboratory comparison.

Methods

Two batches of 30 fortified sera and patient samples from serum or heparinized plasma were sent for duplicate analysis to URMC, University of Minnesota (UMN), Mayo Clinic, and University of Lund. Five proficiency testing materials from Equalis AB were also provided. Iohexol calibration was performed using dilutions of OmnipaqueTM 300 and concentrations measured by HPLC or LC-MS/MS (Mayo).

Results

The 2 batches sent to UMN and University of Lund agreed well. URMC calibration was 11%–13% lower, and Mayo was 4%–8% lower for fortified samples. URMC corrected calibration was 3%–8% higher for these samples. When measured values were adjusted for the results of the Equalis samples, all laboratories agreed within 1%–2% on all iohexol concentrations.

Conclusions

For 12 URMC calibrator lots from November 2006 to March 2016, the factor quantifying the underestimation of measured to true iohexol concentration was 0.89. If each concentration was divided by 0.89, the calculated GFRs would be reduced by 10%–11%. GFR results for CKiD were adjusted for this shift in calibration. Regular examination of iohexol proficiency testing materials, free exchange of samples among laboratories, and standardized dilution of the stock iohexol for calibration would help to bring more universal agreement to this assay.
Severe Isotype-Matched Immunosuppression (IMI) as a Potential Risk Factor for Progression of MGUS Patients
Authors: Jiménez Juana J , Pais Tiago M , Barbosa Nuno , Campos Maria Luisa , Díaz Maria Antonia Peñalver , de Larramendi Carmen H
Published: July 2018

Background

Monoclonal gammopathy of undetermined significance (MGUS) precedes multiple myeloma in virtually every case. However, only a small percentage will progress and at very different rates. In addition, recent data have suggested that MGUS is associated with other comorbidities including infections, suggesting impaired immune function in some MGUS patients. Therefore, we aimed at assessing the value of isotype-matched immunosuppression (IMI; e.g., suppression of an IgAκ in an IgAλ patient), a type of immunosuppression more specific than classical immunoparesis (IP; e.g., IgG and/or IgM suppression in an IgA patient), as a prognostic marker for MGUS progression.

Methods

The Hevylite assay was used to assess IMI and immunoglobulin ratios in 307 serum samples from a cohort of 248 MGUS patients. Follow-up clinical records were available for 154 individuals.

Results

A greater incidence of IMI (51%) over classical IP (37%) was observed, although both show a progressive increase with higher risk groups. Survival analysis of 154 patients showed that severe IMI (>50% suppression) differentiates 2 groups with significantly different time to progression (P = 0.024) while severe IP does not (P = 0.48). Also, a combination of severe IMI and involved monoclonal immunoglobulin >1.5g/dL by Hevylite (both variables found to be independent prognostic markers in multivariate analysis) identified a group of patients with a median time to progression 6-fold shorter than the remaining group (P < 0.0001).

Conclusions

These findings indicate a possible role for IMI in the malignant transformation of MGUS patients and a potential utility as a new risk factor.
Simple HPLC Method with Internal Standard for Evaluation of Vitamin B1 Status By Use of Whole Blood
Authors: Zhang Xiaochun , Tang Xiaoying , Gibson Bill , Daly Thomas M
Published: July 2018

Background

Two primary assays are routinely used for evaluating a patient's vitamin B1 status: plasma free thiamine and whole blood thiamine diphosphate (TDP). TDP is the bioactive form of vitamin B1 and best reflects body stores. Plasma free thiamine levels are driven by recent dietary intake. The objective of this study was to develop a simple HPLC method with an internal standard (IS) that simultaneously measures TDP and thiamine in whole blood, and to assess the use of this single-tube assay to provide comprehensive evaluation of vitamin B1 status.

Methods

The final assay used amprolium thiochrome as an IS, and the sample preparation procedure takes approximately 1 h. Whole blood thiamine and plasma thiamine were concurrently measured for 126 subjects.

Results

The analytical measurement range was 1.7 to 442.3 nmol/L (TDP) and 1.7 to 375.4 nmol/L (thiamine), with interassay precisions of 4.0% to 4.8% (TDP) and 2.9% to 8.0% (thiamine), respectively. Method comparison with a reference laboratory HPLC method showed r = 0.9625, slope = 1.021, and intercept = 0.982 (n = 53) for TDP quantification. Whole blood thiamine correlated closely with plasma thiamine levels but were slightly higher with a mean difference of 1.0 nmol/L (range: −3.0 to 5.0 nmol/L). The reference interval for whole blood TDP and thiamine was 84.3 to 213.3 nmol/L and 1.7 to 21.9 nmol/L, respectively.

Conclusions

This assay provides a simple and reliable HPLC method with a suitable IS for quantification of both TDP and thiamine from whole blood. It also eliminates the need for separate samples for TDP and thiamine measurement, which will allow both short-term and long-term vitamin B1 status to be assessed from a single sample.
Quantitation of Infliximab and Detection of Antidrug Antibodies in Serum by Use of Surface Plasmon Resonance
Authors: Thoren Katie L , Pasi Brian , Delgado Julio C , Wu Alan H B , Lynch Kara L
Published: July 2018

Background

Monitoring infliximab (IFX) concentrations and the presence of antidrug antibodies (ADA) is important for patient management. We developed a method to measure IFX and ADA in serum in a single injection using surface plasmon resonance (SPR).

Methods

Using the Bio-Rad ProteOn XPR36, tumor necrosis factor-α and IFX were covalently immobilized onto separate lanes of a chip surface. Diluted serum was injected over both lanes, followed by an injection of goat antihuman antibody. The binding response was used to quantify IFX or detect ADA. The analytical performance of the assay was determined. Using 50 patient samples, SPR results were compared with results from a reporter gene assay (RGA).

Results

For the quantification of IFX, the functional sensitivity was 0.5 μg/mL. The total precision was <10% for all concentrations tested. IFX concentrations measured by SPR correlated well with RGA (R = 0.862), but a bias was observed (slope = 0.61). SPR detected 14 ADA-positive samples. Compared with RGA for ADA detection, there were 6 true-positive, 8 false-positive, 5 false-negative, and 31 true-negative findings.

Conclusion

SPR can be used to measure biological drug concentrations and detect ADA in serum. This technique may provide complementary information to current methods used to detect ADA.
Comparison of Point-of-Care Activated Clotting Time Methods in Different Clinical Settings in a Large Academic Medical Center
Authors: Korpi-Steiner Nichole L , Walz J Matthias , Schanzer Andres , Rao Lokinendi V
Published: July 2018

Objectives

This study is a comparative analysis of measured activated clotting time (ACT) values by use of 5 different point-of-care (POC) ACT methods spanning the range detected during different clinical procedures at our institution.

Methods

We determined the correlation, imprecision, and differences in measured ACT values with use of 4 POC ACT methods compared with a reference ACT method in 41 venous whole blood samples collected from 25 adult patients undergoing interventional procedures. The POC ACT methods evaluated included the i-STAT with kaolin activator in prewarm mode, i-STAT with Celite activator in prewarm and nonprewarm modes, ACTPlus, and HMSPlus, which was designated the reference method. Each venous whole blood patient sample was tested in duplicate on each POC ACT test system (total n = 410 ACT measurements). Analyses of imprecision and differences in measured ACT values were stratified by moderate (100–299 s) and high (≥300 s) ACT ranges.

Results

In this study population, measured ACT values ranged from 100–835 s with use of the HMSPlus. All methods demonstrated good correlation (r ≥ 0.95) in ACT values compared to the reference method. Imprecision varied by method with ranges of 1.7%–2.7% CV in the moderate ACT range and 2.5%–4.8% CV in the high ACT range. ACTPlus and i-STAT-Celite-prewarm methods exhibited proportional differences in measured ACT values whereas the i-STAT-Celite-nonprewarm and i-STAT-kaolin-prewarm demonstrated constant differences in measured ACT values compared to HMSPlus.

Conclusions

ACT values correlate well between POC methods. Imprecision and difference profiles vary by method; notably, imprecision exceeds systematic differences in the high ACT range and contributes to intermethod differences that are limitations worthy of consideration when contemplating a change in ACT methods.
Accuracy of Two Progesterone Immunoassays for Monitoring In Vitro Fertilization
Authors: Roper Stephen M , Rubin Jessica R , Ali Mahesheema , Gibbons William E , Zarutskie Paul W , Devaraj Sridevi
Published: July 2018

Background

Progesterone concentrations are routinely monitored during in vitro fertilization cycles. Immunoassay-based platforms are used most often in this setting because they are simple to use and amenable to same-day sample collection and result-reporting. However, immunoassay methods are subject to variation in specificity between different assay manufacturers. In this study, a set of unexpectedly high progesterone concentrations led to a method comparison between two in-house immunoassay platforms relative to the reference method.

Methods

Progesterone was measured in 28 serum samples from women undergoing IVF cycles using the Siemens ADVIA Centaur Immunoassay system and the Abbott Architect i1000SR analyzer. A subset of these samples was selected for progesterone measurement by liquid chromatography–tandem mass spectrometry to define the accuracy of each immunoassay.

Results

The Siemens ADVIA Centaur immunoassay system overestimated progesterone concentrations by 19% and the Abbott Architect overestimated progesterone concentrations by 5%.

Conclusions

The Abbott Architect progesterone immunoassay provides a more accurate measurement of serum progesterone than the Centaur immunoassay at concentrations relevant for monitoring in vitro fertilization populations.
Transplant Virus Detection Using Multiplex Targeted Sequencing
Authors: Tan Susanna K , Shen Peidong , Lefterova Martina I , Sahoo Malaya K , Fung Eula , Odegaard Justin I , Davis Ronald W , Pinsky Benjamin A , Scharfe Curt
Published: July 2018

Background

Viral infections are a major cause of complications and death in solid organ and hematopoietic cell transplantation.

Methods

We developed a multiplex viral sequencing assay (mVseq) to simultaneously detect 20 transplant-relevant DNA viruses from small clinical samples. The assay uses a single-tube multiplex PCR to amplify highly conserved virus genomic regions without the need for previous virus enrichment or host nucleic acid subtraction. Multiplex sample sequencing was performed using Illumina MiSeq, and reads were aligned to a database of target sequences. Analytical and clinical performance was evaluated using reference viruses spiked into human plasma, as well as patient plasma and nonplasma samples, including bronchoalveolar lavage fluid, cerebrospinal fluid, urine, and tissue from immunocompromised transplant recipients.

Results

For the virus spike-in samples, mVseq's analytical sensitivity and dynamic range were similar to quantitative PCR (qPCR). In clinical specimens, mVseq showed substantial agreement with single-target qPCR (92%; κ statistic, 0.77; 259 of 282 viral tests); however, clinical sensitivity was reduced (81%), ranging from 62% to 100% for specific viruses. In 12 of the 47 patients tested, mVseq identified previously unknown BK virus, human herpesvirus-7, and Epstein–Barr virus infections that were confirmed by qPCR.

Conclusions

Our results reveal factors that can influence clinical sensitivity, such as high levels of host DNA background and loss of detection in coinfections when 1 virus was at much higher concentration than the others. The mVseq assay is flexible and scalable to incorporate RNA viruses, emerging viruses of interest, and other pathogens important in transplant recipients.