bluuebunny/crossref_metadata_2025 huggingface dataset:
Enabling Large-Scale Pharmacogenetic Studies by High-Throughput Mutation Detection and Genotyping Technologies
Authors: Shi Michael M
Published: January 2020

Background: Pharmacogenetics is a scientific discipline that examines the genetic basis for individual variations in response to therapeutics. Pharmacogenetics promises to develop individualized medicines tailored to patients’ genotypes. However, identifying and genotyping a vast number of genetic polymorphisms in large populations also pose a great challenge.

Approach: This article reviews the recent technology development in mutation detection and genotyping with a focus on genotyping of single nucleotide polymorphisms (SNPs).

Content: Novel mutations/polymorphisms are commonly identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. With a large amount of public sequence information available, in silico SNP mapping has also emerged as a cost-efficient way for new polymorphism identification. Gel electrophoresis-based genotyping methods for known polymorphisms include PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing. Fluorescent dye-based genotyping technologies are emerging as high-throughput genotyping platforms, including oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan®, and molecular beacon genotyping. Rolling circle amplification and InvaderTM assays are able to genotype directly from genomic DNA without PCR amplification. DNA chip-based microarray and mass spectrometry genotyping technologies are the latest development in the genotyping arena.

Summary: Large-scale genotyping is crucial to the identification of the genetic make-ups that underlie the onset of diseases and individual variations in drug responses. Enabling technologies to identify genetic polymorphisms rapidly, accurately, and cost effectively will dramatically impact future drug and development processes.
Capillary and Microchip Electrophoresis for Rapid Detection of Known Mutations by Combining Allele-specific DNA Amplification with Heteroduplex Analysis
Authors: Tian Huijun , Brody Lawrence C , Fan Saijun , Huang Zhili , Landers James P
Published: January 2020

Background: Detection of mutations by gel electrophoresis and allele-specific amplification by PCR (AS-PCR) is not easily scaled to accommodate a large number of samples. Alternative electrophoretic formats, such as capillary electrophoresis (CE) and microchip electrophoresis, may provide powerful platforms for simple, fast, automated, and high-throughput mutation detection after allele-specific amplification.

Methods: DNA samples heterozygous for four mutations (185delAG, 5382insC, 3867G→T, and 6174delT) in BRCA1 and BRCA2, and homozygous for one mutation (5382insC) in BRCA1 and two mutations (16delAA and 822delG) in PTEN were chosen as the model system to evaluate the capillary and microchip electrophoresis methods. To detect each mutation, three primers, of which one was labeled with the fluorescent dye 6-carboxyfluorescein and one was the allele-specific primer (mutation-specific primer), were used to amplify the DNA fragments in the range of 130–320 bp. AS-PCR was combined with heteroduplex (HD) analysis, where the DNA fragments obtained by AS-PCR were analyzed with the conditions developed for CE-based HD analysis (using a fluorocarbon-coated capillary and hydroxyethylcellulose). The CE conditions were transferred into the microchip electrophoresis format.

Results: Three genotypes, homozygous wild type, homozygous mutant, and heterozygous mutant, could be identified by CE-based AS-PCR-HD analysis after 10–25 min of analysis time. Using the conditions optimized with CE, we translated the AS-PCR-HD analysis mutation detection method to the microchip electrophoresis format. The detection of three heterozygous mutations (insertion, deletion, and substitution) in BRCA1 could be accomplished in 180 s or less.

Conclusions: It is possible to develop a CE-based method that exploits both AS-PCR and HD analysis for detecting specific mutations. Fast separation and the capacity for automated operation create the potential for developing a powerful electrophoresis-based mutation detection system. Fabrication of multichannel microchip platforms may enable mutation detection with high throughput.
Development of a Rapid, Quantitative Method for LDL Subfractionation with Use of the Quantimetrix Lipoprint LDL System
Authors: Hoefner Daniel M , Hodel Shannon D , O’Brien John F , Branum Earl L , Sun Deborah , Meissner Irene , McConnell Joseph P
Published: January 2020

Background: Recent evidence suggests that the presence of small, dense LDL is independently associated with increased risk of developing coronary artery disease. Current methods to subfractionate LDL are time-consuming and/or technically demanding. Therefore, we have sought the development of a less complex LDL subfractionation procedure.

Methods: LDL subfractions were separated using the Quantimetrix LipoprintTM LDL System. High-resolution 3% polyacrylamide gel tubes were scanned densitometrically (610 nm) with a Helena EDC system. A computerized method to identify and quantitatively score the resolved LDL subfractions was developed. Results from the Quantimetrix method were compared using 51 plasma samples with values obtained by nondenaturing gradient gel electrophoresis (NDGGE) and nuclear magnetic resonance (NMR) spectroscopy.

Results: LDL subfractionation scores correlated significantly (P <0.05) with triglyceride, HDL-cholesterol, apolipoprotein B100, and LDL-cholesterol/apolipoprotein B100 (r = 0.591, −0.392, 0.454, and −0.411, respectively). For 51 samples, the Quantimetrix method classified 21 with small, 14 with intermediate, and 16 with large LDL. Of the 21 samples classified as small by Quantimetrix, 20 (95%) were classified as small (n = 18) or intermediate (n = 2) by NDGGE. All of the 16 specimens classified as large by Quantimetrix were either large (n = 14) or intermediate (n = 2) by NDGGE. LDL score was inversely correlated (r = −0.674; P <0.0001) with LDL particle size determined by NMR spectroscopy.

Conclusions: A quantitative method for the assessment of LDL particle size phenotype was developed using the Quantimetrix Lipoprint LDL System. The method can be performed in less than 3 h in batch mode and is suitable for routine use in clinical laboratories.
Development of a Routine Method for the Determination of Trace Metals in Whole Blood by Magnetic Sector Inductively Coupled Plasma Mass Spectrometry with Particular Relevance to Patients with Total Hip and Knee Arthroplasty
Authors: Case C Patrick , Ellis Lyndon , Turner Justine C , Fairman Ben
Published: January 2020

Background: Joint-replacement surgery has revolutionized the treatment of osteoarthritis and is still the most effective therapy. A recent clinical trend reintroducing metal-on-metal bearing surfaces has in turn stimulated a requirement for accurate measurement of the concentrations of relevant metals in both pre- and postoperative patients. Thus, there is a need for cost-effective, multielement methods for trace metal analysis in whole blood to monitor possible increases in wear metal concentrations.

Methods: A method was developed to allow routine analysis of whole blood samples for molybdenum, cobalt, chromium, and nickel. Sample preparation consisted of a simple 1:10 dilution of whole blood with a solution of 10 mL/L Triton X-100, 0.0002 mol/L EDTA, and 0.01 mol/L ammonium hydroxide. Final determination was performed by a double-focusing magnetic sector inductively coupled plasma mass spectrometer operated in medium-resolution mode (resolution, 3400). Online addition of rhodium was used for internal standardization.

Results: Detection limits in whole blood were 0.06 μg/L for chromium, cobalt, and molybdenum and 0.30 μg/L for nickel. Base concentrations of 0.22, 0.17, 0.62, and 0.99 μg/L for chromium, cobalt, molybdenum, and nickel, respectively, in whole blood have been found. Polyatomic interferences on all four elements have been shown to be resolved from the analyte masses by use of a resolution of >3000.

Conclusions: The simple, rapid method of sample preparation is effective in minimizing potential contamination and enables 60 samples (run time, 8 h) to be analyzed before cleaning the instrument is necessary. A resolution >3000 was sufficient to separate polyatomic interferences from the masses of interest. The method was used to analyze a large number of blood samples taken from primary patients awaiting total hip arthroplasty. The method is sensitive enough to provide base concentrations for chromium, cobalt, and molybdenum in whole blood. The results for nickel were compromised by high signals for blank samples.
Miniaturization Technologies for Molecular Diagnostics
Authors: McGlennen Ronald C
Published: January 2020

Background: Molecular diagnostics devices are becoming smaller. With the advancement of miniaturization technologies, microchip-based systems will soon be available for genetic testing. The purpose of this review is to highlight the underlying principles in miniaturization, the strategies being developed for bioanalysis, and the potential impact on the practice of this rapidly growing medical discipline.

Approach: The author discusses DNA microchips and their practical importation into the clinical laboratory, based on his background in medical device and microchip design and development. His discussion is supported by a body of literature covering both biomedical and electrical engineering and more recent publications in the field of molecular genetics and pathology.

Content: This review is descriptive and intended to outline the technologic and methodologic approaches to the creation of an integrated genetic analysis instrument based on miniature components. The review draws on published scientific evaluations of these devices without regard to the companies involved in their development.

Summary: The intent of this review is that the reader will better understand the variety of technical approaches toward the miniaturization of molecular genetic testing for the clinical laboratory. With insight into the principles underlying the operation of these chips and the integrated systems, the end user can better evaluate the value to the field in terms of making molecular genetics testing simpler, faster, and less expensive.
Cardiac Troponin T Predicts Long-Term Outcomes in Hemodialysis Patients
Authors: Ooi Daylily S , Zimmerman Deborah , Graham Janet , Wells George A
Published: January 2020

Background: Increased plasma troponin T (cTnT), but not troponin I (cTnI), is frequently observed in end-stage renal failure patients. Although generally considered spurious, we previously reported an associated increased mortality at 12 months.

Methods: We studied long-term outcomes in 244 patients on chronic hemodialysis for up to 34 months, correlating the outcomes to plasma cTnT in routine predialysis samples. In addition, subsequent plasma samples at least 1 year later and within 6 months of data analysis were available in 97 patients and were used to identify patients with increasing plasma cTnT. The endpoints used were death and new or worsening coronary, cerebro-, and peripheral vascular disease and neuropathy.

Results: Transplantation occurred more frequently in patients with low initial cTnT: 31%, 13%, and 3% in the groups with cTnT <0.010, 0.010–0.099, and ≥0.100 μg/L, respectively. In the same groups, total deaths occurred in 6%, 43%, and 59% and cardiac deaths in 0%, 14%, and 24% of patients. In patients with follow-up samples, the group with increasing cTnT had a significantly increased death (relative risk, 2.0; P = 0.028). The increase was mainly in cardiac and sudden deaths.

Conclusions: Higher plasma cTnT predicts long-term all-cause mortality in hemodialysis patients, even at concentrations <0.100 μg/L, as does an increasing cTnT concentration over time.
Clinical and Experimental Results on Cardiac Troponin Expression in Duchenne Muscular Dystrophy
Authors: Hammerer-Lercher Angelika , Erlacher Petra , Bittner Reginald , Korinthenberg Rudolf , Skladal Daniela , Sorichter Stephan , Sperl Wolfgang , Puschendorf Bernd , Mair Johannes
Published: January 2020

Background: Because of controversial earlier studies, the purpose of this study was to provide novel experimental and additional clinical data regarding the possible reexpression of cardiac troponin T (cTnT) in regenerating skeletal muscle in Duchenne muscular dystrophy (DMD).

Methods: Plasma from 14 patients (mean age, 7.5 years; range, 5.7–19.4 years) with DMD was investigated for creatine kinase (CK), the CK MB isoenzyme (CKMB), cTnT and cardiac troponin I (cTnI), and myoglobin. cTnT concentrations were measured by an ELISA (second-generation assay; Roche) using the ES 300 Analyzer. cTnI, myoglobin, and CKMB were measured by an ELISA using the ACCESS System (Beckman Diagnostics). Troponin isoform expression was studied by Western blot analysis in remnants of skeletal muscle biopsies of three patients with DMD and in an animal model of DMD (mdx mice; n = 6).

Results: There was no relation of cTnT and cTnI to clinical evidence for cardiac failure. cTnI concentrations remained below the upper reference limit in all patients. cTnT was increased (median, 0.11 μg/L; range, 0.06–0.16 μg/L) in 50% of patients. The only significant correlation was found for CK (median, 3938 U/L; range, 2763–5030 U/L) with age (median, 7.5 years; range, 6.8–10.9 years; r = −0.762; P = 0.042). Western blot analysis of human or mouse homogenized muscle specimens showed no evidence for cardiac TnT and cTnI expression, despite strong signals for skeletal muscle troponin isoforms.

Conclusions: We found no evidence for cTnT reexpression in human early-stage DMD and in mdx mouse skeletal muscle biopsies. Discrepancies of cTnT and cTnI in plasma samples of DMD patients were found, but neither cTnT nor cTnI plasma concentrations were related with other clinical evidence for cardiac involvement.
Effects of Implantable Cardioverter Defibrillator Implantation and Shock Application on Biochemical Markers of Myocardial Damage
Authors: Schlüter Thomas , Baum Hannsjörg , Plewan Andreas , Neumeier Dieter
Published: January 2020

Background: Implantable cardioverter defibrillator (ICD) implantation is a common approach in patients at high risk of sudden cardiac death. To check for normal function, it is necessary to test the ICD. For this purpose, repetitive induction and termination of ventricular fibrillation by direct current shocks is required. This may lead to minor myocardial damage. Cardiac troponin T (cTnT) and I (cTnI) are specific markers for the detection of myocardial injury. Because these proteins usually are undetectable in healthy individuals, they are excellent markers for detecting minimal myocardial damage. The objective of this study was to evaluate the effect of defibrillation of induced ventricular fibrillation on markers of myocardial damage.

Methods: This study included 14 patients who underwent ICD implantation and intraoperative testing. We measured cTnT, cTnI, creatine kinase MB (CK-MB) mass, CK activity, and myoglobin before and at definite times after intraoperative shock application.

Results: Depending on the effectiveness of shocks and the energy applied, the cardiac-specific markers cTnT and cTnI, as well as CK-MB mass, showed a significant increase compared with the baseline value before testing and peaked for the most part 4 h after shock application. In contrast, the increases in CK activity and myoglobin were predominantly detectable in patients who received additional external shocks.

Conclusions: ICD implantation and testing leads to a short release of cardiac markers into the circulation. This release seems to be of cytoplasmic origin and depends on the number and effectiveness of the shocks applied.
Diagnosis of Dopa-responsive Dystonia and Other Tetrahydrobiopterin Disorders by the Study of Biopterin Metabolism in Fibroblasts
Authors: Bonafé Luisa , Thöny Beat , Leimbacher Walter , Kierat Lucja , Blau Nenad
Published: January 2020

Background: Dopa-responsive dystonia (DRD) and tetrahydrobiopterin (BH4) defects are inherited disorders characterized by monoamine neurotransmitter deficiency with decreased activity of one of the BH4-metabolizing enzymes. The aim of the study was to determine the utility of cultured skin fibroblasts for the diagnosis of these diseases.

Methods: Neopterin and biopterin production and GTP cyclohydrolase I (GTPCH) activity were measured in cytokine-stimulated fibroblasts; 6-pyruvoyltetrahydropterin synthase (PTPS), sepiapterin reductase (SR), and dihydropteridine reductase (DHPR) activities were measured in unstimulated fibroblasts. We examined 8 patients with DRD, 3 with autosomal recessive GTPCH deficiency, 7 with PTPS deficiency, 3 with DHPR deficiency, and 49 controls (35 fibroblast and 14 amniocyte samples).

Results: Fibroblasts from patients with DRD and autosomal recessive GTPCH deficiency showed reduced GTPCH activity (15.4% and 30.7% of normal activity, respectively) compared with controls (P <0.001). Neopterin production was very low and biopterin production was reduced in both disorders. PTPS- and DHPR-deficient cells showed no enzyme activities; in PTPS deficiency the pattern of pterin production was typical (neopterin, 334–734 pmol/mg; controls, 18–98 pmol/mg; biopterin, 0 pmol/mg; controls, 154–303 pmol/mg). Reference values of all enzyme activities and pterin production were measured in fibroblasts and also in amniocytes for prenatal diagnosis.

Conclusions: Cultured skin fibroblasts are a useful tool in the diagnosis of BH4 deficiencies. Intracellular neopterin and biopterin concentrations and GTPCH activity in cytokine-stimulated fibroblasts are particularly helpful in diagnosing patients with DRD.
Triple Lipid Screening Test: A Homogeneous Sequential Assay for HDL-Cholesterol, Total Cholesterol, and Triglycerides
Authors: Sampson Maureen L , Aubry Andrea , Csako Gyorgy , Remaley Alan T
Published: January 2020

Background: The analysis of lipids in serum lipoprotein fractions is useful in assessing the risk for coronary artery disease, but it typically involves performing multiple tests. An automated single-tube assay, referred to as the triple lipid screening (TLS) test, can be used for measuring HDL-cholesterol (HDL-C), total cholesterol, and triglycerides (TGs) with no specimen pretreatment.

Methods: The first part of the assay is based on a homogeneous assay for HDL-C that uses either an anti-apolipoprotein B antibody (TLS-A test) or a polyanion (TLS-B test) that blocks the enzymatic measurement of cholesterol on the non-HDL fraction. After the addition of deoxycholate, which solubilizes the unreacted cholesterol from the non-HDL fraction, the remaining cholesterol in the sample is subsequently measured enzymatically. Using the same enzyme detection system as the cholesterol assay, TGs are measured in the last step, after the addition of the enzymes for the TG assay.

Results: The TLS assay (y) had acceptable analytic performance and compared favorably with standard tests (x) for each analyte: for HDL-C, TLS-A = 0.99x + 0.19 (R = 0.980); TLS-B = 1.00x − 0.15 (R = 0.974); for total cholesterol, TLS-A = 1.03x + 0.12 (R = 0.997); TLS-B = 1.07x − 0.30 (R = 0.965); and for TGs, TLS-A = 1.02x + 0.02 (R = 0.988); TLS-B = 1.04x − 0.28 (R = 0.980).

Conclusions: The TLS test is a single-tube homogeneous assay for the analysis of all of the major serum lipoprotein fractions and can be used as a simple screening test for the detection of hyperlipidemia.
Measurement of Circulating Forms of Prostate-specific Antigen in Whole Blood Immediately after Venipuncture: Implications for Point-of-Care Testing
Authors: Piironen Timo , Nurmi Martti , Irjala Kerttu , Heinonen Olli , Lilja Hans , Lövgren Timo , Pettersson Kim
Published: January 2020

Background: The purpose of this study was to validate the use of whole-blood samples in the determination of circulating forms of prostate-specific antigen (PSA).

Methods: Blood samples of hospitalized prostate cancer and benign prostatic hyperplasia patients were collected and processed to generate whole-blood and serum samples. Three different rapid two-site immunoassays were developed to measure the concentrations of total PSA (PSA-T), free PSA (PSA-F), and PSA-α1-antichymotrypsin complex (PSA-ACT) to detect in vitro changes in whole-blood samples immediately after venipuncture. The possible influence of muscle movement on the release of PSA from prostate gland was studied in healthy men by measuring the rapid in vitro whole-blood kinetics of PSA forms before and after 15 min of physical exercise on a stationary bicycle.

Results: Rapid PSA-T, PSA-F, and PSA-ACT assays were designed using a 10-min sample incubation. No significant changes were detected in the concentrations of PSA-T, PSA-F, and PSA-ACT from the earliest time point of 12–16 min compared with measurements performed up to 4 h after venipuncture. Physical exercise did not influence the concentrations of the circulating forms of PSA. Hematocrit-corrected whole-blood values of PSA-T and PSA-F forms were comparable to the respective serum values. Calculation of the percentage of PSA-F (PSA F/T ratio × 100) was similar irrespective of the sample format used, i.e., whole blood or serum.

Conclusions: We found that immunodetectable PSA forms are likely at steady state immediately after venipuncture, thus enabling the use of anticoagulated whole-blood samples in near-patient settings for point-of-care testing, whereas determinations of PSA (e.g., PSA-T, PSA-F, or PSA-ACT) performed within the time frame of the office visit would provide results equivalent to conventional analyses performed in serum.
Enzymatic Mutation Detection Method Evaluated for Detection of p53 Mutations in cDNA from Breast Cancers
Authors: Norberg Torbjörn , Klaar Sigrid , Lindqvist Lena , Lindahl Thomas , Ahlgren Johan , Bergh Jonas
Published: January 2020

Background: Rapid, reproducible, and easily run methods with high sensitivity and specificity are required for mutation screening of clinical samples. We evaluated the Enzymatic Mutation Detection (EMDTM) method by analysis of archival cDNA from 203 breast cancer patients and comparison with results of cDNA-based sequencing of the tumor suppressor gene p53.

Methods: The EMD technology uses the T4 endonuclease VII, which cleaves double-stranded DNA at sites where a DNA mismatch is present because of mispairing or an insertion/deletion of nucleotides. The EMD analyses were carried out by dividing the p53 gene into two overlapping fragments that were analyzed separately. After PCR amplification, the fragments were hybridized with wild-type p53 and subsequently exposed to the EMD enzyme. Cleavage products were analyzed and scored using an ALFTM automated DNA sequencer and ALFwin Fragment Analyzer software (Ver. 1.02).

Results: The EMD technique had sensitivities of 45% and 64% and specificities of 83% and 84% for the two fragments, respectively. Patients with EMD-positive, wild-type p53 tumors had a survival similar to that of patients with EMD-negative, wild-type p53 tumors. Node-positive patients with p53 mutated tumors according to sequencing had a statistically significantly worse overall survival than those with p53 wild-type tumors (P = 0.016), whereas this difference in survival was not detected when p53 status was determined with EMD (P = 0.47).

Conclusions: EMD had insufficient sensitivity for consideration in screening for the p53 gene in this archival material. Sequencing must still be considered as the standard procedure.
New Polymorphic Short Tandem Repeats for PCR-based Charcot-Marie-Tooth Disease Type 1A Duplication Diagnosis
Authors: Badano Jose L , Inoue Ken , Katsanis Nicholas , Lupski James R
Published: January 2020

Background: Charcot-Marie-Tooth disease type 1A (CMT1A) accounts for 70–90% of cases of CMT1 and is most frequently caused by the tandem duplication of a 1.4-Mb genomic fragment on chromosome 17p12. Molecular diagnosis of CMT1A has been based primarily on pulsed-field electrophoresis, fluorescence in situ hybridization, polymorphic allele dosage analysis, and quantitative PCR. We sought to improve the fidelity and applicability of PCR-based diagnosis by developing a panel of novel, highly polymorphic short tandem repeats (STRs) from within the CMT1A duplicated region.

Methods: We used a recently available genomic sequence to identify potentially polymorphic simple repeats. We then amplified these sequences in a multiethnic cohort of unaffected individuals and assessed the heterozygosity and number of alleles for each STR. Highly informative markers were then tested in a set of previously diagnosed CMT1A duplication patients, and the ability to identify the genomic duplication through the presence of three bands was assessed.

Results: We identified 34 polymorphic markers, 15 of which were suitable for CMT1A diagnosis on the basis of high heterozygosity in different ethnic groups, peak uniformity, and a large number of alleles. On the basis of the fluorescent dye and allele range of each marker, we developed two panels, each of which could be analyzed concurrently. Panel 1, which comprised 10 markers, detected 37 of 39 duplications, whereas panel 2, which comprised the remaining 5 markers, identified 21 of 39 duplications. Through the combination of both panels, we identified 39 of 39 duplications in previously diagnosed CMT1A patients.

Conclusions: The newly developed 15-marker set has the capability of detecting >99% of duplications and thus is a powerful and versatile diagnostic tool.
C-Reactive Protein Concentrations and Angiographic Characteristics of Coronary Lesions
Authors: Katritsis Demosthenes , Korovesis Socrates , Giazitzoglou Eleftherios , Parissis John , Kalivas Pericles , Webb-Peploe Michael M , Ioannidis John P A , Haliassos Alexander
Published: January 2020

Background: C-Reactive protein (CRP) is a strong predictor of clinical outcome in coronary artery disease (CAD), and inflammation has been implicated in the process. We aimed to evaluate whether CRP concentrations measured with a new, automated particle-enhanced immunoturbidimetric method for high-sensitivity CRP may be related to specific high-risk angiographic features of coronary lesions.

Methods: In a cross-sectional study, we examined 103 consecutive patients undergoing cardiac catheterization for suspected CAD. We assessed the association of preprocedural CRP concentrations with clinical presentation (unstable angina) and angiographic features of coronary lesions.

Results: Twenty patients had unstable angina. Independent predictors of unstable angina included increased CRP [odds ratio (OR), 2.93 per 10-fold increase in CRP; 95% confidence interval (CI), 1.28–6.69; P = 0.01] and the presence of macroscopic thrombus (OR, 7.08; 95% CI, 1.33–37.8; P = 0.02). Thirty-two culprit lesions had macroscopic thrombus or eccentric/irregular discrete morphology without total occlusion. Increased CRP was the strongest predictor of such features (OR, 2.04 per 10-fold increase in CRP; 95% CI, 1.03–4.04; P = 0.04), and the effect was independent of the presence of unstable angina.

Conclusions: Among patients with suspected CAD undergoing coronary angiography, increased CRP is strongly associated with unstable angina and with specific high-risk features of the culprit coronary lesions.
Glucuronidation of Prodrug Reactive Site: Isolation and Characterization of Oxymethylglucuronide Metabolite of Fosphenytoin
Authors: Annesley Thomas M , Kurzyniec Stephen , Nordblom Gerald D , Buchanan Nathan , Pool William , Reily Michael , Talaat Rasmy , Roberts William L
Published: January 2020

Background: This investigation was undertaken to identify the structure of a novel immunoreactive metabolite derived from fosphenytoin that has been hypothesized previously as present in sera from renally impaired patients receiving this prodrug.

Methods: The metabolite was isolated from uremic sera using solid-phase extraction and HPLC. Structural analysis was performed using HPLC–tandem mass spectrometry, nuclear magnetic resonance (NMR), deuterium exchange, and chemical derivatization. Immunoreactivity was evaluated using a fluorescence polarization immunoassay.

Results: The metabolite had a parent ion at m/z 457 in the negative-ion mode and fragmented to yield the m/z 251 of phenytoin, as well as other mass fragments of phenytoin. Mass fragments associated with glucuronic acid were also present. The chromatographic peak corresponding to this metabolite demonstrated immunoreactivity sufficient to lead to falsely increased reported values for phenytoin immunoassays. The observed immunoreactivity was also proportional to the relative concentration of the metabolite in collected fractions. Analysis by NMR indicated the presence of phenyl groups with chemical shifts identical to those of phenytoin, as well as the presence of a methylene bridge, which was consistent with the same methylene bridge present on the phosphate ester of fosphenytoin. Comparative analysis of serum samples from renally impaired patients receiving phenytoin vs fosphenytoin using multiple reaction monitoring quantification demonstrated that this metabolite was associated with fosphenytoin administration.

Conclusions: A unique immunoreactive oxymethylglucuronide metabolite derived from fosphenytoin has been isolated from sera from uremic patients receiving this prodrug.
Examination of Postmortem Fluids and Tissues for the Presence of Methylecgonidine, Ecgonidine, Cocaine, and Benzoylecgonine Using Solid-Phase Extraction and Gas Chromatography–Mass Spectrometry2
Authors: Shimomura Eric T , Hodge Gwendolyn D , Paul Buddha D
Published: January 2020

Background: During the smoking of crack cocaine (COC), methyl ecgonidine (MED) is formed as one of the pyrolysis products. Once in the body, MED is converted to ecgonidine (ED) through several processes that include spontaneous hydrolysis and enzymatic hydrolysis. The presence of MED and/or ED could provide valuable information to help determine antemortem conditions in cases where COC is involved. Our goal was to examine postmortem tissues and fluids for the presence of MED, ED, COC, and benzoylecgonine (BZ).

Methods: Liver, brain, blood, and urine specimens obtained from 15 postmortem cases were extracted using solid-phase extraction, derivatized, and analyzed using gas chromatography–mass spectrometry with selective-ion monitoring.

Results: Median concentrations (range) of drugs observed in postmortem liver, brain, blood, and urine were 0 (0–10) ng/g, 7 (0–92) ng/g, 0 (0–42) μg/L, and 62 (0–2030) μg/L, respectively, for MED; 655 (90–3274) ng/g, 22 (0–52) ng/g, 119 (13–773) μg/L, and 456 (109–7452) μg/L, respectively, for ED; 57 (0–503) ng/g, 187 (0–1403) ng/g, 12 (0–88) μg/L, and 1208 (37–28 062) μg/L, respectively, for COC; and 821 (45–4980) ng/g, 524 (46–5153) ng/g, 458 (30–2071) μg/L, and 6768 (917–116 430) μg/L, respectively, for BZ. MED was detected in 12 of 15 postmortem cases. The concentrations were highest in urine compared with liver, brain, and blood. The hydrolysis product ED was detected in all postmortem cases, and the concentrations were substantially higher than MED in all liver, blood, and urine specimens.

Conclusion: ED may be a more useful indicator of crack COC smoking.
No Influence of the MDR-1 C3435T Polymorphism or a CYP3A4 Promoter Polymorphism (CYP3A4-V Allele) on Dose-adjusted Cyclosporin A Trough Concentrations or Rejection Incidence in Stable Renal Transplant Recipients
Authors: von Ahsen Nicolas , Richter Michael , Grupp Clemens , Ringe Burckhardt , Oellerich Michael , Armstrong Victor W
Published: January 2020

Background: A substantial proportion of the variability in the absorption and clearance of cyclosporin A (CsA) after oral administration has been attributed to variability in liver cytochrome P-450 3A4 (CYP3A4) activity and intestinal P-glycoprotein (P-gp) concentration. A polymorphism in the CYP3A4 promoter region, termed “variant” allele CYP3A4-V, was postulated to be associated with altered CYP3A4 enzyme activity. A polymorphism in exon 26 (C3435T) of the multidrug resistance-1 (MDR-1) gene was correlated with intestinal expression and in vivo activity of P-gp.

Methods: We investigated the occurrence of both polymorphisms in 124 stable Caucasian renal transplant recipients (>6 months after transplantation) on CsA as the primary immunosuppressant. Real-time, rapid-cycle PCR methods were developed and used for genotyping.

Results: The estimated allele frequencies for the MDR-1 C3435T allele (54%) and the CYP3A4-V allele (4.8%) were similar to those reported for Caucasian populations. No significant differences were found for the CsA doses needed to maintain similar CsA trough concentrations in patients with and without the CYP3A4-V allele or in patients with different MDR-1 C3435T genotypes. Furthermore, neither of the polymorphisms investigated was associated with renal function as assessed by creatinine plasma concentration or, in a retrospective analysis, the incidence of acute rejection.

Conclusions: These findings suggest that the MDR-1 C3435T mutation and the CYP3A4-V variant are not major determinants of CsA efficacy in renal transplant recipients.
Emerging Homogeneous DNA-based Technologies in the Clinical Laboratory
Authors: Foy Carole A , Parkes Helen C
Published: January 2020

Background: Advances in molecular diagnostic technologies have enabled genetic testing in single closed-tube reactions. The purpose of this review is to highlight some of the platforms and technologies currently available for the homogeneous detection of targets and the application of the technologies in the clinical setting. Validation issues surrounding the technologies, which may need to be addressed before they can become widely accepted, will also be discussed.

Approach: This review discusses the principles of several of the major technologies available for performing homogeneous genetic analyses. Publications arising from the application of the technologies in a wide range of clinical areas are used to highlight and compare the potential advantages and shortcomings of the various technologies.

Content: This review is descriptive and focuses on three areas: the technologies available for performing homogeneous analysis, the clinical applications where the technologies are being used, and validation issues surrounding the acceptance of the technologies in the general clinical setting.

Summary: This review intends to give the reader a greater understanding of the various technologies available for performing homogeneous genetic testing in the clinical laboratory. Through insight into the principles and performance characteristics underlying these technologies, the end user can evaluate their value and limitations in the clinical diagnostic setting.
Immunochemical Assay of Hemoglobin with Nε-(Carboxymethyl)lysine at Lysine 66 of the β Chain
Authors: Iwamoto Hisahiko , Motomiya Yoshihiro , Miura Keisuke , Morisawa Masayo , Yoshimura Yoshimichi , Maruyama Ikuro
Published: January 2020

Background: N ε-(Carboxymethyl)lysine (CML), a well-characterized and major advanced glycation end product structure, is produced via a Maillard reaction by nonenzymatic glycation and/or oxidation. Although few of the carboxymethylation sites of lysine residues on proteins have been identified, it is known that the possible lysine glycation site in hemoglobin (Hb) is Lys-66 on the β chain. We aimed to develop an assay for the Hb with a CML (CML-Hb) site specific to Lys-66 on the Hb β chain and to determine whether the lysine residue at that site is carboxymethylated.

Methods: Ala-His-Gly-Lys-Lys(CM)-Val-Leu-Gly-Ala-Phe-Ser-Cys, the peptide derived from the β chain of human Hb, was synthesized as an immunogen, and a monoclonal antibody against the peptide was prepared. A latex immunoassay method was established using the antibody on an automatic analyzer. In this study, 20 samples from healthy subjects and 80 samples from nondiabetic patients undergoing hemodialysis (HD) were analyzed.

Results: The latex immunoassay method using the antibody correlated significantly with the ELISA method using the antibody (r = 0.95; P <0.001). Between healthy subjects (n = 20) and nondiabetic HD patients (n = 80), a significant difference was seen in circulating CML-Hb (525 ± 76 vs 778 ± 137 pmol CML/mg of Hb; P <0.0001).

Conclusion: The latex method for the CML-Hb site specific to Lys-66 on the β chain can measure large numbers of samples on an automatic analyzer.
Near-Infrared Spectroscopy for Measuring Urea in Hemodialysis Fluids
Authors: Eddy Christopher V , Arnold Mark A
Published: January 2020

Background: Near-infrared spectroscopy is proposed as a method for providing real-time urea concentrations during hemodialysis treatments. The feasibility of such noninvasive urea measurements is evaluated in undiluted dialysate fluid.

Methods: Near-infrared spectra were collected from calibration solutions of urea prepared in dialysate fluid. Spectra were collected over three distinct spectral regions, and partial least-squares calibration models were optimized and compared for each. Selectivity for urea was demonstrated with two-component samples composed of urea and glucose in the dialysate matrix. The clinical significance of this approach was assessed by measuring urea in real hemodialysate samples.

Results: Urea absorptions within the combination and short-wavelength, near-infrared spectral regions provided sufficient spectral information for sound calibration models in the dialysate matrix. The combination spectral region had SEs of calibration (SEC) and prediction (SEP) of 0.38 mmol/L and 0.26 mmol/L, respectively, over the 4720–4600 cm−1 spectral range with 5 partial least-square factors. A second calibration model was established over the combination region from a series of solutions prepared with independently variable concentrations of urea and glucose. The best calibration model for urea in the presence of variable glucose concentrations had a SEC of 0.6 mmol/L and a SEP of 0.4 mmol/L for a 5-factor model over the 4600–4350 cm−1 spectral range. There was no significant decrease in SEP when the 4720–4600 cm−1 calibration model was used to measure urea in real samples collected during actual hemodialysis.

Conclusions: Urea can be determined with sufficient sensitivity and selectivity for clinical measurements within the matrix of the hemodialysis fluid.
Determination of Acid α-Glucosidase Activity in Blood Spots as a Diagnostic Test for Pompe Disease
Authors: Umapathysivam Kandiah , Hopwood John J , Meikle Peter J
Published: January 2020

Background: Pompe disease is an autosomal recessive disorder of glycogen metabolism that is characterized by a deficiency of the lysosomal acid α-glucosidase. Enzyme replacement therapy for the infantile and juvenile forms of Pompe disease currently is undergoing clinical trials. Early diagnosis before the onset of irreversible pathology is thought to be critical for maximum efficacy of current and proposed therapies. In the absence of a family history, the presymptomatic detection of these disorders ideally can be achieved through a newborn-screening program. Currently, the clinical diagnosis of Pompe disease is confirmed by the virtual absence, in infantile onset, or a marked reduction, in juvenile and adult onset, of acid α-glucosidase activity in muscle biopsies and cultured fibroblasts. These assays are invasive and not suited to large-scale screening.

Methods: A sensitive immune-capture enzyme activity assay for the measurement of acid α-glucosidase protein was developed and used to determine the activity of this enzyme in dried-blood spots from newborn and adult controls, Pompe-affected individuals, and obligate heterozygotes.

Results: Pompe-affected individuals showed an almost total absence of acid α-glucosidase activity in blood spots. The assay showed a sensitivity and specificity of 100% for the identification of Pompe-affected individuals.

Conclusions: The determination of acid α-glucosidase activity in dried-blood spots is a useful, noninvasive diagnostic assay for the identification of Pompe disease. With further validation, this procedure could be adapted for use with blood spots collected in newborn-screening programs.
Solid-Phase Amplification for Detection of C282Y and H63D Hemochromatosis (HFE) Gene Mutations
Authors: Turner Mark S , Penning Sarah , Sharp Angela , Hyland Valentine J , Harris Ray , Morris C Phillip , van Daal Angela
Published: January 2020

Background: There is a need for simple, rapid, and inexpensive methods for the detection of single-nucleotide polymorphisms. Our aim was to develop a single-tube ELISA-like PCR assay and evaluate it by detecting the common C282Y and H63D mutations found in the hemochromatosis gene (HFE) by use of clinical samples.

Methods: The method, termed solid-phase amplification (SPA), involves dual liquid- and solid-phase amplification of a target sequence by the use of two PCR primers, one of which is in two forms: the first is covalently immobilized to the wall of a microwell, and the second is free in solution. During allele-specific amplification, both the free and solid-phase amplicons are labeled by incorporation of digoxigenin (DIG)-dUTP. The amount of surface-bound amplicon is determined colorimetrically by the use of an alkaline phosphatase-anti-DIG-Fab conjugate and p-nitrophenyl phosphate.

Results: Two different amplicon-labeling methods were evaluated. Analysis of 173 clinical samples for the C282Y and H63D HFE point mutations with SPA revealed that only one sample was incorrectly diagnosed, apparently because of operator error, when compared with conventional restriction fragment length polymorphism assay results.

Conclusions: The SPA assay has potential for medium-scale mutation detection, having the advantage of being manipulatively simple and immediately adaptable for use in clinical laboratories with existing ELISA instrumentation.
Discrimination of Prostate Cancer from Benign Disease by Plasma Measurement of Intact, Free Prostate-specific Antigen Lacking an Internal Cleavage Site at Lys145-Lys146
Authors: Nurmikko Pauliina , Pettersson Kim , Piironen Timo , Hugosson Jonas , Lilja Hans
Published: January 2020

Background: The proportion of free prostate-specific antigen (PSA) is higher in the sera of patients with benign prostatic hyperplasia compared with patients with prostate cancer (PCa). We developed an immunoassay that measures intact, free PSA forms (fPSA-I), but does not detect free PSA that has been internally cleaved at Lys145-Lys146 (fPSA-N), and investigated whether this form could discriminate patients with PCa from those without PCa.

Methods: The assay for fPSA-I uses a novel monoclonal antibody (MAb) that does not detect PSA that has been internally cleaved at Lys145-Lys146. A MAb specific for free PSA was used as a capture antibody, and purified recombinant proPSA was used as a calibrator. The concentrations of fPSA-I, free PSA (PSA-F), and total PSA (PSA-T) were analyzed in EDTA-plasma samples (n = 276) from patients who participated in a screening program for PCa (PSA-T, 0.83–76.3 μg/L).

Results: The detection limit of the fPSA-I assay was 0.035 μg/L. Both the measured concentrations of fPSA-I and the concentrations of fPSA-N (calculated as PSA-F − fPSA-I) provided statistically significant discrimination of the two clinical groups. By contrast, PSA-F did not discriminate between these groups. Each of the ratios fPSA-I/PSA-F, fPSA-N/PSA-T, and PSA-F/PSA-T separated cancer samples from noncancer samples in a statistically significant manner (P <0.0001). The ratio fPSA-I/PSA-F was significantly higher in cancer (median, 59%) compared with noncancer samples (47%).

Conclusions: The ratio fPSA-I/PSA-F is significantly higher in cancer compared with noncancer. The percentages of both fPSA-N/PSA-T and fPSA-I/PSA-F may provide interesting diagnostic enhancements alone or in combination with other markers and require further studies.
Spectrophotometric Microassay for δ-Aminolevulinate Dehydratase in Dried-Blood Spots as Confirmation for Hereditary Tyrosinemia Type I
Authors: Schulze Andreas , Frommhold David , Hoffmann Georg F , Mayatepek Ertan
Published: January 2020

Background: Hereditary tyrosinemia type I (HT) fulfills the criteria for inclusion in neonatal screening programs, but measurement of tyrosine lacks clinical specificity and quantitative assay of succinylacetone is laborious. We developed a semiquantitative assay based on inhibition of δ-aminolevulinate dehydratase (ALA-D) by succinylacetone.

Methods: Preincubation of 3-mm discs from dried-blood spots and reaction of the enzyme with δ-aminolevulinic acid as substrate were performed in microtiter plates. After separation of the supernatant and 10 min of color reaction with modified Ehrlich reagent, the formation of porphobilinogen was measured at 550 nm in a plate reader.

Results: The detection limit for succinylacetone was 0.3 μmol/L; imprecision (CV) was <5.5% within-run and 10–16% between-run. Storage of blood spots at ambient temperature for several days led to a significant decrease of ALA-D activity. Enzyme activity was lost in filter cards at 45 °C, but remained stable at 2–37 °C. Enzyme activity was decreased in EDTA blood. The absorbance at 550 nm was 0.221 (± 0.073) in healthy neonates and 0.043–0.100 in 11 patients with HT. All neonates with increased tyrosine (above the 99.5th centile) in neonatal screening (97 of 47 000) had normal results by the new assay.

Conclusions: The spectrophotometric microassay for ALA-D is a simple and sensitive test for HT. This represents a basis for further examination of its general reliability as a confirmatory test if tyrosine is found to be increased.
Interlaboratory Variation of Biochemical Markers of Bone Turnover
Authors: Seibel Markus J , Lang Matthias , Geilenkeuser Wolf-Jochen
Published: January 2020

Background: Biochemical markers of bone metabolism are used to assess skeletal turnover, but the variability of marker assays is still an issue of practical concern. We describe the results of an international proficiency testing program for biochemical bone markers among clinical laboratories.

Methods: Two serum and two urine pools (normal and increased marker concentrations) were sent on dry ice to 79 laboratories for analysis within 2 weeks of receipt.

Results: Data were submitted by 73 laboratories. The within-method interlaboratory CVs (CVILs) were as follows: serum bone-specific alkaline phosphatase (n = 47 laboratories), 16–48%; serum osteocalcin (n = 31), 16–42%; urinary free deoxypyridinoline (n = 30), 6.4–12%; urinary total deoxypyridinoline and pyridinoline (n = 29), 27–28%; urinary N-terminal cross-linked telopeptide of type I collagen (n = 10), 39%; serum C-terminal cross-linked telopeptide of type I collagen (ICTP; n = 8), 22–27%; urinary hydroxyproline (n = 13), 12%. Analytical results showed both systematic and nonsystematic deviations. In identical samples, results obtained for the same marker by the same method differed up to 7.3-fold. In urine-based assays, correction for urinary creatinine slightly increased CVs.

Conclusion: Even with identical assays and methods, results for most biochemical markers of bone turnover differ markedly among laboratories.
Successful Testing Protocols in Virology
Authors: Kelley Violet A , Caliendo Angela M
Published: January 2020

Molecular methods have had a significant impact on the diagnosis of viral infections because of their superior sensitivity and rapid turnaround time compared with conventional diagnostic methods. These characteristics have allowed molecular tests to play a central role in the use of testing protocols for managing viral infections. Several examples of such protocols are reviewed in this report, including the use of molecular testing for early disease detection to improve overall disease management and to direct antiviral therapy.