bluuebunny/crossref_metadata_2025 huggingface dataset:
Adulteration of Urine by “Urine Luck”
Authors: Wu Alan HB , Bristol Ben , Sexton Karen , Cassella-McLane Gina , Holtman Verena , Hill Dennis W
Published: January 2020

Background: In vitro adulterants are used to invalidate assays for urine drugs of abuse. The present study examined the effect of pyridinium chlorochromate (PCC) found in the product “Urine Luck”.

Methods: PCC was prepared and added to positive urine controls at concentrations of 0, 10, 50, and 100 g/L. The controls were assayed for methamphetamine, benzoylecgonine (BE), codeine and morphine, tetrahydrocannabinol (THC), and phencyclidine (PCP) with the Emit II (Syva) and Abuscreen Online (Roche) immunoassays, and by gas chromatography/mass spectrometry (GC/MS). Two tests were also developed to detect PCC in urine: a spot test to detect chromate ions using 10 g/L 1,5-diphenylcarbazide as the indicator, and a GC/MS assay for pyridine. We tested 150 samples submitted for routine urinalysis, compliance, and workplace drug testing for PCC, using these assays.

Results: Response rates decreased at 100 g/L PCC for all Emit II drug assays and for the Abuscreen morphine and THC assays. In contrast, the Abuscreen amphetamine assay produced apparently higher results, and no effect was seen on the results for BE or PCP. The PCC did not affect the GC/MS recovery of methamphetamine, BE, PCP, or their deuterated internal standards, but decreased GC/MS recovery of the opiates at both intermediate (50 g/L) and high (100 g/L) PCC concentrations and apparent concentrations of THC and THC-d3 at all PCC concentrations. Two of 50 samples submitted for workplace drug testing under chain-of-custody conditions were positive for PCC, whereas none of the remaining 100 specimens submitted for routine urinalysis or compliance drug testing were positive.

Conclusions: PCC is an effective adulterant for urine drug testing of THC and opiates. Identification of PCC use can be accomplished with use of a spot test for the oxidant.
National Academy of Clinical Biochemistry Standards of Laboratory Practice: Recommendations for the Use of Cardiac Markers in Coronary Artery Diseases
Authors: Wu Alan HB , Apple Fred S , Gibler W Brian , Jesse Robert L , Warshaw Myron M , Valdes Roland
Published: January 2020

The Sixth Conference on the “Standards of Laboratory Practice Series”, sponsored by the National Academy of Clinical Biochemistry (NACB), was held on August 4–5, 1998, at the Annual Meeting of the American Association for Clinical Chemistry, in Chicago, IL. An expert committee was assembled to write recommendations on the use of cardiac markers in coronary artery diseases. The NACB Committee prepared a preliminary draft of the guidelines, made them available on the World Wide Web (www.nacb.org), and distributed them before the presentations. The recommendations were divided into four areas: the use of markers in the triage of patients with chest pain, acute coronary syndromes, clinical applications other than acute myocardial infarction and research, and assay platforms and markers of acute myocardial infarction. The recommendations were revised and subsequently re-presented in part at the “Biomarkers in Acute Cardiac Syndromes Conference”, sponsored by the Jewish Hospital Heart and Lung Institute, Louisville KY, on October 16–17, 1998. This report lists each recommendation, its scientific justification, and a summary of discussions from conference participants and reviewers.

Approximately 100 individuals responded to various versions of these recommendations via direct correspondences, telephone calls to Committee members, electronic mail correspondence to the Committee Chairman, or oral questions and comments raised during one of the two conference presentations. Some of the recommendations were changed to reflect the consensus opinion. In cases in which there was no consensus, the Committee included pertinent discussion without necessarily changing the original recommendations. At times, the Committee members felt that although a particular recommendation might not be the current standard of care today, they anticipate that it likely will be adopted in the near future.
Detection of Five Rare Cystic Fibrosis Mutations Peculiar to Southern Italy: Implications in Screening for the Disease and Phenotype Characterization for Patients with Homozygote Mutations
Authors: Castaldo Giuseppe , Fuccio Antonella , Cazeneuve Cécile , Picci Luigi , Salvatore Donatello , Raia Valeria , Scarpa Maurizio , Goossens Michel , Salvatore Francesco
Published: January 2020

Background: The search for the eight most frequent mutations (i.e., ΔF508, G542X, W1282X, N1303K, 1717-1G→A, R553X, 2183AA→G, and I148T) by allele-specific oligonucleotide dot-blot analysis revealed 78% of 396 cystic fibrosis alleles in Southern Italy. The observation of frequent haplotypes on the unidentified cystic fibrosis alleles suggested that a few mutations could account for a large number of unidentified alleles.

Methods: We screened most of the coding sequence of the cystic fibrosis transmembrane regulator gene by denaturing gradient gel electrophoresis to determine the spectrum of these mutations in 68 unrelated cystic fibrosis patients bearing one or both unidentified mutations.

Results: The screening revealed five mutations, R1158X, 711+1G→T, 4016insT, L1065P, and G1244E, each of which had a frequency of 1.3–1.8% (7% collectively). The 7% increase in the detection rate (85% vs 78%) reduces by >50% the residual risk of being cystic fibrosis carriers for couples who had tested negative by molecular analysis. We therefore designed a second allele-specific oligonucleotide set to analyze the five mutations. Among the patients analyzed, one patient homozygous for the L1065P mutation expressed a mild pulmonary and intestinal form of the disease with pancreatic insufficiency. Two other patients, homozygous for mutations R1158X and 4016insT, both expressed a severe cystic fibrosis phenotype.

Conclusions: Five cystic fibrosis mutations are peculiar to patients from Southern Italy. The method described for their analysis is efficient, inexpensive, and can be semi-automated by use of a robotic workstation. The results obtained in patients from Southern Italy may have an impact on laboratories in other countries, given the large migrations of populations from Southern Italy to other countries in the last two centuries.
BRCA1 Gene Mutations in Sporadic Ovarian Carcinomas: Detection by PCR and Reverse Allele-specific Oligonucleotide Hybridization
Authors: Tong Dan , Stimpfl Margit , Reinthaller Alexander , Vavra Norbert , Müllauer-Ertl Silvia , Leodolter Sepp , Zeillinger Robert
Published: January 2020

Background: Although germline mutations in BRCA1 play a central role in familial breast and ovarian cancers, to date, no somatic mutations in BRCA1 have been reported in sporadic breast cancer, and only five somatic mutations have been identified in the sporadic ovarian carcinomas. Because loss of heterozygosity appears frequently at the BRCA1 locus in nonfamilial breast and ovarian carcinomas, we searched for mutations in the BRCA1 gene in sporadic ovarian tumors.

Methods: We developed a detection system based on PCR and reverse allele-specific oligonucleotide hybridization on membrane strips for the simultaneous detection of 17 frequently occurring mutations in the BRCA1 gene.

Results: As little as 2% mutant DNA in a sample could be detected. Two of 122 DNA samples isolated from sporadic ovarian tumor biopsies contained the Cys61Gly mutation. Both mutations were germline mutations. One of these was an ovarian metastasis of a primary fallopian tube carcinoma. The tubal carcinoma was also confirmed to contain the Cys61Gly mutation.

Conclusions: This is the first report that a germline BRCA1 mutation is associated with primary tubal carcinoma. The 17 specific mutations in the BRCA1 gene do not play a major role in the tumorigenesis and progression of sporadic ovarian cancer.
Optimal Temperature Selection for Mutation Detection by Denaturing HPLC and Comparison to Single-Stranded Conformation Polymorphism and Heteroduplex Analysis
Authors: Jones Alistair C , Austin Jehannine , Hansen Nancy , Hoogendoorn Bastiaan , Oefner Peter J , Cheadle Jeremy P , O’Donovan Michael C
Published: January 2020

Background: Denaturing HPLC (DHPLC) is a semi-automated method for detecting unknown DNA sequence variants. The sensitivity of the method is dependent on the temperature at which the analysis is undertaken, the selection of which is dependent on operator experience. To circumvent this, software has been developed for predicting the optimal temperature for DHPLC analysis. We examined the utility of this software.

Methods: To maximize the relevance of our data for other investigators, we have screened 42 different amplimers from CFTR, TSC1, and TSC2. The samples consisted of 103 unique sequence heterozygotes and 126 wild-type homozygous controls.

Results: At the temperature recommended by the software, 96% (99 of 103) of heterozygotes and all of the wild-type controls were correctly classified. This compares favorably with sensitivities of 85% for single-stranded conformation polymorphism and 82% for gel-based heteroduplex analyses of the same fragments.

Conclusions: Software-optimized DHPLC is a highly sensitive method for mutation detection. However, where sensitivity >96% is required, our data suggest that in addition to the recommended temperature, fragments should also be run at the recommended temperature plus 2 °C.
Real-Time Reverse Transcription-PCR Assay for Future Management of ERBB2-based Clinical Applications
Authors: Bièche Ivan , Onody Peter , Laurendeau Ingrid , Olivi Martine , Vidaud Dominique , Lidereau Rosette , Vidaud Michel
Published: January 2020

Background: Gene amplification/overexpression of ERBB2 (HER2, neu) is a major event in human breast tumorigenesis. ERBB2-based therapeutic agents and ERBB2-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen breast cancer patients for ERBB2 alterations.

Methods: We have developed and validated a real-time quantitative reverse transcription (RT)-PCR assay, based on fluorescent TaqMan methodology, to quantify ERBB2 gene expression at the mRNA level in breast tumors. This recently developed method of nucleic acid quantification in homogeneous solutions has the potential for a wide dynamic range, interlaboratory agreement, and high-throughput capacity without tedious post-PCR processing. The ERBB2 mRNA signal was normalized to the signal for TATA box-binding protein mRNA.

Results: The dynamic range was >1000-fold. The relationship between Ct and log starting concentration was linear (r2 ≥0.99). The mean (SD) normalized expression of ERBB2 in healthy breast tissue was 0.95 (0.37). Overexpression (>5 SD above mean for healthy breast) of the ERBB2 gene was observed (at 3.2- to 135-fold) in 23 (17%) of 134 breast tumor RNA samples. As expected, ERBB2 overexpression was present in all tumors with ERBB2 gene amplification but was uncommon and at a low ratio (<5) in breast cancers without gene amplification.

Conclusions: This new simple, rapid, semi-automated assay is a major alternative to fluorescence in situ hybridization and immunochemistry for gene alteration analysis in human tumors and may be a powerful tool for large randomized, prospective cooperative group trials and to support future ERBB2-based biological and gene therapy approaches.
Detection of Mitochondrial DNA Mutations by Temporal Temperature Gradient Gel Electrophoresis
Authors: Chen Tian-Jian , Boles Richard G , Wong Lee-Jun C
Published: January 2020

Background: A unique requirement for the molecular diagnosis of mitochondrial DNA (mtDNA) disorders is the ability to detect heteroplasmic mtDNA mutations and to distinguish them from homoplasmic sequence variations before further testing (e.g., sequencing) is performed. We evaluated the potential utility of temporal temperature gradient gel electrophoresis (TTGE) for these purposes in patients with suspected mtDNA mutations.

Methods: DNA samples were selected from patients with known mtDNA mutations and patients suspected of mtDNA disorders without detectable mutations by routine analysis. Six regions of mtDNA were PCR amplified and analyzed by TTGE. Electrophoresis was carried out at 145 V with a constant temperature increment of 1.2 °C/h. Mutations were identified by direct sequencing of the PCR products and confirmed by PCR/allele-specific oligonucleotide or PCR/restriction fragment length polymorphism analysis.

Results: In the experiments using patient samples containing various amounts of mutant mtDNA, TTGE detected as little as 4% mutant heteroplasmy and identified heteroplasmy in the presence of a homoplasmic polymorphism. In 109 specimens with 15 different known mutations, TTGE detected the presence of all mutations and distinguished heteroplasmic mutations from homoplasmic polymorphisms. When 11% of the mtDNA genome was analyzed by TTGE in 104 patients with clinically suspected mitochondrial disorders, 7 cases of heteroplasmy (≈7%) were detected.

Conclusions: TTGE distinguishes heteroplasmic mutation from homoplasmic polymorphisms and appears to be a sensitive tool for detection of sequence variations and heteroplasmy in patients suspected of having mtDNA disorders.
The Influence of an Expert System for Test Ordering and Interpretation on Laboratory Investigations
Authors: Smith Beverly J , McNeely Michael DD
Published: January 2020

Background: The Laboratory Advisory System (LAS) is an expert system interface that works interactively with clinicians to assist them with test selection and result interpretation throughout the laboratory investigation of a patient.

Methods: To study the influence of the LAS on laboratory investigations, a repeated-measures experiment using clinical vignettes was conducted. To collect baseline data on how laboratory investigations are currently conducted, clinicians investigated one-half of the vignettes using a conventional (noncomputer) approach. To determine the influence of the LAS on clinicians’ behavior, the other half of the vignettes were investigated using the LAS.

Results: Clinicians using the LAS (compared with conventional practice) ordered fewer laboratory tests during the diagnostic process (mean, 17.8 vs 32.7), completed the diagnostic workup with fewer sample collections (mean, 5.8 vs 7.5), generated lower laboratory costs (mean, $194 vs $232), shortened the time required to reach a diagnosis (mean, 1 day vs 3.2 days), showed closer adherence to established clinical practice guidelines, and exhibited a more uniform and diagnostically successful investigation.

Conclusion: The LAS enhances the outcome of the investigation and improves laboratory utilization.
Estrogen Replacement Therapy, Serum Lipids, and Polymorphism of the Apolipoprotein E Gene
Authors: Garry Philip J , Baumgartner Richard N , Brodie Steven G , Montoya George D , Liang Hwa Chi , Lindeman Robert D , Williams Thomas M
Published: January 2020

Background: Pharmacogenomics, the study of genetic loci that modulate drug responsiveness, may help to explain why estrogen replacement therapy (ERT) has differential effects on serum lipid and lipoprotein concentrations in postmenopausal women who inherit distinct alleles of the apolipoprotein E gene (APOE).

Methods: We compared total-cholesterol, triglyceride, and lipoprotein (LDL and HDL) concentrations in 66 postmenopausal women receiving ERT ([+]ERT) with 174 postmenopausal women not receiving ERT ([−]ERT), controlling for three APOE genotypes divided into three groups: E2 (ε2/ε3, n = 31), E3 (ε3/ε3, n = 160), and E4 (ε3/ε4 + ε4/ε4, n = 49).

Results: Mean total-cholesterol concentrations were lower in all three [+]ERT groups compared with their [−]ERT counterparts but were statistically significant only for women in group E4 (P = 0.014). The mean LDL-cholesterol concentrations were significantly lower in all three [+]ERT groups compared with their [−]ERT counterparts (P ≤0.005). Although all three groups of [+]ERT women tended to have higher mean HDL-cholesterol concentrations compared with their [−]ERT counterparts, the differences were not statistically significant. [+]ERT women in groups E2 and E3 had significantly higher (P <0.05) triglyceride concentrations than their [−]ERT counterparts. In [+]ERT women, the ratios of total and LDL-cholesterol to HDL-cholesterol were significantly higher in group E3 and E4 women compared with E2 women (P <0.006). Group E4 [+]ERT women had ratios of total and LDL-cholesterol to HDL-cholesterol that were comparable to group E2 [−]ERT women.

Conclusions: Triglyceride concentrations in group E2 [+]ERT women may need to be monitored more closely than those in E3 or E4 [+]ERT women. Group E4 women should probably be targeted for ERT. Results suggest that APOE genotypes have a differential effect on serum lipids and lipoproteins in [+]ERT postmenopausal women.
Differential Patterns of Bone Turnover in Relation to Bone Pain and Disease Extent in Bone in Cancer Patients with Skeletal Metastases
Authors: Berruti Alfredo , Dogliotti Luigi , Gorzegno Gabriella , Torta Mirella , Tampellini Marco , Tucci Marcello , Cerutti Stefania , Mosca Frezet Massimo , Stivanello Michela , Sacchetto Giovanna , Angeli Alberto
Published: January 2020

Background: The alteration of the bone microenvironment as a consequence of skeletal metastases is poorly understood. The aim of this study was to search for patterns of bone markers in relation to primary tumor type, bone pain, and number of sites involved in patients with bone metastases.

Methods: We studied 323 patients with bone metastases from various primary malignancies. We sequentially measured the serum concentrations of bone alkaline phosphatase [by an electrophoretic technique (BALP)], carboxy-terminal telopeptide of type I collagen (ICTP), calcium (CaS), intact parathyroid hormone (PTH), and the fasting urinary excretion of calcium (Ca:Cr). Immunoradiometric serum bone alkaline phosphatase (I-BALP) and urinary excretion of deoxypyridinoline (DPYD) were also assessed in the 175 cases. Data were analyzed as a function of bone pain (assessed by a validated pain questionnaire), the number of radiographically confirmed sites of bone involvement, and the most frequent primary tumor types: breast cancer (BC; 124 patients), prostate cancer (PC; 90 patients), and non-small cell lung cancer (LC; 49 patients).

Results: Serum BALP and I-BALP correlated with the number of radiologically identified blastic bone lesions. BALP and I-BALP were more frequently increased in PC (72% for both measurements) than in BC (50% and 60%, respectively) or LC (3% and 5%, respectively; P <0.001 for BALP and P = 0.001 for I-BALP). ICTP and DPYD values did not differ among PC, BC, and LC, but they did show a direct relationship with the disease extent in bone (P <0.001). CaS and Ca:Cr did not vary significantly according to the bone tumor burden. Bone pain directly correlated with ICTP (P <0.001), DPYD (P = 0.002), CaS (P <0.002), and Ca:Cr (P = 0.001), whereas the relationship was inverse for serum PTH (P = 0.002). When patients were stratified according to the primary tumor, ICTP correlated with the bone pain in all subsets (P <0.005, <0.005, and <0.001 for BC, PC, and LC, respectively), as did CaS and Ca:Cr in LC patients (P = 0.01 and 0.02, respectively) but not in PC and BC patients.

Conclusions: The patterns of bone turnover markers differ among the primary tumor types. Both resorption and formation markers reflect the number of radiographically identified sites of bone metastases, whereas resorption markers and serum calcium but not formation markers correlate with bone pain.
Structural Investigations of a New Familial Dysalbuminemic Hyperthyroxinemia Genotype
Authors: Petersen Charles E , Ha Chung-Eun , Harohalli Krishna , Park David S , Feix Jimmy B , Isozaki Osamu , Bhagavan Nadhipuram V
Published: January 2020

Background: In a previous study, we found that the amino acid substitution R218H in human serum albumin (HSA) was the cause of familial dysalbuminemic hyperthyroxinemia (FDH) in several Caucasian patients. Subsequently the substitution R218P was shown to be the cause of FDH in several members of a Japanese family. This study attempts to resolve discrepancies in the only other study of R218P HSA and identifies two new Japanese R218P FDH patients unrelated to those described previously.

Methods and Results: Recombinant R218H, R218P, and wild-type HSA were synthesized in yeast, and the affinities of these HSA species for l- and d-thyroxine were determined using fluorescence spectroscopy. The dissociation constants for the binding of wild-type, R218P, and R218H HSA to l-thyroxine were 1.44 × 10−6, 2.64 × 10−7, and 2.49 × 10−7 mol/L, respectively. The circular dichroism spectra of thyroxine bound to R218H and R218P HSA were markedly different, indicating that the structure of the thyroxine/HSA complex is different for either protein.

Conclusions: The Kd values for l-thyroxine bound to R218P and R218H HSA determined in this study were similar. The extremely high serum total-thyroxine concentrations reported previously for R218P FDH patients (10-fold higher than those reported for R218H FDH patients) are not consistent with the Kd values determined in this study. Possible explanations for these discrepancies are discussed.
Type 1 Diabetes
Authors: Lernmark Åke
Published: January 2020

Type 1 (insulin-dependent) diabetes occurs worldwide and can appear at any age. The genetic susceptibility is strongly associated with HLA-DQ and DR on chromosome 6, but genetic factors on other chromosomes such as the insulin gene on chromosome 11 and the cytotoxic T-lymphocyte antigen gene on chromosome 2 may modulate disease risk. Numerous studies further support the view that environmental factors are important. Gestational infections may contribute to initiation, whereas later infections may accelerate islet β-cell autoimmunity. The pathogenesis is strongly related to autoimmunity against the islet β cells. Markers of autoimmunity include autoantibodies against glutamic acid decarboxylase, insulin, and islet cell antigen-2, a tyrosine phosphatase-like protein. Molecular techniques are used to establish reproducible and precise autoantibody assays, which have been subject to worldwide standardization. The diagnostic sensitivity (40–80%) and specificity (99%) of all three autoantibodies for type 1 diabetes are high, and double or triple positivity among first-degree relatives predicts disease. Combined genetic and antibody testing improved prediction in the general population despite the transient nature of these autoantibodies. Classification of diabetes has also been improved by autoantibody testing and may be used in type 2 diabetes to predict secondary failure and insulin requirement. Islet autoantibodies do not seem to be related to late complications but rather to metabolic control, perhaps because the presence of islet cell autoantibodies marks different residual β-cell function. Combined genetic and autoantibody screening permit rational approaches to identify subjects for secondary and tertiary intervention trials.
Microheterogeneity of Serum Glycoproteins in Patients with Chronic Alcohol Abuse Compared with Carbohydrate-deficient Glycoprotein Syndrome Type I
Authors: Henry Hugues , Froehlich Florian , Perret Renaud , Tissot Jean-Daniel , Eilers-Messerli Barbara , Lavanchy Daniel , Dionisi-Vici Carlo , Gonvers Jean-Jacques , Bachmann Claude
Published: January 2020

Background: Chronic alcohol abuse alters the normal N-glycosylation of transferrin, producing the carbohydrate-deficient transferrin isoforms. This alteration could be similar to that present in patients with carbohydrate-deficient glycoprotein syndrome type 1 (CDG1). We thus compared the alterations of N-glycans present in patients with alcoholism and patients with CDG1.

Methods: The N-glycans of serum glycoproteins were compared in sera of patients with alcoholism, patients with CDG1, and controls by two-dimensional electrophoresis, neuraminidase, peptide:N-glycosidase F, and endoglycosidase F2 treatments. A specific antibody directed against the amino acid sequence surrounding the N-432 N-glycosylation site of transferrin was prepared (SZ-350 antibody).

Results: In patients with alcoholism, the abnormal transferrin and α1-antitrypsin isoforms were devoid of a variable number of entire N-glycan moieties and were identical with those present in CDG1. In the serum of patients with alcoholism, this finding was less pronounced than in CDG1. In contrast to CDG1, there was no decrease in clusterin or serum amyloid P in patients with alcoholism. The SZ-350 antibody recognized only transferrin isoforms with one or no N-glycan moieties.

Conclusion: Antibodies directed against specific N-glycosylation sites of glycoproteins could be useful for developing more specific immunochemical tests for the diagnosis of chronic alcohol abuse.
Standardization of Creatine Kinase-MB (CK-MB) Mass Assays: The Use of Recombinant CK-MB as a Reference Material
Authors: Christenson Robert H , Vaidya Hemant , Landt Yvonne , Bauer Roger S , Green Sol F , Apple Fred A , Jacob Ann , Magneson Gerald R , Nag Sumitra , Wu Alan HB , Azzazy Hassan ME
Published: January 2020

Background: The AACC assembled a committee to identify and validate a standard creatine kinase MB isoenzyme (CK-MB) material to improve the comparability of CK-MB mass assays.

Methods: Three protocols were used. In protocol I, various CK-MB materials prepared in different matrices were screened as candidate standards. In protocol II, participating manufacturers calibrated their systems with concentrates of human heart CK-MB and then tested 20 patient samples to evaluate calibration bias. In protocol III, participating manufacturers calibrated their immunoassay systems using recombinant CK-MB2 (rCK-MB2) diluted into their respective sample diluents and measured 50 samples.

Results: Candidate materials showed high recovery in stripped human serum, but bias improved only from 59% to 38%. These data led to the use of human heart CK-MB diluted in each manufacturer’s sample diluent. This strategy reduced bias from 31% to 15%. Because human heart CK-MB is difficult to provide, a lyophilized source of CK-MB2 was identified. rCK-MB2 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase HPLC, intrinsic protein fluorescence, circular dichroism, agarose gel electrophoresis, immunoreactivity studies, high and low temperature stability, and reconstituted stability to be equivalent to human heart CK-MB. Calibration of immunoassay systems with rCK-MB2 added into each respective manufacturer’s sample diluent showed a 13% between-manufacturer bias.

Conclusion: Lyophilized rCK-MB2 was determined suitable for use as a reference material for CK-MB mass assays.
Method for the Determination of Total Homocysteine in Plasma and Urine by Stable Isotope Dilution and Electrospray Tandem Mass Spectrometry
Authors: Magera Mark J , Lacey Jean M , Casetta Bruno , Rinaldo Piero
Published: January 2020

Background: Total homocysteine (tHcy) has emerged as an important independent risk factor for cardiovascular disease. Analytical methods are needed to accommodate the high testing volumes for tHcy and provide rapid turnaround.

Methods: We developed liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) method based on the analysis of 100 μL of either plasma or urine with homocystine-d8 (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the multiple reaction monitoring mode in which tHcy and Hcy-d4 were detected through the transition from the precursor to the product ion (m/z 136 to m/z 90 and m/z 140 to m/z 94, respectively). The retention time of tHcy and Hcy-d4 was 1.5 min in a 2.5-min analysis.

Results: Daily calibrations between 2.5 and 60 μmol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 μmol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for the comparison between our previous HPLC method (y) and the LC-MS/MS method (x) was y = 1.097x − 1.377 (r = 0.975; Sy|x =1.595 μmol/L; n = 367), and for comparison between a fluorescence polarization immunoassay (Abbott IMx; y) and LC-MS/MS (x) was y = 1.039x + 0.025 (r = 0.969; Sy|x =1.146 μmol/L; n = 367). Inter- and intraassay CVs were 2.9–5.9% and 3.6–5.3%, respectively, at mean concentrations of 3.9, 22.7, and 52.8 μmol/L. Mean recovery of tHcy was 94.2% (20 μmol/L) and 97.8% (50 μmol/L).

Conclusions: The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy. Reagents are inexpensive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation.
Evaluation of a Particle-enhanced Turbidimetric Immunoassay for the Measurement of Immunoglobulin E in an ILab 900 Analyzer
Authors: Guaita Sandra , Simó Josep MA , Ferré Natàlia , Joven Jorge , Camps Jordi
Published: January 2020

Background: The measurement of immunoglobulin E (IgE) in serum is widely used in the diagnosis of allergic reactions and parasitic infections. We describe here a fully automated assay for human IgE suitable for routine application in a general chemistry analyzer.

Methods: We used an ILab 900® analyzer. This instrument automates a particle-enhanced immunoturbidimetric assay with an analysis time of 9 min.

Results: The assay was linear in the range 4–1000 kIU/L (r = 0.9998). The intra- and interassay CVs at 57, 235, and 434 kIU/L were <3.5% and <7.4%, respectively. The detection limit was 4 kIU/L. Hemoglobin (≤16 g/L), bilirubin (≤250 μmol/L), and myeloma paraproteins did not interfere with the assay. The assay showed good correlation with a microparticle enzyme immunoassay (r = 0.998) with a mean difference between methods of −6 ± 26 kIU/L.

Conclusion: The new automated serum assay for IgE is an attractive alternative that avoids the need for dedicated instrumentation.
Evaluation of Measurement Sites for Noninvasive Blood Glucose Sensing with Near-Infrared Transmission Spectroscopy
Authors: Burmeister Jason J , Arnold Mark A
Published: January 2020

Six putative measurement sites were evaluated for noninvasive sensing of blood glucose by first-overtone near-infrared spectroscopy. The cheek, lower lip, upper lip, nasal septum, tongue, and webbing tissue between the thumb and forefinger were examined. These sites were evaluated on the basis of their chemical and physical properties as they pertain to the noninvasive measurement of glucose. Critical features included the effective optical pathlength of aqueous material within the tissue and the percentage of body fat within the optical path. Aqueous optical paths of 5 mm are required to measure clinically relevant concentrations of glucose in the first-overtone region. All of the tested sites met this requirement. The percentage of body fat affects the signal-to-noise ratio of the measurement and must be minimized for reliable glucose sensing. The webbing tissue contains a considerable amount of fat tissue and is clearly the worse measurement site. All other sites possess substantially less fat, with the least amount of fat in tongue tissue. For this reason, the tongue provides spectra with the highest signal-to-noise ratio and is, therefore, the site of choice on the basis of spectral quality.
Thin Film Biosensor for Rapid Visual Detection of Nucleic Acid Targets
Authors: Ostroff Rachel M , Hopkins Deborah , Haeberli Ayla B , Baouchi Wahab , Polisky Barry
Published: January 2020

Background: We have developed a silicon-based biosensor that generates a visual signal in response to nucleic acid targets.

Methods: In this system, capture oligonucleotide probes are immobilized on the surface of the biosensor. Interaction of the capture probes with a complementary target and a biotinylated detector oligonucleotide allows initiation of formation of an organic thin film on the biosensor. Thin film formation is completed by enzymatic activity of peroxidase conjugated to an anti-biotin antibody. Peroxidase catalyzes deposition of an insoluble product onto the silicon surface, generating a uniform thin film. The increased thickness on the surface alters the perceived color of the biosensor through changes in the interference patterns of reflected light from the surface, causing a color change from gold to purple.

Results: The biosensor results may be evaluated by direct visual inspection or quantified by ellipsometry. Results are obtained in 25 min with a detection limit of 5 pmol/L (150 amol/sample). Selectivity of the biosensor is demonstrated by discrimination of single nucleotide mismatches. Multitarget arrays are also analyzed with the thin film biosensor, and the system is capable of detecting targets from human serum and urine.

Conclusions: The biosensor surface is inexpensive to produce, and the assay format is simple and rapid. The thin film biosensor is adaptable to a wide variety of nucleic acid detection applications, including rapid diagnostic testing for infectious disease panels, antibiotic resistance panels, or allelic discrimination of specific genetic markers.
Molecular Forms of Prostate-specific Antigen in Malignant and Benign Prostatic Tissue: Biochemical and Diagnostic Implications
Authors: Jung Klaus , Brux Brigitte , Lein Michael , Rudolph Birgit , Kristiansen Glen , Hauptmann Steffen , Schnorr Dietmar , Loening Stefan A , Sinha Pranav
Published: January 2020

Background: Patients with prostate cancer (PCa) show a lower ratio of free prostate-specific antigen (fPSA) to total PSA (tPSA) in serum than patients with benign prostatic hyperplasia (BPH). The patterns of the intracellular PSA isoforms in malignant and benign prostatic tissue have been studied as potential molecular reasons for this phenomenon.

Methods: Prostatic tissue samples were obtained after cystoprostatectomy from patients with bladder cancer (n = 10), from BPH patients (transurethral resection of the prostate, n = 10; adenomectomy, n = 10), and from the cancerous and noncancerous parts of the same prostates removed surgically by prostatectomy because of PCa (n = 20). PSA pattern was characterized by gel filtration, immunoblotting, and immunoassays for tPSA, fPSA, α1-antichymotrypsin-PSA (ACT-PSA), and complexed PSA (Bayer Immuno 1 assay). Comparisons were made with the PSA concentrations in serum.

Results: The major portion of tPSA in all tissue samples was fPSA; complexed PSA forms were <2%. Samples from cystoprostatectomy patients had the lowest and those from adenomectomy patients the highest values of tPSA and fPSA. PSA concentrations were lower in cancerous than in the noncancerous parts of the prostate. No significant correlations were found between tumor stage or grade and the amounts of tPSA, fPSA, and ACT-PSA in tissue. Tissue PSA values were not correlated with the serum PSA concentrations nor with the ratios fPSA/tPSA and ACT-PSA/tPSA in sera.

Conclusions: The amounts of tPSA and the PSA isoforms in prostatic tissue explain neither the concentrations of tPSA and PSA isoforms in serum nor the behavior of the ratio fPSA/tPSA in patients with BPH and PCa.
Ratio of Free or Complexed Prostate-specific Antigen (PSA) to Total PSA: Which Ratio Improves Differentiation between Benign Prostatic Hyperplasia and Prostate Cancer?
Authors: Jung Klaus , Elgeti Ulrike , Lein Michael , Brux Brigitte , Sinha Pranav , Rudolph Birgit , Hauptmann Steffen , Schnorr Dietmar , Loening Stefan A
Published: January 2020

Background: The aim of this study was to compare the diagnostic utility of a new assay that measures all forms of prostate-specific antigen complexed (cPSA) to serum proteins except α2-macroglobulin with the assay of free PSA (fPSA) and the corresponding ratios to total PSA (tPSA) to improve the differentiation between benign prostatic hyperplasia (BPH) and prostate cancer (PCa).

Methods: Serum samples were collected from 91 men without prostate disease and with normal digital rectal examination (controls), 144 untreated patients with PCa, and 89 patients with BPH. tPSA and cPSA were measured using the Bayer Immuno 1 system; fPSA and the additional tPSA were measured with the Roche Elecsys system.

Results: The median cPSA/tPSA, fPSA/tPSA, and fPSA/cPSA ratios were significantly different between patients with BPH and PCa (78.7% vs 90.7%, 25.5% vs 12.1%, and 36.8% vs 14.3%, respectively; P <0.001). No correlations of cPSA and its ratios to tumor stage and grade were found. ROC analysis showed that cPSA was not different from tPSA (areas under the curve, 0.632 vs 0.568), whereas the cPSA/tPSA ratio was similar to the fPSA/tPSA ratio in increasing discrimination between BPH and PCa patients with tPSA concentrations in the tPSA gray zone between 2 and 10 μg/L (areas under the curve, 0.851 vs 0.838).

Conclusions: Compared with tPSA, the fPSA/tPSA and cPSA/tPSA ratios both improve the differentiation between BPH and PCa comparably and are similarly effective in reducing the rate of unnecessary biopsies, whereas cPSA alone does not have any effect.
Assessment of Between-Instrument Variations in a HPLC Method for Serum Lipoproteins and Its Traceability to Reference Methods for Total Cholesterol and HDL-Cholesterol
Authors: Usui Shinichi , Nakamura Masakazu , Jitsukata Kazuhiro , Nara Masayuki , Hosaki Seijin , Okazaki Mitsuyo
Published: January 2020

Background: The main purpose of this study was to evaluate the between-instrument variation of the HPLC method for the measurement of total cholesterol (TC), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), VLDL-cholesterol (VLDL-C), chylomicron cholesterol (CM-C), LDL size, and HDL size. Furthermore, the accuracy of the HPLC was assessed for the determination of TC and HDL-C, compared with CDC reference methods.

Methods: We used four HPLC instruments with different column-load numbers from 250 to 5000. For accuracy assessment of TC and HDL-C, we used the reference methods recommended by the CDC.

Results: The values measured by the four instruments were highly correlated with each other (mean r = 0.965), and the absolute mean differences were 4–43 mg/L for TC, 4–30 mg/L for HDL-C, 0–48 mg/L for LDL-C, 7–66 mg/L for VLDL-C, 0–7 mg/L for CM-C, 0.1–0.3 nm for LDL size, and 0–0.1 nm for HDL size. For TC, the HPLC instruments showed high correlation and good agreement with the reference method: r = 0.997; total error <6.6%; absolute mean bias <1.2%. For HDL-C, the results from the HPLC method were significantly higher (10.8% absolute mean bias) than those of the CDC reference method, in spite of good correlation between the two methods (r = 0.998).

Conclusions: The between-instrument variation in serum lipoprotein analysis by HPLC was confirmed to be very small. This method met the US National Cholesterol Education Program’s performance criteria for TC but not for HDL-C.
Transferrin Polymorphism Influences Iron Status in Blacks
Authors: Kasvosve Ishmael , Delanghe Joris R , Gomo Zvenyika A R , Gangaidzo Innocent T , Khumalo Hlosukwazi , Wuyts Birgitte , Mvundura Elisha , Saungweme Thokozile , Moyo Victor M , Boelaert Johan R , Gordeuk Victor R
Published: January 2020

Background: Genetic variants of human transferrin (TF) have been described, but little is known about their functional differences. We studied iron status according to TF phenotype in a healthy Zimbabwean population and in subjects at risk of African iron overload.

Methods: The study population consisted of 483 nondrinkers, 31 drinking spouse pairs, and 5 family pedigrees (n = 88) with index cases of iron overload. TF phenotypes were determined using starch gel electrophoresis. To evaluate iron status, serum iron, total iron-binding capacity (TIBC), ferritin, and soluble TF receptors were measured, and the percentage of saturation and the serum iron:TF ratio were calculated. The binding of the TF variants was studied by equilibrium dialysis.

Results: The reference population was characterized by a high TF D allele frequency (0.050) and a complete absence of homozygous TF DD individuals. Similar allele frequencies were observed in subjects at risk of African iron overload. In the reference population, male TF CD heterozygotes had significantly lower (P <0.01) values for serum iron, TIBC, TF saturation, and serum iron:TF ratio than the TF CC homozygotes; in females, only TIBC was significantly different. Overall red blood cell indices did not differ according to TF phenotype. In the population at risk of African iron overload, only serum iron:TF ratio was consistently significantly lower in TF CD phenotypes (P <0.05). After equilibrium dialysis, the amount of iron bound by TF was significantly lower (P <0.01) in TF CD individuals.

Conclusions: The present data demonstrate a functional difference between TF phenotypes in blacks.
Rapid Single-Tube Screening of the C282Y Hemochromatosis Mutation by Real-Time Multiplex Allele-specific PCR without Fluorescent Probes
Authors: Donohoe Gerard G , Laaksonen Maija , Pulkki Kari , Rönnemaa Tapani , Kairisto Veli
Published: January 2020

Background: An accurate determination of the major HFE mutation (C282Y), which is associated with hereditary hemochromatosis, is important in diagnosis and risk assessment for this disease. We report a single-tube high-throughput PCR method for the detection of C282Y.

Methods: We combined three previously described principles: allele-specific PCR, mutagenically separated PCR, and amplicon identification by specific dissociation curves. PCR amplification was performed with fluorescence detection or conventional thermocycler using the same primers, reactant constituents, and cycling protocol. Primer cross-reactions were prevented by deliberate primer:primer and primer:template mismatches.

Results: PCR products were identified by their characteristic melting temperatures based on SYBR Green I fluorescence. For each of the 256 random and 17 known HFE C282Y samples, mutant homozygous, wild-type, and heterozygous samples were unequivocally distinguished.

Conclusions: This homogeneous assay is rapid, reproducible, does not require fluorescent oligonucleotide probes, and correctly identifies HFE genotypes.
Multicenter Evaluation of Five Assays for Myoglobin Determination
Authors: Zaninotto Martina , Pagani Franca , Altinier Sara , Amboni Paolo , Bonora Roberto , Dolci Alberto , Pergolini Patrizia , Vernocchi Arialdo , Plebani Mario , Panteghini Mauro ,   for the Italian Society of Clinical Biochemistry and Clinical Molecular Biology–Italian Society of Laboratory Medicine Working Group on Markers of Myocardial Damage
Published: January 2020

Background: Lacking assay standardization, different myoglobin methods may produce results that differ significantly.

Methods: A multicenter study was carried out to compare the analytical performance of five commercially available assays for myoglobin measurement. Linearity, imprecision, interferences, and method comparison were studied according to NCCLS guidelines, whereas reference values were determined following IFCC recommendations.

Results: The BNA and Opus showed relatively high imprecision (all but one total CV >7.4%). Other assays showed lower CVs, but they varied among laboratories, particularly at a normal myoglobin concentration (Access, 6.0–11%; Hitachi, 3.8–5.8%; Stratus, 3.4–6.5%). Results were lower in anticoagulated samples on the Access, in heparin and citrate samples on the Stratus, and in citrate samples on the BNA and Opus, and increased in heparin and EDTA samples on the Hitachi. Use of separator gel produced results significantly lower (P <0.001) on the Hitachi and higher (P = 0.016) on the Opus. Bilirubin, turbidity, and hemoglobin had no effect on evaluated methods, but rheumatoid factor affected the Access. In method comparisons, high correlation coefficients (≥0.98) were obtained. The Stratus gave higher results; however, the Access and BNA gave the lowest. The following upper reference limits (μg/L) for men and women, respectively, were obtained: Access, 70 and 52; BNA, 51 and 49; Hitachi, 67 and 58; Opus, 80 and 50; and Stratus, 86 and 63.

Conclusion: The possibility of high imprecision and marked disagreement among commercial myoglobin assays should be carefully considered in clinical practice.
Determination of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma and Cerebrospinal Fluid by Stable-Isotope Dilution Tandem Mass Spectrometry
Authors: Struys Eduard A , Jansen Erwin E W , de Meer Kees , Jakobs Cornelis
Published: January 2020

Background: Available methods for the determination of nanomolar concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma and cerebrospinal fluid (CSF) are time-consuming. We wished to develop a method for their rapid and simultaneous measurement.

Methods: We used tandem mass spectrometry (MS/MS) for the simultaneous determination of SAM and SAH, with stable-isotope-labeled internal standards. The 13C5-SAH internal standard was enzymatically prepared using SAH-hydrolase and [13C5]adenosine. The method comprises a weak anion-exchange solid-phase extraction procedure serving as clean-up step for the deproteinized plasma and CSF samples. After clean-up, samples were injected on a C18 HPLC column, which was connected directly to the tandem mass spectrometer, operating in MS/MS mode.

Results: In plasma samples, the intraassay CVs for SAM and SAH were 4.2% and 4.0%, respectively, and the interassay CVs were 7.6% and 5.9%, respectively. In CSF, the intraassay CVs for SAM and SAH were 6.8% and 6.9%, respectively, and the interassay CVs were 4.2% and 5.5%, respectively. Mean recovery of SAM and SAH for both matrices at two concentrations was 93%. Detection limits for SAM and SAH in samples were 7.5 and 2.5 nmol/L, respectively. Concentrations of SAM and SAH in plasma from healthy subjects were within the previously reported ranges. In 10 CSF samples, the mean concentrations (range) were 248 (137–385) nmol/L for SAM and 11.3 (8.9–14.1) nmol/L for SAH.

Conclusions: SAM and SAH can be analyzed by MS/MS, taking optimal advantage of the speed and high sensitivity and specificity of this relatively new analytical technique.
Association of Serum Bilirubin Concentration with Risk of Coronary Artery Disease
Authors: Mayer Michael
Published: January 2020

Background: Lipid oxidation and formation of oxygen radicals are important elements of arterial plaque formation and atherosclerosis, and are involved in the pathophysiology of coronary artery disease (CAD). Because bilirubin has antioxidant properties, it has been suggested that it may have a protective role in the atherosclerotic process.

Approach: This review examines in vitro and in vivo studies indicating that bilirubin inhibits lipid oxidation and oxygen radical formation. Experimental and epidemiological evidence is presented that suggests that bilirubin may serve as a physiological antioxidant providing protection against atherosclerosis and CAD. Special attention is focused on studies that noted an inverse relationship between plasma bilirubin concentration and cardiovascular morbidity.

Content: Serum bilirubin concentrations in the upper portion of the reference interval reportedly reduce atherogenic risk and provide protection against CAD. In contrast, serum bilirubin concentrations in the lower portion of the reference interval may be associated with increased risk of ischemic heart disease.

Summary: Taken together, the evidence presented in this review supports the concept that bilirubin, via its antioxidant potential, has antiatherogenic properties and that an inverse relationship exists between circulating bilirubin concentrations and risk of CAD.