bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of DNA Target Melting Behavior on Real-Time PCR Quantification
Authors: Wilhelm Jochen , Hahn Meinhard , Pingoud Alfred
Published: January 2020

Background: Quantitative real-time PCR is increasingly used to quantify copy numbers of nucleic acids for clinical applications. We observed that the measurements of allele imbalances of the tumor suppressor gene p16 and the oncogene ErbB-2 yielded results with variable precision under certain experimental conditions.

Methods: We used the LightCyclerTM real-time PCR system to quantify different genomic target sequences using hybridization probes or SYBR Green for detection.

Results: With two primer/template systems (p16 and ErbB-2), we observed sinusoidal scattering of the threshold cycle values depending on the capillary position in the thermostated reaction chamber. This scattering depended on the denaturation temperature only when complete genomic DNA was used as template and did not occur when PCR product or restricted or boiled genomic DNA was used or the denaturation temperature in the first cycles was increased (and other targets, such as p53, HBB, IGF-1, GAPDH, and PBGD, did not show this behavior).

Conclusions: Before a primer system is used for precise quantitative real-time PCR, the dependence of the quantification results on the positions of reaction tubes in the thermocycler should be tested. Our data indicate that amplification efficiencies, especially in the first cycles, depend not only on the priming efficiencies of the primers and the melting temperature of the amplicon, but also on the melting behavior of the amplicon’s genomic vicinity. Complete denaturation of genomic DNA is necessary to maximize precision of quantitative PCR. Higher denaturation temperatures in the initial cycles or boiling of DNA before the PCR can improve the accuracy of quantification in some cases.
Evaluation of Indicators of Cobalamin Deficiency Defined as Cobalamin-induced Reduction in Increased Serum Methylmalonic Acid
Authors: Bolann Bjørn J , Solli Jan Dag , Schneede Jörn , Grøttum Kjell A , Loraas Arne , Stokkeland Morgan , Stallemo Asbjørn , Schjøth Arne , Bie Rolf Bruun , Refsum Helga , Ueland Per Magne
Published: January 2020

Background: Early detection of cobalamin deficiency is clinically important, and there is evidence that such deficiency occurs more frequently than previously anticipated. However, serum cobalamin and other commonly used tests have limited ability to diagnose a deficiency state.

Methods: We investigated the ability of hematological variables, serum cobalamin, plasma total homocysteine (tHcy), serum and erythrocyte folate, gastroscopy, age, and gender to predict cobalamin deficiency. Patients (n = 196; age range, 17–87 years) who had been referred from general practice for determination of serum cobalamin were studied. Cobalamin deficiency was defined as serum methylmalonic acid (MMA) >0.26 μmol/L with at least 50% reduction after cobalamin supplementation. ROC and logistic regression analyses were used.

Results: Serum cobalamin and tHcy were the best predictors, with areas under the ROC curve (SE) of 0.810 (0.034) and 0.768 (0.037), respectively, but age, intrinsic factor antibodies, and gastroscopy gave additional information.

Conclusions: When cobalamin deficiency is suspected in general practice, serum cobalamin should be the first diagnostic test, and the result should be interpreted in relation to the age of the patient. When a definite diagnosis cannot be reached, MMA and tHcy determination will provide additional discriminative information, but MMA, being more specific, is preferable for assessment of cobalamin status.
A Reference Method Laboratory Network for Cholesterol: A Model for Standardization and Improvement of Clinical Laboratory Measurements
Authors: Myers Gary L , Kimberly Mary M , Waymack Parvin P , Smith S Jay , Cooper Gerald R , Sampson Eric J
Published: January 2020

Background: Accurate and precise measurement of blood cholesterol plays a central role in the National Cholesterol Education Program’s strategy to reduce the morbidity and mortality attributable to coronary heart disease. Matrix effects hamper the ability of manufacturers to adequately calibrate and validate traceability to the National Reference System for Cholesterol (NRS/CHOL). CDC created the Cholesterol Reference Method Laboratory Network (CRMLN) to improve cholesterol measurement by assisting manufacturers of in vitro diagnostic products with validation of the traceability of their assays to the NRS/CHOL.

Methods: CRMLN laboratories established the CDC cholesterol reference method (modification of the Abell-Levy-Brodie-Kendall chemical method) and are standardized using CDC frozen serum reference materials. CRMLN laboratories use common quality-control materials and participate in monthly external performance evaluations conducted by CDC. The CRMLN performance criteria require member laboratories to agree with CDC within ± 1.0% and maintain a CV ≤2.0%.

Results: From 1995 to 2000, the CRMLN laboratories met the accuracy criterion 97% of the time and the precision criterion 99% of the time. During this time period, the CRMLN maintained an average bias to CDC of 0.01% and an average collective CV of 0.33%.

Conclusions: CDC established the CRMLN as the first international reference method laboratory network. The CRMLN assists manufacturers in the validation of the calibration of their diagnostic products so that clinical laboratories can measure blood cholesterol more reliably. The CRMLN can serve as a model for other clinical analytes where traceability to a hierarchy of methods is needed and matrix effects of the field methods with processed calibrators or reference materials are present.
Influence of Increased Fruit and Vegetable Intake on Plasma and Lipoprotein Carotenoids and LDL Oxidation in Smokers and Nonsmokers
Authors: Chopra Mridula , O’Neill Maura E , Keogh Nora , Wortley Gary , Southon Susan , Thurnham David I
Published: January 2020

Background: Epidemiological studies suggest a cardioprotective role for carotenoid-rich foods. Smokers have a high risk of cardiovascular disease and low dietary intake and plasma concentrations of carotenoids. The aim of this study was to determine the carotenoid response of smokers and nonsmokers to increased intake of 300–400 g of vegetables and its effect on LDL oxidation.

Methods: After a depletion period of 8 days, 34 healthy females (18 nonsmokers, 16 smokers) were supplemented with β-carotene- and lutein-rich (green) and lycopene-rich (red) vegetable foods, each for 7 days.

Results: Baseline concentrations (mean ± SD) of plasma β-carotene (0.203 ± 0.28 μmol/L vs 0.412 ± 0.34 μmol/L; P <0.005) and lutein (0.180 ± 0.10 vs 0.242 ± 0.11 μmol/L; P <0.05) but not lycopene (0.296 ± 0.10 vs 0.319 ± 0.33 μmol/L) were significantly lower in smokers compared with nonsmokers. After supplementation, the change (supplementation minus depletion) in plasma β-carotene (0.152 ± 0.43 vs 0.363 ± 0.29 μmol/L in smokers vs nonsmokers; P = 0.002) and LDL lutein (0.015 ± 0.03 vs 0.029 ± 0.03 μmol/mmol cholesterol; P = 0.01) was significantly lower in smokers than nonsmokers. Green-vegetable supplementation had no effect on the resistance of LDL to oxidation (lag-phase) in either group. After red-vegetable supplementation, plasma and LDL lycopene concentrations were increased in both groups, but only nonsmokers showed a significant increase in the lag-phase (44.9 ± 9.5 min at baseline, 41.4 ± 6.5 min after depletion, and 49.0 ± 8.9 min after supplementation; P <0.01) compared with depletion.

Conclusions: In this short-term intervention study, a dietary intake of >40 mg/day of lycopene by a group of nonsmoking individuals significantly reduced the susceptibility of LDL to oxidation, whereas an equivalent increase in lycopene by a group of smokers showed no such effect.
Use of a Reference Material Proposed by the International Federation of Clinical Chemistry and Laboratory Medicine to Evaluate Analytical Methods for the Determination of Plasma Lipoprotein(a)
Authors: Marcovina Santica M , Albers John J , Scanu Angelo M , Kennedy Hal , Giaculli Federico , Berg Kåre , Couderc Rémy , Dati Francesco , Rifai Nader , Sakurabayashi Ikunosuke , Tate Jillian R , Steinmetz Armin
Published: January 2020

Background: As part of the NIH/National Heart, Lung and Blood Institute Contract for the Standardization of Lipoprotein(a) [Lp(a)] Measurements, a study was performed in collaboration with the IFCC Working Group for the Standardization of Lp(a) Assays. The aims of the study, performed with the participation of 16 manufacturers and 6 research laboratories, were to evaluate the IFCC proposed reference material (PRM) for its ability to transfer an accuracy-based value to the immunoassay calibrators and to assess concordance in results among different methods.

Methods: Two different purified Lp(a) preparations with protein mass concentrations determined by amino acid analysis were used to calibrate the reference method. A Lp(a) value of 107 nmol/L was assigned to PRM. After uniformity of calibration was demonstrated in the 22 evaluated systems, Lp(a) was measured on 30 fresh-frozen sera covering a wide range of Lp(a) values and apolipoprotein(a) [apo(a)] sizes.

Results: The among-laboratory CVs for these samples (6–31%) were, in general, higher than those obtained for PRM (2.8%) and the quality-control samples (14%, 12%, and 9%, respectively), reflecting the broad range of apo(a) sizes in the 30 samples and the sensitivity of most methods to apo(a) size heterogeneity. Thus, although all of the assays were uniformly calibrated through the use of PRM, no uniformity in results was achieved for the isoform-sensitive methods.

Conclusions: Linear regression analyses indicated that to various degrees, apo(a) size heterogeneity affects the outcome of the immunochemical methods used to measure Lp(a). We have also shown that the inaccuracy of Lp(a) values determined by methods sensitive to apo(a) size significantly affects the assessment of individual risk status for coronary artery disease.
Erythrocyte Folate Analysis: Saponin Added During Lysis of Whole Blood Can Increase Apparent Folate Concentrations, Depending on Hemolysate pH
Authors: Wright Anthony J A , Finglas Paul M , Southon Susan
Published: January 2020

Background: The analysis of red cell folate (RCF) depends on complete hemolysis of erythrocytes, and it is assumed that complete hemolysis is achieved by 10-fold dilution of whole blood with hypotonic solutions of 10 g/L ascorbic acid/ascorbate. This report challenges this assumption.

Methods: The conventional method of erythrocyte lysis was modified to include saponin, a known effective hemolyzing agent. The influence of saponin was determined at various lysate pHs, using the microbiological (Lactobacillus rhamnosus) folate assay. The effect of saponin during lysate preparation was subsequently compared with either the effect of 30 s of sonication or a single 1-h freeze-thaw cycle.

Results: Saponin addition was found to increase assayable RCF up to ninefold, depending on lysate pH. Sonication of lysates had no effect, and freezing-thawing lysates once did not always guarantee complete hemolysis. Lysates created with 10 g/L ascorbic acid (a historically widely used diluent) without pH adjustment produced assayable folate concentrations significantly lower than optimal.

Conclusions: A lysing agent should be incorporated into RCF assays to guarantee complete hemolysis. Ten-fold dilution of blood with 10 g/L ascorbic acid, without pH adjustment, produces lysates with pHs (pH 4.0) below the point (pH 4.7) at which hemoglobin can denature irreversibly. The optimum pH for hemolysates is ∼5.0.
Real-Time Quantification of Human Telomerase Reverse Transcriptase mRNA in Tumors and Healthy Tissues
Authors: de Kok Jacques B , Ruers Theo J M , van Muijen Goos N P , van Bokhoven Adrie , Willems Hans L , Swinkels Dorine W
Published: January 2020

Background: Expression of the hTERT gene, which codes for the catalytic subunit of telomerase, is associated with malignancy. We recently developed a real-time reverse transcription-PCR assay, based on TaqMan technology, for accurate and reproducible determination of hTERT mRNA expression (Lab Investig 1999;79:911–2). This method may be of interest for molecular tumor diagnostics in tissues and corresponding body fluids, washings, or brushes.

Methods: In this study, we measured hTERT expression in a subset of healthy tissues and tumors to select those tumor types with the best potential for quantification of hTERT in corresponding body fluids. To demonstrate the use of the method in body fluids, we quantified hTERT expression in voided urine of patients with bladder cancer and controls.

Results: Real-time measurement of hTERT expression could discriminate between all healthy and malignant tissue samples from pancreas, lung, esophagus, and bladder, but not for colon tissues. Moreover, in five of nine (55%) urine samples, hTERT could be quantified.

Conclusions: The present study demonstrates that accurate quantitative measurement of hTERT expression has high potential for discrimination between healthy and tumor cells in tissues and urine and supports future measurements in pancreatic fluid, bronchoalveolar lavage fluid, esophageal brushings, and urine or bladder washings.
Development of Conventional and Real-Time PCR Assays for the Rapid Detection of Group B Streptococci
Authors: Ke Danbing , Ménard Christian , Picard François J , Boissinot Maurice , Ouellette Marc , Roy Paul H , Bergeron Michel G
Published: January 2020

Background: Group B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity.

Methods: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCyclerTM. For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays.

Results: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only ∼30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays.

Conclusion: These assays provide promising tools for the rapid detection and identification of GBS.
Correlation between Plasma Total Homocysteine and Copper in Patients with Peripheral Vascular Disease
Authors: Mansoor Mohammad A , Bergmark Claes , Haswell Steve J , Savage Ian F , Evans Peter H , Berge Rolf K , Svardal Asbjørn M , Kristensen Ole
Published: January 2020

Background: Increased concentrations of both plasma total homocysteine and copper are separately associated with cardiovascular disease. Correlations between plasma total homocysteine, trace elements, and vitamins in patients with peripheral vascular disease have not been investigated.

Methods: The concentrations of trace elements in plasma were determined by the multielement analytical technique of total-reflection x-ray fluorescence spectrometry. Plasma total homocysteine was determined by HPLC.

Results: In the univariate and multivariate regression analyses, copper was positively correlated with plasma total homocysteine in all subjects (coefficient ± SE, 0.347 ± 0.113; P = 0.0026 and coefficient ± SE, 0.422 ± 0.108; P = 0.0002, respectively), and in patients with peripheral vascular disease (coefficient ± SE, 0.370 ± 0.150; P = 0.016; and coefficient ± SE, 0.490 ± 0.151; P = 0.0025, respectively). Correlation between copper and plasma total homocysteine was not detected in healthy control subjects. The concentration of calcium in plasma (67.5 vs 80.8 μg/g) was significantly lower in the patients than in the control subjects (P = 0.02). When the patients were divided into groups, the patients with suprainguinal lesions had significantly higher copper concentrations (P = 0.04) and significantly lower selenium and calcium concentrations (P = 0.01 and 0.008, respectively) than the healthy subjects. Patients had higher concentrations of autoantibodies against oxidized LDL and concentrations of thiobarbituric acid-reactive substance than the healthy subjects (P <0.0001 and P = 0.001, respectively). The concentrations of plasma total homocysteine and α-tocopherol were significantly higher, and the concentrations of vitamin B6 and β-carotene were lower in the patients than the healthy subjects.

Conclusion: Our findings suggest that the atherogenicity of homocysteine may be related to copper-dependent interactions.
Cerebrospinal Fluid Protein Concentrations in Children: Age-related Values in Patients without Disorders of the Central Nervous System
Authors: Biou Daniel , Benoist Jean-François , Nguyen-Thi Claire , Huong Xuan , Morel Philippe , Marchand Martine
Published: January 2020

Background: The published reference values for cerebrospinal fluid (CSF) total protein concentrations in children suffer from two major drawbacks: (a) the age-related range often is too broad when applied to the steeply falling concentrations in early infancy; and (b) no values have been published for widely used dry chemistry methods.

Methods: We conducted a 2-year retrospective survey of CSF results obtained in a children’s hospital with a dry chemistry-based method set up on the Vitros 700 analyzer.

Results: The data related to ambulatory children up to 16 years of age and term neonates with no clinical or biological signs of brain disease (n = 1074). Seven age groups with significantly different CSF protein values were identified, and their age-related percentiles (5th, 50th, and 95th) were determined. On the basis of the upper 95th percentile, from age 0 to 6 months the CSF protein concentrations fell rapidly from 1.08 to 0.40 g/L. A plateau (0.32 g/L) was reached from age 6 months to 10 years, followed by a slight increase (0.41 g/L) in the 10–16 years age range.

Conclusions: These results imply that CSF total protein concentrations in the pediatric setting, particularly in infants, must always be interpreted with regard to narrow age-related reference values to avoid false-positive results.
Rapid Screening of High-Risk Patients for Disorders of Purine and Pyrimidine Metabolism Using HPLC-Electrospray Tandem Mass Spectrometry of Liquid Urine or Urine-soaked Filter Paper Strips
Authors: Ito Tetsuya , van Kuilenburg André B P , Bootsma Albert H , Haasnoot Anja J , van Cruchten Arno , Wada Yoshiro , van Gennip Albert H
Published: January 2020

Background: A rapid and specific screening method for patients at risk of inherited disorders of purine and pyrimidine metabolism is desirable because symptoms are varied and nonspecific. The aim of this study was to develop a rapid and specific method for screening with use of liquid urine samples or urine-soaked filter paper strips.

Methods: Reverse-phase HPLC was combined with electrospray ionization (ESI), tandem mass spectrometry (MS/MS), and detection performed by multiple reaction monitoring. Transitions and instrument settings were established for 17 purines or pyrimidines. Stable-isotope-labeled reference compounds were used as internal standards when available.

Results: Total analysis time of this method was 15 min, approximately one-third that of conventional HPLC with ultraviolet detection. Recoveries were 96–107% in urine with added analyte, with two exceptions (hypoxanthine, 64%; xanthine, 79%), and 89–110% in urine-soaked filter paper strips, with three exceptions (hypoxanthine, 65%; xanthine, 77%; 5-hydroxymethyluracil, 80%). The expected abnormalities were easily found in samples from patients with purine nucleoside phosphorylase deficiency, ornithine transcarbamylase deficiency, molybdenum cofactor deficiency, adenylosuccinase deficiency, or dihydropyrimidine dehydrogenase deficiency.

Conclusions: HPLC-ESI MS/MS of urine allows rapid screening for disorders of purine and pyrimidine metabolism. The filter paper strips offer the advantage of easy collection, transport, and storage of the urine samples.
Evaluation of Four Automated High-Sensitivity C-Reactive Protein Methods: Implications for Clinical and Epidemiological Applications
Authors: Roberts William L , Sedrick Rachel , Moulton Linda , Spencer Anthony , Rifai Nader
Published: January 2020

Background: C-reactive protein (CRP) can provide prognostic information about the risk of developing atherosclerotic complications in apparently healthy patients. This new clinical application requires quantification of CRP concentrations below those traditionally measured in the clinical laboratory.

Methods: The Dade Behring BN II, the Abbott IMx, the Diagnostic Products Corporation IMMULITE, and the Beckman Coulter IMMAGE are four automated analyzers with high-sensitivity CRP (hs-CRP) methods. We evaluated these assays for precision, linearity, and comparability with samples from 322 apparently healthy blood donors.

Results: The imprecision (CV) of the BN II, IMx, IMMULITE, and IMMAGE methods was ≤7.6%, ≤12%, ≤9.8%, and ≤9.7% at 3.5 mg/L, respectively. The BN II, IMx, IMMULITE, and IMMAGE methods were linear down to ≤0.30, ≤0.32, ≤0.85, and 2.26 mg/L, respectively. CRP concentrations demarcating each quartile in a healthy population were method dependent. The IMx method gave results comparable to the BN II method for values in the reference interval. The IMMULITE method had a positive intercept compared with the BN II method. The IMMAGE method demonstrated more scatter and a positive intercept compared with the BN II method, which may reflect the fact that it is a less sensitive assay.

Conclusions: The four hs-CRP methods exhibited differences in results for a healthy population. Additional standardization efforts are required to ensure that hs-CRP results can be related to large-scale epidemiologic studies.
Simple Microplate Method for Determination of Urinary Iodine
Authors: Ohashi Toshinori , Yamaki Mitsuo , Pandav Chandrakant S , Karmarkar Madhu G , Irie Minoru
Published: January 2020

Background: Urinary iodine is a good biochemical marker for control of iodine deficiency disorders. Our aim was to develop and validate a simple, rapid, and quantitative method based on the Sandell–Kolthoff reaction, incorporating both the reaction and the digestion process into a microplate format.

Methods: Using a specially designed sealing cassette to prevent loss of vapor and cross-contamination among wells, ammonium persulfate digestion was performed in a microplate in an oven at 110 °C for 60 min. After the digestion mixture was transferred to a transparent microplate and the Sandell–Kolthoff reaction was performed at 25 °C for 30 min, urinary iodine was measured by a microplate reader at 405 nm.

Results: The mean recovery of iodine added to urine was 98% (range, 89–109%). The theoretical detection limit, defined as 2 SD from the zero calibrator, was 0.11 μmol/L (14 μg/L iodine). The mean intra- and interassay CVs for samples with iodine concentrations of 0.30–3.15 μmol/L were ≤10%. The new method agreed well with the conventional chloric acid digestion method (n = 70; r = 0.991; y = 0.944x + 0.04; Sy|x = 0.10) and with the inductively coupled plasma mass spectrometry method (n = 61; r = 0.979; y = 0.962x + 0.03; Sy|x = 0.20). The agreement was confirmed by difference plots. The distributions of iodine concentrations for samples from endemic areas of iodine deficiency diseases showed similar patterns among the above three methods.

Conclusions: Our new method, incorporating the whole process into a microplate format, is readily applicable and allows rapid monitoring of urinary iodine.
Current Concepts in Biomarker Technology for Bladder Cancers
Authors: Burchardt Martin , Burchardt Tatjana , Shabsigh Ahmad , De La Taille Alexandre , Benson Mitchell C , Sawczuk Ihor
Published: January 2020

Background: Transitional cell carcinoma of the bladder (TCC) is the second most common malignancy of the urinary tract. More than 70% of treated tumors recur, and 30% of recurrent tumors progress. Currently, pathologic staging and grading are valuable prognostic factors for detecting and monitoring TCC. Urinalysis, cystoscopy, and cytology are either invasive or lack sensitivity and specificity. The availability of a noninvasive, reliable, and simple test would greatly improve the detection and monitoring of patients with TCC. Several biomarkers for bladder cancer have been proposed, but no single marker has emerged as the test of choice.

Approach: We undertook a comprehensive literature search using Medline to identify all publications from 1980 to 1999. Articles that discussed potential biomarkers for TCC were screened. Only compounds that demonstrated high sensitivity or specificity, significant correlation with TCC diagnosis and staging, and extensive investigation were included in this review.

Content: Potential biomarkers of disease progression and prognosis include nuclear matrix protein, fibrin/fibrinogen product, bladder tumor antigen, blood group-related antigens, tumor-associated antigens, proliferating antigens, oncogenes, growth factors, cell adhesion molecules, and cell cycle regulatory proteins. The properties of the biomarkers and the methods for detecting or quantifying them are presented. Their sensitivities and specificities for detecting and monitoring disease were 54–100% and 61–97%, respectively, compared with 20–40% and 90% for urinalysis and cytology.

Summary: Although urine cytology and cystoscopy are still the standard of practice, many candidate biomarkers for TCC are emerging and being adopted into clinical practice. Further research and better understanding of the biology of bladder cancer, improved diagnostic techniques, and standardized interpretation are essential steps to develop reliable biomarkers. It is possible that using the current biomarkers as an adjuvant modality will improve our ability to diagnose and monitor bladder cancer.
Detection of Rare Mutant Alleles by Restriction Endonuclease-mediated Selective-PCR: Assay Design and Optimization
Authors: Fuery Caroline J , Impey Helen L , Roberts Natalie J , Applegate Tanya L , Ward Robyn L , Hawkins Nicholas J , Sheehan Catherine A , O’Grady Roslyn , Todd Alison V
Published: January 2020

Background: Restriction endonuclease-mediated selective (REMS)-PCR, allows detection of point mutations, deletions, and insertions. Reactions require concurrent activity of a restriction endonuclease (RE) and a DNA polymerase, both of which must be sufficiently thermostable to retain activity during thermocycling. The inclusion of the RE in REMS-PCR inhibits amplification of sequences containing the RE recognition site, thus producing selective amplification of sequences that lack the RE site.

Methods: Assays were used that allowed the selection of conditions that produce concurrent RE/DNA polymerase activity. The RE thermostability assay involved thermocycling a RE under various conditions and assessing residual cleavage activity at various time points. Conditions found to preserve RE activity during thermocyling were then tested for their compatibility with DNA polymerase-mediated PCR.

Results: A range of conditions that preserve activity of the RE BstNI over 30 cycles of PCR was identified. A subset of these conditions was subsequently found to mediate specific amplification using Taq DNA polymerase. These conditions were used to develop a REMS-PCR protocol for the detection of mutations at codon 12 of the K-ras gene. This protocol allowed the detection of 1 mutant allele in a background of 1000 wild-type alleles. The presence of primer sets for RE and PCR control amplicons provided unambiguous assessment of mutant status.

Conclusion: Implementation of the assays described may facilitate development of REMS-PCR assays targeted to other loci associated with disease.
The Plasma Homocysteine Concentration Is Better Than That of Serum Methylmalonic Acid as a Marker for Sociopsychological Performance in a Psychogeriatric Population
Authors: Nilsson Karin , Gustafson Lars , Hultberg Björn
Published: January 2020

Background: Cobalamin/folate deficiency in elderly subjects may lead to psychiatric symptoms, but more often it increases the severity of various organic and nonorganic mental diseases. A major clinical problem, however, is the uncertainty and controversy concerning biochemical markers of cobalamin/folate deficiency to be used in the diagnostic evaluation of suspected cobalamin/folate deficiency.

Methods: We measured plasma homocysteine (tHcy), blood folate, serum methylmalonic acid, and serum cobalamin in 80 psychogeriatric patients (age, 77.3 ± 8.6 years) and 50 controls (age, 76.1 ± 8.0 years). We assessed associations of these tests with measures of cognitive and behavior performance by use of regression analyses.

Results: Plasma tHcy was increased in 45% of the psychogeriatric population (mean, 20.5 ± 9.2 μmol/L vs 15.3 ± 4.7 μmol/L for controls; P <0.01). Plasma tHcy correlated with severity of dementia (r = 0.36; P <0.01), the Katz ADL index (r = 0.29; P <0.05), the Berger scale (r = 0.29; P <0.05), and the score of symptoms (r = 0.39; P <0.001) in the psychogeriatric population. Similarly, blood folate was significantly correlated with these measures, but the concentrations of serum cobalamin and methylmalonic acid were not. In a stepwise multiple regression analysis including the biochemical markers, tHcy was the only significant predictor of the severity of dementia (r2 = 0.11; P <0.01) and the score of symptoms (r2 = 0.16; P <0.001).

Conclusion: Plasma tHcy is the best marker of those measured to investigate suspected tissue deficiency of cobalamin/folate.
Vitamin B1 Status Assessed by Direct Measurement of Thiamin Pyrophosphate in Erythrocytes or Whole Blood by HPLC: Comparison with Erythrocyte Transketolase Activation Assay
Authors: Talwar Dinesh , Davidson Helen , Cooney Josephine , St. JO’Reilly Denis
Published: January 2020

Background: The concentration of thiamin diphosphate (TDP) in erythrocytes is a useful index of thiamin status. We describe an HPLC method for TDP and its results in patients at risk of thiamin deficiency.

Methods: We used reversed-phase HPLC with postcolumn derivatization with alkaline potassium ferricyanide and fluorescence detection. Samples were deproteinized and injected directly onto a C18 column. TDP concentrations in erythrocytes were compared with those in whole blood. Reference intervals for erythrocyte TDP (n = 147; 79 males and 68 females; mean age, 54 years) and whole blood TDP (n = 124; 68 males and 56 females; mean age, 54 years) were determined in an apparently healthy population. We compared erythrocyte TDP with results of the erythrocyte transketolase activation test in 63 patients who were considered at risk of thiamin deficiency.

Results: The method was linear to at least 200 μg/L. The between-run CV was <8%. The lower limit of quantification for both whole blood and packed erythrocytes was 300 pg on column with a detection limit of 130 pg on column. Recovery of TDP from blood samples was >90%. TDP in erythrocytes correlated strongly with that in whole blood (r = 0.97). Reference intervals for erythrocyte and whole blood TDP were 280–590 ng/g hemoglobin and 275–675 ng/g hemoglobin, respectively. Of the 63 patients suspected of thiamin deficiency, 46 were normal by both TDP and activation tests, 13 were deficient by both tests, 1 was deficient by the activation test but had normal erythrocyte TDP concentrations, and 4 were normal by the activation test but had low TDP.

Conclusions: The HPLC method is precise and yields results similar to the erythrocyte activation assay.
Developing an Automation Concept That Is Right for Your Laboratory
Authors: Middleton Stephen R
Published: January 2020

Background: Trends in laboratory automation and critical project principles and design concepts are presented.

Approach: MDS AutoLab technology development and automation projects were reviewed. Successful methods and approaches were extracted.

Issues: Continued pressure on the laboratory to reduce costs and increase productivity has catalyzed dramatic development in laboratory automation. Today, laboratories can choose from a wide range of options. The most effective choices are not always the most obvious and will not be the same for all laboratories. Laboratory automation projects are highly complex and must be planned and managed across clinical, technical, operational, financial, and human dimensions.

Conclusions: Success requires excellent communications, an understanding of the risks and barriers, and a dedicated team supported by strong champions throughout the organization. The automation project team will need to use a variety of skills and techniques to evaluate and reengineer processes to identify the highest value targets for automation.
Protein Glycosylation and Diseases: Blood and Urinary Oligosaccharides as Markers for Diagnosis and Therapeutic Monitoring
Authors: Durand Geneviève , Seta Nathalie
Published: January 2020

Background: N- and O-oligosaccharide variants on glycoproteins (glycoforms) can lead to alterations in protein activity or function that may manifest themselves as overt disease.

Approach: This review summarizes those diseases that are known to be the result of an inherited or acquired glycoprotein oligosaccharide structural alteration and that are diagnosed in blood or urine by chemical characterization of that oligosaccharide alteration.

Content: The biochemical synthesis steps and catabolic pathways important in determining glycoprotein function are outlined with emphasis on alterations that lead to modified function. Clinical and biochemical aspects of the diagnosis are described for inherited diseases such as I-cell disease, congenital disorders of glycosylation, leukocyte adhesion deficiency type II, hereditary erythroblastic multinuclearity with a positive acidified serum test, and Wiskott-Aldrich syndrome. We also review the laboratory use of measurements of glycoforms related to acquired diseases such as alcoholism and cancer.

Conclusions: Identification of glycoprotein glycoforms is becoming an increasingly important laboratory contribution to the diagnosis and management of human diseases as more diseases are found to result from glycan structural alterations.
Troponin T and I Assays Show Decreased Concentrations in Heparin Plasma Compared with Serum: Lower Recoveries in Early than in Late Phases of Myocardial Injury
Authors: Gerhardt Willie , Nordin Gunnar , Herbert Ann-Katrin , Linåker Burzell Birgitta , Isaksson Anders , Gustavsson Elisabeth , Haglund Sofie , Müller-Bardorff Margit , Katus Hugo A
Published: January 2020

Background: Heparinized plasma samples allow more rapid analysis than serum samples, but preliminary studies showed lower cardiac troponin T (cTnT) results in plasma. We undertook a multicenter study to characterize this effect for cTnT and cardiac troponin I (cTnI).

Methods: Blood samples were collected with and without heparin at five hospitals. cTnT was measured by a “third generation” assay (Elecsys®), and cTnI was measured by a commercial immunoassay (IMMULITE®).

Results: Mean cTnT was 15% lower in heparin sampling tubes than in serum. Measured concentrations of cardiac troponins also decreased with increasing heparin concentrations added to sera. Heparin-induced losses were greater in early than in late phases after onset of chest pain. Addition of heparin (∼100 IU/mL) to serial samples from nine acute myocardial infarction patients produced mean cTnT losses of 33% at 1–12 h after onset of chest pain, 17% at 13–48 h, and 7% after 48 h. The changing heparin effects were seen for both cTnT and cTnI during time courses of individual patients with myocardial infarction.

Conclusion: We suggest that binding of heparin to troponins decreases immunoreactivity, especially in early phases of myocardial injury. The resulting losses may depend on the antibodies used in each troponin assay.
Age-related Increases in Plasma Phosphatidylcholine Hydroperoxide Concentrations in Control Subjects and Patients with Hyperlipidemia
Authors: Kinoshita Mikio , Oikawa Shinichi , Hayasaka Kyoko , Sekikawa Akihiro , Nagashima Tazuko , Toyota Takayoshi , Miyazawa Teruo
Published: January 2020

Background: The basal lipid peroxide concentration in the plasma of patients with hyperlipidemia may be related to atherosclerosis. Quantitative determination of lipid peroxides in the plasma is an important step in the overall evaluation of the biochemical processes leading to oxidative injury. Unfortunately, the currently available methods for lipid peroxidation lack specificity and sensitivity.

Methods: Hyperlipidemic patients (44 males and 50 females), ages 12–82 years (mean ± SE, 53 ± 2.3 years for males, 58 ± 2.0 years for females, and 56 ± 14 years for total cases), and normolipidemic volunteers (controls, 32 males and 15 females), ages 13–90 years (49 ± 4 years for males, 65 ± 4 years for females, and 55 ± 24 years for total cases), were recruited in the present study. Plasma phosphatidylcholine hydroperoxide (PCOOH) was determined by chemiluminescence-HPLC (CL-HPLC).

Results: Plasma PCOOH concentrations increased with age in both controls and hyperlipidemic patients. However, the mean plasma PCOOH concentration in patients with hyperlipidemia (331 ± 19 nmol/L; n = 94) was significantly (P <0.001) higher than in the controls (160 ± 65 nmol/L; n = 47). Plasma PCOOH concentrations were similar in three hyperlipidemic phenotypes: hypercholesterolemia (IIa), hypertriglyceridemia (IV), and combined hyperlipidemia (IIb). The mean plasma PCOOH in patients with treatment-induced normalized plasma lipids was 202 ± 17 nmol/L. There was no significant correlation between plasma PCOOH concentration and total cholesterol, triglycerides, or phospholipids in hyperlipidemic patients. For all subjects, there was a significantly positive correlation between plasma PCOOH and each lipid (total cholesterol, P = 0.0002; triglycerides, P = 0.0137; and phospholipids, P <0.0001). Analysis of fatty acids composition of plasma phosphatidylcholine showed significantly low concentrations of n-6 fatty acids moieties (linoleic acid and arachidonic acid) in patients compared with controls.

Conclusions: Our results suggest that an increase in plasma PCOOH in patients with hyperlipidemia may be related to the development and progression of atherosclerosis, particularly in the elderly. Measurement of plasma PCOOH is useful for in vivo evaluation of oxidative stress.
Prostate-specific Antigen: A Cancer Fighter and a Valuable Messenger?
Authors: Diamandis Eleftherios P
Published: January 2020

Background: Prostate-specific antigen (PSA) is a valuable prostatic cancer biomarker that is now widely used for population screening, diagnosis, and monitoring of patients with prostate cancer. Despite the voluminous literature on this biomarker, relatively few reports have addressed the issue of its physiological function and its connection to the pathogenesis and progression of prostate and other cancers.

Approach: I here review literature dealing with PSA physiology and pathobiology and discuss reports that either suggest that PSA is a beneficial molecule with tumor suppressor activity or that PSA has deleterious effects in prostate, breast, and possibly other cancers.

Content: The present scientific literature on PSA physiology and pathobiology is confusing. A group of reports have suggested that PSA may act as a tumor suppressor, a negative regulator of cell growth, and an apoptotic molecule, whereas others suggest that PSA may, through its chymotrypsin-like activity, promote tumor progression and metastasis.

Summary: The physiological function of PSA is still not well understood. Because PSA is just one member of the human kallikrein gene family, it is possible that its biological functions are related to the activity of other related kallikreins. Only when the physiological functions of PSA and other kallikreins are elucidated will we be able to explain the currently apparently conflicting experimental data.
Prevention of in Vitro Lipolysis by Tetrahydrolipstatin
Authors: Krebs Michael , Stingl Harald , Nowotny Peter , Weghuber Daniel , Bischof Martin , Waldhäusl Werner , Roden Michael
Published: January 2020

Background: Metabolic effects of free fatty acids (FFAs) frequently are tested using combined infusion of triglycerides and heparin, which stimulates lipolysis in vivo. Ongoing in vitro lipolysis, however, probably produces falsely high plasma FFA concentrations under these conditions. Therefore, this study aims to assess the efficacy of tetrahydrolipstatin (THL) in inhibiting plasma lipolytic activity and to improve plasma FFA determination.

Methods: Plasma concentrations of FFAs and glycerol were measured in five healthy subjects in the presence and absence of THL. Blood was drawn at baseline, during infusion of a triglyceride emulsion (1.5 mL/min), and during infusion of triglycerides plus heparin (0.2 IU · kg−1 · min−1). In addition, the effects of storage temperature of the samples were analyzed.

Results: In samples frozen immediately after collection, plasma FFAs were 28% lower in the presence of THL than in its absence (P = 0.008). When THL-free plasma was incubated for 3 h on ice or at room temperature, plasma FFAs were 22% (P = 0.02) and 91% (P = 0.0004) higher, respectively, than in samples frozen immediately. The addition of THL blunted temperature-dependent in vitro lipolysis by 88% (P <0.01) and 89% (P <0.001) after incubation on ice and at room temperature, respectively. Changes in plasma glycerol concentrations exhibited similar behavior.

Conclusions: THL, which is safe and easy to handle, is a potent inhibitor of in vitro lipolysis and could, therefore, be added to blood samples drawn during triglyceride/heparin infusions to allow more accurate determination of plasma FFA concentrations.
Genotyping of Factor V G1691A (Leiden) without the Use of PCR by Invasive Cleavage of Oligonucleotide Probes
Authors: Hessner Martin J , Budish Mary Ann , Friedman Kenneth D
Published: January 2020

Background: The factor V G1691A Leiden (FVL) mutation is the most common known hereditary risk factor for venous thrombosis.

Methods: Third Wave Technologies, Inc. (Madison, WI) has developed a new microtiter plate-based assay that does not require PCR, restriction digestion, or gel electrophoresis. This technology system, termed the InvaderTM assay, utilizes a 5′ “invading” oligonucleotide and a partially overlapping 3′ “signal” oligonucleotide, which together form a specific structure when bound to a complementary genomic DNA template. A thermostable flap endonuclease cleaves this structure, releasing the 5′ flap from the signal oligonucleotide. Increased temperature and an excess of the signal probe enable multiple probes to be cleaved for each target sequence present without temperature cycling. The cleaved probes then direct cleavage of a secondary probe, which is 5′ end-labeled with fluorescein but is quenched by an internal dye. Upon cleavage, the fluorescein-labeled product is detected using a standard fluorescence plate reader. Genotypes are determined by net wild-type/mutant signal ratio.

Results: Complete concordance was observed, after resolution of four discordances, when 1369 individuals (1264 wild type, 102 heterozygous, 3 homozygous) were FVL genotyped by both the Invader assay and by allele-specific PCR.

Conclusion: We conclude that FVL genotyping using invasive cleavage of oligonucleotide probes is a rapid and reliable alternative to genotyping by more traditional PCR-based methods.
Genetic Analysis of DNA Excreted in Urine: A New Approach for Detecting Specific Genomic DNA Sequences from Cells Dying in an Organism
Authors: Botezatu Irina , Serdyuk Ol’ga , Potapova Galina , Shelepov Valery , Alechina Raisa , Molyaka Yuriy , Anan’ev Vitaliy , Bazin Igor , Garin August , Narimanov Mehti , Knysh Vasiliy , Melkonyan Hovsep , Umansky Samuil , Lichtenstein Anatoly
Published: January 2020

Background: Cell-free DNA from dying cells recently has been discovered in human blood plasma. In experiments performed on animals and humans, we examined whether this cell-free DNA can cross the kidney barrier and be used as a diagnostic tool.

Methods: Mice received subcutaneous injections of either human Raji cells or purified 32P-labeled DNA. DNA was isolated from urine and analyzed by measurement of radioactivity, agarose gel electrophoresis, and PCR. In humans, the permeability of the kidney barrier to polymeric DNA was assessed by detection in urine of sequences that were different from an organism bulk nuclear DNA.

Results: In the experiments on laboratory animals, we found that ∼0.06% of injected DNA was excreted into urine within 3 days in a polymeric form and that human-specific Alu sequences that passed through the kidneys could be amplified by PCR. In humans, male-specific sequences could be detected in the urine of females who had been transfused with male blood as well as in DNA isolated from urine of women pregnant with male fetuses. K-ras mutations were detected in the urine of patients with colon adenocarcinomas and pancreatic carcinomas.

Conclusions: The data suggest that the kidney barrier in rodents and humans is permeable to DNA molecules large enough to be analyzed by standard genetic methodologies.