bluuebunny/crossref_metadata_2025 huggingface dataset:
Leptin in Cerebrospinal Fluid from Children: Correlation with Plasma Leptin, Sexual Dimorphism, and Lack of Protein Binding
Authors: Landt Michael , Parvin Curtis A , Wong Michael
Published: January 2020

Background: Previous studies in adults have established that leptin is present at very low concentrations in cerebrospinal fluid (CSF), but few data exist concerning CSF leptin in children. Current evidence suggests that CSF leptin concentrations interact with hypothalamic centers controlling food intake. Serum leptin concentrations manifest a sexual dimorphism that arises during puberty.

Methods: Leptin concentrations were determined in CSF from 42 pre- and postpubertal children who had been objectively classified into non-neurological disease or aseptic meningitis groups. Multivariate analysis of the dependence of CSF leptin on gender, pubertal state, body mass index (BMI), presence of aseptic meningitis, and CSF protein concentration was performed.

Results: CSF leptin concentrations correlated with log-transformed plasma leptin concentrations in concomitantly collected samples (r = 0.582; P = 0.029). BMI and gender were significant determinants of CSF leptin in postpubertal children, but only BMI was significant in prepubertal children. Analysis with HPLC to separate protein-bound and free forms of leptin found only free leptin in CSF.

Conclusions: CSF leptin concentrations in children reflect plasma leptin concentrations, including the advent of sexual dimorphism at puberty. Only free leptin is detectable in CSF, suggesting that it is the biologically active form.
Accurate and Rapid “Multiplex Heteroduplexing” Method for Genotyping Key Enzymes Involved in Folate/Homocysteine Metabolism
Authors: Barbaux Sandrine , Kluijtmans Leo A J , Whitehead Alexander S
Published: January 2020

Background: Hyperhomocysteinemia, which is often associated with low folate status, is an independent risk factor for cardiovascular diseases and several other pathologies. The four most common functional polymorphisms in genes involved in folate/homocysteine metabolism are methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, methionine synthase (MS) A2756G, and cystathionine β-synthase (CBS) 844ins68. The pathogenic impact of these variants is under active investigation in many laboratories. However, conventional genotyping methods, mostly using PCR followed by restriction enzyme digestion, often are compromised by partial fragment digestion. There is, therefore, a need to develop more reliable approaches to genotyping the above polymorphisms that may be applied in large-scale studies.

Methods: Sequence-specific heteroduplex generators for each of the MTHFR and MS single nucleotide polymorphisms were generated by site-directed mutagenesis. These were subcloned into a single construct, pHcyHG-1, which could be multiplexed with a simple PCR amplification across the CBS 844ins68 polymorphic site to generate composite genotype-specific banding patterns from individual genomic DNA samples that could be electrophoretically resolved.

Results: The “multiplex heteroduplexing” method yielded unambiguous MTHFR, MS, and CBS genotypes in a single-tube reaction that could be analyzed in a single gel run.

Conclusions: This method permits unambiguous genotyping of the four most common functional variants of enzymes involved in folate/homocysteine metabolism. It is rapid, reproducible, and inexpensive, and requires no special preparative or analytic facilities; consequently, it will facilitate large-scale studies of the genetic basis of hyperhomocysteinemia and the many pathologies that have been associated with this phenotype.
Total and Pancreatic Amylase Measured with 2-Chloro-4-nitrophenyl-4-O-β-d-galactopyranosylmaltoside
Authors: Morishita Yoshitaka , Iinuma Yoshitsugu , Nakashima Nobuo , Majima Keiichi , Mizuguchi Katsuhiko , Kawamura Yoshihisa
Published: January 2020

Background: Many different methods have been used to assay amylase activity, using nitrophenylated oligosaccharides as substrate; however, the hydrolysis steps in these methods are complex.

Methods: We developed a new continuously monitoring assay for amylase activity in biological fluids, using 2-chloro-4-nitrophenyl-4-O-β-d-galactopyranosylmaltoside (GalG2CNP) as the substrate; this assay was used with anti-human salivary amylase monoclonal antibodies for specific determination of the pancreatic isoenzyme. Amylase converted GalG2CNP into β-d-galactopyranosylmaltose and 2-chloro-4-nitrophenol, which was measured at 405 nm.

Results: GalG2CNP was cleaved between 2-chloro-4-nitrophenol and β-d-galactopyranosylmaltose and did not undergo transfer reactions. The within-assay CVs (n = 20) for total amylase (T-AMY) and pancreatic amylase (P-AMY) were 0.6–1.6% and 0.5–2.5%, respectively; and day-to-day CVs (n = 10) for T-AMY and P-AMY were 0.8–3.7% and 0.6–4.1%, respectively. T-AMY and P-AMY activities in serum or urine obtained by the proposed method correlated well with those determined by the 2-chloro-4-nitrophenyl 4-O-β-d-galactopyranosyl-β-maltotetraoside method or the modified IFCC method.

Conclusions: This novel assay for T-AMY and P-AMY measures both activities stoichiometrically, directly, and easily, and may be suitable for routine procedures.
Immunoradiometric Assay of Circulating C-Reactive Protein: Age-related Values in the Adult General Population
Authors: Hutchinson Winston L , Koenig Wolfgang , Fröhlich Margit , Sund Malte , Lowe Gordon D O , Pepys Mark B
Published: January 2020

Background: Increased values of C-reactive protein (CRP), the classical acute phase protein, within the range below 5 mg/L, previously considered to be within the reference interval, are strongly associated with increased risk of atherothrombotic events, and are clinically significant in osteoarthritis and neonatal infection.

Methods: A robust new polyclonal-monoclonal solid- phase IRMA for CRP was developed, with a range of 0.05–10.0 mg/L.

Results: Plasma CRP values in general adult populations from Augsburg, Germany (2291 males and 2203 females; ages, 25–74 years) and Glasgow, Scotland (604 males and 650 females; ages, 25–64 years) were very similar. The median CRP approximately doubled with age, from ∼1 mg/L in the youngest decade to ∼2 mg/L in the oldest, and tended to be higher in females.

Conclusion: This extensive data set, the largest such study of CRP, provides valuable reference information for future clinical and epidemiological investigations.
Laboratory Costs in the Context of Disease
Authors: Young Donald S , Sachais Bruce S , Jefferies Leigh C
Published: January 2020

Background: To determine the contribution of laboratory costs to the overall costs of managing hospital patients with different diseases, we studied the costs of laboratory testing overall and in relation to the other costs incurred during hospitalization.

Methods: We used a database developed by the University HealthSystems Consortium containing >1 million patients in 60 University Hospitals with diseases included in 486 diagnosis-related groups (DRGs). Laboratory costs included in the database comprised those associated with testing in the clinical laboratory together with those incurred in point-of-care testing and anatomic pathology but not those involving blood products and their transfusion.

Results: The mean laboratory costs to manage surgical patients were greater than those to manage medical patients in 19 of the 25 major diagnostic categories. The median laboratory costs for patients with liver transplants exceeded $8000, and the laboratory costs to support other organ transplants were among the highest. The highest proportion of total costs attributable to the laboratory was 18.3% for acute leukemia and kidney and urinary tract signs and symptoms, both in children. Laboratory costs were <1.0% of the total costs for only 15 DRGs. The highest median daily laboratory cost, $416, was attributable to liver transplant patients. Several conditions had median laboratory costs less than $30 per day, in spite of lengths of stay that exceeded 10 days in some cases.

Conclusions: Although laboratory costs generally average 6% of the total costs for surgical conditions and 9% of the total costs for medical conditions, there is considerable variability. In general, laboratory costs were relatively poorly correlated with total costs. However, observation of high daily laboratory costs for many DRGs suggests that reducing length of stay would reduce both laboratory and total costs.
Evidence-based Laboratory Medicine: Supporting Decision-Making
Authors: Price Christopher P
Published: January 2020

There is an implicit acceptance that an evidence-based culture underpins the practice of laboratory medicine, in part because it is perceived as the scientific foundation of medicine. However, several reviews of specific test procedures or technologies have shown that the evidence base is limited and in many cases flawed. One of the key deficiencies in the scientific literature on diagnostic tests often is the absence of an explicit statement of the clinical need, i.e., the clinical or operational question that the use of the test is seeking to answer. Several reviews of the literature on specific procedures have also demonstrated that the experimental methodology used is flawed with, in some cases, significant bias being introduced. Despite these limitations it is recognized that a more evidence-based approach will help in the education and training of health professionals, in the creation of a research agenda, in the production of guidelines, in the support of clinical decision-making, and in resource allocation. Furthermore, as knowledge and technologies continue to be developed, an evidence-based strategy will be critical to harnessing these developments.
Riboflavin as a Determinant of Plasma Total Homocysteine: Effect Modification by the Methylenetetrahydrofolate Reductase C677T Polymorphism
Authors: Hustad Steinar , Ueland Per Magne , Vollset Stein Emil , Zhang Ying , Bjørke-Monsen Anne Lise , Schneede Jørn
Published: January 2020

Background: Plasma total homocysteine (tHcy) is a risk factor for cardiovascular disease. tHcy concentrations are partly determined by folate, cobalamin, and vitamin B6 status, and methylenetetrahydrofolate reductase (MTHFR) and other flavoenzymes are important for the biotransformation of these vitamins. This motivates the investigation of the possible relationship between riboflavin status and tHcy.

Methods: The study had a cross-sectional design and included 423 healthy blood donors, ages 19–69 years. We determined plasma tHcy, serum folate, serum cobalamin, serum creatinine, and MTHFR C677T genotype. In addition, we measured riboflavin and its two coenzyme forms, flavin mononucleotide and flavin adenine dinucleotide, in EDTA plasma by capillary electrophoresis and laser-induced fluorescence detection.

Results: Riboflavin determined tHcy independently in a multiple linear regression model with adjustment for sex, age, folate, cobalamin, creatinine, and MTHFR genotype (P = 0.008). tHcy was 1.4 μmol/L higher in the lowest compared with the highest riboflavin quartile. The riboflavin-tHcy relationship was modified by genotype (P = 0.004) and was essentially confined to subjects with the C677T transition of the MTHFR gene.

Conclusions: Plasma riboflavin is an independent determinant of plasma tHcy. Studies on deficient populations are needed to evaluate the utility of riboflavin supplementation in hyperhomocysteinemia.
Prognostic Significance of Low Serum Cholesterol after Cardiothoracic Surgery
Authors: Stachon Axel , Böning Andreas , Weisser Heike , Laczkovics Axel , Skipka Guido , Krieg Michael
Published: January 2020

Background: The precise prognostic significance of critically low cholesterol concentrations in patients undergoing cardiothoracic surgery is unknown.

Methods: In a retrospective case-control study, we analyzed the database of 2074 patients, of whom 87 died postoperatively in hospital. All patients underwent cardiothoracic surgery using a heart-lung machine. Age, sex, body mass index, preoperative ejection fraction, smoking, diabetes mellitus, type of operation, emergency surgery, renal deficiency, pulmonary hypertension, and endocarditis were considered together with serum concentrations of cholesterol, C-reactive protein, alanine aminotransferase, and triglycerides. The statistics included sensitivity, specificity, predictive value, odds ratio, and the ROC curve.

Results: Cholesterol decreased sharply immediately after surgery in both the deceased and the survivors. In the deceased, the mean cholesterol concentration (± SE) remained rather low between days 4 and 7 after surgery [2.46 ± 0.16 mmol/L (95 ± 6 mg/dL)]. In the survivors at that time, the mean cholesterol concentration was significantly (P <0.001) higher [4.37 ± 0.03 mmol/L (169 ± 1 mg/dL)]. The positive predictive value of a critically low cholesterol concentration [<3.10 mmol/L (<120 mg/dL)] was 25.4%, increasing to 66.6% at a cutoff value of 1.55 mmol/L (60 mg/dL). The odds ratio under those circumstances was 15.5, and the area under curve (C-statistic) was 0.90.

Conclusions: The cholesterol concentration between days 4 and 7 after cardiothoracic surgery possesses a high prognostic significance in terms of in-hospital mortality.
Glyceraldehyde Preserves Glucose Concentrations in Whole Blood Specimens
Authors: Landt Michael
Published: January 2020

Background: Glucose concentrations decrease in blood specimens during transport/processing, primarily because of continuing metabolism (glycolysis) by erythrocytes. Several means to reduce the loss of glucose in blood specimens have been developed, but all have major drawbacks. Glyceraldehyde, which has antiglycolytic activity, was assessed for potential in preserving glucose in blood specimens.

Methods: Heparinized blood from volunteers was treated with glyceraldehyde and other agents. After incubation for various times, plasma concentrations of glucose and other common analytes were determined with prevalent commercial analyzers.

Results: The racemic mixture of glyceraldehyde (d,l-GA) preserved glucose concentrations for up to 8 h at room temperature. Half-maximal effect was attained with 0.9 mmol/L d,l-GA. Trials of the d and l stereoisomers individually indicated that the l isomer (l-GA) was responsible for all or most of the antiglycolytic activity of the racemic mixture. Other related compounds were ineffective. Measurements of most common clinical laboratory analytes were unaffected by the presence of d,l-GA or l-GA.

Conclusions: Glyceraldehyde (d,l-GA or l-GA) effectively preserves glucose concentrations in whole blood specimens for up to 8 h. Specimens collected with d,l-GA or l-GA are suitable for analysis of many analytes commonly comeasured with glucose.
Evaluation of Novel Assays in Clinical Chemistry: Quantification of Plasma Total Homocysteine
Authors: Nexo Ebba , Engbaek Frode , Ueland Per Magne , Westby Christina , O’Gorman Paudy , Johnston Carole , Kase Bengt Frode , Guttormsen Anne B , Alfheim Ingrid , McPartlin Joseph , Smith David , Møller Jan , Rasmussen Karsten , Clarke Robert , Scott John M , Refsum Helga
Published: January 2020

Background: There is a need for systematic evaluation of methods before their release to the market. We addressed this problem in novel homocysteine assays as part of an European Demonstration Project involving six centers in four countries.

Methods: Two immunological methods for measurement of plasma total homocysteine (P-tHcy), the fluorescence polarization immunoassay (FPIA) and the enzyme immunoassay (EIA), were compared with two comparison methods, HPLC and gas chromatography–mass spectrometry (GC-MS). All laboratories performed the following procedures: (a) familiarization; (b) determination of linearity and precision by analyzing five plasma samples with interrelated concentrations for 20 days; (c) correlation using patients’ samples; and (d) assessment of long-term performance.

Results: Both immunological methods were linear for P-tHcy between 5 and 45 μmol/L. The intralaboratory imprecision (CV) was <5% for FPIA and <9% for EIA used with a sample processor. The bias was −2% to 3% for FPIA and 2–4% for EIA used with a sample processor.

Conclusions: The immunological methods provide results with little bias compared with HPLC and GC-MS. The imprecision of the assays must be considered in the context of their intended use(s).
Nonalcoholic Red Wine Extract and Quercetin Inhibit LDL Oxidation without Affecting Plasma Antioxidant Vitamin and Carotenoid Concentrations
Authors: Chopra Mridula , Fitzsimons Patricia E E , Strain John J , Thurnham David I , Howard Alan N
Published: January 2020

Background: Antioxidant enrichment of LDL can increase its resistance to oxidation and hence reduce its atherogenicity. The objective of the present study was to investigate whether in vivo supplementation with nonalcoholic red wine extract and quercetin can increase the oxidative resistance of LDL, and also whether the supplementation has any effect on other antioxidative micronutrients present in the blood.

Methods: Twenty-one male subjects were supplemented with a placebo drink for 2 weeks and randomized into two groups. One group (n = 11) received the red wine extract (1 g/day, equivalent to 375 mL of red wine) and the other group (n = 10) quercetin (30 mg/day) for 2 weeks, followed by a 5-week washout period.

Results: In the red wine extract-supplemented group, ex vivo copper-initiated oxidation of LDL (lag phase, mean ± SD) was 40 ± 11 min at the baseline, and increased significantly to 47 ± 6 min [P <0.05 compared with placebo (38 ± 4 min) and the washout values (40 ± 5 min)]. In the quercetin-supplemented group, the lag phase was 44 ± 11 and 40 ± 5 min for the baseline and placebo, respectively, and increased significantly to 51 ± 7 min [P <0.05 compared with placebo and washout (41 ± 9 min)] after supplementation. Plasma lipids (triglycerides, total cholesterol, LDL- and HDL-cholesterol) did not change during the study period. Supplementation with red wine extract or quercetin had no effect on plasma vitamin C and E, retinol, and carotenoid concentrations.

Conclusions: Alcohol-free red wine extract and one of its components, quercetin, can inhibit LDL oxidation after in vivo supplementation; such “inhibition” is unrelated to changes in antioxidant vitamin and carotenoid concentrations.
Bone Marrow and Peripheral Blood Hematopoietic Stem Cell Transplantation: Focus on Autografting
Authors: Baynes Roy D , Hamm Caroline , Dansey Roger , Klein Jared , Cassells Lucinda , Karanes Chatchada , Abella Esteban , Peters William P
Published: January 2020

This review focuses on certain of the principles involved in high-dose chemotherapy and radiation therapy along with autologous hematopoietic stem cell transplantation for the treatment of certain malignancies. In addition, the evidence, wherever possible based on randomized data, for the application of this approach in certain malignancies is reviewed. The malignancies highlighted include acute myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma, Hodgkin disease, and breast cancer.
Coagulation and Bleeding Disorders: Review and Update
Authors: Triplett Douglas A
Published: January 2020

Hemostasis is initiated by injury to the vascular wall, leading to the deposition of platelets adhering to components of the subendothelium. Platelet adhesion requires the presence of von Willebrand factor and platelet receptors (IIb/IIIa and Ib/IX). Additional platelets are recruited to the site of injury by release of platelet granular contents, including ADP. The “platelet plug” is stabilized by interaction with fibrinogen. In this review, I consider laboratory tests used to evaluate coagulation, including prothrombin time, activated partial thromboplastin time, thrombin time, and platelet count. I discuss hereditary disorders of platelets and/or coagulation proteins that lead to clinical bleeding as well as acquired disorders, including disseminated intravascular coagulation and acquired circulating anticoagulants.
Transfusion Medicine: An Overview and Update
Authors: Ness Paul M
Published: January 2020

The discovery of AIDS in the 1980s and its rapid evolution as a major concern for physicians and their patients have led to many questions about the safety of the blood supply. The attention placed on AIDS has led to new discoveries and technologies to reduce the risk of other transfusion complications such as hepatitis, bacterial contamination, and transfusion-associated graft-vs-host disease. Concerns about blood safety have focused much attention on alternative blood transfusion strategies such as autologous blood, viral inactivation, and artificial blood substitutes. This review describes the transfusion medicine delivery system in the United States, with special emphasis on evolving developments and their implications for the discipline of chemical pathology.
Quantitative Analysis of the Bidirectional Fetomaternal Transfer of Nucleated Cells and Plasma DNA
Authors: Lo Y M Dennis , Lau Tze K , Chan Lisa Y S , Leung Tse N , Chang Allan M Z
Published: January 2020

Background: Recently, much interest has been generated on the fetomaternal transfer of nucleated cells and plasma DNA. However, there has been no systematic quantitative comparison of these two directions and two modalities of trafficking within the same study population.

Methods: The fetus-to-mother transfer of nucleated cells and plasma DNA in pregnant women carrying male babies was studied using a real-time quantitative PCR assay for the SRY gene. For mother-to-fetus transfer, real-time quantitative PCR assays for the insertion/deletion polymorphisms involving the glutathione S-transferase M1 and angiotensin-converting enzyme genes were used.

Results: Of the 50 informative mother-baby pairs, maternal DNA was detected in the cellular fraction of umbilical cord blood in 24% of cases (12 of 50), at a median fractional concentration of 2.6 × 10−4 (interquartile range, 1.7 × 10−4 to 3.6 × 10−4). In the plasma fraction of cord blood, maternal DNA was detected in 30% (15 of 50) of cases at a median fractional concentration of 3 × 10−3 (interquartile range, 1 × 10−3 to 1.6 × 10−2). For the other direction of trafficking, fetus-to-mother transfer of nucleated cells was detected in 26% of cases (13 of 50) at a median fractional concentration of 3.2 × 10−4 (interquartile range, 0.6 × 10−4 to 7.6 × 10−4). In the plasma fraction, fetal DNA was detected in 100% of maternal plasma (50 of 50) at a median fractional concentration of 3 × 10−2 (interquartile range, 1.4 × 10−2 to 5.3 × 10−2).

Conclusions: This study indicated that significantly more fetal DNA is present in the plasma of pregnant women compared with DNA from the cellular fraction of maternal blood. In addition, maternal DNA was demonstrated in both the cellular and plasma fractions of cord blood after delivery. This study has therefore determined the fundamental quantitative values for the bidirectional fetomaternal cellular and plasma DNA traffic.
Assessment of Mycophenolic Acid-induced Immunosuppression: A New Approach
Authors: Millan Olga , Oppenheimer Federic , Brunet Merce , Vilardell Jordi , Rojo Isabel , Vives Jordi , Martorell Jaume
Published: January 2020

Background: Mycophenolic acid (MPA), a metabolite of mycophenolate mofetil (MMF), is an immunosuppressive agent that inhibits inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the ex novo synthesis of GTP. We measured IMPDH activity in peripheral blood mononuclear cells (PBMCs) from MMF-treated patients to evaluate the efficacy of MMF in individual patients.

Methods: IMPDH activity was measured by 3H released from [2,8-3H]IMP that had been formed in the cells from added [2,8-3H]hypoxanthine in PBMCs of 35 renal transplant recipients treated with cyclosporin A and corticoids plus MMF: 2 g (n = 10), 1.5 g (n = 7), 1 g (n = 10), or 0 g (n = 8) per day. An alternative method, based on the capacity of the patients’ sera to inhibit spontaneous proliferation of the CEM cell line, was also analyzed.

Results: The IMPDH activity of PBMCs in transplanted patients was highly variable. For the method based on CEM cell line proliferation: (a) cell proliferation was inhibited only in MMF-treated patients; (b) there was a clear postdose increase in inhibition; (c) inhibition was not affected by other immunosuppressants in vitro or in vivo; (d) inhibition from predose to predose sample was correlated; and (e) when the MMF dosage was <20 mg · kg−1 · day−1, two groups of patients were identified, one that maintained a high inhibitory capacity in all dose intervals, and one with periods of low inhibitory capacity.

Conclusions: Measurement of the inhibition of CEM cell line proliferation by sera from MMF-treated patients may be useful for evaluating the relative efficacy of MMF treatment in individual patients, especially those receiving low doses of MMF.
Stability of Common Analytes in Urine Refrigerated for 24 h before Automated Analysis by Test Strips
Authors: Froom Paul , Bieganiec Barbara , Ehrenrich Zahava , Barak Mira
Published: January 2020

Background: Central outpatient laboratories might find processing large numbers of urinary samples that arrive in the late afternoon inconvenient and refrigerate them overnight before testing. Furthermore, in certain settings clinics might have difficulty assuring that the urine arrives at the laboratory during the same day as the collection. Because the stability of urine samples for delayed automated dipstick analysis (Supertron) is unknown, after defining precision, we retested urines refrigerated for 24 h to determine stability.

Methods: Urinalysis was done twice on the same day and repeated after the sample was refrigerated for 24 h. Combur-10S (Roche Diagnostics) dipsticks were read automatically by a Supertron analyzer. Repeat tests on the same day were compared with tests after storage.

Results: Leukocyte esterase had high precision, but after storage ∼25% of the positive samples were less reactive (P <0.005). Precision of hemoglobin retests was also high but declined significantly after storage for 24 h. Urine protein values increased after storage. The precision and stability were excellent for nitrites, glucose, and ketones.

Conclusions: The stability of the automated dipstick urinalysis varies with the substance tested. After refrigeration for 24 h, there is a risk of false-positive results for protein, false-negative results for leukocytes and erythrocytes, and little effect on glucose, nitrite, and ketone values.
Screening for Single-Nucleotide Polymorphisms Using Branch Migration Inhibition in PCR-amplified DNA
Authors: Lishanski Alla
Published: January 2020

Background: New methods are required for the exploration of the human genome by discovering sequence variations. This study evaluated the performance of a new method for screening a large number of samples for several DNA polymorphisms.

Methods: We used a homogeneous method based on inhibition of spontaneous branch migration by any sequence difference between two molecules of PCR-amplified DNA. A set of four PCR primers is required: a forward primer, either biotinylated or labeled with digoxigenin, and two reverse primers that share a priming domain but have different “tail” sequences at their 5′ ends. After PCR amplification, denaturation and reannealing of the single DNA strands produce doubly labeled cruciform structures, which dissociate by strand exchange. The presence of two different alleles in a sample causes complete inhibition of dissociation, and the association of biotin and digoxigenin is homogeneously detected using luminescent oxygen channeling immunoassay.

Results: The 90 samples of the Human Variation Panel (Coriell Cell Repositories) were screened for nine known single-nucleotide polymorphisms (SNPs) and one 5-bp deletion. The average signal-to-background ratio varied from ∼10 to 20. The frequency of the predominant allele for different SNPs varied from 51% to 88% overall. For some SNPs, it varied among the nine ethnic groups, e.g., 25–85% (average, 51%) for one SNP. The average heterozygosity varied from 0.17 to 0.54 and as much as 0.2–0.9 (average, 0.54) for one of the SNPs.

Conclusion: The method allows simple and rapid screening of a large number of samples for the presence of multiple alleles.
Carbohydrate-deficient Transferrin as a Marker of Chronic Alcohol Abuse: A Critical Review of Preanalysis, Analysis, and Interpretation
Authors: Arndt Torsten
Published: January 2020

Background: Carbohydrate-deficient transferrin (CDT) is used for diagnosis of chronic alcohol abuse. Some 200–300 reports on CDT have been published in impact factor-listed journals. The aims of this review were to condense the current knowledge and to resolve remaining issues on CDT.

Approach: The literature (1976–2000) was searched using MEDLINE and Knowledge Server with “alcohol and CDT” as the search items. The data were reviewed systematically, checked for redundancy, and organized in sequence based on the steps involved in CDT analysis.

Content: The review is divided into sections based on microheterogeneity of human serum transferrin (Tf), definition of CDT, structure of human serum CDT, pathomechanisms of ethanol-induced CDT increase, preanalysis, analysis, and medical interpretation (postanalysis). Test-specific cutoff values for serum CDT and causes of false positives and negatives for chronic alcohol abuse are discussed and summarized.

Summary: Asialo- and disialo-Fe2-Tf, which lack one or two complete N-glycans, and monosialo-Fe2-Tf (structure remains unclear) are collectively referred to as CDT. Diminished mRNA concentration and glycoprotein glycosyltransferase activities involved in Tf N-glycan synthesis and increased sialidase activity most likely account for alcohol-induced increases in CDT. Knowledge about in vivo and in vitro effects on serum CDT is poor. Reliable CDT and non-CDT fractionation is needed for CDT measurement. Analysis methods with different analytical specificities and recoveries decreased the comparability of values and statistical parameters of the diagnostic efficiency of CDT. CDT is the most specific marker of chronic alcohol abuse to date. Efforts should concentrate on the pathomechanisms (in vivo), preanalysis, and standardization of CDT analysis.
Successful Diagnosis of Fetal Gender Using Conventional PCR Analysis of Maternal Serum
Authors: Honda Hiroshi , Miharu Norio , Ohashi Yoko , Ohama Koso
Published: January 2020

Background: Fetal DNA has been found in maternal plasma and serum. Diagnosis of fetal gender using maternal plasma and serum has been attempted in an effort to develop a new noninvasive method of prenatal diagnosis.

Methods: Peripheral blood samples were obtained from 61 pregnant women at 10–17 weeks of gestation before amniocentesis. DNA was extracted from 800 μL of each plasma or serum sample. To detect the Y-chromosome-specific sequences DYS14 and DYZ3 in the maternal plasma and serum, 40 cycles of PCR were carried out for each DNA extract. The PCR products were analyzed by 2.5% agarose gel electrophoresis and ethidium bromide staining, and the results were compared with the results of the cytogenetic analyses of amniocentesis.

Results: Cytogenetic analysis of amniocentesis revealed that 31 pregnant women had a male fetus and the remaining 30 pregnant women had a female fetus. Both DYS14 and DYZ3 were detected in 27 of the 31 plasma samples obtained from pregnant women carrying a male fetus and in all of 31 serum samples obtained from the same women. Neither DYS14 nor DYZ3 was detected in either the plasma or serum samples obtained from any of the 30 pregnant women carrying a female fetus.

Conclusion: PCR analysis of maternal serum can be used to diagnose fetal gender.
Tartrate-resistant Acid Phosphatase Isoform 5b as Serum Marker for Osteoclastic Activity
Authors: Janckila Anthony J , Takahashi Karen , Sun Susan Z , Yam Lung T
Published: January 2020

Background: Tartrate-resistant acid phosphatase (AcP) 5b is a marker of osteoclastic activity and bone resorption. Immunoassays for serum TRAcP may lack sensitivity and specificity because of the presence of non-bone isoform 5a. The purpose of this study was to isolate the serum isoforms, quantify their disease-related expressions, and test an improved immunoassay for TRAcP 5b.

Methods: We separated TRAcP isoforms chromatographically from pooled sera of healthy, rheumatoid arthritis (RA) and endstage renal disease (ESRD) subjects. TRAcP isoforms were identified by electrophoresis and quantified by biochemical and immunochemical assays. Serum TRAcP activity in healthy, RA, and ESRD cohorts was assessed at pH 5.5 and 6.1, and compared with bone alkaline phosphatase (BAP) and N-telopeptides of type I collagen (NTx).

Results: TRAcP isoforms 5a and 5b were present in all sera; 5b was identical to osteoclastic TRAcP. In serum from healthy subjects, 5a accounted for 87% of the enzyme protein but only 55% of the activity. In RA, both isoforms were increased two- to threefold in protein, but their specific activities were subnormal. In ESRD, only 5b was abnormal, being increased fivefold in protein and threefold in activity. In RA sera, TRAcP activity did not correlate with either BAP or NTx. In ESRD sera, TRAcP activity correlated with BAP and NTx only when measured at pH 6.1.

Conclusions: All sera contained both TRAcP isoforms 5a and 5b, but only 5b was present in bone. TRAcP isoform expression was variable in different diseases. Measurement of TRAcP activity at pH 6.1 improves the specificity of immunoassay for isoform 5b.
Evidence for a Substantial Genetic Influence on Biochemical Liver Function Tests: Results from a Population-based Danish Twin Study
Authors: Bathum Lise , Petersen Hans Christian , Rosholm Jens-Ulrik , Hyltoft Petersen Per , Vaupel James , Christensen Kaare
Published: January 2020

Background: Biochemical liver function tests are widely used in the clinic and are some of the most frequently used tests in screening for diseases in older age groups. The aim of the present study was to estimate the relative importance of genetic and environmental factors to variations in these tests among the elderly.

Methods: We conducted a survey among Danish twins, 73–102 years of age, identified in the population-based Danish Twin Registry. Among the 2749 individuals in the study population, an interview was conducted with 79%. From these, a blood sample was collected from 290 same-sex twin pairs, total of 580 subjects, within a 6-month period and analyzed for alanine aminotransferase (ALT), lactate dehydrogenase (LDH), γ-glutamyltransferase (GGT), bilirubin, and albumin. The interview included questions about alcohol consumption and body mass index (BMI; self-calculated height and weight). Heritability (proportion of the population variance attributable to genetic variation) was estimated using structural-equation analyses before and after correction for alcohol consumption and BMI.

Results: Structural-equation analyses revealed a substantial heritability (35–61%) for the four biochemical liver function tests: ALT, GGT, LDH, and bilirubin. The remaining variation could be attributed to individuals’ nonfamilial environments. Adjustment for alcohol consumption and BMI had no influence on the heritability for ALT, GGT, LDH, and bilirubin. For albumin, two models fit equally well before adjustment for alcohol and BMI: a model including additive genetic and nonshared environmental factors (AE), and a model including shared and nonshared environmental factors (CE). After adjustment, the model including shared and nonshared environment was clearly the best fitting model.

Conclusions: For both males and females, the effect of genetic factors on the biochemical liver function tests ALT, GGT, LDH, and bilirubin is substantial and accounts for one-third to two-thirds of the variation among individuals 73–102 years of age. The heritability is equal for males and females and does not change notably after controlling for alcohol consumption and BMI. For albumin, no major impact of genetic factors was found independent of BMI and alcohol consumption. An understanding of the genetic mechanisms underlying biochemical liver function tests among the very old may be of value in the interpretation of these tests in this age group.
Correlation of Mycophenolic Acid Pharmacokinetic Parameters with Side Effects in Kidney Transplant Patients Treated with Mycophenolate Mofetil
Authors: Mourad Michel , Malaise Jacques , Chaib Eddour Djamila , De Meyer Martine , König Josiane , Schepers Raf , Squifflet Jean Paul , Wallemacq Pierre
Published: January 2020

Background: Mycophenolate mofetil (MMF) is widely used in organ transplantation to prevent acute rejection. Because MMF can produce hematologic and/or gastrointestinal toxicity, therapeutic monitoring is becoming mandatory. This study was designed to investigate the relationship between the clinical events and the pharmacokinetics of mycophenolic acid (MPA) in adult renal transplantation.

Methods: Thirty-one adult kidney recipients were prospectively included in the study. MPA pharmacokinetic profiles (blood sampling at 0, 0.5, 1, 2, 4, 6, and 12 h after MMF oral dose) were obtained after transplantation (desired creatinine clearance, 40 mL/min), at 3 months after grafting, and at every clinical event (e.g., side effect or rejection). All patients received a 10-day course of anti-thymocyte globulin, cyclosporine, MMF (1 g twice daily), and steroids.

Results: We divided the 31 patients into two groups (groups 1 and 2). Ten patients (32%; group 1) had uneventful outcomes, and 21 patients (68%; group 2) presented with MPA-related side effects. For groups 1 and 2, the MPA trough concentrations (Cmin) were 1.63 ± 1.07 and 2.29 ± 1.16 mg/L, respectively (P = 0.06), and the areas under the curve (AUCs) for MPA from t0 to t12 h (MPA-AUC0–12h) were 39.80 ± 15.29 and 62.10 ± 21.07 mg · h/L, respectively (P = 0.0005, two-sample t-test). Three patients experienced acute graft rejection after the oral MMF dose was reduced because of side effects. In this group, the MPA-Cmin and MPA-AUC were significantly lower by the time acute rejection occurred (1.00 ± 0.45 mg/L and 25.00 ± 6.20 mg · h/L, respectively). At a fixed dose (1 g twice per day), we compared the pharmacokinetic parameters of MPA [Cmin, the MPA concentration 30 min after the oral dose of MMF (C30), and AUC] according to the presence or absence of side effects in the two groups. Cmin and AUC did not differ between the two groups [Cmin = 2.22 ± 1.13 vs 2.17 ± 1.13 mg/L (P = 0.9); AUC = 66.82 ± 29.87 vs 55.70 ± 11.74 mg · h/L (P = 0.11)]; and C30 was significantly higher in group 2 than in group 1 (C30 = 32.99 ± 12.59 vs 7.45 ± 5.40 mg/L; P <0.0001).

Conclusions: Our results demonstrate a pharmacokinetic/pharmacodynamic relationship between MPA and clinical events. At a fixed dose of 2 g/day, a high C30 is associated with increased risk for side effects. This study suggests that dividing the MMF daily oral dose into more than two divided doses might prevent early MPA toxicity.
Measurement of Homocysteine and Other Aminothiols in Plasma: Advantages of Using Tris(2-carboxyethyl)phosphine as Reductant Compared with Tri-n-butylphosphine
Authors: Krijt Jakub , Vacková Martina , Kožich Viktor
Published: January 2020

Background: Aminothiols have been implicated in the pathogenesis of arteriosclerosis, and reliable methods are needed to determine their concentrations in body fluids. We present a comparison of two analytical methods and focus on the reduction of low-molecular weight and protein-mixed disulfides of homocysteine, cysteine, cysteinyl-glycine, and glutathione.

Methods: The plasma total aminothiol profile was determined by HPLC with fluorescence detection after derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate. Disulfides and protein-bound aminothiols were reduced by either tri-n-butylphosphine (the TBP method) or tris(2-carboxyethyl)phosphine (the TCEP method); the effects of temperature, time of reduction, and concentration of reductants were evaluated.

Results: The intraassay imprecision (CV) was <3% for all aminothiols using both methods. The interassay CVs for total cysteine (tCys), total cysteinyl-glycine (tCys-Gly), and total homocysteine (tHcy) were <4% and <8% for the TCEP and TBP methods, respectively, whereas for total glutathione (tGSH) the interassay CV was >12% for both methods. Deming regression and Bland–Altman difference plots showed positive biases for total aminothiol concentrations determined by the TCEP method relative to the TBP method. The mean proportional biases were 65%, 27%, 6%, and 60% for tCys, tCys-Gly, tHcy, and tGSH, respectively. The calculated concentrations of total aminothiols by the TCEP method were less influenced by changes in temperature and concentration of reducing agent or by calibrator matrix.

Conclusions: The agreement between the TCEP and TBP methods was considerably lower for the determination of tCys, tCys-Gly, and tGSH than for tHcy. For total-aminothiol determination, the TCEP method yields better reproducibility and is more robust than the TBP method.
Proteomics in Early Detection of Cancer
Authors: Srinivas Pothur R , Srivastava Sudhir , Hanash Sam , Wright George L
Published: January 2020

Early detection is critical in cancer control and prevention. Biomarkers help in this process by providing valuable information about a the status of a cell at any given point in time. As a cell transforms from nondiseased to neoplastic, distinct changes occur that could be potentially detected through the identification of the appropriate biomarkers. Biomarker research has benefited from advances in technology such as proteomics. We discuss here ongoing research in this field, focusing on proteomic technologies. The advances in two-dimensional electrophoresis and mass spectrometry are discussed in light of their contribution to biomarker research. Chip-based techniques, such as surface-enhanced laser desorption, and ionization and emerging methods, such as tissue and antibody arrays, are also discussed. The development of bioinformatic tools that have and are being developed in parallel to proteomics is also addressed. This report brings into focus the efforts of the Early Detection Research Network at the National Cancer Institute in harnessing scientific expertise from leading institutions to identify and validate biomarkers for early detection and risk assessment.
Divergence between LDL Oxidative Susceptibility and Urinary F2-Isoprostanes as Measures of Oxidative Stress in Type 2 Diabetes
Authors: Devaraj Sridevi , Hirany Shaina V , Burk Raymond F , Jialal Ishwarlal
Published: January 2020

Background: Oxidative stress is pivotal in atherogenesis. Although the most widely used indirect assay to quantify oxidative stress is LDL oxidative susceptibility, direct assays such as urinary F2-isoprostanes have shown great promise.

Methods: We evaluated the utility of both a direct measure of oxidative stress (urinary F2-isoprostanes) and an indirect measure of copper-catalyzed, LDL oxidation in a model of increased oxidative stress (diabetes). We also evaluated an enzyme immunoassay (EIA) method for urinary F2-isoprostanes with a gas chromatography–mass spectrometry method.

Results: Excellent intraassay and interassay CVs of <4% were obtained with our EIA method. A good correlation was obtained between the two methods (r = 0.80; n = 68) of F2-isoprostane measurement. An excellent correlation for F2-isoprostane concentrations was obtained between a timed collection vs 24-h urine (r = 0.96; n = 46). Baseline F2-isoprostane concentrations by EIA were significantly higher in both type 2 diabetics with and without macrovascular complications compared with controls (P <0.001). Supplementation with α-tocopherol led to a significant reduction in F2-isoprostane concentrations in all diabetic patients compared with baseline values (2.51 ± 1.76 compared with 1.69 ± 1.32 ng/mg creatinine; P <0.001). There were no significant differences in LDL oxidation in both diabetic groups compared with controls. α-Tocopherol supplementation led to significant increases in the lag phase of oxidation as measured by 3 indices in all groups.

Conclusions: The measurement of urinary F2-isoprostanes provides a direct measure of in vivo lipid peroxidation and oxidative stress and appears to be superior to an indirect measure, e.g., LDL oxidative susceptibility, in type 2 diabetes.
Predicting the Severity of Acute Pancreatitis by Rapid Measurement of Trypsinogen-2 in Urine
Authors: Lempinen Marko , Kylänpää-Bäck Marja-Leena , Stenman Ulf-Håkan , Puolakkainen Pauli , Haapiainen Reijo , Finne Patrik , Korvuo Armi , Kemppainen Esko
Published: January 2020

Background: Early identification of patients at risk of developing a severe attack of acute pancreatitis (AP) is of great importance because rapid therapeutic interventions improve outcome. At a cutoff of 50 μg/L, trypsinogen-2 measured by a rapid urinary dipstick is a sensitive and specific diagnostic test in AP. The trypsinogen-2 concentration correlates with the severity of the disease, and a test with a higher cutoff might therefore be useful for prediction of disease severity.

Methods: We increased the detection limit of the urinary trypsinogen-2 test strip (Actim Pancreatitis) from 50 μg/L to 2000 μg/L and evaluated the prognostic value of this test. The results were compared with those obtained with serum C-reactive protein and the acute physiology and chronic health evaluation II (APACHE II) score. The study population consisted of 150 consecutive patients with AP (42 with severe disease).

Results: The sensitivity of the rapid urinary test strip (detection limit, 2000 μg/L) for prediction of severe AP, both on admission and at 24 h, was 62%; specificities were 87% and 85%, respectively, positive predictive values were 65% and 62%, and negative predictive values were 85% and 85%. C-Reactive protein had a sensitivity of only 38% on admission, but at 24 h, it was 83%; specificities were 90% and 70%, respectively, whereas positive predictive values were 59% and 52%, and NPVs were 79% and 91%, respectively. On admission the positive-likelihood ratio for the urinary trypsinogen-2 test strip was 4.8, and at 24 h it was 4.2; for C-reactive protein, the values were 3.7 and 2.7, respectively.

Conclusions: The urinary trypsinogen-2 dipstick is a simple and rapid method for prediction of severe acute pancreatitis.
Detection of Early Pregnancy Forms of Human Chorionic Gonadotropin by Home Pregnancy Test Devices
Authors: Butler Stephen A , Khanlian Sarah A , Cole Laurence A
Published: January 2020

Background: Home pregnancy testing devices claim >99% diagnostic accuracy for pregnancy and utility on the first day of the missed menses or earlier. We investigated the forms of human chorionic gonadotropin (hCG) in early pregnancy urines, the diagnostic accuracy claim, and the abilities of 15 devices to detect the different forms of hCG.

Methods: We measured the concentrations of regular hCG and hyperglycosylated hCG (H-hCG, a large hCG variant) in 592 urines. Fifteen home devices were tested according to manufacturers’ instructions with regular hCG and H-hCG diluted in urine.

Results: H-hCG was the principal hCG-related molecule in pregnancy urine in the 2 weeks following the missed menses (61% and 50% of total immunoreactivity in the 4th and 5th completed weeks of pregnancy, respectively). Of 15 home test devices, 2 had a detection limit of 6.3 IU/L for regular hCG, but poorer detection of H-hCG. Two devices detected 13 IU/L regular hCG, one with similar detection and one with poorer detection of H-hCG. Ten devices detected 25 IU/L regular hCG, 6 with poorer detection of H-hCG. One device detected 50 IU/L regular hCG, but better detected H-hCG. Overall, 9 of 15 devices did not detect H-hCG as well as regular hCG.

Conclusions: H-hCG is the principal hCG immunoreactivity in early pregnancy urine. Home tests vary widely in detection limits for regular hCG (6.3–50 IU/L), and 9 of 15 devices (60%) had poorer detection limits for H-hCG than for hCG. The variation in analytical detection limits appears contradictory to the common claim for all devices of >99% detection of pregnancy on the first day of the missed menses or earlier. We suggest that manufacturers calibrate devices for both hCG and H-hCG and determine the detection rates for pregnancy rather than the proportion of positive results at arbitrary hCG concentrations.