bluuebunny/crossref_metadata_2025 huggingface dataset:
Arsenic Metabolites in Human Urine after Ingestion of an Arsenosugar
Authors: Francesconi Kevin A , Tanggaar René , McKenzie Christine J , Goessler Walter
Published: January 2020

Background: Arsenic-containing carbohydrates (arsenosugars) are common constituents of marine algae, including those species used as human food. The toxicology of these compounds has not been fully evaluated.

Methods: Arsenic metabolites in human urine were monitored over a 4-day period after ingestion of a synthetic specimen of arsenosugar. The metabolites were determined by HPLC-inductively coupled plasma mass spectrometry, and structural assignments were confirmed with liquid chromatography-electrospray ionization mass spectrometry.

Results: Approximately 80% of the total ingested arsenic was excreted in the urine during the 4 days of the experiment. There was a lag-period of ∼13 h before substantial quantities of arsenic appeared in the urine, and the excretion rate peaked between 22 and 31 h. At least 12 arsenic metabolites were detected, only 3 of which could be positively identified. Dimethylarsinate (DMA) was the major metabolite, constituting 67% of the total arsenicals excreted. A new urinary arsenic metabolite, dimethylarsinoylethanol, represented 5% of the total arsenicals, whereas trimethylarsine oxide was present as a trace (0.5%) constituent. One other significant metabolite cochromatographed with a reduced DMA standard, and hence was possibly dimethylarsinous acid. The second most abundant metabolite in the urine (20% of the total arsenic) remained unidentified, whereas the rest of the excreted arsenic was made up of several trace metabolites and small amounts of unchanged arsenosugar.

Conclusions: Arsenosugars are biotransformed by humans to at least 12 arsenic metabolites, the toxicologies of which are currently unknown.
Boosted Decision Tree Analysis of Surface-enhanced Laser Desorption/Ionization Mass Spectral Serum Profiles Discriminates Prostate Cancer from Noncancer Patients
Authors: Qu Yinsheng , Adam Bao-Ling , Yasui Yutaka , Ward Michael D , Cazares Lisa H , Schellhammer Paul F , Feng Ziding , Semmes O John , Wright George L
Published: January 2020

Background: The low specificity of the prostate-specific antigen (PSA) test makes it a poor biomarker for early detection of prostate cancer (PCA). Because single biomarkers most likely will not be found that are expressed by all genetic forms of PCA, we evaluated and developed a proteomic approach for the simultaneous detection and analysis of multiple proteins for the differentiation of PCA from noncancer patients.

Methods: Serum samples from 386 men [197 with PCA, 92 with benign prostatic hyperplasia (BPH), and 96 healthy individuals], randomly divided into training (n = 326) and test (n = 60) sets, were analyzed by surface-enhanced laser desorption/ionization (SELDI) mass spectrometry. The 124 peaks detected by computer analyses were analyzed in the training set by a boosting tree algorithm to develop a classifier for separating PCA from the noncancer groups. The classifier was then challenged with the test set (30 PCA samples, 15 BPH samples, 15 samples from healthy men) to determine the validity and accuracy of the classification system.

Results: Two classifiers were developed. The AdaBoost classifier completely separated the PCA from the noncancer samples, achieving 100% sensitivity and specificity. The second classifier, the Boosted Decision Stump Feature Selection classifier, was easier to interpret and used only 21 (compared with 74) peaks and a combination of 21 (vs 500) base classifiers to achieve a sensitivity and specificity of 97% for the test set.

Conclusions: The high sensitivity and specificity achieved in this study provides support of the potential for SELDI, coupled with a bioinformatics learning algorithm, to improve the early detection/diagnosis of PCA.
Effect of Analytical Error on the Assessment of Cardiac Risk by the High-Sensitivity C-Reactive Protein and Lipid Screening Model
Authors: Middleton John
Published: January 2020

Background: Several prospective epidemiologic studies have demonstrated that high-sensitivity C-reactive protein (hsCRP) is an effective serum marker for cardiac risk assessment. When hsCRP is considered in conjunction with traditional lipid screening, its clinical utility is further increased. In this report, hsCRP, HDL-cholesterol (HDLC), and total cholesterol (TC) assay imprecision is evaluated in terms of the impact on the cardiac risk assessment process.

Methods: Cardiac risk assessment events were simulated using software written in Visual Basic for Applications with Microsoft Excel. Representative sets of analyte concentrations were used for true patient cardiac marker values. Monte Carlo simulations about the marker values were run using assay SD estimates based on College of American Pathologists surveys and journal articles.

Results: Risk distributions for reasonable assay imprecision showed clinically significant variation. Estimated relative risks of cardiovascular disease using the Ridker–Rifai quintile model varied by as much as 2.5-, 3.5-, and 6-fold for conditions of low, medium, and high laboratory test imprecision, respectively. The true relative risks were underestimated by >25% in 3%, 6.7%, and 10.5% of cases under conditions of low, medium, and high laboratory test imprecision, respectively, and overestimated by >25% in 4.4%, 8.5%, and 11.1% of cases under those conditions.

Conclusions: Use of the Monte Carlo simulation method as a tool to assess the impact of analytical variation on the clinical decision-making process is valuable. From this analysis, it is shown that multiple measurements of HDLC may reduce misclassifications that result from assay imprecision. This is most important when using HDLC assays that include sample preparation with 500 000 molecular weight dextran sulfate. For these assays the total assay method SDs are higher than for other assay methods. In addition, as demonstrated by a propagation of error analysis, HDLC has the largest component of overall error in the relative risk estimate. Under conditions of typical TC and hsCRP assay performance, replication of these assays is less important.
Effects of Temperature on Stability of Blood Homocysteine in Collection Tubes Containing 3-Deazaadenosine
Authors: Hill Diane M , Johnson Lisa J , Burns Paul J , Neale Angela M , Harmening Denise M , Kenney Andrew C
Published: January 2020

Background: The accuracy of homocysteine (Hcy) results is currently compromised by the requirement to separate the plasma within 1 h of sample collection. We studied the effect of temperature on the stability of plasma Hcy over a 72-h time course in blood collected into evacuated tubes containing either EDTA alone or both EDTA and 3-deazaadenosine (3DA).

Methods: We recruited 100 volunteers, including both diseased and healthy individuals with a range of baseline plasma Hcy values, from two centers. Blood samples were collected into tubes containing EDTA, and EDTA plus 3DA and stored at ambient temperature (20–25 °C) or refrigerated (2–8 °C). Aliquots of blood were centrifuged at various times up to 72 h, the plasma was removed, and Hcy was measured by HPLC.

Results: Plasma Hcy measurement covering the sample collection and storage conditions during the whole time course was possible on samples from 59 of those recruited. One-way ANOVA for repeated measures within subjects revealed that only samples that were collected into tubes containing EDTA plus 3DA and stored refrigerated were stable over 72 h (P = 0.2761).

Conclusions: A combination of 3DA and storage at 2–8 °C will allow collection of samples for plasma Hcy measurement outside of the hospital setting and wider population screening.
Fetal DNA Clearance from Maternal Plasma Is Impaired in Preeclampsia
Authors: Lau Tai-Wah , Leung Tse N , Chan Lisa YS , Lau Tze K , Chan KC Allen , Tam Wing H , Lo YM Dennis
Published: January 2020

Background: Increased fetal DNA in maternal plasma/serum has been reported in pregnancies complicated by preeclampsia. We hypothesized that impaired clearance of fetal DNA might contribute, at least in part, to the above-mentioned phenomenon.

Methods: We studied 7 preeclamptic and 10 control pregnant women. All had male fetuses. Serial blood samples were obtained from before delivery to 6 h postpartum. Male fetal DNA in maternal plasma was measured by real-time quantitative PCR for the SRY gene on the Y chromosome.

Results: The median fetal DNA concentrations before delivery were significantly higher in the preeclamptic women than in the controls (521 vs 227 genome-equivalents/mL for preeclamptic and control women, respectively; Mann–Whitney rank-sum test, P = 0.017). The median fetal DNA concentrations at 6 h after delivery were also significantly different between the two groups (208 vs 0 genome-equivalents/mL for preeclamptic and control women, respectively; Mann–Whitney rank-sum test, P = 0.002). A first-order clearance model was found to best describe the kinetics of maternal plasma fetal DNA clearance. Moreover, we observed a significant difference in the median apparent clearance half-lives of fetal DNA between the preeclamptic women (114 min) and controls (28 min; Mann–Whitney rank-sum test, P = 0.007).

Conclusions: This study represents the first documentation of impaired fetal DNA clearance from maternal plasma in preeclampsia. Such an abnormality in circulating DNA clearance may also be present in other medical conditions associated with quantitative aberrations in circulating DNA concentrations.
High-Throughput Quantification of Lysophosphatidylcholine by Electrospray Ionization Tandem Mass Spectrometry
Authors: Liebisch Gerhard , Drobnik Wolfgang , Lieser Bernd , Schmitz Gerd
Published: January 2020

Background: Lysophosphatidylcholine (LPC) has been suggested to play a functional role in various diseases, including atherosclerosis, diabetes, and cancer mediated by LPC-specific G-protein-coupled receptors. Initial studies provided evidence for a potential use of LPC as diagnostic maker. However, existing methodologies are of limited value for a systematic evaluation of LPC species concentrations because of complicated, time-consuming procedures. We describe a methodology based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) applicable for high-throughput LPC quantification.

Methods: Crude lipid extracts of EDTA-plasma samples were used for direct flow injection analysis. LPC 13:0 and LPC 19:0 were added as internal standards, and the ESI-MS/MS was operated in the parent-scan mode for m/z 184. Quantification was achieved by standard addition. Data processing was highly automated by use of the mass spectrometer software and self-programmed Excel macros.

Results: The calibrators LPC 16:0, LPC 18:0, and LPC 22:0 showed a linear response independent of sample dilution and plasma cholesterol concentration for both internal standards. The within-run imprecision (CV) was 3% for the major and 12% for the minor species, whereas the total imprecision was ∼12% for the major and 25% for the minor species. The detection limit was <1 μmol/L.

Conclusion: The developed ESI-MS/MS methodology with an analysis time of 2 min/sample, simple sample preparation, and automated data analysis allows high-throughput quantification of distinct LPC species from plasma samples, which could be a valuable tool for the evaluation of LPC as diagnostic marker.
Stability Studies of Twenty-Four Analytes in Human Plasma and Serum
Authors: Boyanton Bobby L , Blick Kenneth E
Published: January 2020

Background: The stability and stoichiometric changes of analytes in plasma and serum after prolonged contact with blood cells in uncentrifuged Vacutainer® tubes were studied.

Methods: We simultaneously investigated the stability of 24 analytes (a) after prolonged contact of plasma and serum with blood cells and (b) after immediate separation of plasma and serum (centrifuged twice at 2000g for 5 min). We verified biochemical mechanisms of observed analyte change by concomitant measurement of pH, Pco2, and Po2. Hemolysis was qualitatively and semiquantitatively assessed. All specimens were maintained at room temperature (25 °C) and analyzed in duplicate 0.5, 4, 8, 16, 24, 32, 40, 48, and 56 h after collection. Statistically significant changes from the 0.5 h mean were determined using repeated-measures ANOVA. The significant change limit was applied to determine clinically significant changes in measured analytes.

Results: Fifteen of 24 analytes in plasma and serum maintained in contact with cells showed clinically relevant changes, with the degree of change more pronounced in most plasma specimens. All analytes in plasma and serum immediately separated from cells after collection were stable.

Conclusion: Storage of uncentrifuged specimens beyond 24 h caused significant changes in most analytes investigated because of (a) glucose depletion and Na+,K+-ATPase pump failure; (b) the movement of water into cells, causing hemoconcentration; and (c) leakage of intracellular constituents and metabolites. Immediate separation of plasma or serum from cells provides optimal analyte stability at room temperature. When prolonged contact of plasma or serum with cells is unavoidable, use of serum is recommended because of the higher instability of plasma analytes.
Markers of Triglyceride-rich Lipoprotein Remnant Metabolism in Visceral Obesity
Authors: Chan Dick C , Watts Gerald F , Barrett P Hugh , Mamo John CL , Redgrave Trevor G
Published: January 2020

Background: Triglyceride-rich lipoprotein remnants are atherogenic, and this may be particularly important in visceral obesity. We investigated remnant metabolism in obese men by measuring remnant-like particle-cholesterol (RLP-C), apolipoprotein (apo) B-48, apoC-III, and the clearance of a labeled remnant-like emulsion.

Methods: Fasting RLP-C, apoB-48, and apoC-III concentrations were measured in 48 viscerally obese men and 10 lean controls. RLP-C was determined by immunoseparation assay, apoB-48 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enhanced chemiluminescence, and apoC-III by immunoturbidimetric assay. The catabolism of chylomicron remnants was measured by intravenous injection of a remnant-like emulsion containing cholesteryl [13C]oleate, with isotopic enrichment of 13CO2 in breath determined by isotope-ratio mass spectrometry and a multicompartmental model to estimate fractional catabolic rate (FCR) of the emulsion.

Results: Compared with controls, obese men had significantly increased plasma concentrations of RLP-C, apoB-48, and apoC-III (P <0.001 for all). Plasma total apoB-100, non-HDL-cholesterol, LDL-cholesterol, triglycerides, and insulin resistance (HOMA score) were also significantly higher in the obese group (P <0.001 for all). Obese men had a significantly lower FCR of the remnant-like emulsion compared with controls (P = 0.020).

Conclusions: Viscerally obese individuals have insulin resistance and increased plasma concentrations of triglyceride-rich lipoprotein remnants, which may be attributable to decreased catabolism of these particles.
Rapid Analysis of Metanephrine and Normetanephrine in Urine by Gas Chromatography-Mass Spectrometry
Authors: Crockett David K , Frank Elizabeth L , Roberts William L
Published: January 2020

Background: Widely used HPLC methods for quantification of metanephrine and normetanephrine in urine often have long analysis times and are frequently plagued by drug interferences. We describe a gas chromatography-mass spectrometry method designed to overcome these limitations.

Methods: Metanephrine and normetanephrine conjugates were converted to unconjugated metanephrine and normetanephrine by acid hydrolysis. To avoid the rapid decomposition of the deuterated internal standards (metanephrine-d3 and normetanephrine-d3) under hydrolysis conditions, the internal standards were added after hydrolysis. Solid-phase extraction was used to isolate the hydrolyzed metanephrines from urine. Samples were concentrated by evaporation, then derivatized simultaneously with N-methyl-N-(trimethylsilyl)trifluoroacetamide and N-methyl-bis-heptafluoro-butryamide at room temperature.

Results: The assay was linear from 25 to 7000 μg/L. The intraassay CVs were <5% and the interassay CVs <12%. Comparison with a routine HPLC method (n = 192) by Deming regression yielded a slope of 1.00 ± 0.02 μg/L, an intercept of −5.8 ± 7.8 μg/L, and Sy|x = 50.6 μg/L for metanephrine and a slope of 0.94 ± 0.03, intercept of 19 ± 11 μg/L, and Sy|x = 60 μg/L for normetanephrine. The correlation coefficients (r) were calculated after log transformation of the data and gave r = 0.97 for metanephrine and r = 0.97 for normetanephrine. Interference from common medications or drug metabolites was seen in <1% of samples. The time between sequential injections was <7 min.

Conclusions: This new gas chromatography-mass spectrometry assay for total fractionated metanephrines is rapid, compares well with a standard HPLC assay, and avoids most drug interferences that commonly affect HPLC assays for urine metanephrines.
Synthetic Peptides Identified from Phage-displayed Combinatorial Libraries as Immunodiagnostic Assay Surrogate Quality-Control Targets
Authors: Sompuram Seshi R , Kodela Vani , Ramanathan Halasya , Wescott Charles , Radcliffe Gail , Bogen Steven A
Published: January 2020

Background: Quantitative immunohistochemical (IHC) assays currently lack optimal reference quality-control material for cellular protein targets. To address this problem, we identified peptides that mimic the site on the native analyte to which the primary (monoclonal) antibody binds and used them as surrogate peptide controls.

Methods: We identified peptide candidates from a combinatorial peptide phage-display library that mimic the epitope for the 1D5 estrogen receptor (ER) monoclonal antibody (mAb). The peptide inserts of the phage clones were sequenced. Several phage-encoded peptides were then synthesized and analyzed for affinity and specificity.

Results: We identified phage clones that specifically bound to the ER 1D5 mAb. The binding was specific, in that the phage clones did not bind to two other isotype-matched mAbs. Their ability to bind the ER 1D5 mAb was related to the presence of a consensus sequence. Binding analysis revealed a Kd of 8.3 × 10−8 mol/L. The peptide was not recognized by any of 15 other mAbs commonly used for clinical IHC testing. Moreover, the peptide was able to inhibit the binding of ER 1D5 mAb to native ER, indicating that the peptide bound to ER 1D5 mAb at or close to the antigen-binding site.

Conclusions: Surrogate peptide controls behave like the native analyte in terms of affinity and specificity. This technology may be especially useful when the native analyte is in short supply.
Can Measurement of Serum Apolipoprotein B Replace the Lipid Profile Monitoring of Patients with Lipoprotein Disorders?
Authors: Miremadi Saman , Sniderman Allan , Frohlich Jiri
Published: January 2020

Background: Current clinical guidelines require that five indices (total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, and the total/HDL cholesterol ratio) be measured or calculated to assess the lipid-related risk of vascular disease. All five are also targets of therapy and therefore all must be measured initially and at follow-up. Considerable evidence indicates that apolipoprotein B (apo B) is a better index of reaching or not reaching treatment targets than total or LDL cholesterol.

Methods: The objective of this study was to examine whether measurement of a single marker (apo B) led to the same categorization of risk as the traditional five indices (lipid profile). If both apo B and lipid profile indicated that the patient was either within or outside their respective treatment targets, the indices were considered concordant. If not, the indices were considered discordant. Concordance/discordance was examined in 215 patients at their first and last clinic visit.

Results: Concordance was high in both higher (88% at the first and 92% at the last clinic visit) and lower (76% at the first and 78% at the last clinic visit) risk groups at both the initial and final visits. Discordance was virtually restricted to the group with hypertriglyceridemia with normal concentrations of apo B, a group in which little independent evidence points to any substantially increased risk of vascular disease.

Conclusions: These data raise the possibility that at least for high risk patients treated with statins, follow-up could be simplified and expenses reduced if only apo B were measured. They also raise the possibility that outcome might be improved if the therapeutic algorithm were simplified.
Validation of Liquid Chromatography–Tandem Mass Spectrometry Method for Analysis of Urinary Conjugated Metanephrine and Normetanephrine for Screening of Pheochromocytoma
Authors: Taylor Robert L , Singh Ravinder J
Published: January 2020

Background: Metanephrines are biochemical markers for tumors of the adrenal medulla (e.g., pheochromocytoma) and other tumors derived from neural crest cells (e.g., paragangliomas and neuroblastomas). We describe a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the measurement of urinary conjugated metanephrines.

Methods: We added 250 ng of d3-metanephrine (d3-MN) and 500 ng of d3-normetanephrine (d3-NMN) to 1 mL of urine samples as stable isotope internal standards. The samples were then acidified, hydrolyzed for 20 min in a 100 °C water bath, neutralized, and prepared by solid-phase extraction. The methanol eluates were analyzed by LC-MS/MS in the selected-reaction-monitoring mode after separation on a reversed-phase amide C16 column.

Results: Multiple calibration curves for the analysis of urine MN and NMN exhibited consistent linearity and reproducibility in the range of 10–5000 μg/L. Interassay CVs were 5.7–8.6% at mean concentrations of 90–4854 μg/L for MN and NMN. The detection limit was 10 μg/L. Recovery of MN and NMN (144–2300 μg/L) added to urine was 91–114%. The regression equation for the LC-MS/MS (x) and colorimetric (y) methods was: y = 0.81x − 0.006 (r = 0.822; n = 110). The equation for the HPLC (x) and LC-MS/MS (y) methods was: y = 1.09x + 0.05 (r = 0.998; n = 40).

Conclusions: The sensitivity and specificity of the MS/MS method for urinary conjugated metanephrines offer advantages over colorimetric, immunoassay, HPLC, and gas chromatography–mass spectrometry methods because of elimination of drug interferences, high throughput, and short chromatographic run time.
Errors in Laboratory Medicine
Authors: Bonini Pierangelo , Plebani Mario , Ceriotti Ferruccio , Rubboli Francesca
Published: January 2020

Background: The problem of medical errors has recently received a great deal of attention, which will probably increase. In this minireview, we focus on this issue in the fields of laboratory medicine and blood transfusion.

Methods: We conducted several MEDLINE queries and searched the literature by hand. Searches were limited to the last 8 years to identify results that were not biased by obsolete technology. In addition, data on the frequency and type of preanalytical errors in our institution were collected.

Results: Our search revealed large heterogeneity in study designs and quality on this topic as well as relatively few available data and the lack of a shared definition of “laboratory error” (also referred to as “blunder”, “mistake”, “problem”, or “defect”). Despite these limitations, there was considerable concordance on the distribution of errors throughout the laboratory working process: most occurred in the pre- or postanalytical phases, whereas a minority (13–32% according to the studies) occurred in the analytical portion. The reported frequency of errors was related to how they were identified: when a careful process analysis was performed, substantially more errors were discovered than when studies relied on complaints or report of near accidents.

Conclusions: The large heterogeneity of literature on laboratory errors together with the prevalence of evidence that most errors occur in the preanalytical phase suggest the implementation of a more rigorous methodology for error detection and classification and the adoption of proper technologies for error reduction. Clinical audits should be used as a tool to detect errors caused by organizational problems outside the laboratory.
β-Trace Protein, Cystatin C, β2-Microglobulin, and Creatinine Compared for Detecting Impaired Glomerular Filtration Rates in Children
Authors: Filler Guido , Priem Friedrich , Lepage Nathalie , Sinha Pranav , Vollmer Ilka , Clark Heather , Keely Erin , Matzinger Mary , Akbari Ayub , Althaus Harald , Jung Klaus
Published: January 2020

Background: Because of the limitations of serum creatinine as a marker of glomerular filtration rate (GFR) in children, we assessed the diagnostic accuracy of the novel marker β-trace protein (BTP) in comparison with cystatin C (Cys-C), β2-microglobulin (β2-MG), and creatinine as conventional indicators of reduced GFR.

Methods: We obtained serum samples from 225 children (age range, 0.2–18 years) with various renal pathologies who were referred for nuclear medicine clearance investigations (technetium-diethylenetriamine pentaacetic acid or chromium-EDTA). We measured Cys-C, BTP (nephelometric tests; Dade Behring), β2-MG (Tinaquant; Roche), and creatinine (enzymatic assay; Creatinine-PAP; Roche).

Results: Seventy-five children had reduced GFR (<90 mL · min−1 · 1.73 m−2). One hundred fifty children (independent of gender and age) with values >90 mL · min−1 · 1.73 m−2 comprised the control group with gaussian distributions of BTP and Cys-C concentrations. The upper reference limits (97.5 percentile) were 1.01 mg/L for BTP and 1.20 mg/L for Cys-C. The correlations of nuclear medicine clearance with the reciprocals of BTP, Cys-C, and the Schwartz GFR estimate were significantly higher (r = 0.653, 0.765, and 0.706, respectively; P <0.05) than with the reciprocal of creatinine or β2-MG (r = 0.500 and 0.557, respectively). ROC analysis showed a significantly higher diagnostic accuracy of BTP, Cys-C, and the GFR estimate for the detection of impaired GFR than serum creatinine (P <0.05). Compared to creatinine, BTP increased the diagnostic sensitivity by ∼30%, but it was not more sensitive than Cys-C or the Schwartz GFR estimate.

Conclusions: BTP is superior to serum creatinine and an alternative for Cys-C to detect mildly reduced GFR in children, but it is not better than the Schwartz GFR estimate.
Changes in Bone Turnover in Patients with Anorexia Nervosa during Eleven Weeks of Inpatient Dietary Treatment
Authors: Heer Martina , Mika Claudia , Grzella Ina , Drummer Christian , Herpertz-Dahlmann Beate
Published: January 2020

Background: Many adolescents with anorexia nervosa suffer from severe osteopenia and osteoporosis. We hypothesized that individualized nutrition therapy may improve bone turnover in anorectic patients.

Methods: We studied 19 female patients [mean age, 14.2 ± 1.4 years; mean body weight, 39.3 ± 5.4 kg; mean body mass index (BMI), 14.2 ± 1.4 kg/m2] with anorexia nervosa (International Classification of Diseases-10: F50.0, F50.1) for a period of 3 months. Nutrition therapy began at the end of the first week and included individualized hypercaloric diets, high calcium intake (2000 mg/day), and administration of vitamin D (400 IU/day). Blood samples were taken at baseline and again in weeks 3, 7, and 11. We measured serum calcium, parathyroid hormone, bone formation and resorption markers, insulin-like growth factor 1 (IGF-1), and leptin.

Results: Mean BMI increased significantly, from 14.2 ± 1.4 to 17.1 ± 0.7 kg/m2 (P = 0.000001), during the course of treatment, whereas serum total calcium and phosphate concentrations remained unchanged. The bone formation markers procollagen-I carboxy-terminal propeptide and bone alkaline phosphatase almost doubled (P = 0.006). Both IGF-1 (P = 0.00001) and leptin (P = 0.000005) increased significantly by week 11. Parallel to this, the serum concentration of C-telopeptide, a bone resorption marker, decreased significantly (P = 0.009).

Conclusions: Nutritional rehabilitation, possibly as a result of increasing IGF-1 and leptin concentrations, may increase bone formation. It therefore provides additional objective evidence of the importance of nutrition for bone.
5,10-Methylenetetrahydrofolate Reductase (MTHFR) Assay in the Forward Direction: Residual Activity in MTHFR Deficiency
Authors: Suormala Terttu , Gamse Gertraud , Fowler Brian
Published: January 2020

Background: Assay of methylenetetrahydrofolate reductase (MTHFR), a key enzyme in homocysteine metabolism, is important for the study of severe and mild deficiency states. Because the conventional assay measures in the reverse direction, lacks sensitivity, and uses nonphysiologic substrates, the exact measurement and characterization of residual activity in easily accessible tissues have been difficult.

Methods: To measure MTHFR in the physiologic direction, we determined the NADPH-dependent conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate by use of HPLC with fluorescence detection.

Results: MTHFR activity in control fibroblast in the presence of FAD was maximal between pH 6.3 and 6.9, increased linearly up to 40 min and 80 μg protein/assay, and showed Kms of 30 μmol/L for NADPH and 26 μmol/L for 5,10-methylenetetrahydrofolate. Intraassay variation (CV) was 10%, interassay variation was 7.2%, and variation among 10 subcultures of the same cell line was 18%. Mean (SD) control activity was 431 (150) μU/mg protein (range, 242–910; n = 75), which is 2.5-fold higher than that with the reverse assay. After heat treatment (46 °C for 5 min), the activity showed a trimodal distribution corresponding to the 677TT (thermolabile; 15%), 677CT (35%), and 677CC (51%) genotypes. We found clearly measurable activity ranging from 2.6% to 25.6% of the mean control value in 15 patients with MTHFR deficiency, including 11 cell lines with zero activity in the reverse assay. Ten patients had complete enzyme deficiency.

Conclusion: This assay allows reliable determination of residual activity in mutant fibroblasts and characterization of kinetic parameters for natural substrates.
Evaluation of the Anticoagulants EDTA and Citrate, Theophylline, Adenosine, and Dipyridamole (CTAD) for Assessing Platelet Activation on the ADVIA 120 Hematology System
Authors: Macey Marion , Azam Urooj , McCarthy Desmond , Webb Lee , Chapman E Sabrinah , Okrongly David , Zelmanovic David , Newland Adrian
Published: January 2020

Background: Monitoring of platelet activation by the ADVIA® 120 Hematology System requires an anticoagulant and protocol that ensures that platelets are sphered and their activation status is not altered artifactually in vitro.

Methods: Blood from healthy controls was collected into tripotassium EDTA; citrate, theophylline, adenosine, and dipyridamole (CTAD); or a combination of both (E/C) and stored at ambient temperature or at 4 °C (E/C only) and then analyzed between 0 and 180 min later on the ADVIA 120. In addition, immunofluorescent flow cytometry was used to identify activated platelets and platelet-leukocyte aggregates.

Results: In blood stored with all three anticoagulants, the platelet count changed little, but the mean platelet volume (MPV) at first decreased and then increased, whereas the mean platelet component (MPC; an indicator of activation) changed in a reciprocal manner. The changes in MPV and MPC, which reflect platelet sphering and swelling, were greatest between 30 and 60 min in blood stored at ambient temperature, irrespective of which anticoagulant was used, and between 60 and 180 min when blood anticoagulated with E/C was stored at 4 °C. In all anticoagulants, the percentages of platelets expressing CD62P and of leukocytes in platelet-leukocyte aggregates increased significantly (P <0.01) over 180 min at ambient temperature. Only minimal (<2%) increases occurred when blood with E/C was stored at 4 °C.

Conclusions: When determining platelet activation ex vivo on the ADVIA 120, blood should be collected into E/C, stored at 4 °C, and analyzed between 60 and 180 min later; these conditions ensure maximum platelet sphering without concurrent artifactual platelet activation.
Determination of α-Fetoprotein in Human Serum by a Quartz Crystal Microbalance-based Immunosensor
Authors: Chou Shu-Fen , Hsu Win-Lin , Hwang Jing-Min , Chen Chien-Yuan
Published: January 2020

Background: Increased α-fetoprotein (AFP) in adult plasma is considered an early indication of hepatocellular carcinoma and teratoblastoma. The aim of this study was to develop a rapid method to measure AFP in human serum by use of a direct immunosensor based on a quartz crystal microbalance (QCM).

Methods: A self-assembled monolayer prepared by the cystamine method was applied to immobilize anti-AFP monoclonal antibodies on the gold surface of a quartz crystal. The frequency shifts of the QCM were measured and related to AFP concentrations. The piazoimmunosensor used no labeled reagent and no pretreatment of samples.

Results: Ten cycles of measurements could be performed on the gold surface of the same crystal regenerated with a solution of glycine-HCl. A linear relationship existed between the frequency shifts (Hz) and the log values of AFP concentrations from 0.1 to 100 μg/L in buffer and human serum. When used for 15 days, the frequency shifts were all >95% of those on the response at the first day. The regression equation was y = 1.03x − 0.06 (Sy|x = 3.92; r = 0.9987) for this QCM method and RIA in 29 clinical human serum samples.

Conclusions: The QCM sensor can measure AFP in buffer and human serum and offers advantages of high specificity, reusability, low detection limit, no label or sample pretreatment, and low sample requirement. The assay format of the immunosensor was more rapid and simpler than conventional methods.
Performance of a Revised Cardiac Troponin Method That Minimizes Interferences from Heterophilic Antibodies
Authors: Kim Wesley J , Laterza Omar F , Hock Karl G , Pierson-Perry James F , Kaminski Delores M , Mesguich Martine , Braconnier François , Zimmermann Régine , Zaninotto Martina , Plebani Mario , Hanna Atef , Cembrowski George S , Scott Mitchell G
Published: January 2020

Background: Recent guidelines for use of cardiac troponin to detect cardiac damage and for cardiovascular risk stratification have made increasingly sensitive troponin assays important. Troponin assays continue to be plagued by interferences caused by heterophilic antibodies (HAs). We evaluated the performance of a revised cardiac troponin I (cTnI) assay designed to have increased analytical sensitivity and to minimize the effect of HAs.

Methods: The revised Dade Behring Dimension® cTnI assay was evaluated according to NCCLS EP5-A at five institutions. Plasma samples from 14 309 patients were assayed by the original Dimension cTnI assay. To identify samples that may have interfering HAs, samples with values >1.4 μg/L were reanalyzed on the Dade Behring Stratus® CS cTnI assay. Samples with possible interfering antibodies were also analyzed before and after selective absorbance studies on the revised Dade Behring Dimension cTnI assay.

Results: The limit of quantification in the revised method was 0.1 μg/L with imprecision (CV) of 11–17% at 0.1 μg/L. Values correlated well with the Stratus CS cTnI method: revised = 1.06(original) + 0.01; r = 0.98, Sy|x = 0.25 μg/L). Falsely increased results consistent with myocardial infarction by the original Dimension cTnI assay and presumably attributable to HAs were identified in 0.17% of all patients with samples submitted for cTnI analysis. The revised Dimension cTnI assay eliminated the interference in 17 of 25 samples identified and greatly decreased the interference in the other 8.

Conclusions: The revised Dimension cTnI method greatly minimizes the effect of interfering HAs. It also exhibits analytical performance characteristics consistent with recent guidelines for use of this assay to detect cardiac damage.
Biochemical Markers and Hematologic Indices in the Diagnosis of Functional Iron Deficiency
Authors: Thomas Christian , Thomas Lothar
Published: January 2020

Background: The hypochromic red cell is a direct indicator of functional iron deficiency (ID) in contrast to the majority of biochemical markers, which measure functional ID indirectly via iron-deficient erythropoiesis. The aim of this study was to evaluate the extent to which these biochemical markers can distinguish ID from anemia of chronic disease (ACD) as well as from the combined state of functional ID/ACD, using red cell hemoglobinization as the gold standard.

Methods: We studied 442 patients with various disease-specific anemias and 154 nonanemic patients. As indicators of red cell hemoglobinization, we measured the reticulocyte hemoglobin content (CHr) and the proportion of hypochromic red cells (HYPO), using an Advia 120 hematology analyzer. Ferritin, transferrin, transferrin saturation, and the concentration of the soluble transferrin receptor (sTfR) were determined by ELISA and immunoturbidimetric assay. The sTfR/log ferritin ratio (sTfR-F index) was used as an additional marker for biochemical identification of iron-deficient erythropoiesis.

Results: In a control group (n = 71), the 2.5 percentile values were 28 pg for CHr and 5% for HYPO. These values were used to indicate unimpaired red cell hemoglobinization and absence of functional ID. In patients with deficient red cell hemoglobinization but no acute-phase response (APR), iron-deficient erythropoiesis was indicated by serum ferritin and sTfR-F index values ≤20.8 μg/L and >1.5, respectively. Corresponding values in patients with APR were ≤61.7 μg/L and >0.8, respectively. The positive likelihood ratios for the biochemical markers and the sTfR-F index for identifying iron-restricted erythropoiesis in patients with and without APR were 2.6–6.9 and 4.3–16.5, respectively.

Conclusion: In APR patients, biochemical markers demonstrate weaknesses in the diagnosis of functional ID as defined by hematologic indices. Use of diagnostic plots to illustrate the relationship between the sTfR-F index and CHr allows the progression of ID to be identified, regardless of whether an APR is present.
Real-Time PCR Technology for Cancer Diagnostics
Authors: Bernard Philip S , Wittwer Carl T
Published: January 2020

Background: Advances in the biological sciences and technology are providing molecular targets for diagnosing and treating cancer. Current classifications in surgical pathology for staging malignancies are based primarily on anatomic features (e.g., tumor-node-metastasis) and histopathology (e.g., grade). Microarrays together with clustering algorithms are revealing a molecular diversity among cancers that promises to form a new taxonomy with prognostic and, more importantly, therapeutic significance. The challenge for pathology will be the development and implementation of these molecular classifications for routine clinical practice.

Approach: This article discusses the benefits, challenges, and possibilities for solid-tumor profiling in the clinical laboratory with an emphasis on DNA-based PCR techniques.

Content: Molecular markers can be used to provide accurate prognosis and to predict response, resistance, or toxicity to therapy. The diversity of genomic alterations involved in malignancy necessitates a variety of assays for complete tumor profiling. Some new molecular classifications of tumors are based on gene expression, requiring a paradigm shift in specimen processing to preserve the integrity of RNA for analysis. More stable markers (i.e., DNA and protein) are readily handled in the clinical laboratory. Quantitative real-time PCR can determine gene duplications or deletions. Furthermore, melting curve analysis immediately after PCR can identify small mutations, down to single base changes. These techniques are becoming easier and faster and can be multiplexed. Real-time PCR methods are a favorable option for the analysis of cancer markers.

Summary: There is a need to translate recent discoveries in oncology research into clinical practice. This requires objective, robust, and cost-effective molecular techniques for clinical trials and, eventually, routine use. Real-time PCR has attractive features for tumor profiling in the clinical laboratory.
Human Kallikrein Gene 5 (KLK5) Expression by Quantitative PCR: An Independent Indicator of Poor Prognosis in Breast Cancer
Authors: Yousef George M , Scorilas Andreas , Kyriakopoulou Lianna G , Rendl Laura , Diamandis Maria , Ponzone Riccardo , Biglia Nicoletta , Giai Maurizia , Roagna Riccardo , Sismondi Piero , Diamandis Eleftherios P
Published: January 2020

Background: KLK5 is a newly discovered human kallikrein gene. Many kallikrein genes have been found to be differentially expressed in various malignancies, and prostate-specific antigen (PSA; encoded by the KLK3 gene) is the best tumor marker for prostate cancer. Like the genes that encode PSA and other kallikreins, the KLK5 gene was found to be regulated by steroid hormones in the BT-474 breast cancer cell line.

Methods: We studied KLK5 expression in 179 patients with different stages and grades of epithelial breast carcinoma by quantitative reverse transcription-PCR (RT-PCR), using LightCycler® technology. An optimal cutoff point equal to the detection limit (65th percentile) was used. KLK5 values were then compared with other established prognostic factors in terms of disease-free (DFS) and overall survival (OS).

Results: High KLK5 expression was found more frequently in pre-/perimenopausal (P = 0.026), node-positive (P = 0.029), and estrogen receptor-negative (P = 0.038) patients. In univariate analysis, KLK5 overexpression was a significant predictor of reduced DFS (P <0.001) and OS (P <0.001). Cox multivariate analysis indicated that KLK5 was an independent prognostic factor for DFS and OS. KLK5 remained an independent prognostic variable in the subgroups of patients with large tumors (>2 cm) and positive nodes. Hazard ratios derived from Cox analysis and related to DFS and OS were 2.48 (P = 0.005) and 2.37 (P = 0.009), respectively, for the node-positive group and 3.03 (P = 0.002) and 2.94 (P = 0.002), respectively, for patients with tumor sizes >2 cm. KLK5 expression was also associated with statistically significantly shorter DFS (P = 0.006) and OS (P = 0.004) in the subgroup of patients with grade I and II tumors.

Conclusions: KLK5 expression as assessed by quantitative RT-PCR is an independent and unfavorable prognostic marker for breast carcinoma.
Effect of Aspirin Treatment on Serum Concentrations of Lipoprotein(a) in Patients with Atherosclerotic Diseases
Authors: Akaike Masashi , Azuma Hiroyuki , Kagawa Ayako , Matsumoto Kazuya , Hayashi Ikuro , Tamura Katsuya , Nishiuchi Takeshi , Iuchi Takahiko , Takamori Nobuyuki , Aihara Ken-ichi , Yoshida Tomonori , Kanagawa Yasuhiko , Matsumoto Toshio
Published: January 2020

Background: Increased serum lipoprotein(a) [Lp(a)] is an independent risk factor for atherosclerosis. We previously reported that aspirin reduced Lp(a) production by cultured hepatocytes via the reduction of apolipoprotein(a) [apo(a)] gene transcription.

Methods: We evaluated both the effect of aspirin treatment (81 mg/day) on serum Lp(a) concentrations and the correlation between the degree of reduction in serum Lp(a) and the type of apo(a) isoform in 70 patients with coronary artery disease or cerebral infarction.

Results: Aspirin lowered serum Lp(a) concentrations to ∼80% of the baseline values in patients with high Lp(a) concentrations (>300 mg/L). The percentage of decrease in serum Lp(a) was larger in patients with high Lp(a) than in patients with low Lp(a) (<300 mg/L), irrespective of apo(a) isoform size. The decreases in serum Lp(a) in high Lp(a) patients with both the high-molecular-weight and the low-molecular-weight isoforms were positively correlated with the baseline Lp(a) concentrations.

Conclusions: Because the secretory efficiencies of apo(a) in the same isoform are likely to be similar, the difference in serum Lp(a) concentrations in patients having the same apo(a) isoform depends on the transcriptional activity of the apo(a) gene. These findings suggest that aspirin decreases serum Lp(a) concentrations via a decrease in apo(a) gene transcription more effectively in patients with high transcriptional activity of this gene.
Limited Sampling Strategy for the Estimation of Mycophenolic Acid Area under the Curve in Adult Renal Transplant Patients Treated with Concomitant Tacrolimus
Authors: Pawinski Tomasz , Hale Mike , Korecka Magda , Fitzsimmons William E , Shaw Leslie M
Published: January 2020

Background: Significant relationships between the mycophenolic acid (MPA) area under the concentration–time curve (AUC0–12h) and the risks for acute rejection and side effects have been reported. We developed a practical method for estimation of MPA AUCs. Regression equations were developed using repeated cross-validation for randomly chosen subsets, characterized statistically, and verified for acceptable performance.

Methods: Twenty-one renal transplant patients receiving 0.5 or 1.0 g of mycophenolate mofetil twice daily and concomitant tacrolimus provided a total of 50 pharmacokinetic profiles. MPA concentrations were measured by a validated HPLC method in 12 plasma samples collected at predose and at 30 and 60 min; 2, 3, 4, 6, 8, 9, 10, 11, and 12 h; 1 and 2 weeks; and 3 months after transplantation. Twenty-six 1-, 2-, or 3-sample estimation models were fit (r2 = 0.341–0.862) to a randomly selected subset of the profiles using linear regression and were used to estimate AUC0–12h for the profiles not included in the regression fit, comparing those estimates with the corresponding AUC0–12h values, calculated with the linear trapezoidal rule, including all 12 timed MPA concentrations. The 3-sample models were constrained to include no samples past 2 h.

Results: The model using c0h, c0.5h, and c2h was superior to all other models tested (r2 = 0.862), minimizing prediction error for the AUC0–12h values not included in the fit (i.e., the cross-validation error). The regression equation for AUC estimation that gave the best performance for this model was: 7.75 + 6.49c0h + 0.76c0.5h + 2.43c2h. When we applied this model to the full data set, 41 of the 50 (82%) estimated AUC values were within 15% of the value of AUC0–12h calculated using all 12 concentrations.

Conclusions: This limited sampling strategy provides an effective approach for estimation of the full MPA AUC0–12h in renal transplant patients receiving concomitant tacrolimus therapy.