bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of Erythrocyte Glutathione S-Transferase in Uremia and Dialysis
Authors: Galli Francesco , Rovidati Simona , Benedetti Serena , Buoncristiani Umberto , Covarelli Carla , Floridi Ardesio , Canestrari Franco
Published: January 2020

Background: Overexpression of glutathione S-transferase (GST; EC 2.5.1.18) has been documented in the erythrocytes of patients with chronic renal failure, and this event may well be of relevance from a clinical standpoint. In fact, it could serve as a marker of uremic toxicity overall, which can contribute to impair the function and survival of the erythrocytes. However, the biochemical details of this phenomenon are poorly understood.

Methods: In this study, we characterized the expression of GST in erythrocytes of 118 uremic patients under different clinical conditions. The mechanisms responsible for the regulation of protein expression and enzyme activity were investigated in light of different dialysis approaches, oxidative stress, uremic toxins, erythrocyte age, and erythropoietin (EPO) supplementation.

Results: Mean GST activity in uremic patients was highly overexpressed with respect to controls, and this phenomenon was exclusively attributable to an increased expression of GST. Overexpression of GST did not appear to be dependent on oxidative stress and was not influenced by vitamin E supplementation. In the same manner, both erythrocyte age and EPO supplementation apparently did not interfere with the GST concentrations, which were the same in controls and patients. Preliminary experiments suggested that high-molecular weight or protein-bound toxins could play some role in the overexpression of GST.

Conclusions: GST expression may be a useful marker for the individual accumulation of uremic toxins as well as of the efficiency of new dialysis strategies in removing them.
Human Pharmacokinetics of l-3,4-Dihydroxyphenylalanine Studied with Microdialysis
Authors: Dizdar Nil , Kullman Anita , Norlander Björn , Olsson Jan-Edvin , Kågedal Bertil
Published: January 2020

Background: Intravenous and subcutaneous microdialysis was performed to compare the free concentrations and pharmacokinetics of l-3,4-dihyroxyphenylalanine (l-dopa) in blood and tissue in healthy subjects and in patients with Parkinson disease.

Methods: Nine healthy volunteers and 10 patients with Parkinson disease, stage 1.5–2 according to the Hoehn-Yahr rating scale, took part of the study. In the patient group subcutaneous microdialysis and ordinary blood sampling were performed, whereas in the control group intravenous microdialysis was also performed. Microdialysis samples were collected in fractions of 15 min. The first two fractions were collected for analysis of basal concentrations. A blood sample was also taken. The patients were then given one tablet of Madopar® (100 mg of l-dopa and 25 mg of benserazide), and the microdialysis was continued for another 210 min. Blood samples were obtained at 30-min intervals.

Results: The serum samples gave a significantly higher mean area under the curve (AUC; 491 ± 139 μmol · min/L) than that for intravenous dialysates (235 ± 55.3 μmol · min/L), suggesting a protein binding of 50%. The l-dopa concentrations from the subcutaneous dialysates matched those from the intravenous dialysates, indicating rapid distribution of l-dopa to the tissues.

Conclusions: Parkinsonian patients in early stages of the disease have a pharmacokinetic pattern of free l-dopa similar to that of healthy subjects. Comparison of AUCs from microdialysis with ordinary serum analysis revealed data indicating significant protein binding. Microdialysis is a suitable and easily applied tool in pharmacokinetic studies.
Multicenter Evaluation of Tosoh Glycohemoglobin Analyzer
Authors: Gibb Ian , Parnham Angela , Fonfrède Michele , Lecock Freddy
Published: January 2020

Background: We describe an Anglo-French evaluation of a new analyzer.

Methods: The Tosoh HLC-723 GHb V, A1c2.2 glycohemoglobin analyzer is an HPLC instrument with primary blood tube sampling, bar-code reading, cap piercing, and the ability to chromatographically separate labile hemoglobin A1c (HbA1c). We evaluated two analytical protocols, 2.2 and 3.0 min, and compared results for blood samples collected from diabetic and nondiabetic subjects with those obtained with Bio-Rad Diamat and Variant analyzers.

Results: Within- and between batch-imprecision (CVs) was <2% with linearity to at least 15.9% HbA1c. Although some hemoglobinopathies were detected in the 2.2-min chromatography, clearer evidence of abnormality was visible in the 3.0-min version. Comparison with established methods showed good correlation (r = 0.993; n = 316 with Diamat; and r = 0.995; n = 133 with Variant) but highlighted calibration differences.

Conclusions: The problems of manual blood sample preparation, labile HbA1c, and carbamylated hemoglobin interference associated with the older instruments have been eliminated in the new Tosoh analyzer. The 3.0-min protocol is preferred for routine use.
Different Real-Time PCR Formats Compared for the Quantitative Detection of Human Cytomegalovirus DNA
Authors: Nitsche Andreas , Steuer Nina , Schmidt Christian Andreas , Landt Olfert , Siegert Wolfgang
Published: January 2020

Background: The aim of this study was to compare the ABI PRISM 7700 Sequence Detection System and the LightCycler to develop a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA suitable for routine hospital application.

Methods: We used one exonuclease probe and five different hybridization probe sets as sequence-specific fluorescence detection formats. For the exonuclease assay and two hybridization probe sets, reproducibility and the detection limit were determined. To keep the total assay time to a minimum, we gradually shortened individual reaction steps on both instruments.

Results: The exonuclease assay can be interchangeably performed on the 7700 and the LightCycler. No change of reaction conditions is required, except for the addition of bovine serum albumin to the LightCycler reaction. The shortest possible total assay time is 80 min for the ABI PRISM 7700 Sequence Detection System and 20 min for the LightCycler. When the LightCycler is used, the exonuclease probe can be replaced by a set of hybridization probes. All assays presented here detected HCMV DNA in a linear range from 101 to 107 HCMV genome equivalents/assay (r >0.995) with low intraassay (<5%) and interassay (<10%) variation.

Conclusions: The ABI PRISM 7700 Sequence Detection System as well as the LightCycler are useful instruments for rapid and precise online PCR detection. Moreover, the two principles of fluorescence signal production allow HCMV quantification with the same accuracy.
Two-Site Expression Immunoassay Using a Firefly Luciferase-coding DNA Label
Authors: Chiu Norman HL , Christopoulos Theodore K
Published: January 2020

Background: We report the first two-site, “sandwich type” expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase.

Methods: The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3′ end of the DNA label was biotinylated and complexed with streptavidin. A sandwich immunoassay for prostate-specific antigen (PSA) was developed in which the antigen was first bound to an immobilized monoclonal antibody and then reacted with a biotinylated polyclonal antibody. The streptavidin-luciferase-coding DNA complex was then bound to the immunocomplex. The DNA label was subsequently expressed in vitro by coupled transcription and translation. The generated luciferase was measured by its characteristic bioluminescent reaction.

Results: The bioluminescence was linearly related to the concentration of PSA in the sample. As low as 30 ng/L PSA was measured (12.5-μL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 μg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 μg/L; r = 0.971; n = 44).

Conclusions: The use of the newly developed DNA label in a two-site immunoassay was demonstrated for the first time. The assay was applied successfully to the measurement of serum PSA.
Endpoint Colorimetric Method for Assaying Total Cholesterol in Serum with Cholesterol Dehydrogenase
Authors: Kayamori Yuzo , Hatsuyama Hiroyuki , Tsujioka Tadayoshi , Nasu Masato , Katayama Yoshiaki
Published: January 2020

Background: Various methods are available to measure serum cholesterol concentrations. Of these, the cholesterol ester hydrolase (CEH)-cholesterol oxidase-peroxidase chromogenic method is widely used. However, this method has the disadvantage of interference by reducing substances. We developed and evaluated an endpoint assay for serum cholesterol, based on a CEH-cholesterol dehydrogenase (CDH)-ultraviolet method.

Methods: Cholesterol esters are first hydrolyzed to free cholesterol by CEH. The free cholesterol is then reduced by CDH to cholest-4-ene-3-one with the simultaneous production of β-NADH from β-NAD+. At equilibrium, the CDH reaction gives incomplete conversion of cholesterol to cholest-4-ene-3-one. To overcome this disadvantage, we added hydrazine monohydrate to the reaction mixture to remove cholest-4-ene-3-one, which allowed the reaction to proceed to completion and gave stoichiometric production of β-NADH from the reaction of β-NAD+ with cholesterol.

Results: We tested whether the amount of cholesterol added was equivalent to the absorbance change of NADH at 340 nm with six aqueous samples. Recoveries were 97.1–100.3%. The reaction was linear up to 20.28 mmol/L. The mean within-day (n = 20) and between-day (n = 10) imprecision (CV) was 0.29–0.43% and 0.22–0.61%, respectively. No interference by bilirubin, hemoglobin, ascorbic acid, and other reducing agents was observed. The equation obtained in comparison with the modified Abell-Levy-Brodie-Kendall method was: y = 0.992x − 0.0058 mmol/L; r = 0.997; Sy|x = 0.117 mmol/L; n = 50.

Conclusion: This method is an accurate, reliable method for serum cholesterol analysis and is amenable to automation.
Preanalytical Variables Affecting the Quantification of Fatty Acid Ethyl Esters in Plasma and Serum Samples
Authors: Soderberg Britt L , Sicinska Ewa T , Blodget Emily , Cluette-Brown Joanne E , Suter Paolo M , Schuppisser Theresa , Vetter Wilhem , Laposata Michael
Published: January 2020

Background: Fatty acid ethyl esters (FAEEs) are cytotoxic nonoxidative ethanol metabolites produced by esterification of fatty acids and ethanol. FAEEs are detectable in blood up to 24 h after ethanol consumption. The objective of this study was to assess the impact of gender, serum or plasma triglyceride concentration, time and temperature of specimen storage, type of alcoholic beverage ingested, and the rate of ethanol consumption on FAEE concentrations in plasma or serum.

Methods: For some studies, subject were recruited volunteers; in others, residual blood samples after ethanol quantification were used. FAEEs were isolated by solid-phase extraction and quantified by gas chromatography–mass spectrometry.

Results: For weight-adjusted amounts of ethanol intake, FAEE concentrations were twofold greater for men than women (P ≤0.05). Accounting for triglycerides improved the correlation between blood ethanol concentrations and FAEE concentrations for both men (from r = 0.640 to r = 0.874) and women (from r = 0.619 to r = 0.673). FAEE concentrations did not change when samples were stored at or below 4 °C, but doubled when stored at room temperature for ≥24 h. The type of alcoholic beverage and rate of consumption did not affect FAEE concentrations.

Conclusion: These studies advance plasma and serum FAEE measurements closer to implementation as a clinical test for ethanol intake.
Arsenic Speciation in Human Organs following Fatal Arsenic Trioxide Poisoning—A Case Report
Authors: Benramdane Larbi , Accominotti Michele , Fanton Laurent , Malicier Daniel , Vallon Jean-Jacques
Published: January 2020

The aim of this investigation was to study the distribution of arsenic species in human organs following fatal acute intoxication by arsenic trioxide. The collected autopsy samples of most organs were ground and dried, and the total arsenic was measured by electrothermal atomic absorption spectrometry (ETAAS). The arsenic species—inorganic arsenic, in the form of arsenite [As(III)] and arsenate [As(V)], and its metabolites [monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)]—were quantified by ETAAS after extraction with methanol/water (1:1, by volume) and separation by HPLC. The results indicate that after acute intoxication, the liver and kidneys show the highest concentrations of total arsenic and that the total concentration in blood is 7- to 350-fold less concentrated than in organs. In all organs, As(III) is the predominant species, and MMA is more concentrated than DMA. MMA and DMA are more prevalent in lipidic organs (49% of total arsenic) compared with other organs (25% of total arsenic). As(V) was found in small quantities in the liver, kidneys, and blood.
Characterization of Monoclonal Antibodies for Prostate-specific Antigen and Development of Highly Sensitive Free Prostate-specific Antigen Assays
Authors: Black Margot H , Grass C Linda , Leinonen Jari , Stenman Ulf-Håkan , Diamandis Eleftherios P
Published: January 2020

Background: The recent elucidation of the importance of serological free prostate-specific antigen (PSA) in the diagnosis of prostate cancer has created a demand for immunoassays specific for free PSA.

Methods: We developed and characterized 11 monoclonal antibodies with high affinities for PSA (Kavalues from 1.1 × 108 to 1.8 × 1010L/mol), only 3 of which cross-react with human glandular kallikrein (hK2). Using these antibodies and PSA antibodies developed by others, in conjunction with time-resolved fluorometry, we developed ultrasensitive sandwich immunoassays specific for the free form of PSA.

Results: The analytical detection limit of these immunoassays is 0.001 μg/L. To our knowledge, this is the most sensitive free PSA assay reported to date. The free PSA immunoassays exhibit <1% cross-reactivity with PSA-α1-antichymotrypsin, show no cross-reactivity with hK2, and correlate well with established free PSA kits. The 11 antibodies developed by our group, in conjunction with 4 commercially available antibodies, were used to generate a putative epitope map of the PSA molecule.

Conclusion: The highly sensitive free PSA immunoassays may be used for measuring PSA subfractions in female serum, an application currently impossible with other reported free PSA immunoassays.
Evaluation of a New Method for the Analysis of Free Catecholamines in Plasma Using Automated Sample Trace Enrichment with Dialysis and HPLC
Authors: Dutton John , Hodgkinson Andrew J , Hutchinson George , Roberts Norman B
Published: January 2020

Background: Analysis of urinary free catecholamines was automated recently, but analysis of plasma samples posed special difficulties. The present study was undertaken to evaluate a new method for the automated analysis of plasma catecholamines.

Methods: The procedure is based on an improved sample handling system that includes dialysis and sample clean-up on a strong cation trace-enrichment cartridge. The catecholamines norepinephrine, epinephrine, and dopamine are then separated by reversed-phase ion-pair chromatography and quantified by electrochemical detection.

Results: Use of a 740-μL sample is required to give the catecholamine detection limit of 0.05 nmol/L and analytical imprecision (CV) between 1.1% and 9.3%. The assay can be run unattended, although >12 h of analysis time is not recommended without cooling of the autosampler rack. Comparison (n = 68) of the automated cation-exchange clean-up with the well-established manual alumina procedure gave excellent agreement (mean, 3.78 ± 2.76 and 3.8 ± 2.89 nmol/L for norepinephrine and 0.99 ± 1.72 and 1.08 ± 1.78 nmol/L for epinephrine). Hemodialysis had no clear effect on plasma norepinephrine. Epinephrine concentrations were similar (0.05 < P < 0.1) in chronic renal failure patients (0.24 ± 0.3 nmol/L; n = 15) and healthy controls (0.5 ± 0.24 nmol/L; n = 31). Dopamine was not quantified, being usually <0.2 nmol/L.

Conclusion: The availability of such a fully automated procedure should encourage the more widespread use of plasma catecholamine estimation, e.g., after dialysis, exercise, or trauma/surgery and in the investigation of catecholamine-secreting tumors, particularly in the anuric patient.
The Future of Molecular Genetic Testing
Authors: Leonard Debra GB
Published: January 2020

The potential applications for genetic testing are immense, with most diseases having some aspect influenced by, if not directly caused by, changes in the genome of the patient. The translation of genetic information into medical applications will be influenced by our understanding of the human genome, technological advances, and social, ethical, and legal issues surrounding genetic testing. With time, new genetic information will be translated into clinical tests for the diagnosis of current illness and prediction of future disease risk, and will be used for the development of genetically directed therapies and preventive interventions. Most genetic testing will be highly automated, with only rare genetic disease tests performed manually. The challenge for the clinical genetic laboratory is to keep pace with this information explosion to provide state-of-the-art genetic testing and to ensure that the genetic test results are used in a morally, ethically, and socially responsible way.
Highly Sensitive Automated Chemiluminometric Assay for Measuring Free Human Glandular Kallikrein-2
Authors: Klee George G , Goodmanson Marcia K , Jacobsen Steven J , Young Charles YF , Finlay Judith A , Rittenhouse Harry G , Wolfert Robert L , Tindall Donald J
Published: January 2020

Background: Human glandular kallikrein (hK2) is a serine protease that has 79% amino acid identity with prostate-specific antigen (PSA). Both free hK2 and hK2 complexed to α1-antichymotrypsin (ACT) are present in the blood in low concentrations. We wished to measure hK2 in serum with limited contribution from hK2-ACT for the results.

Methods: We developed an automated assay for hK2 with use of a select pair of monoclonal antibodies. The prototype assay was implemented on a Beckman Coulter ACCESS® analyzer.

Results: The detection limit of the assay was 1.5 ng/L, the “functional sensitivity” (day-to-day CV <15%) was <4 ng/L, cross-reactivity with PSA and PSA-ACT was negligible, and cross-reactivity with hK2-ACT was 2%. After surgical removal of prostate glands, serum hK2 was <7 ng/L and was <15 ng/L in most healthy women. The median serum concentration of hK2 in healthy men without prostate cancer was 26 ng/L. The median concentration of hK2 was 72 ng/L for men having prostate cancer with lower Gleason scores compared with 116 ng/L for men with more advanced cancer. The concentration of hK2 correlated weakly with PSA, with the mean hK2 concentrations generally 30- to 80-fold lower than PSA concentrations.

Conclusion: The availability of a robust, high sensitivity automated assay for hK2 should facilitate further investigations of the role of hK2 measurements in the management of patients with prostate disease.
Lipoprotein(a)-Cholesterol and Coronary Heart Disease in the Framingham Heart Study
Authors: Seman Leo J , DeLuca Carl , Jenner Jennifer L , Cupples L Adrienne , McNamara Judith R , Wilson Peter WF , Castelli William P , Ordovas Jose M , Schaefer Ernst J
Published: January 2020

Background: Increased plasma lipoprotein(a) [Lp(a)] concentrations have been reported to be an independent risk factor for coronary heart disease (CHD) in some prospective studies, but not in others. These inconsistencies may relate to a lack of standardization and the failure of some immunoassays to measure all apolipoprotein(a) isoforms equally.

Methods: We measured plasma Lp(a)-cholesterol [Lp(a)-C] in a Caucasian population of offspring and spouses of the Framingham Heart Study participants, using a lectin-based assay (LipoproTM). We compared the prevalence of increased Lp(a)-C to the presence of sinking pre-β-lipoprotein (SPB). We also related Lp(a)-C concentrations to the prevalence of CHD risk in the entire population.

Results: The mean (± SD) Lp(a)-C concentration in the Framingham population (n = 3121) was 0.186 ± 0.160 mmol/L, with no significant gender or age differences. The mean Lp(a)-C concentrations in the absence or presence of SPB were 0.158 ± 0.132 mmol/L and 0.453 ± 0.220 mmol/L, respectively (P <0.0001). The mean Lp(a)-C concentration in men with CHD (n = 156) was 0.241 ± 0.204 mmol/L, which was significantly (P <0.001) higher, by 34%, than in controls. The odds ratio for CHD risk in men with Lp(a)-C ≥0.259 mmol/L (≥10 mg/dL), after adjusting for age, HDL-cholesterol, LDL-cholesterol, smoking, diabetes, blood pressure, and body mass index, was 2.293 (confidence interval, 1.55–3.94; P <0.0005). Lp(a)-C values correlated highly with a Lp(a)-mass immunoassay [ApotekTM Lp(a); r = 0.832; P <0.0001; n = 1000].

Conclusions: An increased Lp(a)-C value ≥0.259 mmol/L (≥10 mg/dL) is an independent CHD risk factor in men with a relative risk of more than 2, but was inconclusive in women. Lp(a)-C measurements offer an alternative to Lp(a)-mass immunoassays and can be performed on automated analyzers.
Broad Spectrum Drug Identification Directly from Urine, Using Liquid Chromatography-Tandem Mass Spectrometry
Authors: Fitzgerald Robert L , Rivera Jeffrey D , Herold David A
Published: January 2020

Background: Currently the rate-limiting step for mass spectrometric analysis of drugs in biological samples is sample preparation. Many gas chromatography/mass spectrometry (GC/MS) methods are specific for a certain class of compounds, requiring extraction and/or derivatization before analysis. The purpose of this study was to develop a broad spectrum liquid chromatography/mass spectrometry (LC/MS) procedure that allowed for direct analysis of urine specimens with potential for quantitative analysis.

Methods: We modified a commercially available column-switching instrument, the REMEDi HS from Bio-Rad Diagnostics, to make it compatible with atmospheric pressure ionization. The system we developed was based on electrospray ionization and used three LC columns to extract, purify, and separate drugs directly from urine specimens. Drugs and metabolites were tentatively identified on the basis of retention times and (M+H)+ ions. Tandem mass spectrometry (MS/MS) was used to confirm the qualitative identification of suspected drugs, using data-dependent acquisition. For quantitative analysis, the cocaine metabolite benzoylecgonine was analyzed using isotope dilution and selected reaction monitoring.

Results: Seventeen basic drugs from a variety of classes of compounds were identified directly from urine without the need for prior sample extraction, using LC and MS/MS. Quantitative analysis was demonstrated for benzoylecgonine. When benzoylecgonine-d3 was used as the internal standard, the method was linear from 30 to 10 000 μg/L (range tested). At these concentrations, the within-run accuracy was ± 10% of the target concentration, with CVs <10%. Analytical results by LC/MS/MS compared favorably with GC/MS values for 50 benzoylecgonine-containing specimens and for 25 negative specimens.

Conclusions: The ability to directly analyze urine for a wide variety of drug classes, combined with the sensitivity and specificity of LC/MS/MS makes this technique attractive for many clinical, forensic, and biotechnology applications.
Determination of the Sum of Bilirubin Sugar Conjugates in Plasma by Bilirubin Oxidase
Authors: Doumas Basil T , Yein Fred , Perry Billy , Jendrzejczak Bernadine , Kessner Arthur
Published: January 2020

Background: A reliable indicator of cholestasis is the presence of abnormal concentrations of bilirubin mono- and diglucuronide [conjugated bilirubin (CB)] in blood. A routine assay of CB is available only to those who possess a certain type of clinical analyzer. We describe a two-point manual method for CB that could be adapted as a rate assay to automated clinical analyzers.

Methods: The measurement of CB is based on its oxidation to biliverdin by bilirubin oxidase. The resulting decrease in absorbance at 460 nm is proportional to the CB concentration. The assay is calibrated with solutions of ditaurobilirubin in human serum.

Results: Under the conditions of the assay (0.1 mol/L glycine buffer, pH 10.0; reaction time, 2 min), only 5% of unconjugated bilirubin is oxidized and δ-bilirubin is not oxidized at all. Results obtained with the bilirubin oxidase method agreed well with those obtained by HPLC. The long-term CVs at CB concentrations of 6 and 63.4 mg/L were 20% and 2.6%, respectively. The reference values, established by analyzing 51 plasma specimens from healthy adults, were 0.0–1.2 mg/L, with a mean value of 0.2 mg/L.

Conclusions: The proposed method for CB has good analytical specificity and obviates the requirement for HPLC or a dry chemistry analyzer. The measurement of CB in blood is superior to the measurement of direct bilirubin because an abnormal concentration of direct bilirubin does not necessarily indicate the presence of cholestasis.
Comparison of Plasma Total Homocysteine Measurements in 14 Laboratories: An International Study
Authors: Pfeiffer Christine M , Huff Dan L , Smith S Jay , Miller Dayton T , Gunter Elaine W
Published: January 2020

Background: Information on interlaboratory variation and especially on methodological differences for plasma total homocysteine is lacking.

Methods: We studied 14 laboratories that used eight different method types: HPLC with electrochemical detection (HPLC-ED); HPLC with fluorescence detection (HPLC-FD) further subdivided by type of reducing/derivatizing agent; gas chromatography/mass spectrometry (GC/MS); enzyme immunoassay (EIA); and fluorescence polarization immunoassay (FPIA). Three of these laboratories used two methods. The laboratories participated in a 2-day analysis of 46 plasma samples, 4 additional plasma samples with added homocystine, and 3 plasma quality-control (QC) pools. Results were analyzed for imprecision, recovery, and methodological differences.

Results: The mean among-laboratory and among-run within-laboratory imprecision (CV) was 9.3% and 5.6% for plasma samples, 8.8% and 4.9% for samples with added homocystine, and 7.6% and 4.2% for the QC pools, respectively. Difference plots showed values systematically higher than GC/MS for HPLC-ED, HPLC-FD using sodium borohydride/monobromobimane (however, for only one laboratory), and EIA, and lower values for HPLC-FD using trialkylphosphine/4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. The two HPLC-FD methods using tris(2-carboxyethyl) phosphine/ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) or tributyl phosphine/SBD-F, and the FPIA method showed no detectable systematic difference from GC/MS.

Conclusions: Among-laboratory variations within one method can exceed among-method variations. Some of the methods tested could be used interchangeably, but there is an urgent need to improve analytical imprecision and to decrease differences among methods.
Maximizing Efficacy of Endocrine Tests: Importance of Decision-focused Testing Strategies and Appropriate Patient Preparation
Authors: Klee George G
Published: January 2020

The efficacy of endocrine tests depends on the choice of tests, the preparation of the patients, the integrity of the specimens, the quality of the measurements, and the validity of the reference data. Close dialogue among the clinicians, the laboratory, and the patients is a key factor for optimal patient care. The characteristics of urine and plasma samples and the advantages and limitations of paired test measurements are presented. The importance of test sequence strategies, provocative or inhibitory procedures, and elimination of drug interferences is illustrated with four cases involving Cushing syndrome, pheochromocytoma, primary aldosteronism, and hypercalcemia. For each of these scenarios, key clinical issues are highlighted, along with discussions of the best test strategies, including which medications are likely to interfere. The importance of targeting laboratory tests to answer well-focused clinical decisions is emphasized. The roles of some time-honored provocative procedures are questioned in light of more sensitive and specific analytic methods. The importance of decision-focused analytical tolerance limits is emphasized by demonstrating the impact of analytic bias on downstream medical resource utilization. User-friendly support systems to facilitate the implementation of test strategies and postanalytic tracking of patient outcomes are presented as essential requirements for quality medical practice.
Development of a Chemiluminescence Competitive PCR for the Detection and Quantification of Parvovirus B19 DNA Using a Microplate Luminometer
Authors: Fini Fabiana , Gallinella Giorgio , Girotti Stefano , Zerbini Marialuisa , Musiani Monica
Published: January 2020

Background: Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk assessment, and monitoring of antiviral therapy. We wished to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B19.

Methods: Parvovirus DNA target sequences and competitor sequences were coamplified and directly labeled. Amplified products were then separately hybridized by specific biotin-labeled probes, captured onto streptavidin-coated ELISA microplates, and detected immunoenzymatically using chemiluminescent substrates of peroxidase. Chemiluminescent signals were quantitatively analyzed by a microplate luminometer and were correlated to the amounts of amplified products.

Results: Luminol-based systems displayed constant emission but had a higher detection limit (100–1000 genome copies) than the acridan-based system (20 genome copies). The detection limit of chemiluminescent substrates was lower (20 genome copies) than colorimetric substrates (50 genome copies). In chemiluminescence cPCR, the titration curves showed linear correlation above 100 target genome copies. Chemiluminescence cPCR was positive in six serum samples from patients with parvovirus infections and negative in six control sera.

Conclusions: The chemiluminescence cPCR appears to be a sensitive and specific method for the quantitative detection of viral DNAs.
Increased Ability of LDL from Normolipidemic Type 2 Diabetic Women to Generate Peroxides
Authors: Guerci Bruno , Antebi Helena , Meyer Laurent , Durlach Vincent , Ziegler Olivier , Nicolas Jean-Pierre , Alcindor Louis-Gerald , Drouin Pierre
Published: January 2020

Background: We assessed the ability of LDL from 30 type 1 diabetic patients (18 men, 12 women), 65 type 2 diabetic patients (35 men, 30 women), and 35 controls (19 men, 16 women) to generate peroxides. The men and women in the diabetic groups were studied separately and matched for age, body mass index, duration of diabetes, glycohemoglobin, and conventional lipid characteristics according to the presence or absence of hyperlipidemia.

Methods: The ability of LDL to form peroxides was assessed by measuring the thiobarbituric acid-reactive substances corrected for LDL-cholesterol [ratio of malondialdehyde (MDA) to LDL-cholesterol]. LDL particle size was expressed as the ratio of LDL-cholesterol to apolipoprotein B (LDL-cholesterol/apoB).

Results: The MDA/LDL-cholesterol ratio was higher in type 1 and type 2 diabetic patients with hyperlipidemia than in controls. The MDA/LDL-cholesterol ratio was also higher in type 2 normolipidemic women than in controls (P <0.01). The LDL-cholesterol/apoB ratio was lower in type 2 diabetic women than in type 2 diabetic men (P <0.05). The MDA/LDL-cholesterol ratio was negatively correlated with the LDL-cholesterol/apoB ratio (r = −0.78, P <0.001) in hyperlipidemic type 1 (not type 2) diabetic patients. In normolipidemic type 2 diabetic patients, the MDA/LDL-cholesterol ratio was also negatively correlated with the LDL-cholesterol/apoB ratio (r = −0.75, P <0.001) because of the highly significant negative correlation in type 2 diabetic women (r = −0.89, P <0.01).

Conclusions: LDL from well-controlled type 2 diabetic women is smaller and more prone to form peroxides. This could explain why diabetic women are at greater risk of cardiovascular disease.
Measurement of Urea in Human Serum by Isotope Dilution Mass Spectrometry: A Reference Procedure
Authors: Kessler Anja , Siekmann Lothar
Published: January 2020

Background: A reference measurement procedure is needed to demonstrate the traceability of results of urea measurements in human serum. We developed a measurement procedure using the principle of isotope dilution gas chromatography/mass spectrometry.

Methods: [13C,15N2]Urea as internal standard was added to a serum sample and equilibrated with endogenous nonlabeled urea. For the preparation of calibrators, the same amount of labeled urea was mixed with known amounts of nonlabeled urea. The serum samples were treated with ethanol to remove proteins by precipitation. The labeled and nonlabeled urea of the samples was converted into a trimethylsilyl derivative of 2-hydroxypyrimidine. The gas chromatography/mass spectrometry system was adjusted to monitor m/z 153 and 168 for the nonlabeled urea derivative and m/z 156 and 171 for the isotopically labeled analogs. The results of the determination were calculated from peak ratios by a hyperbolic calculation function based on the theory of isotope dilution analysis.

Results: The procedure was applied to control samples and patient samples and evaluated with respect to its trueness and precision. The standard uncertainty of the results was 0.47–1.72%.

Conclusions:This reference measurement procedure allows values to be assigned to controls and calibrators that are traceable to the primary urea reference material of NIST and, therefore, to the Système International unit “mole” with a low degree of uncertainty. This procedure provides a tool for the highly accurate determination of urea in control materials as well as in patient sera.
Standardized Comparison of Processing Capacity and Efficiency of Five New-Generation Immunoassay Analyzers
Authors: Hendriks Henriëtte Alice , Kortlandt Wouter , Verweij Willem Marius
Published: January 2020

Background: With the trend toward laboratory and workstation consolidation, more studies are necessary to evaluate instrumentation, solutions for coping with workflow and test diversity, and opportunities for increasing the overall efficiency of laboratory testing. We assessed the processing capacity and efficiency of new-generation immunoassay analyzers by determining productivity parameters of five commercially available systems.

Methods: A workload protocol was developed and used to assess processing capacity and efficiency parameters of five immunoassay analyzers under standardized conditions in a real-life routine situation. We studied the ACS:Centaur® (analyzer A), ArchitectTMi2000 (analyzer B), Elecsys® 2010 tandem (analyzer C), Immulite® 2000 (analyzer D), and Vitros ECi (analyzer E) on the basis of a standardized workload protocol that reflected a routine laboratory situation. This workload encompassed reflex and STAT testing, dilutions, and in-run calibration of a new reagent lot number. The analyzers were compared for hands-on labor time, unattended time (UT), throughput, and differentiated relative productivity indexes [RPI(UT); number of reportable results/(processing time − sum of unattended time)]. The RPI data for analyzers linked to an automated (aut) sample-handling system [RPI(aut)] were also calculated.

Results: The evaluation produced a set of parameters for the productivity of the instruments. An overview of the most important parameters revealed the following: the throughput was 193, 123, 97, 109, and 46 tests/hour for instruments A, B, C, D and E, respectively; the RPI(10) was 425, 238, 161, 445, and 151 tests/operator-hour; the RPI(30) was 229, 136, 118, 264, and 86 tests/operator-hour; the RPI(10,aut) was 1701, 637, 235, 964, and 223 tests/operator-hour; and the RPI(30,aut) was 298, 150, 174, 400, and 114 tests/operator-hour.

Conclusions: The combination of a standardized workload protocol and determination of parameters for productivity and labor efficiency, especially the differentiated RPIs, made it possible to make an objective comparison of the organizational consequences of the use of these instruments. The described parameters allow for a scientifically based choice, given a certain workflow and a particular laboratory organization.
Measurement of Different Forms of Tissue Plasminogen Activator in Plasma
Authors: Chandler Wayne L , Jascur Marcy L , Henderson Paul J
Published: January 2020

Background: We evaluated assays to measure both total tissue plasminogen activator (tPA) and the three principle forms of tPA in plasma: active tPA, tPA complexed with plasminogen activator inhibitor type 1 (PAI-1), and tPA complexed with C1-inhibitor.

Methods: Active tPA was measured by use of an indirect amidolytic assay and immunofunctional assays. tPA/PAI-1, tPA/C1-inhibitor, and total tPA antigen were measured by use of microtiter plates coated with anti-tPA antibodies and, respectively, anti-PAI-1, anti-C1-inhibitor, and anti-tPA antibodies conjugated to peroxidase.

Results: The immunofunctional tPA assay detected 1 U/L (0.001 U/mL) tPA and recovered 108% ± 12% of active tPA added to samples containing high (mean, 60 000 IU/L) PAI-1 activities vs a detection limit of 10 U/L (0.01 U/mL) and 13% ± 25% recovery for the indirect amidolytic tPA activity assay. For measurement of tPA/PAI-1 complex, polyclonal anti-PAI-1 conjugates recovered 112% ± 20% of the expected tPA/PAI-1 vs recovery of only 38% ± 16% when monoclonal anti-PAI-1 conjugates were used. Of three methods tested, two total tPA antigen assays correlated well (r2 = 0.85) and showed recoveries near 100%, whereas the third method showed lower correlations, higher intercepts, and falsely high recovery. A single anti-tPA capture antibody that performed the best in the individual assay evaluations was used to measure the different forms of tPA in 22 samples with a range of tPA and PAI-1 values. The sum of the molar concentrations of active tPA, tPA/PAI-1, and tPA/C1-inhibitor using the optimized methods was equal to 94% ± 7% of measured total tPA.

Conclusion: Optimized assays based on a single anti-tPA capture antibody can be used to accurately measure the major forms of tPA in plasma.
Simultaneous HPLC Assay for Quantification of Indinavir, Nelfinavir, Ritonavir, and Saquinavir in Human Plasma
Authors: Remmel Rory P , Kawle Sagar P , Weller Dennis , Fletcher Courtney V
Published: January 2020

Background: HIV protease inhibitors are recommended as part of combination antiretroviral therapy. Dual protease inhibitor therapy is also being used clinically. Consequently, a simultaneous assay for indinavir, nelfinavir, ritonavir, and saquinavir was developed.

Methods: Indinavir, nelfinavir, ritonavir, and saquinavir were extracted from plasma (250 μL) with methyl-t-butyl ether at basic pH after addition of an internal standard (A-86093). The compounds were separated on a Keystone BetaBasic C4 column (250 × 3 mm i.d.) at 40 °C with a mobile phase of acetonitrile-50 mmol/L ammonium formate buffer, pH 4.1 (52:48, by volume) at a flow rate of 0.5 mL/min. Indinavir, nelfinavir, ritonavir, and the internal standard (A-86093) were detected at 218 nm, and saquinavir was detected at 235 nm. The method was validated by analysis of five triplicate analyses of calibrators along with quality-control samples at three different concentrations prepared in human plasma.

Results: The extraction recovery was 87–92%. Within-run accuracy for quality-control samples was 6–8%, with CVs of 2–8%. Limits of quantification were 40–50 μg/L for indinavir, nelfinavir, and ritonavir, and 20 μg/L for saquinavir. Cross-validation with a liquid chromatography-mass spectroscopy method for saquinavir and nelfinavir was conducted with patient samples. Regression analysis revealed a good correlation (r2 >0.94) between methods. Larger variations at concentrations >4000 μg/L were observed with nelfinavir. Interference with drugs commonly used in AIDS patients was not observed. Pharmacokinetic profiles for two patients on dual protease therapy were determined.

Conclusions: A reliable and rugged simultaneous HPLC assay for four HIV protease inhibitors was developed. The assay method is convenient for clinical laboratories involved in therapeutic drug monitoring for HIV protease inhibitors. The assay has enough sensitivity to conduct pharmacokinetic studies in patients taking more than one HIV protease inhibitor along with other antiretroviral medications.
Pitfalls in the Diagnosis of Patients with a Partial Dihydropyrimidine Dehydrogenase Deficiency
Authors: Van Kuilenburg André B P , Van Lenthe Henk , Tromp Annelies , Veltman Patricia C J , Van Gennip Albert H
Published: January 2020

Background: Dihydropyrimidine dehydrogenase (DPD) catalyzes the degradation of thymine, uracil, and the chemotherapeutic drug 5-fluorouracil. To identify patients suffering from complete or partial DPD deficiency and to identify pitfalls that can preclude the proper diagnosis of patients with partial DPD deficiency, a sensitive and accurate assay is necessary.

Methods: The activity of DPD was measured using [4-14C]thymine followed by separation of substrate and products with reversed-phase HPLC with on-line detection of the radioactivity.

Results: Complete baseline separation of radiolabeled thymine and all degradation products was achieved within 15 min. The detection limit for dihydrothymine was 0.4 pmol. In lymphocytes, the DPD activity deviated from linearity at low protein concentrations (<0.2 g/L). Profoundly decreased activity of DPD was detected in the peripheral blood mononuclear cells (PBM cells) of two tumor patients when measured at low protein concentrations. Low DPD activity comparable to that observed in obligate heterozygotes was initially detected in PBM cells, containing substantial amounts of myeloid cells, from a patient suffering from 5-fluorouracil toxicity. However, after the patient experienced full clinical recovery, normal DPD activity was observed in the PBM cells. No significant differences in DPD activity were observed between exponentially growing fibroblasts and those at confluence. The range of DPD activities of obligate heterozygotes overlaps the range of DPD activities of controls.

Conclusions: The low activity of DPD measured in PBM cells containing myeloid cells or that measured at a low protein concentration in the assay mixture is not indicative of heterozygosity for a mutant DPD allele. Although fibroblasts are suitable to establish a complete deficiency of DPD, unambiguous detection of heterozygotes is not possible.
Enzymatic Mutation Detection in the P53 Gene
Authors: Inganäs Mats , Byding Sara , Eckersten Ann , Eriksson Solveig , Hultman Tomas , Jorsback Anneli , Löfman Esfir , Sabounchi Fariba , Kressner Ulf , Lindmark Gudrun , Tooke Nigel
Published: January 2020

Background: The enzymatic mutation detection (EMD) assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves preformed heteroduplex molecules at mismatch sites, forming two shorter fragments that can be resolved by gel electrophoresis. The method can be used to detect single and multiple base changes, as well as insertions and deletions.

Methods: The sensitivity, specificity, and positional accuracy of mutation detection by EMD with the PASSPORTTM Mutation Scanning Kit were assessed in a blind fashion for three analytical platforms (radioactive detection and automated laser sequencers ALFexpress and ABI PRISM 377). PCR products of 703 bp covering codons 188–393 of the P53 gene were prepared from colorectal tumor samples and analyzed by EMD; the results were compared to data from cDNA sequencing. A 1362-bp PCR product prepared from IL4r gene was used to test detection of multiple base changes in long PCR products.

Results: The sensitivity for detection of mutations using EMD exceeded 90%, and the specificity exceeded 80% on all analysis platforms. The method localized 90% of mutations to within two codons and four codons for automated laser sequencers and detection by radioactivity, respectively. The method detected at least five mismatches in heteroduplexes >1 kb.

Conclusions: The EMD system facilitates efficient detection of genetic variation in fragments exceeding 1 kb irrespective of location and type. The technology is particularly well suited to the detection of mutations in genes frequently mutated at unpredictable locations.
Production and Characterization of Novel Anti-Prostate-specific Antigen (PSA) Monoclonal Antibodies That Do Not Detect Internally Cleaved Lys145-Lys146 Inactive PSA
Authors: Nurmikko Pauliina , Väisänen Ville , Piironen Timo , Lindgren Sari , Lilja Hans , Pettersson Kim
Published: January 2020

Background: The nature of free, uncomplexed prostate-specific antigen (PSA) in the circulation is still unknown. In this study, we developed novel anti-PSA antibodies using PSA produced by a metastasized cancer cell line, LNCaP, as an immunogen.

Methods: Hybridoma cell lines were screened with different methods that aimed at finding antibodies specific for the forms of free PSA produced by LNCaP cell line. Obtained antibodies were further studied for their characteristics related to previously characterized monoclonal antibodies.

Results: Numerous anti-PSA antibodies were obtained, of which four represented unique epitopes previously unrecognized by us. One free-PSA-specific antibody was bound to PSA on two distinct epitopes, and one antibody was bound to the carboxyl-terminal peptide of PSA. Two antibodies were found to bind to the peptide sequence adjacent to the internal cleavage site Lys145-Lys146. These antibodies failed to recognize internally cleaved PSA at Lys145-Lys146. We could not find anti-proPSA antibodies despite the fact that LNCaP PSA contained more than one-half of the zymogen form of PSA.

Conclusions: We report, for the first time, novel anti-PSA antibodies that do not recognize internally cleaved PSA at Lys145-Lys146 and thus are specific for intact, unclipped PSA.
Testing Claimed Minimal Detectable Concentrations of in Vitro Medical Diagnostic Devices
Authors: Davenport James M , Schlain Brian
Published: January 2020

Manufacturers and users of medical diagnostic devices are provided a statistical decision tool for investigating a claimed minimal detectable concentration (MDC). The MDC is defined by setting two respective probabilities: that the blank sample being analyzed is determined to have analyte and that the device fails to determine a low concentration of analyte at the MDC. The statistical procedure for simultaneously testing the two aforementioned analytical decision errors assumes that signal responses follow a gaussian distribution but does not require a fitted calibration curve, knowledge of distribution parameters, or the assumption of constant variance in the low assay range. Evaluation of the operating characteristics of the procedure requires knowledge only of the variance ratio between the MDC and zero-dose signal distributions, which usually is well known.