bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of the Clinical Laboratory in Managing Chemical or Biological Terrorism
Authors: Jortani Saeed A , Snyder James W , Valdes Jr Roland
Published: January 2020

Background: Domestic and international acts of terrorism using chemicals and pathogens as weapons have recently attracted much attention because of several hoaxes and real incidents. Clinical laboratories, especially those affiliated with major trauma centers, should be prepared to respond rapidly by providing diagnostic tests for the detection and identification of specific agents, so that specific therapy and victim management can be initiated in a timely manner. As first-line responders, clinical laboratory personnel should become familiar with the various chemical or biological agents and be active participants in their local defense programs.

Approach: We review the selected agents previously considered or used in chemical and biological warfare, outline their poisonous and pathogenic effects, describe techniques used in their identification, address some of the logistical and technical difficulties in maintaining such tests in clinical laboratories, and comment on some of the analytical issues, such as specimen handling and personal protective equipment.

Content: The chemical agents discussed include nerve, blistering, and pulmonary agents and cyanides. Biological agents, including anthrax and smallpox, are also discussed as examples for organisms with potential use in bioterrorism. Available therapies for each agent are outlined to assist clinical laboratory personnel in making intelligent decisions regarding implementation of diagnostic tests as a part of a comprehensive defense program.

Summary: As the civilian medical community prepares for biological and chemical terrorist attacks, improvement in the capabilities of clinical laboratories is essential in supporting counterterrorism programs designed to respond to such attacks. Accurate assessment of resources in clinical laboratories is important because it will provide local authorities with an alternative resource for immediate diagnostic analysis. It is, therefore, recommended that clinical laboratories identify their current resources and the extent of support they can provide, and inform the authorities of their state of readiness.
Synergistic Effect between Apolipoprotein E and Angiotensinogen Gene Polymorphisms in the Risk for Early Myocardial Infarction
Authors: Batalla Alberto , Alvarez Ruth , Reguero Julián R , Hevia Sergio , Iglesias-Cubero Gustavo , Alvarez Victoria , Cortina Arturo , González Pelayo , Celada Mateo M , Medina Alfonso , Coto Eliecer
Published: January 2020

Background: Several studies based on different populations worldwide have described an association between cardiovascular diseases and genetic variations in the apolipoprotein E (APOE), angiotensinogen (AGT), angiotensin receptor type 1 (AT1R), and angiotensin-converting enzyme (ACE) genes. In addition, there is growing evidence of an interaction between hypercholesterolemia and the renin-angiotensin system in the risk for hypertension and atherosclerosis.

Methods: To determine whether the DNA polymorphisms in APOE (ε2, ε3, and ε4 alleles), AGT (M235T), AT1R (1166 A/C), and ACE (I/D) are associated with early onset of myocardial infarction (MI), we genotyped 220 patients and 200 controls <55 years of age. Patients and controls were males from the same homogeneous Caucasian population. Data concerning hypertension, diabetes, and tobacco consumption were recorded. The lipid profiles of patients and controls were also determined.

Results: APOE, ACE, AGT, and AT1R allele and genotype frequencies did not differ between patients and controls. None of these polymorphisms was related to the biochemical values in patients or controls. The frequency of individuals who were both APOEε4 allele carriers and AGT-TT homozygotes was significantly higher in patients than in controls (11% vs 3.5%; P = 0.0037). In patients, the frequency of ε4 carriers was significantly higher (P <0.00001) in those who were AGT-TT (46%) than those who were AGT-MT/MM (14%). Mean cholesterol was significantly higher in AGT-TT + APOE ε34/44 patients than in the TM/MM + ε34/44 or TT + ε23/33 genotypes (P = 0.029).

Conclusions: Our data suggest a synergistic effect between the APOE and AGT polymorphisms and early MI. The increased risk could be mediated in part through higher cholesterol concentrations among individuals who are AGT-TT + APOEε4 allele carriers.
A Modified, Optimized Kinetic Photometric Assay for the Determination of Blood Coagulation Factor XIII Activity in Plasma
Authors: Kárpáti Levente , Penke Botond , Katona Éva , Balogh István , Vámosi György , Muszbek László
Published: January 2020

Background: Blood coagulation factor XIII (FXIII) is a zymogen that is transformed into an active transglutaminase by thrombin and Ca2+. FXIII plays an essential role in fibrin stabilization and in the protection of fibrin from proteolytic degradation. No convenient method has been available for the measurement of FXIII activity in plasma. The aim of the present study was to improve and optimize a kinetic photometric FXIII assay originally developed in our laboratory.

Methods: In the assay, FXIII was activated by thrombin and Ca2+. Fibrin polymerization was prevented by an inhibitory tetrapeptide. Glycine-ethyl ester and a glutamine residue of a synthetic dodecapeptide served as acyl acceptor and acyl donor transglutaminase substrates, respectively. The amount of ammonia released during the reaction was monitored using glutamate dehydrogenase and NADPH.

Results: The use of a new glutamine substrate and optimization of activator and substrate concentrations increased sensitivity. Substitution of NADPH for NADH and introduction of an appropriate blank eliminated systemic overestimation of FXIII activity. The recovery of FXIII was 96%, the assay was linear up to 470 U/L, the detection limit was 1 U/L, and the imprecision (CV) was <8% even at very low FXIII activities. A reference interval of 108–224 U/L (69–143%) was established. The results correlated well with results obtained by an immunoassay specific for plasma FXIII.

Conclusions: The optimized FXIII assay is a simple, rapid method for the diagnosis of inherited or acquired FXIII deficiencies and increased FXIII concentrations. It can be easily adapted to clinical chemistry analyzers.
Prolonged Hyperlipasemia Attributable to a Novel Type of Macrolipase
Authors: Taes Youri E , Louagie Henk , Yvergneaux Jean-Pierre , De Buyzere Marc L , De Puydt Hendrik , Delanghe Joris R , Lott John A
Published: January 2020

Background: We present the case of an 80-year-old woman who was admitted to hospital with an intermittent volvulus of the right colon. A total colectomy was performed. Initially, serum amylase and lipase increased concordantly, but after a few weeks amylase normalized (85 U/L), whereas lipase increased to 3764 U/L. This discrepancy and persistence of hyperlipasemia suggested a macromolecular form of lipase.

Methods: The nature of the macromolecular complex was studied using high-pressure liquid gel-permeation chromatography, affinity chromatography, (immuno)electrophoresis, and immunodiffusion.

Results: Gel-permeation chromatography revealed a macrolipase, with a molecular mass >900 kDa, that contributed up to 56% of total serum lipase activity. Butanol extraction of the specimen did not alter the elution profile. The thermostabilities of pancreatic lipase and the macroform were similar, whereas activation energy (Ea) was lower in the macromolecular lipase (28 ± 4 kJ · mol−1 · K−1 vs 48 ± 7 kJ · mol−1 · K−1 (P = 0.02). Agarose electrophoresis showed a broad band of lipase activity at the application site. Protein A-Sepharose affinity gel chromatography excluded IgG-linked lipase. Agarose electrophoresis and immunofixation excluded linkage to other immunoglobulins. Radial immunodiffusion did not show lipase activity in the immunoglobulin precipitation bands. Radial immunodiffusion with α2-macroglobulin (α2-MG) antibodies showed a diffuse spot of lipase activity within the precipitation band, suggesting a macromolecular association between lipase and α2-MG. Affinity gel chromatography against α2-MG showed lipase activity in the α2-MG-bound fractions.

Conclusion: This is the first report of a macrolipase in which an association between α2-MG and lipase is described.
Improved Stable Isotope Dilution-Gas Chromatography-Mass Spectrometry Method for Serum or Plasma Free 3-Hydroxy-Fatty Acids and Its Utility for the Study of Disorders of Mitochondrial Fatty Acid β-Oxidation
Authors: Jones Patricia M , Quinn Rebecca , Fennessey Paul V , Tjoa Susan , Goodman Stephen I , Fiore Stephany , Burlina Alberto B , Rinaldo Piero , Boriack Richard L , Bennett Michael J
Published: January 2020

Background: Disorders of fatty acid oxidation (FAO) are difficult to diagnose, primarily because in many of the FAO disorders measurable biochemical intermediates accumulate in body fluids only during acute illness. Increased concentrations of 3-hydroxy-fatty acids (3-OH-FAs) in the blood are indicative of FAO disorders of the long- and short-chain 3-hydroxy-acyl-CoA dehydrogenases, LCHAD and SCHAD. We describe a serum/plasma assay for the measurement of 3-OH-FAs with carbon chain lengths from C6 to C16.

Methods: We used stable isotope dilution gas chromatography-mass spectrometry (GC-MS) with electron impact ionization and selected ion monitoring. Natural and isotope-labeled compounds were synthesized for the assay.

Results: The assay was linear from 0.2 to 50 μmol/L for all six 3-OH-FAs. CVs were 5–15% at concentrations near the upper limits seen in healthy subjects. In 43 subjects, the medians (and ranges) in μmol/L were as follows: 3-OH-C6, 0.8 (0.3–2.2); 3-OH-C8, 0.4 (0.2–1.0); 3-OH-C10, 0.3 (0.2–0.6); 3-OH-C12, 0.3 (0.2–0.6); 3-OH-C14, 0.2 (0.0–0.4); and 3-OH-C16, 0.2 (0.0–0.5). 3-OH-FAs were increased in infants receiving formula containing medium chain triglycerides. Two patients diagnosed with LCHAD deficiency showed marked increases in 3-OH-C14 and 3-OH-C16 concentrations. Two patients diagnosed with SCHAD deficiency showed increased shorter chain 3-OH-FAs but no increases in 3-OH-C14 to 3-OH-C16.

Conclusion: Measuring blood concentrations of the 3-OH-FAs with this assay may be a valuable tool for helping to rapidly identify deficiencies in LCHAD and SCHAD and may also provide useful information about the status of the FAO pathway.
Evaluation of a Bead-based Enzyme Immunoassay for the Rapid Detection of Osteocalcin in Human Serum
Authors: Crăciun Alexandra M , Vermeer Cees , Eisenwiener Hans-Georg , Drees Norbert , Knapen Marjo H J
Published: January 2020

Background: Circulating osteocalcin is a well-known marker for bone formation, but none of the commercial kits currently available can be used in automated systems. Here we present the first semiautomated assay for human serum osteocalcin.

Methods: Polystyrene beads were coated with antibodies against the COOH terminus of osteocalcin and used in the COBAS® EIA System. Osteocalcin was detected with peroxidase-conjugated antibodies against the osteocalcin NH2 terminus.

Results: The time required to analyze an unknown sample was 60 min, with a lower detection limit of 4.5 μg/L and a linear dose–response curve between 4.5 and 100 μg/L. The intraassay imprecision (CV) was 5–8% (n = 21); the interassay variation was 6–9% (n = 14). In samples from human volunteers and patients, data generated with the newly developed assay were comparable to those obtained with standard microtiter plate-based assays.

Conclusions: The coated beads assay may be implemented on fully automated analyzers, which not only may further reduce imprecision but may also substantially increase the applicability of osteocalcin as a marker for bone metabolism in the routine clinical setting.
Measurement of Plasma and Intracellular S-Adenosylmethionine and S-Adenosylhomocysteine Utilizing Coulometric Electrochemical Detection: Alterations with Plasma Homocysteine and Pyridoxal 5′-Phosphate Concentrations
Authors: Melnyk Stepan , Pogribna Marta , Pogribny Igor P , Yi Ping , James S Jill
Published: January 2020

Background: The relative changes in plasma and intracellular concentrations of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) may be important predictors of cellular methylation potential and metabolic alterations associated with specific genetic polymorphisms and/or nutritional deficiencies. Because these metabolites are present in nanomolar concentrations in plasma, methods of detection generally require time-consuming precolumn processing or metabolite derivatization.

Methods: We used HPLC with coulometric electrochemical detection for the simultaneous measurement of SAM and SAH in 200 μL of plasma, 106 lymphocytes, or 10 mg of tissue. Filtered trichloroacetic acid extracts were injected directly into the HPLC system without additional processing and were eluted isocratically.

Results: The limits of detection were 200 fmol/L for SAM and 40 fmol/L SAH. In plasma extracts, the interassay CV was 3.4–5.5% and the intraassay CV was 2.8–5.6%. The analytical recoveries were 96.8% and 97.3% for SAM and SAH, respectively. In a cohort of healthy adult women with mean total homocysteine concentrations of 7.3 μmol/L, the mean plasma value was 156 nmol/L for SAM and 20 nmol/L for SAH. In women with increased homocysteine concentrations (mean, 12.1 μmol/L), plasma SAH, but not SAM, was increased (P <0.001), and plasma pyridoxal 5′-phosphate concentrations were reduced (P <0.001). Plasma SAM/SAH ratios were inversely correlated with homocysteine concentrations (r = 0.73; P <0.01), and the SAM/SAH ratio in plasma was directly correlated with the intracellular SAM/SAH ratio in lymphocytes (r = 0.70; P <0.01).

Conclusions: Increased homocysteine in serum is associated with an increase in SAH and a decrease in the SAM/SAH ratio that could negatively affect cellular methylation potential. Accurate and sensitive detection of these essential metabolites in plasma and in specific tissues should provide new insights into the regulation of one-carbon metabolism under different nutritional and pathologic conditions.
Correlation of Antemortem Serum Creatine Kinase, Creatine Kinase-MB, Troponin I, and Troponin T with Cardiac Pathology
Authors: Ooi Daylily S , Isotalo Phillip A , Veinot John P
Published: January 2020

Background: Spurious increases in serum troponins, especially troponin T, have been reported in patients with and without acute myocardial syndromes.

Methods: We studied 78 autopsied patients without clinical myocardial infarction (MI) and correlated histologic cardiac findings with antemortem serum creatine kinase (CK), its MB isoenzyme (CK-MB), cardiac troponin I (cTnI), and cardiac troponin T (cTnT).

Results: There was no significant myocardial pathology in 15 patients. Cardiac pathologies were in five groups: scarring from previous MI or patchy ventricular fibrosis (n = 9), recent MI (n = 27), healing MI (n = 7), degenerative myocyte changes consistent with congestive heart failure (CHF; n = 12), and other cardiac pathologies (n = 8). The median concentrations in the five groups were not significantly different for either CK or CK-MB. Compared with the no-pathology group, only the MI group was significantly different for cTnI, and the MI and other pathology groups were significantly different for cTnT. For patients with MI, 22%, 19%, 48%, and 65% had increased CK, CK-MB, cTnI, and cTnT, respectively; for CHF and other cardiac pathologies combined, the percentages were 28%, 17%, 22%, and 50%. For patients with increased cTnI, 72% and 28% had MI and other myocardial pathologies, respectively; patients with increased cTnT had 64% and 36%, respectively. Patients without myocardial pathology had no increases in CK-MB, cTnI, or cTnT.

Conclusions: All patients with increased serum CK-MB, cTnI, and cTnT had significant cardiac histologic changes. The second-generation cTnT assay appears to be a more sensitive indicator of MI and other myocardial pathologies than the cTnI assay used in this study.
Determination of the Acyl Glucuronide Metabolite of Mycophenolic Acid in Human Plasma by HPLC and Emit
Authors: Shipkova Maria , Schütz Ekkehard , Armstrong Victor William , Niedmann Paul Dieter , Oellerich Michael , Wieland Eberhard
Published: January 2020

Background: The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) has been found to possess pharmacologic and potentially proinflammatory activity in vitro. To establish its pharmacologic and toxicologic relevance in vivo, a reversed-phase HPLC method was modified to simultaneously determine MPA, the phenolic MPA-glucuronide (7-O-MPAG), and AcMPAG. In addition, cross-reactivity of AcMPAG in the Emit assay for MPA was investigated.

Methods: The procedure used simple sample preparation, separation with a Zorbax Eclipse-XDB-C8 column, and gradient elution. AcMPAG was quantified as 7-O-MPAG-equivalents.

Results: The assay was linear up to 50 mg/L for MPA, 250 mg/L for 7-O-MPAG, and 10 mg/L for AcMPAG (r >0.999). Detection limits were 0.01, 0.03, and 0.04 mg/L for MPA, 7-O-MPAG, and AcMPAG, respectively. The recoveries were 99–103% for MPA, 95–103% for 7-O-MPAG, and 104–107% for AcMPAG. The within-day imprecision was <5.0% for MPA (0.2–25 mg/L), <4.4% for 7-O-MPAG (10–250 mg/L), and ≤14% for AcMPAG (0.1–5 mg/L). The between-day imprecision was <6.2%, <4.5%, and ≤14% for MPA, 7-O-MPAG, and AcMPAG, respectively. When isolated from microsomes, purified AcMPAG (1–10 mg/L) revealed a concentration-dependent cross-reactivity in an Emit assay for the determination of MPA ranging from 135% to 185%. This is in accordance with the bias between HPLC and Emit calculated in 270 samples from kidney transplant recipients receiving mycophenolate mofetil therapy, which was greater (median, 151.2%) than the respective AcMPAG concentrations determined by HPLC. AcMPAG was found to undergo hydrolysis when samples were stored up to 24 h at room temperature or up to 30 days at 4 °C or −20 °C. Acidified samples (pH 2.5) were stable up to 30 days at −20 °C.

Conclusions: The HPLC and Emit methods for AcMPAG described here may allow investigation of its relevance for the immunosuppression and side effects associated with mycophenolate mofetil therapy.
Detection and Measurement of Urinary 2-Hydroxyestradiol 17-Sulfate, a Potential Placental Antioxidant during Pregnancy
Authors: Takanashi Kaori , Honma Takashi , Kashiwagi Tomohiro , Honjo Hideo , Yoshizawa Itsuo
Published: January 2020

Background: Preeclampsia is associated with a quantitative imbalance between lipid peroxide and an antioxidant coproduced in the placenta. To investigate our hypothesis that 2-hydroxyestradiol 17-sulfate (2-OH-ES) is the placental antioxidant during pregnancy, we developed an assay for 2-OH-ES in urine and studied samples from women with and without preeclampsia.

Methods: The detection and measurement of 2-OH-ES in the urine of pregnant women were performed by RIA using highly specific antiserum to 2-OH-ES. To confirm the reliability of the RIA method, the same samples were analyzed by HPLC using an electrochemical detector.

Results: Urinary 2-OH-ES values obtained by RIA showed a close relationship to those obtained by HPLC (y = 1.1x − 0.01; r = 0.96). The urinary 2-OH-ES concentrations during the first, second, and third trimesters were 2.0 ± 0.6 (mean ± SE, n = 13), 5.3 ± 1.3 (n = 21), and 15.3 ± 2.0 μg/mg creatinine (n = 54), respectively, and <0.15 μg/mg creatinine (n = 10) at 2–24 h after delivery. The concentrations in preeclamptic women during the third trimester were significantly lower, 3.9 ± 1.9 μg/mg creatinine (mean ± SE, n = 12).

Conclusions: RIA can be used to measure urinary 2-OH-ES during pregnancy. The increase in urinary 2-OH-ES during gestation, its decrease after delivery, and the lower values in preeclampsia are consistent with a role of 2-OH-ES as a placental antioxidant.
Leptin Binding and Binding Capacity in Serum
Authors: Landt Michael
Published: January 2020

Background: Leptin, a hormone produced primarily by adipose tissue, is known to be present in serum as both monomeric (free) and higher molecular mass (bound) forms, but little is known about the nature of the bound forms or physiological variation in binding capacity.

Methods: A new method to quantify the free and bound forms was developed, based on HPLC separation and RIA quantification in chromatography fractions. Reanalysis of specimens after addition of exogenous leptin allowed direct determination of leptin-binding capacity and the degree of saturation of leptin-binding capacity.

Results: HPLC chromatography fractionated serum leptin into both the free form and as a broad peak of 59–130 kDa. Several experiments were conducted to validate the new method. The concentrations of bound leptin in serum were 0.45–3.94 μg/L, and they increased as total leptin (reflecting adiposity) increased in 24 lean and obese volunteers. Leptin was readily dissociated from the bound fraction by competition from exogenous leptin. Rechromatography of the bound fraction led to dissociation of leptin, which was promoted by warming the sera before chromatography. Leptin-binding capacity was 1.8–5.3 μg/L; binding capacity was nearly constant over a range of total leptin concentrations of 2–10 μg/L, and slowly increased at higher total leptin concentrations. Saturation of binding capacity was low (15%) at very low total leptin concentrations (<5 μg/L), but rose quickly to a plateau near 80% at higher total leptin concentrations.

Conclusions: The new method facilitates measurement of free and bound fractions of serum leptin, and is the first method measuring leptin-binding capacity. These experiments demonstrate that the concentration of bound leptin and leptin-binding capacity vary physiologically, with binding/binding capacity increasing with adiposity. Except in very lean individuals, binding capacity is nearly completely saturated.
Investigation by Isoelectric Focusing of the Initial Carbohydrate-deficient Transferrin (CDT) and non-CDT Transferrin Isoform Fractionation Step Involved in Determination of CDT by the ChronAlcoI.D. Assay
Authors: Hackler Rolf , Arndt Torsten , Helwig-Rolig Angelika , Kropf Juergen , Steinmetz Armin , Schaefer Juergen R
Published: January 2020

Background: The introduction of a new set of reagents for the determination of carbohydrate-deficient transferrin (CDT) as a marker of chronic alcohol abuse requires an independent evaluation of the analytic specificity of the test. This information is needed for correct interpretation and classification of test results.

Methods: Isoelectric focusing on the PhastSystemTM followed by immunofixation, silver staining, and densitometry was used to validate the initial transferrin isoform fractionation step on anion-exchange microcolumns involved in the ChronAlcoI.D.TM assay.

Results: The in vitro transferrin iron load was complete and stable. The CDT and non-CDT transferrin fractionation on anion-exchange microcolumns was reliable and reproducible (CV ≤10%). Except for quantitatively unimportant traces of trisialo-Fe2-transferrin (<5% of total CDT), only asialo-, mono-, and disialo-Fe2-transferrin were detected in the microcolumn eluates (n = 170). There was a loss of proportionally similar amounts of asialo-Fe2-transferrin (during column rinsing) and disialo-Fe2-transferrin (on the anion exchanger). Thus, the peak height ratios for disialo- and asialo-Fe2-transferrin did not change from >1 (serum) to <1 (eluates) as described for the CDTect assays. The transferrin patterns in the ChronAlcoI.D. eluates were representative of those in serum. Transferrin D variants with isoelectric points close to that of trisialo-Fe2-transferrin C1 did not cause overdetermination of CDT by the ChronAlcoI.D. test.

Conclusions: The initial CDT and non-CDT fractionation step involved in determination of CDT by the ChronAlcoI.D. assay is efficient for eliminating non-CDT transferrins from serum before quantification of CDT in the final turbidimetric immunoassay. We recommend IEF for validation of other (commercial) CDT analysis methods and of odd CDT results.
Seasonal and Biological Variation of Blood Concentrations of Total Cholesterol, Dehydroepiandrosterone Sulfate, Hemoglobin A1c, IgA, Prolactin, and Free Testosterone in Healthy Women
Authors: Garde Anne Helene , Hansen Åse Marie , Skovgaard Lene Theil , Christensen Jytte Molin
Published: January 2020

Background: Concentrations of physiological response variables fluctuate over time. The present study describes within-day and seasonal fluctuations for total cholesterol, dehydroepiandrosterone sulfate (DHEA-S), hemoglobin A1c (HbA1c), IgA, prolactin, and free testosterone in blood, and estimates within- (CVi) and between-subject (CVg) CVs for healthy women. In addition, the index of individuality, prediction intervals, and power calculations were derived.

Methods: A total of 21 healthy female subjects participated in the study. Using a random effects analysis of variance, we estimated CVg and total within-subject variation (CVti), i.e., the combined within-subject and analytical variation, from logarithmically transformed data. Analytical variation was subtracted from CVti to give CVi. CVi was estimated from samples taken monthly during 1 year (CViy), weekly during 1 month (CVim), and six times within 1 day (CVid).

Results: A cyclic seasonal variation was demonstrated for total cholesterol, DHEA-S, HbA1c, prolactin, and free testosterone. Within-day variation was shown for prolactin and free testosterone. The overall mean values for the group and the variability (CViy and CVg) were: 5.1 mmol/L, 5.5%, and 5.0% for total cholesterol; 6.6 μmol/L, 7.1%, and 21% for DHEA-S; 4.3%, 2.6%, and 3.3% for HbA1c/hemoglobintotal; 2.1 g/L, 5.9%, and 13% for IgA; 136 mIU/L, 23%, and 27% for prolactin; and 5.4 pmol/L, 21%, and 29% for free testosterone.

Conclusions: Collecting samples at specific hours of the day or times of the year may reduce high biological variation. Alternatively, the number of individuals may be increased and a paired study design chosen to obtain adequate statistical power.
Analysis of Concentration and 13C Enrichment of d-Galactose in Human Plasma
Authors: Schadewaldt Peter , Hammen Hans-Werner , Loganathan Kamalanathan , Bodner-Leidecker Annette , Wendel Udo
Published: January 2020

Background: A stable-isotope dilution method for the sensitive determination of d-galactose in human plasma was established.

Methods: d-[13C]Galactose was added to plasma, and the concentration was measured after d-glucose was removed from the plasma by treatment with d-glucose oxidase and the sample was purified by ion-exchange chromatography. For gas chromatographic–mass spectrometric analysis, aldononitrile pentaacetate derivatives were prepared. Monitoring of the [MH-60]+ ion intensities at m/z 328, 329, and 334 in the positive chemical ionization mode allowed the assessment of 1-12C-, 1-13C-, and U-13C6-labeled d-galactose, respectively. The d-galactose concentration was quantified on the basis of the 13C-labeled internal standard.

Results: The method was linear (range examined, 0.1–5 μmol/L) and of good repeatability in the low and high concentration ranges (within- and between-run CVs <15%). The limit of quantification for plasma d-galactose was <0.02 μmol/L. Measurements in plasma of postabsorptive subjects yielded d-galactose concentrations (mean ± SD) of 0.12 ± 0.03 (n = 16), 0.11 ± 0.04 (n = 15), 1.44 ± 0.54 (n = 10), and 0.17 ± 0.07 (n = 5) μmol/L in healthy adults, diabetic patients, patients with classical galactosemia, and obligate heterozygous parents thereof, respectively. These data were considerably lower (3- to 18-fold) than the values of a conventional enzymatic assay. The procedure was also applied successfully in a stable-isotope turnover study to evaluate endogenous d-galactose formation.

Conclusions: The present findings establish that detection of d-galactose from endogenous sources is feasible in human plasma and show that erroneously high results may be obtained by enzymatic methods.
DzyNA-PCR: Use of DNAzymes to Detect and Quantify Nucleic Acid Sequences in a Real-Time Fluorescent Format
Authors: Todd Alison V , Fuery Caroline J , Impey Helen L , Applegate Tanya L , Haughton Margaret A
Published: January 2020

Background: DzyNA-PCR is a general strategy for the detection and quantification of specific genetic sequences associated with disease or the presence of foreign agents. The method allows homogeneous gene amplification coupled with signal detection in a single closed vessel.

Methods: The strategy involves in vitro amplification of genetic sequences using a DzyNA primer that harbors the complementary (antisense) sequence of a 10-23 DNAzyme. During amplification, amplicons are produced that contain active (sense) copies of DNAzymes that cleave a reporter substrate included in the reaction mixture. The accumulation of amplicons during PCR can be monitored in real time by changes in fluorescence produced by separation of fluoro/quencher dye molecules incorporated into opposite sides of a DNAzyme cleavage site within the reporter substrate. The DNAzyme and reporter substrate sequences can be generic and hence can be adapted for use with primer sets targeting various genes or transcripts.

Results: Experiments using K-ras plasmid as template demonstrated that DzyNA-PCR allows quantification of DNA over at least six orders of magnitude (r = 0.992). Studies with human genomic DNA demonstrated the ability to resolve as little as twofold differences in the amount of starting template. DzyNA-PCR allowed the detection of 10 or fewer copies of the target. The clinical utility of the assay was demonstrated using DzyNA-PCR to analyze DNA that was isolated from human serum.

Conclusion: DzyNA-PCR is a simple, rapid, and sensitive technique for homogeneous amplification and quantification of nucleic acids in clinical specimens.
Rapid Detection of Point Mutations by Fluorescence Resonance Energy Transfer and Probe Melting Curves in Candida Species
Authors: Loeffler Juergen , Hagmeyer Lars , Hebart Holger , Henke Norbert , Schumacher Ulrike , Einsele Hermann
Published: January 2020

Background: The LightCyclerTM combines rapid amplification of nucleic acids in glass capillaries with melting curve analysis based on fluorescence resonance energy transfer for the sensitive detection of point mutations in various settings, such as drug resistance and hereditary diseases. Point mutations leading to an altered structure of lanosteroldemethylase, the target enzyme of the fungistatic azoles, are an important mechanism of acquired resistance in Candida albicans.

Methods: We screened 13 fluconazole-resistant C. albicans and 21 fluconazole-resistant C. tropicalis strains (minimum inhibitory concentration >128 mg/L), isolated from patients with AIDS, for the presence of defined point mutations by comparing conventional cycle sequencing with a newly designed LightCycler-based assay.

Results: In C. tropicalis, 5 of 21 isolates showed the wild-type sequence, and 8 of 21 showed the homozygous nucleotide exchange thymine to cytosine at position 1554 (T1554C). A heterozygous genotype was detected in 8 of 21 isolates by the LightCycler, but in only 3 of 21 isolates by conventional cycle sequencing. In 2 of 13 C. albicans isolates, a homozygous point mutation leading to an amino acid exchange at position 464 (glycine to serine) was detected in both assays.

Conclusion: The LightCycler technique offers standardized, fast, sensitive, and reproducible detection of point mutations in different Candida spp.
ELISA for Urinary Trehalase with Monoclonal Antibodies: A Technique for Assessment of Renal Tubular Damage
Authors: Ishihara Reiko , Taketani Shigeru , Sasai-Takedatsu Misa , Adachi Yasushi , Kino Minoru , Furuya Akiko , Hanai Nobuo , Tokunaga Rikio , Kobayashi Yohnosuke
Published: January 2020

Background: α,α-Trehalase, located on renal proximal tubules, is a glycoprotein that hydrolyses α,α-trehalose to two glucose molecules. Urinary trehalase reflects damage to renal proximal tubules, but its activity has not been measured routinely because measurement of catalytic activity is rather complicated and because conventional assays for enzyme activity might not reflect all of the trehalase protein because of enzyme inactivation in urinary samples.

Methods: We established novel monoclonal antibodies for human trehalase and a sandwich ELISA for quantification of urinary trehalase. We determined the urinary trehalase protein concentration with this ELISA and trehalase catalytic activity, and the results of these two methods were compared.

Results: The ELISA system was more sensitive than the detection of enzyme activity and could detect a subtle difference in the amount of trehalase present in renal diseases. The within- and between-assay CVs in the ELISA were 6.7–7.6% and 6.2–8.2%, respectively. Highly significant increases in both the quantity and activity were seen in patients with nephrotic syndrome (acute phase), Lowe syndrome, and Dent disease. The quantities were 70- to 200-fold greater, whereas enzyme activities were, at most, 10-fold higher than those of control subjects. In the detection of small amounts of trehalase in patients with chronic glomerulonephritis and renal anomalies, quantities were better than enzyme activities.

Conclusions: We have established an ELISA system for quantification of urinary trehalase that uses novel monoclonal antibodies. Our ELISA system is simpler and more sensitive than a conventional activity assay and reflects trehalase protein. This ELISA can be a useful as a common tool for clinical assessment of renal proximal tubular damage.
Laboratory Automation: Trajectory, Technology, and Tactics
Authors: Markin Rodney S , Whalen Scott A
Published: January 2020

Laboratory automation is in its infancy, following a path parallel to the development of laboratory information systems in the late 1970s and early 1980s. Changes on the horizon in healthcare and clinical laboratory service that affect the delivery of laboratory results include the increasing age of the population in North America, the implementation of the Balanced Budget Act (1997), and the creation of disease management companies. Major technology drivers include outcomes optimization and phenotypically targeted drugs. Constant cost pressures in the clinical laboratory have forced diagnostic manufacturers into less than optimal profitability states. Laboratory automation can be a tool for the improvement of laboratory services and may decrease costs. The key to improvement of laboratory services is implementation of the correct automation technology. The design of this technology should be driven by required functionality. Automation design issues should be centered on the understanding of the laboratory and its relationship to healthcare delivery and the business and operational processes in the clinical laboratory. Automation design philosophy has evolved from a hardware-based approach to a software-based approach. Process control software to support repeat testing, reflex testing, and transportation management, and overall computer-integrated manufacturing approaches to laboratory automation implementation are rapidly expanding areas. It is clear that hardware and software are functionally interdependent and that the interface between the laboratory automation system and the laboratory information system is a key component. The cost-effectiveness of automation solutions suggested by vendors, however, has been difficult to evaluate because the number of automation installations are few and the precision with which operational data have been collected to determine payback is suboptimal. The trend in automation has moved from total laboratory automation to a modular approach, from a hardware-driven system to process control, from a one-of-a-kind novelty toward a standardized product, and from an in vitro diagnostics novelty to a marketing tool. Multiple vendors are present in the marketplace, many of whom are in vitro diagnostics manufacturers providing an automation solution coupled with their instruments, whereas others are focused automation companies. Automation technology continues to advance, acceptance continues to climb, and payback and cost justification methods are developing.
Branched-Chain Keto-Acids and Pyruvate in Blood: Measurement by HPLC with Fluorimetric Detection and Changes in Older Subjects
Authors: Pailla Karine , Blonde-Cynober Françoise , Aussel Christian , De Bandt Jean-Pascal , Cynober Luc
Published: January 2020

Background: Measurement of keto-acids is important in various clinical situations. The aim of the present work was to develop a rapid HPLC method for the determination of keto-acids in human serum and to assess the concentrations of these acids in young adults and institutionalized elderly adults. This method was applied to the determination of blood keto-acid concentrations of young adults and institutionalized elderly people, divided into age groups

Methods: Four keto-acids (α-ketoisocaproate, α-ketoisovalerate, α-keto-β-methylvalerate, and pyruvate) were derivatized with o-phenylenediamine to give fluorescent derivatives. After the sample preparation step (75 min to prepare 20 samples), the derivatives were separated chromatographically on a reversed-phase column using a binary gradient.

Results: The fluorometric detection of the four keto-acids was rapid, <12 min. The method is repeatable and reproducible: the CVs were <6% and <11%, respectively, for each of the keto-acids. We found no significant difference between males and females. Concentrations of the branched-chain keto-acids decreased after age 60 years, especially α-ketoisocaproate, which decreased ∼40%.

Conclusions: The proposed method allows rapid and reliable measurement of keto-acids. The data demonstrate that changes in branched-chain keto-acids concentrations in serum occur with age.
Serum and Urinary Prostate-specific Antigen and Urinary Human Glandular Kallikrein Concentrations Are Significantly Increased after Testosterone Administration in Female-to-Male Transsexuals
Authors: Obiezu Chrisitna V , Giltay Erik J , Magklara Angeliki , Scorilas Andreas , Gooren Louis J G , Yu He , Howarth David J C , Diamandis Eleftherios P
Published: January 2020

Background: The genes that encode prostate-specific antigen (PSA) and human glandular kallikrein (hK2) are up-regulated by androgens and progestins in cultured cells, but no published studies have described the effect of androgen administration in women on serum and urinary PSA or hK2.

Methods: We measured serum and urinary PSA and hK2 before, and 4 and 12 months post testosterone treatment by immunofluorometric methods in 32 female-to-male transsexuals.

Results: Mean serum PSA increased from 1.1 ng/L to 11.1 ng/L and then to 22 ng/L by 4 and 12 months post treatment, respectively; the corresponding mean values in urine were 17, 1420, and 18 130 ng/L, respectively. Serum hK2, another kallikrein closely related to PSA, remained undetectable at the three time points. However, urinary hK2 concentration rose from below the detection limit (<6 ng/L) before treatment to 18 and 179 ng/L by the 4th and the 12th month of treatment, respectively. All changes were statistically significant (P <0.001) at 4 months.

Conclusions: Testosterone administration increases serum and urinary PSA and urinary hK2 in women. These measurements may be useful as indicators of androgenic stimulation in women.
Manifold-assisted Reverse Transcription-PCR with Real-Time Detection for Measurement of the BCR-ABL Fusion Transcript in Chronic Myeloid Leukemia Patients
Authors: Barbany Gisela , Hagberg Anette , Olsson-Strömberg Ulla , Simonsson Bengt , Syvänen Ann-Christine , Landegren Ulf
Published: January 2020

Background: BCR-ABL fusion mRNA expression in bone marrow or peripheral blood can be used as a measure of minimal residual disease in patients with chronic myeloid leukemia (CML).

Methods: We used an oligo(dT)-coated manifold support to capture the mRNA directly from the cell lysate. After reverse transcription, the cDNA was eluted from the manifold support, and BCR-ABL and GAPDH mRNAs were quantified in real time using the TaqMan fluorogenic detection system.

Results: The detection limit of the method was one positive K562 cell among 105 negative cells. GAPDH was chosen as a reference gene based on the low variation between samples from different stages of the disease and the low signal in the absence of reverse transcription. The day-to-day variation of the method (CV) was 32%. In 43 blood samples from 13 CML patients, mRNA quantification agreed well with cytogenetic data.

Conclusions: The proposed procedure constitutes a reproducible and sensitive BCR-ABL mRNA quantification method and is suitable to monitor minimal residual disease in CML patients.
Identification of Lignans as Major Components in the Phenolic Fraction of Olive Oil
Authors: Owen Robert W , Mier Walter , Giacosa Attilio , Hull William E , Spiegelhalder Bertold , Bartsch Helmut
Published: January 2020

Background: Because olive oil is an important component of the Mediterranean diet, it is necessary to establish unequivocal identification of the major potential antioxidant phenolic compounds it contains.

Methods: The major phenolic antioxidants in extra virgin olive oil were isolated and purified. Structural analysis was conducted using several spectroscopic techniques, including mass spectrometry and nuclear magnetic resonance (NMR). In particular, detailed 1H and 13C NMR data are presented, and several assignment errors in the literature are corrected.

Results: The data show for the first time that the lignans (+)-1-acetoxypinoresinol and (+)-pinoresinol are major components of the phenolic fraction of olive oils. These lignans, which are potent antioxidants, are absent in seed oils and virtually absent in refined virgin oils but are present at concentrations of up to 100 mg/kg (mean ± SE, 41.53 ± 3.93 mg/kg; range, 0.65–99.97 mg/kg) in extra virgin oils. As with the simple phenols and secoiridoids, there is considerable interoil variation in lignan concentrations. Foods containing high amounts of lignan precursors have been found to be protective against breast, colon, and prostate cancer.

Conclusion: Lignans, as natural components of the diet, may be important modulators of cancer chemopreventive activity.
Rapid Detection of the CYP2D6*3, CYP2D6*4, and CYP2D6*6 Alleles by Tetra-Primer PCR and of the CYP2D6*5 Allele by Multiplex Long PCR
Authors: Hersberger Martin , Marti-Jaun Jacqueline , Rentsch Katharina , Hänseler Edgar
Published: January 2020

Background: Interindividual differences in CYP2D6 activity range from total absence of metabolism of certain drugs to ultrafast metabolism and can produce adverse effects or lack of therapeutic effect under standard therapy. Several mutations have been described in the CYP2D6 gene that abolish CYP2D6 activity. However, four mutations explain the majority of the poor metabolizers. We describe four single-tube assays to detect these mutations.

Methods: Three tetra-primer PCR assays were developed to detect the mutations in the CYP2D6*3, *4, and *6 alleles. In these single-tube assays, the CYP2D6 locus is amplified directly, followed by the allele-specific amplification on this new template. In addition, a multiplex long PCR was developed to genotype the CYP2D6*5 allele. Two long PCR amplifications for detection of the deletion of CYP2D6 (*5) and for detection of the CYP2D6 gene region were combined in one tube.

Results: Analysis of 114 alleles showed no CYP2D6*3 allele, and allele frequencies of 28.1% for CYP2D6*4, 2.6% for CYP2D6*5, and 0.9% for CYP2D6*6. Re-analysis of the DNA samples by restriction fragment length polymorphism and sequencing analysis confirmed these results. Furthermore, re-analysis of sequenced genomic DNA by tetra-primer PCR analysis (7–11 times) always showed identical results.

Conclusions: Our set of single-tube assays allows rapid and reproducible genotyping of the majority of CYP2D6 poor metabolizers.
Analytical Quality of Near-Patient Blood Cholesterol and Glucose Determinations
Authors: du Plessis Marita , Ubbink Johan B , Vermaak W J Hayward
Published: January 2020

Background: Screening for diabetes and hypercholesterolemia is widely advocated, and extra-laboratory testing could play a major role in cost-effective population screening. We wished to assess the analytical quality and interchangeability of capillary blood cholesterol and glucose assays as performed on near-patient devices in pharmacies in Pretoria, South Africa.

Methods: Accuracy of near-patient and laboratory analyzers was assessed by analyses of human-serum-based reference material. To assess interchangeability in routine use, six volunteers visited each of 12 randomly selected pharmacies consecutively during a 3-week period to have their fasting blood glucose and cholesterol concentrations determined. For comparison purposes, a similar procedure was followed to evaluate the eight clinical chemistry laboratories servicing Pretoria and surroundings.

Results: The analytical performances in our laboratory of a single point-of-care instrument and of a laboratory analyzer compared well. Nevertheless, between-pharmacy analytical variation was larger than between-laboratory variation (11% vs 6.1% for cholesterol; 10% vs 7.6% for glucose). For glucose measurements, near-patient testing in pharmacies demonstrated a bias of −48.1% to 16.2%, whereas bias for laboratory measurements was −1.0% to 7.4%. Cholesterol assays showed a bias of −5.6% to 16.6% in pharmacies compared with −10.6% to 3.7% in laboratories. The percentage of closeness to the homeostatic set point for a single glucose and cholesterol determination done in any pharmacy was 24.6% and 23.6%, respectively. The corresponding values for laboratories were 16.9% and 15.6%, respectively.

Conclusions: Although modern point-of-care instruments allow high-quality blood analyses under ideal conditions, performance goals often are not achieved in practice as indicated by a higher uncertainty of cholesterol and glucose blood results when determined in pharmacies. Nonuniformity of calibration procedures, deficiencies in training, a lack of internal quality control, and the absence of an external quality assessment program may all contribute to the current state of affairs.