bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Urine Certified Reference Material for Arsenic Speciation
Authors: Yoshinaga Jun , Chatterjee Amit , Shibata Yasuyuki , Morita Masatoshi , Edmonds John S
Published: January 2020

Background: Chemical speciation analysis is essential for the biological monitoring of inorganic arsenic exposure using urine as indicator medium. There is increasing demand for a certified reference material (CRM) of urine matrix for arsenic speciation.

Methods: Urine (10 L) was collected from non-occupationally exposed Japanese males. We prepared 954 bottles of urine, each containing ∼10 mL, after filtering and blending the urine stock. The urine in each bottle was freeze-dried. Between-bottle homogeneity was confirmed by measuring the concentrations of selected minor and trace elements in the material and subsequent statistical analysis. Certification was based on a collaborative analysis involving 15 laboratories.

Results: Certified values were determined for arsenobetaine (0.069 ± 0.012 mg As/L), dimethylarsinic acid (0.036 ± 0.009 mg As/L), and total arsenic (0.134 ± 0.011 mg/L) as well as for total selenium (0.059 ± 0.005 mg/L) and zinc (0.62 ± 0.05 mg/L), based on the analytical values from the collaborating laboratories. Reference values are given for copper (0.010 mg/L) and lead (0.0011 mg/L), based on definitive analysis at the National Institute for Environmental Studies (NIES).

Conclusions: The present CRM, NIES CRM No. 18 Human Urine, is the first human urine CRM for arsenic speciation and will be of value for analytical quality assurance of the biological monitoring of arsenic exposure.
Stability of Methylecgonidine and Ecgonidine in Sheep Plasma in Vitro
Authors: Scheidweiler Karl B , Shojaie Jalil , Plessinger Mark A , Wood Ronald W , Kwong Tai C
Published: January 2020

Background: Crack smokers are exposed to a pyrolysis product, methylecgonidine (MEG), which can be used as an analytical marker for crack smoking. Ecgonidine (EC), a hydrolytic product of MEG, has been identified in urine of crack smokers. MEG undergoes conversion to EC, complicating analysis and perhaps explaining a lack of forensic blood specimens containing MEG.

Methods: We developed gas chromatography–mass spectrometry (GC-MS) assays for MEG and EC. Plasma was collected from sheep blood containing 0, 0.06, or 0.24 mol/L (0%, 0.25%, or 1%) NaF. MEG was added to these plasmas, and they were incubated at −80, 1, 21, or 37 °C to determine whether there were temporal, temperature, or storage effects on MEG stability over 48 h.

Results: Decreased temperature and increased NaF concentrations limited MEG degradation and EC formation. MEG stored in plasma at −80 °C was stable up to 1 month, even in the absence of NaF.

Conclusions: MEG is stable in sheep plasma collected in commercially available, evacuated blood-collection tubes containing NaF and stored at −80 °C. In vitro formation of EC can be minimized with appropriate sample handling, and its in vivo formation may provide a better marker of crack smoking than its parent pyrolysis product.
DNA Base Bulge vs Unmatched End Formation in Probe-based Diagnostic Insertion/Deletion Genotyping: Genotyping the UGT1A1 (TA)n Polymorphism by Real-Time Fluorescence PCR
Authors: von Ahsen Nicolas , Oellerich Michael , Schütz Ekkehard
Published: January 2020

Background: Gilbert syndrome is a clinically inconsequential entity of mild unconjugated hyperbilirubinemia caused by an A(TA)nTAA insertion polymorphism (UGT1A1*28) in the promoter region of the gene coding for the enzyme UDP-glucuronosyltransferase 1 (EC 2.4.1.17; UGT1A1). Present methods for genotyping this polymorphism are laborious.

Methods: Hybridization probes were designed complementary to the wild type (TA)6 and to alleles with (TA)7 and (TA)8 repeats in the promoter region. Melting points were measured in samples representing all currently known alleles with (TA)5 to (TA)8 repeats. Probe melting points were predicted with a thermodynamic nearest-neighbor model for Watson-Crick paired probes. The dominant secondary structures resulting from probe hybridization were predicted by thermodynamic free energy calculations. Alternatively samples were genotyped based on amplicon size resolved by high-resolution polyacrylamide gel electrophoresis.

Results: Only short probes (22–24 bases) could be successfully used for genotyping this locus because of the very low stability of this TA repeat. Assays based on (TA)7 or (TA)8 genotype-compatible hybridization probes effectively discriminated five to eight TA repeats. The consecutive use of two different detection probes was necessary for better discrimination of some heterozygous genotypes. All results were in concordance with the alternative genotyping method. Of 100 investigated Caucasians (50 males, 50 females), 9 (9%) were homozygous for the (TA)7 allele.

Conclusions: The presented method for genotyping the (TA)n promoter polymorphism of the UGT1A1 gene with the LightCycler has the potential to genotype all currently known (TA)n repeats in a single assay and is sensitive toward possible new genotypes. Our findings also show that thermodynamic calculations are of practical value for the design of hybridization probe assays for the genotyping of insertion/deletion polymorphisms.
Sensitive and Specific Immunodetection of Human Glandular Kallikrein 2 in Serum
Authors: Becker Charlotte , Piironen Timo , Kiviniemi Johanna , Lilja Hans , Pettersson Kim
Published: January 2020

Background: Human glandular kallikrein 2 (hK2) is expressed in the prostate and is present in serum from men with prostate cancer. Specific detection in serum is difficult mainly because of low concentrations and immunological cross-reactivity with prostate-specific antigen (PSA). Our objectives were to design an assay with improved analytical detection and functional sensitivity and nonsignificant cross-reactivity with PSA, and to characterize different immunoreactive forms of hK2.

Methods: In the assay, critical PSA epitopes were blocked with four monoclonal antibodies (MAbs) specific for PSA. Subsequently, hK2 was captured using a MAb against hK2 (5% cross-reactivity with PSA), and after washing, hK2 was detected by a europium-labeled MAb with identical affinity for hK2 and PSA.

Results: The analytical detection limit was <10 ng/L, and functional sensitivity was 30 ng/L. Cross-reaction with PSA was <0.01%. Between-assay imprecision was 3.1% for 1600 ng/L hK2 and 4.8% for 160 ng/L hK2; corresponding values for within-assay precision were 1.9% and 4.5%, respectively. Complexes of hK2-α1-antichymotrypsin (ACT) were detected in vitro with −6% bias compared with the free form of hK2. Gel filtration of patient samples showed that hK2 correlated in size mainly with free hK2; only 4–19% corresponded to hK2 possibly complexed with ACT or protein C inhibitor.

Conclusions: Our assay had extremely low cross-reactivity with PSA, provided a very low detection limit, and allowed close to equimolar detection of the free and complexed forms of hK2. Moreover, we found that free hK2 is the predominant immunoreactive form of hK2 in serum.
Sodium Pump Isoforms in Xenotransplantation: Importance of Biochemical Compatibility
Authors: Rose Andrea M , Qazzaz Hassan M , Zolotarjova Nina , Mellett Brenda J , Martin Alvin W , Valdes Jr Roland
Published: January 2020

Background: Xenotransplantation of pig hearts to humans could be hampered by the reportedly reduced affinity for digoxin of pig heart. We examined the hypothesis that expression of the individual α-subunit isoforms of the sodium pump [Na+,K+-ATPase (NKA)], the receptor for the plant-derived cardiac glycosides, may be responsible for this difference.

Methods: We used a NKA-inhibition assay in combination with Western analysis, immunohistochemistry, and phosphorylation of the NKA α subunit to identify the distribution and expression of α isoforms in four chambers of porcine and human hearts.

Results: We confirmed that tissue from porcine heart is less sensitive to digitalis (IC50 = 1740 nmol/L) when compared with human heart (IC50 = 840 nmol/L), whereas porcine cerebral cortex-mix had an affinity comparable to that of human heart (IC50 = 910 nmol/L). Our data show that porcine cerebral cortex-mix and human heart contain all three α isoforms, whereas porcine heart expresses only the α1 isoform.

Conclusions: The different expressions of sodium pump isoforms in human vs porcine cardiac tissues suggests that porcine hearts may not be pharmacologically or endocrinologically compatible when used in humans. Studies of both pharmacologic and endocrinologic tissue compatibility are needed prior to selection of organs for xenotransplantation.
Development of a New Assay for Complex I of the Respiratory Chain
Authors: Brooks Hilary , Krähenbühl Stephan
Published: January 2020

Background: Measurement of complex I activity has been hampered by the large amounts of tissue required and the resulting turbidity of the assay solution, which makes spectrophotometric analysis difficult. We have developed a new assay for measuring the activity of complex I in isolated mitochondria that is also applicable to skeletal muscle homogenate in patients with suspected mitochondrial diseases.

Methods: The method was a radioenzymatic assay based on the preferential oxidation of the 4B hydrogen of NADH by complex I. We prepared tritiated isoforms of NADH for both the respective 4A-3H and 4B-3H positions. Enzyme in the form of purified mitochondria or homogenate was prepared from rat or human skeletal muscle and incubated with the respective radioisotopes. The product (3H2O) was collected after charcoal adsorption of unreacted NADH and taken as an indicator of NADH oxidation. Sensitivity to rotenone was used as a measure of complex I specific activity.

Results: The assay was linear with time and protein for isolated mitochondria and tissue homogenates from rats and humans. The Vmax and Km values obtained for 4B-NADH with isolated rat skeletal muscle mitochondria were 35 μmol/L and 90 μmol · min−1 · mg protein−1, respectively. The assay was reproducible and useable for routine measurements in human skeletal muscle. The sensitivity was >10-fold higher than the sensitivities of spectrophotometric techniques.

Conclusions: The results of our studies demonstrate the successful development of a new assay for complex I that is rapid, easy to perform, and that enables the processing of multiple samples at one time.
Estimating the Long-Term Effects of Storage at −70 °C on Cholesterol, Triglyceride, and HDL-Cholesterol Measurements in Stored Sera
Authors: Shih Weichung Joe , Bachorik Paul S , Haga Jo A , Myers Gary L , Stein Evan A
Published: January 2020

We estimated the effects of long-term storage at −70 °C on serum total cholesterol, HDL-cholesterol, and triglycerides in specimens that had been stored for up to 7 years. These estimates were made using measurements in serial specimens collected from the placebo control group of the Air Force/Texas Coronary Atherosclerosis Prevention Study over a period of ∼5 years. We compared the group means for pairs of serial specimens taken at 6- and 12-month intervals, assuming that (a) a negligible placebo effect occurred between the serial specimen pairs; (b) in the absence of storage effects, the variation in the group means would reflect only normal biological variation and would not materially affect the group means for the serial specimens; (c) any systematic changes in these group means would reflect storage-related changes; and (d) storage-related changes are cumulative, i.e., the overall changes for a given storage period are the sum of the changes during previous storage periods. We observed average decreases of 2.0% per year for total cholesterol over 7 years and 2.8% per year in triglycerides for the first 5 years. HDL-cholesterol decreased by 1.3% per year, but this change was not statistically significant. This approach may be useful for estimating storage-related changes for studies in specimens stored for a period of years and for which stability data may not be available.
Total Protein Determination in Urine: Elimination of a Differential Response between the Coomassie Blue and Pyrogallol Red Protein Dye-binding Assays
Authors: Marshall Thomas , Williams Katherine M
Published: January 2020

Background: The total protein content of urine is a good index of renal function, but its determination is unreliable. Protein dye-binding assays are simple, but they characteristically lack a uniform response to different proteins.

Methods: We investigated a differential response of the Sigma Microprotein Coomassie Brilliant Blue (CBB) and Pyrogallol Red-molybdate (PRM) protein dye-binding assays to urine, using human albumin, albumin/globulin, or urinary protein as calibrator.

Results: The urine protein values (n = 60) obtained with the CBB assay were 110–13 500 mg/L (mean, 2390 mg/L) compared with 160–18 300 mg/L (mean, 3470 mg/L) obtained with the PRM assay (CBB:PRM protein concentration ratio, 0.46–0.88, mean, 0.69 ± 0.10). The differential response was highly reproducible as indicated by Sigma urine control Level 1 (within-day CBB:PRM ratio, 0.68 ± 0.02; between-day CBB:PRM ratio, 0.67 ± 0.04) and Sigma urine control Level 2 (within-day CBB:PRM ratio, 0.60 ± 0.01; between-day CBB:PRM ratio, 0.59 ± 0.02). The use of urinary protein as a calibrator (rather than human albumin) greatly improved the agreement between the assays when applied to urine (yCBB = 0.972xPRM − 16 vs yCBB = 0.685xPRM + 17). In studies using urine controls, this calibrator also improved agreement between the CBB, PRM, trichloroacetic acid (TCA), and benzethonium chloride protein methods and, to a lesser extent, agreement with the TCA-Ponceau S method.

Conclusion: The use of a urinary protein calibrator improves the agreement between different methods used to determine total protein in urine.
Development of a Kinetic Assay for Band 5b Tartrate-resistant Acid Phosphatase Activity in Serum
Authors: Nakanishi Mamoru , Yoh Kousei , Miura Toshihide , Ohasi Tatsuya , Rai Shiba Kumar , Uchida Kagehiro
Published: January 2020

Background: Band 5 tartrate-resistant acid phosphatase (TrACP; EC 3.1.3.2) consists of two isoenzymes, bands 5a and 5b, of which band 5b TrACP is considered to be derived from bone. However, no kinetic method for the specific measurement of band 5b TrACP in serum is available. Our aim was to develop a kinetic assay method for the specific measurement of band 5b TrACP in serum.

Methods: Band 5b TrACP was measured kinetically in serum as tartrate-resistant fluoride-sensitive heparin-resistant ACP with 2,6-dichloro-4-acetylphenyl phosphate as substrate at pH 6.6.

Results: Heparin inhibited band 5a TrACP but had no effect on band 5b TrACP in serum or in bone extract. The presence of EDTA or ascorbic acid had no effect, but dithiothreitol inhibited enzyme activity. The within-run (n = 20) and between-run (n = 20) CVs of band 5b TrACP activity were 3.3–5.8% and 5.0–7.3%, respectively. The mean ± SD values of band 5b TrACP activity in males (n = 25) and females (n = 57) 20–29 years of age by this method were 8.0 ± 2.2 U/L and 6.4 ± 1.8 U/L, respectively. The band 5b TrACP value was significantly higher in females >50 years of age compared with the younger subjects (20–29 years). The highest band 5b TrACP values were among children younger than 15 years.

Conclusions: This kinetic assay is a simple and specific method for the measurement of band 5b TrACP in serum samples and is useful in the evaluation of bone turnover activity.
Analysis of Free Prostate-specific Antigen (PSA) after Chemical Release from the Complex with α1-Antichymotrypsin (PSA-ACT)
Authors: Peter Jochen , Unverzagt Carlo , Hoesel Wolfgang
Published: January 2020

Background: Prostate-specific antigen (PSA), a marker for prostate cancer (CaP), forms a covalent complex with α1-antichymotrypsin (ACT) in human blood. Structural analysis of the PSA-ACT complex is difficult, and complexation may be a reason for biased immunological assays when compared with the analysis of free PSA. We developed a method to cleave the PSA-ACT complex chemically. The liberated PSA was thus available for analysis as free PSA (F-PSA).

Methods: PSA was released from the PSA-ACT complex by cleaving the interprotein ester bond with ethanolamine under alkaline conditions. The release was followed by reversed-phase HPLC and an immunoassay for F-PSA. Released PSA obtained from human blood was further immunopurified and analyzed by matrix-assisted laser desorption-induced time of flight (MALDI-TOF) mass spectrometry.

Results: In vitro-prepared PSA-ACT complex was completely cleaved by treatment with nucleophilic compounds such as ethanolamine at pH 9–10. The released PSA was stable under these conditions and could be measured by reversed-phase HPLC as well as the ENZYMUN® immunoassay for F-PSA. When plasma from a CaP patient [containing 190 μg/L F-PSA and 1890 μg/L total PSA (T-PSA)] was treated under similar conditions, a concentration of ∼1600 μg/L F-PSA was measured at the end of the incubation, indicating that the PSA-ACT complex was completely cleaved. Two benign prostatic hyperplasia and CaP sera panels (12 and 13 sera, respectively) containing 4–45 μg/L T-PSA were similarly treated. The concentrations of F-PSA measured after incubation were, on average, 85% of the T-PSA values of the untreated sera. Finally, the PSA released from the complex of the CaP plasma was isolated by immunosorption, analyzed by MALDI-TOF mass spectrometry, and compared to PSA obtained from semen. The intact PSA as well as the peptides observed after digestion with endoproteinase Lys C did not reveal any structural difference between the PSA from these two sources.

Conclusions: PSA complexed to ACT in plasma of a CaP patient seems to be structurally very similar to the PSA reference material from semen. The release of PSA from the PSA-ACT complex allows F-PSA and T-PSA to be measured by the same immunological assay, thus eliminating any possible bias between two different assays.
Analytical and Clinical Performance of a Detergent-based Homogeneous LDL-Cholesterol Assay: A Multicenter Evaluation
Authors: Nauck Matthias , Graziani Maria Stella , Bruton Deborah , Cobbaert Christa , Cole Thomas G , Lefevre Fabrice , Riesen Walter , Bachorik Paul S , Rifai Nader
Published: January 2020

Background: LDL-cholesterol (LDL-C) concentrations currently are determined in most clinical laboratories using the Friedewald calculation. This approach has several limitations and may not always meet the current total error recommendation in LDL-C measurement of ≤12% established by the National Cholesterol Education Program.

Methods: In a multicenter study, we evaluated the analytical and clinical performance of a homogeneous LDL-C assay (LDL-CRoche; Roche Diagnostics, Indianapolis, IN) in a comparison with a β-quantification method.

Results: This direct assay correlated highly with a β-quantification method (r = 0.968; y = 1.037x − 95.8 mg/L; n = 355; 95% confidence intervals, 1.011–1.063 for the slope and −129.5 to 62.0 mg/L for the y-intercept) and met the current total error requirement. The assay was not affected significantly by concentrations of hemoglobin up to 6000 mg/L or bilirubin up to 500 mg/L. However, a negative bias of 10% was seen when triglyceride concentrations exceeded 10 000 mg/L. At the medical decision cut-point range, the LDL-CRoche assay showed positive predictive values of 91–100% and negative predictive values of 80–99%. Furthermore, the clinical utility of the assay seemed unaffected in samples obtained postprandially.

Conclusions: The homogeneous LDL-CRoche assay meets the currently established analytical performance goals and may be useful for the diagnosis and management of hyperlipidemic patients.
Oral Testosterone Administration Detected by Testosterone Glucuronidation Measured in Blood Spots Dried on Filter Paper
Authors: Peng Shi-Hua , Segura Jordi , Farré Magí , de la Torre Xavier
Published: January 2020

Background: Blood sampling is not a common practice for sports drug testing. Our aim was to investigate whether dried blood spots on filter paper could be an alternative to plasma samples for monitoring steroid profiles in dope testing.

Methods: We collected dried blood spots and plasma from six healthy Caucasian subjects after an oral 120-mg dose of testosterone undecanoate (TU). Nonconjugated testosterone, testosterone glucuronide (TG), androsterone glucuronide (AG), and etiocholanolone glucuronide (EtG) were measured by gas chromatography–mass spectrometry in both matrices. 17α-Hydroxyprogesterone (17αOHP) and luteinizing hormone (LH) also were measured in the plasma samples. For comparison, similar measurements were done on samples obtained from the same subjects given 25 mg of testosterone propionate (TP) plus 110 mg of testosterone enanthate (TE) intramuscularly after a wash-out period.

Results: After oral TU intake, TG, AG, and EtG increased sharply, whereas nonconjugated testosterone did not change significantly. Results on dried blood spots correlated well with those on plasma. The TG/testosterone ratio in blood or plasma was verified to be a sensitive and specific marker (significantly increased for up to 8 h after intake; P <0.05) for oral TU intake but not for intramuscular administration of TP plus TE. Little suppression of plasma LH and 17αOHP was observed after a single oral dose of TU. One subject did not show a significant increase of blood TG after oral TU intake.

Conclusions: The measurement of glucuronide conjugates in blood and plasma samples is relevant for sports drug testing when analyzing the steroid profile. Dried blood spots collected on filter paper are a suitable alternative to plasma for detecting testosterone abuse.
Remnant-like Particle-Cholesterol Concentrations in Patients with Type 2 Diabetes Mellitus and End-Stage Renal Disease
Authors: Hirany Shaina , O’Byrne Dawn , Devaraj Sridevi , Jialal Ishwarlal
Published: January 2020

Background: Lipid abnormalities contribute significantly to the increased risk of cardiovascular disease in diabetic and end-stage renal disease (ESRD) patients. Accumulating evidence supports a proatherogenic role for remnant lipoproteins. Thus, the aim of the present study was to compare remnant-like particle-cholesterol (RLP-C) in type 2 diabetic and ESRD patients with age- and gender-matched controls.

Methods: Using an immunoaffinity assay, we measured RLP-C concentrations in 48 type 2 diabetic patients with (n = 24) and without (n = 24) macrovascular complications, and 24 age- and gender-matched controls, as well as in 38 ESRD patients on hemodialysis (n = 19) and peritoneal dialysis (n = 19), and 19 age- and gender-matched controls.

Results: RLP-C correlated significantly with plasma triglycerides (TGs; r = 0.8). When compared with controls, RLP-C concentrations were significantly higher in type 2 diabetic patients with and without macrovascular complications (median, 0.22 and 0.17 mmol/L vs 0.14 mmol/L; P <0.0002 and <0.01, respectively); diabetic patients with macrovascular complications also had significantly higher RLP-C than diabetic patients without macrovascular complications (P <0.05). However, when RLP-C/TG ratios were computed, only diabetic patients with macrovascular complications showed significantly higher RLP-C/TG ratios compared with controls (P <0.05). Regarding ESRD, RLP-C concentrations were significantly increased in patients on both hemodialysis and peritoneal dialysis compared with controls (median, 0.23 and 0.21 mmol/L vs 0.13 mmol/L; P <0.0001). Whereas RLP-C was increased in ESRD patients on hemodialysis with TGs <2.26 mmol/L compared with controls, RLP-C/TG ratios were not significantly increased in these patients.

Conclusions: Type 2 diabetic patients with macrovascular disease demonstrated increased RLP-C and RLP-C/TG ratios, whereas ESRD patients showed only increased RLP-C concentrations.
Influence of Glomerular Filtration Rate on Non-(1-84) Parathyroid Hormone (PTH) Detected by Intact PTH Assays
Authors: Brossard Jean-Hugues , Lepage Raymond , Cardinal Héloïse , Roy Louise , Rousseau Louise , Dorais Claude , D’Amour Pierre
Published: January 2020

Background: Commercial intact parathyroid hormone (I-PTH) assays detect molecular form(s) of human PTH, non-(1-84) PTH, different from the 84-amino acid native molecule. These molecular form(s) accumulate in hemodialyzed patients. We investigated the importance of non-(1-84) PTH in the interpretation of the increased I-PTH in progressive renal failure.

Methods: Five groups were studied: 26 healthy individuals, 12 hemodialyzed patients, and 31 patients with progressive renal failure subdivided according to their glomerular filtration rate (GFR) into 11 with a GFR between 60 and 100 mL · min−1 · 1.73 m−2, 12 with a GFR between 30 and 60 mL · min−1 · 1.73 m−2, and 8 with a GFR between 5 and 30 mL · min−1 · 1.73 m−2. We evaluated indicators of calcium and phosphorus metabolism and creatinine clearance (CrCl) in the progressive renal failure groups, and the HPLC profile of I-PTH and C-terminal PTH in all groups.

Results: Only patients with a GFR <30 mL · min−1 · 1.73 m−2 and hemodialyzed patients had decreased Ca2+ and 1,25-dihydroxyvitamin D, and increased phosphate. In patients with progressive renal failure, I-PTH was related to Ca2+ (r = −0.66; P <0.0001), CrCl (r = −0.61; P <0.001), 1,25-dihydroxyvitamin D (r = −0.40; P <0.05), and 25-hydroxyvitamin D (r = −0.49; P <0.01) by simple linear regression. The importance of non-(1-84) PTH in the composition of I-PTH increased with each GFR decrease, being 21% in healthy individuals, 32% in progressive renal failure patients with a GFR <30 mL · min−1 · 1.73 m−2, and 50% in hemodialyzed patients, with PTH(1-84) making up the difference.

Conclusions: As I-PTH increases progressively with GFR decrease, part of the increase is associated with the accumulation of non-(1-84) PTH, particularly when the GFR is <30 mL · min−1 · 1.73 m−2. Concentrations of I-PTH 1.6-fold higher than in healthy individuals are necessary in hemodialyzed patients to achieve PTH(1-84) concentrations similar to those in the absence of renal failure.
Modular Robotic Workcell for Coagulation Analysis
Authors: Graves Sean , Holman Bill , Felder Robin A
Published: January 2020

Background: Total laboratory automation (TLA) has been shown to increase laboratory efficiency and quality. However, modular automation is smaller, requires less initial capital, and requires less planning than TLA. We engineered and performed clinical trials on a modular robotic preanalytical workcell for coagulation analysis.

Methods: Timing studies were used to quantify the efficiency of the manual processes and to identify areas in the processing of coagulation specimens where bottlenecks and long waiting periods were encountered. We then designed our modular robotic system to eliminate these bottlenecks. Our robotic modular workcell was engineered to allow a choice of specimen introduction manually, by conveyor, or by mobile robot. Additional timing studies were performed during clinical trials of the robotic system.

Results: Prior to automation, the time required for preanalytical processing time was 18–107 min; after automation, it was 45–50 min. Additional improvements in workcell efficiency could be realized when high quality, prelabeled specimens were introduced into the system.

Conclusion: Compared with manual methods, modular automation provides more predictable variation in specimen processing.
Detection of the Finnish-Type Congenital Nephrotic Syndrome by Restriction Fragment Length Polymorphism and Dual-Color Oligonucleotide Ligation Assays
Authors: Romppanen Eeva-Liisa , Mononen Ilkka
Published: January 2020

Background: Congenital nephrotic syndrome of Finnish type (NPHS1) is an autosomal recessive disorder characterized by severe proteinuria of intrauterine onset. Ninety-four percent of the Finnish NPHS1 chromosomes have been reported to carry either a 2-bp deletion in exon 2 (FinMajor) or a nonsense mutation in exon 26 (FinMinor) of the NPHS1 gene. The high prevalence of only two mutations in the Finnish population enables the use of molecular techniques in the diagnosis of NPHS1 and for carrier screening.

Methods and Results: We describe two different molecular methods for the detection of the NPHS1 mutations: a PCR-restriction fragment length polymorphism (PCR-RFLP) and a dual-color oligonucleotide ligation assay (OLA). The dual-color OLA, which enables simultaneous detection of the NPHS1 FinMajor and FinMinor mutations, can be used for rapid analysis of large sets of samples. The analysis of 2004 Finnish blood samples revealed 34 carriers of the FinMajor mutation and 1 carrier of the FinMinor mutation, indicating a carrier frequency of 1:59 (95% confidence interval, 1:89–1:44) for the NPHS1 FinMajor mutation and 1:2004 (95% confidence interval, 0 to 1:677) for the NPHS1 FinMinor mutation, respectively.

Conclusion: PCR-RFLP and dual-color OLA are suitable for molecular diagnosis and carrier screening of the major mutations that cause NPHS1.
Circulating Immunoreactive proANP(1-30) and proANP(31-67) in Sedentary Subjects and Athletes
Authors: De Palo Elio F , Woloszczuk Wolfgang , Meneghetti Martina , De Palo Carlo B , Nielsen Henning B , Secher Niels H
Published: January 2020

Background: Atrial natriuretic peptide (ANP) is synthesized and stored in myocytes as prohormone(1-126), which upon release is cleaved into proANP(1-98) and α-ANP(99-126). In addition, cleavage of proANP(1-98) produces proANP(1-30), proANP(31-67), and proANP(79-98) fragments. ProANP(1-30) and proANP(31-67) have roles in fluid and electrolyte homeostasis. The aim of the present study was to develop a plasma assay for proANP(1-30) and proANP(31-67) and to compare results in trained athletes and sedentary subjects.

Methods: Two competitive enzyme immunoassays were established with affinity-purified sheep antiserum against synthetic ANP fragments. The immunoreactivity (ir) of proANP(1-30) and proANP(31-67) was measured in 10-μL plasma samples without extraction in a microwell-based assay. Plasma concentrations in sedentary male subjects (n = 22) and male endurance athletes (n = 14) were examined.

Results: In the assay for ir-proANP(1-30) and ir-proANP(31-67), the concentrations at 95% B/B0 were 4.7 and 14.2 pmol/L, respectively. Within-run CVs were 4–6% and 5–6%, and between-run CVs were 9% for both assays. Both assays were linear on dilution (y = 0.9945x − 0.7291 and y = 1.0001x − 3.428), and the recoveries were 102–112% and 102–106%, respectively. In the sedentary and athletic groups, the ir-proANP(1-30) concentrations were similar: 318 ± 38 pmol/L and 312 ± 25 pmol/L (mean ± SE), respectively, whereas the ir-proANP(31-67) was higher in the rowers (713 ± 81 pmol/L) than in the sedentary subjects (387 ± 71 pmol/L; P <0.005).

Conclusions: The proANP fragment assays are precise (CV <10%) and exhibit nearly quantitative recovery (102–112%). Only ir-proANP(31-67) responds to physical training.
Description of a Computer Program to Assess Cancer Antigen 15.3, Carcinoembryonic Antigen, and Tissue Polypeptide Antigen Information during Monitoring of Metastatic Breast Cancer
Authors: Sölétormos György , Schiøler Vibeke
Published: January 2020

It is time-consuming to process and compare the clinical and marker information registered during monitoring of breast cancer patients. To facilitate the assessment, we developed a computer program for interpreting consecutive measurements. The intraindividual biological variation, the analytical precision profile, the cutoff limit, and the detection limit for each marker are entered and stored in the program. The assessment procedure for marker signals considers the analytical and biological variation of the applied markers. The software package contains a database that can store the interpretation of the measurements as evaluation codes together with patient demographics, information about treatment type, dates for treatment periods, control periods, and evaluation codes for clinical activity of disease. The consecutive concentrations for a patient are imported temporarily into the program from outside sources and presented graphically. Marker concentrations to be compared are selected with the computer mouse and the significance of the difference is calculated by the program. The program has an option for calculating the lead time of marker signals vs clinical information. The program facilitates the monitoring of individual breast cancer patients with tumor marker measurements. It may also be implemented in trials investigating the utility of potential new markers in breast cancer as well as in other malignancies.
Space Flight Is Associated with Rapid Decreases of Undercarboxylated Osteocalcin and Increases of Markers of Bone Resorption without Changes in Their Circadian Variation: Observations in Two Cosmonauts
Authors: Caillot-Augusseau Anne , Vico Laurence , Heer Martina , Voroviev Dimitri , Souberbielle Jean-Claude , Zitterman Armin , Alexandre Christian , Lafage-Proust Marie-Hélène
Published: January 2020

Background: Microgravity induces bone loss by mechanism(s) that remain largely unknown.

Methods: We measured biochemical markers related to bone remodeling in two cosmonauts before, during, and after 21- and 180-day space flights, respectively.

Results: During both flights, type I procollagen propeptide and bone alkaline phosphatase decreased as early as 8 days after launch. Undercarboxylated osteocalcin percentage increased early and remained high during both flights. Vitamin K supplementation restored carboxylation of osteocalcin during the long-term flight. Urinary and serum C-telopeptide of type I collagen (CTX) increased as early as day 8 of the flights; the increase was greater in serum than in urine. Pyridinoline, free deoxypyridinoline, and N-telopeptide increased less than CTX during the short-term space flight. The circadian rhythm of bone resorption assessed by urine CTX and free deoxypyridinoline was not altered by microgravity.

Conclusion: Vitamin K metabolism or action and bone remodeling may be altered in cosmonauts.
Rapid Method to Characterize Mutations in Transthyretin in Cerebrospinal Fluid from Familial Amyloidotic Polyneuropathy Patients by Use of Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Authors: Bergquist Jonas , Andersen Oluf , Westman Ann
Published: January 2020

Background: Familial amyloidotic polyneuropathy (FAP) type I, the most common dominantly inherited form of amyloidosis, is caused by a Val-to-Met point mutation at position 30 (Val30→Met) in the protein transthyretin. Mass spectrometric analysis can identify modification of proteins, such as point mutations, acetylation, phosphorylation, sulfation, oxidation, and glycosylation.

Methods: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra from cerebrospinal fluid (CSF) drawn from a patient with FAP were compared with CSF from controls. We also isolated transthyretin with a Centrisart molecular size cutoff filter and performed high-accuracy peptide mass mapping to localize the site of the amino acid substitution (Val30→Met).

Results: Mass spectra of transthyretin were produced directly from human CSF as well as from CSF after a simple prepurification method without immunoprecipitation. On-target tryptic digestion and MALDI-MS verified mass spectrometric peak identification. The point mutation was still detectable in CSF after hepatic transplantation.

Conclusions: It is possible to diagnose FAP by a rapid MALDI-TOF MS analysis using only 100 μL of CSF, with only 250 nL actually consumed on target. The approach may also be useful to monitor production of mutated transthyretin by choroid plexus, especially after liver transplantation.
Mechanism of Interference of a Polymerized Hemoglobin Blood Substitute in an Alkaline Phosphatase Method
Authors: Chance Jeffrey J , Norris Edward J , Kroll Martin H
Published: January 2020

Background: Hemoglobin-based oxygen carriers can cause profound interferences in many analytical procedures. We determined the mechanism of interference in the assay of alkaline phosphatase activity and identified approaches that might be used to correct for this interference.

Methods: Interference of a polymerized hemoglobin blood substitute with the assay of alkaline phosphatase was examined with a Hitachi 917 analyzer and ultraviolet-visible spectrophotometry.

Results: Hemoglobin-based oxygen carrier solutions had substantial absorbance at 415 nm, the wavelength of analysis used to measure the formation of 4-nitrophenol. In addition to offsetting the initial absorbance at the analytical wavelength, polymerized hemoglobin gave rise to a strong negative interference plot because of alkali denaturation of the substitute. The same interference mechanism was also observed for native hemoglobin (hemolysate), indicating that the interference was not derived from the polymerization process. The interference can be corrected by implementing a rate-correction procedure, or the interference can be avoided by measurement at 450 nm.

Conclusions: The interference of polymerized hemoglobin in the alkaline phosphatase assay is a result of an absorbance offset caused by alkali denaturation of hemoglobin. The interference can be corrected or avoided by modifying the calculation or the analytical wavelength. The correction strategy may also be applicable to improving the hemolysis index for this method.
Measurement of Human Plasma Phospholipid Transfer Protein by Sandwich ELISA
Authors: Oka Tomoichiro , Kujiraoka Takeshi , Ito Mayumi , Nagano Makoto , Ishihara Mitsuaki , Iwasaki Tadao , Egashira Tohru , Miller Norman E , Hattori Hiroaki
Published: January 2020

Background: Plasma phospholipid transfer protein (PLTP) plays a central role in the remodeling of HDLs. Reliable and accurate methods for assaying PLTP concentration are required.

Methods: A sandwich ELISA for PLTP has been developed, using two monoclonal antibodies against recombinant human PLTP (rhPLTP) expressed in Chinese hamster ovary cells. The ELISA allows for the quantification of PLTP in the range 0.625–15.0 ng/assay (1.2–30.0 mg/L). Intra- and interassay CVs were <3.0% and <4.2% respectively. The assay was used to quantify plasma PLTP concentrations in 132 Japanese subjects (75 males and 57 females).

Results: PLTP concentrations were 12.0 ± 3.0 mg/L (mean ± SD; range, 4.9–20.5 mg/L). No sex difference was observed. Plasma PLTP concentration was positively correlated with HDL-cholesterol (r = 0.72; P <0.001), apolipoprotein (apo) A-I (r = 0.62; P <0.001) and HDL2-cholesterol (r = 0.72; P <0.001), and was negatively correlated with triacylglycerol (r = −0.45; P <0.001). There was no correlation with plasma apo A-II. These results agree with other evidence that plasma PLTP is associated with large apo A-I-containing lipoproteins. There was no correlation (r = −0.01) between plasma PLTP and plasma phosphatidylcholine transfer activity (range, 3.5–10.5 μmol · mL−1 · h−1), suggesting that PLTP may exist in active and inactive forms.

Conclusion: This new ELISA will be of value for further studies of PLTP in health and disease.
Bioconjugates of Intelligent Polymers and Recognition Proteins for Use in Diagnostics and Affinity Separations
Authors: Hoffman Allan S
Published: January 2020

Polymers that respond to small changes in environmental stimuli with large, sometimes discontinuous changes in their physical state or properties are often called “intelligent” or “smart” polymers. We have conjugated these polymers to different recognition proteins, including antibodies, protein A, streptavidin, and enzymes. These bioconjugates have been prepared by random polymer conjugation to lysine amino groups on the protein surface, and also by site-specific conjugation of the polymer to specific amino acid sites, such as cysteine sulfhydryl groups, that are genetically engineered into the known amino acid sequence of the protein. We have conjugated several different smart polymers to streptavidin, including temperature-, pH-, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed here.
Interlaboratory Study on Thermal Cycler Performance in Controlled PCR and Random Amplified Polymorphic DNA Analyses
Authors: Saunders Ginny C , Dukes Juliet , Parkes Helen C , Cornett Johanne H
Published: January 2020

Background: Intercomparisons of PCR-based data between laboratories require an assurance of assay reproducibility. We performed an interlaboratory study to investigate the contribution made by a variety of thermal cyclers to PCR performance as measured by interblock reproducibility and intrablock repeatability.

Methods: Two standardized assays designed to minimize the introduction of non-thermal-cycler-dependent variations were evaluated by 18 laboratories in the United Kingdom, using 33 thermal cyclers of various makes and models. We used a single-product (590 bp) PCR, established in our laboratory as a robust and specific reaction. The second reaction, a multiproduct random amplified polymorphic DNA (RAPD) PCR, was known to be more susceptible to small changes in block temperature and was therefore considered a way of assessing block uniformity with respect to temperature. Assay repeatability data were analyzed with respect to temperature calibration status, the type of temperature control mechanism, thermal cycler age, and the presence of oil overlay or heated lid systems.

Results: All (100%) of the laboratories produced the correct target for the single-product PCR assay, although substantial variation in yield in replicate reactions was observed in 9.4% of these. The RAPD reaction generated results that varied extensively both within the same block and between different thermal cyclers. For eight replicates of a positive sample, 88% intrablock repeatability was demonstrated in calibrated thermal cyclers, which decreased to 63% in noncalibrated instruments.

Conclusions: Irrespective of the make and model of thermal cycler, temperature-calibrated instruments consistently generated more repeatable RAPD data than noncalibrated instruments. Guidelines are offered on optimizing and monitoring thermal cycler performance.
Effect of Urgent Clinician Notification of Low Hemoglobin Values
Authors: Froom Paul , Mahameed Tayser , Havis Rosa , Barak Mira
Published: January 2020

Background: Urgent clinician notification of low hemoglobin values is a common practice. The effect of such notification for samples obtained during an office visit is uncertain.

Methods: We notified the attending physicians in 100 consecutive cases of outpatients with hemoglobin <80 g/L. We reviewed the medical charts of these patients with their personal physicians 1–2 months later. We considered transfusions unnecessary only when given to a clinically stable patient with probable iron deficiency anemia in the absence of chronic diseases that produce the anemia.

Results: Overall, 47 (47%) of the patients were referred to the emergency room (ER), and 31 of the 47 (66%) were transfused. Increasing age and decreasing hemoglobin were associated with increased referral of patients to the ER. Six of 31 transfusions were unnecessary. An emergency transfusion was clearly indicated in only 3 of the 31 patients; the other patients were clinically stable. A modified notification strategy excluding microcytic samples >50 g/L would have detected these three patients. Over the follow-up period, no patient not referred to the ER died from complications of anemia, nor were any patients hospitalized with acute symptoms of low hemoglobin.

Conclusion: In some settings, urgent notification of medical outpatient clinics of patients with hemoglobin <80 g/L may lead to unnecessary ER visits.
Novel Technique for Scanning of Codon 634 of the RET Protooncogene with Fluorescence Resonance Energy Transfer and Real-Time PCR in Patients with Medullary Thyroid Carcinoma
Authors: Ruiz Agustín , Antiñolo Guillermo , Marcos Irene , Borrego Salud
Published: January 2020

Background: The multiple endocrine neoplasia 2 (MEN 2) syndromes [MEN 2A, MEN 2B, and familial medullary thyroid carcinoma (FMTC)] are caused by germline mutations of the RET protooncogene. Because 85% of MEN 2A patients and 30% of FMTC patients have mutations at codon 634, the recommended molecular analyses begin at exon 11, where codon 634 is located.

Methods: We scanned codon 634 of the RET protooncogene with real-time PCR and fluorescence resonance energy transfer (FRET), using a unique pair of internal probes to detect mutations localized at codon 634. We compared results with sequencing results in 66 patients.

Results: The method detected all codon 634 mutations available in our laboratory (Cys634Tyr, Cys634Arg, Cys634Phe, Cys634Trp). Comparing this method with the direct sequencing of exon 11 in a cohort of 66 patients with MTC, the system identified all 14 MTC patients carrying germline mutations at codon 634. One apparent false-positive result occurred among 52 patients.

Conclusions: The simultaneous scanning of multiple mutations is possible with the FRET system. The method allows rapid characterization of germline mutations at codon 634 in MTC patients.
Commutability of Calibration and Control Materials for Serum Lipase
Authors: Cattozzo Giampaolo , Franzini Carlo , Melzi d’Eril Gianvico
Published: January 2020

Background: To effectively assess and correct for intermethod variability, calibration and control materials (CCMs) must show the same intermethod behavior as patient sera, i.e., they must be commutable. We describe the commutability of selected CCMs for lipase assays, the impact of noncommutability of CCMs in normalizing patient results, and characteristics of reagents that affect assay specificity and commutability.

Methods: Lipase was measured in 98 patient sera and in 29 commercial CCMs, with 2 commercial methods using different substrates and with 4 experimental methods using 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin) ester as substrate and colipase as cofactor, but differing in the stabilizing proteins used and in the size of the substrate micelles.

Results: The noncommutability rate, i.e., the frequency of aberrant intermethod behavior of CCMs in comparison with patient sera, was 27% for liquid CCMs and 47% for lyophilized CCMs. The normalized residuals, measuring the degree of noncommutability, were −2.3 to 2.4 for CCMs with “normal” lipase activity, and −3.5 to 21.7 for CCMs with higher lipase activity. Recalculation of patient results with CCMs as calibrators decreased or increased the original bias according to whether the CCMs were commutable.

Conclusions: For the lipase methods in this study, the frequency of noncommutability of CCMs is affected by assay-specific characteristics, including size of substrate micelles and the presence or absence of added proteins.