bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid, automated assay for progesterone on the Abbott AxSYM™ analyzer
Authors: Wilson David H , Groskopf William , Hsu Stephen , Caplan Diane , Langner Tom , Baumann Michael , DeManno Deborah , Williams Gregg , Payette Don , Dagel Cheryl , Lynch Don , Manderino George
Published: January 2020

We describe an automated assay for progesterone (P4) in human serum and plasma with the Abbott AxSYMTM random-access immunoassay analyzer. In this one-step competitive assay, P4 immobilized onto latex microparticles competes with sample P4 for binding to a conjugate of alkaline phosphatase (AP) and anti-P4 antibody. Total CVs ranged from 3.4% to 8.2% in multiple precision studies conducted according to the 20-day NCCLS EP5-T protocol. The detection limit (zero calibrator + 2 SD) was 0.10 μg/L across 36 experiments. Values for diluted samples were 83–116% of expected. Recovery of P4 added to serum specimens was 92–115%. Cross-reactivities with 43 natural and synthetic steroids were 0–6.3%. No significant interference was detected from bilirubin, protein, erythrocytes, hemoglobin, triglycerides, or cholesterol. In a multisite correlation study, AxSYM P4 results compared well with results from a commercial RIA method (n = 1156; r = 0.976; slope = 1.03; y-intercept = 0.04). Assay throughput is >80 tests per hour in batch mode, 60 tests per hour with mixed load list configurations.
Cyclosporin whole blood immunoassays (AxSYM, CEDIA, and Emit): a critical overview of performance characteristics and comparison with HPLC
Authors: Schütz Ekkehard , Svinarov Dobrin , Shipkova Maria , Niedmann Paul-Dieter , Armstrong Victor W , Wieland Eberhard , Oellerich Michael
Published: January 2020

Assays with different specificity are used for cyclosporin monitoring in clinical transplantation. A recent survey of 35 centers showed that 86% used immunoassays for cyclosporin A (CsA). In consensus documents the following performance criteria were recommended: (a) imprecision ≤10% at 50 μg/L and ≤5% at 300 μg/L; and (b) comparison with the reference method (HPLC) should yield a slope of 0.9–1.1, an intercept of −15 to 15 μg/L, and Sy‖x ≤15 μg/L. The newly developed CsA assays for the AxSYM (Abbott) and the CEDIATM (Boehringer Mannheim) as well as the EmitTM assay (Behring Diagnostica) were evaluated. Results from samples of heart, kidney, and liver recipients (100 specimens each) were compared with a validated HPLC-ultraviolet detection method. Between-series imprecision (CV) with commercial controls was 5.8% and 1.7% for AxSYM (70 and 300 μg/L), 11% and 5.5% for CEDIA (90 and 200 μg/L), and 8.1% and 4.5% for Emit (63 and 172 μg/L). In the presence of 300 μg/L parent CsA, cross-reactivities were (for AxSYM, CEDIA, and Emit, respectively) 7%, 4%, and none for AM1 (1 mg/L) and 12.6%, 25%, and 6% for AM9 (0.5 mg/L). Comparison with HPLC showed in heart and kidney recipients an average overestimation with the Emit and the CEDIA of ∼22%, with overestimation in the AxSYM of 32%. In liver recipients, the most challenging patient group, the CEDIA and the AxSYM showed a mean overestimation of 43% and 47%, respectively, and the Emit differed by 31% compared with HPLC. None of the immunoassays fully satisfied the performance criteria recommended in the consensus documents. In terms of specificity, Emit ranks before CEDIA, which ranks before AxSYM. Regarding imprecision, the ranking is AxSYM < Emit < CEDIA. These limitations must be considered when using these assays for therapeutic drug monitoring of CsA in clinical transplantation.
Electrospray ionization mass analysis of normal and genetic variants of human serum albumin
Authors: Brennan Stephen O
Published: January 2020

Both normal albumin (Al A) and genetically modified forms were isolated from six heterozygous subjects. Albumins from each individual were analyzed by electrospray ionization mass spectrometry (ESI MS), and the mass was compared with that predicted from the protein sequence. In all cases, the Al A was heterogeneous, with components of mass (± SE) 66 463 ± 4, 66 586 ± 3, and 66 718 ± 5 Da. Each genetic variant showed similar heterogeneity. The mass increase in Al Casebrook (2214 Da) was very close to that predicted (2205 Da) from protein and carbohydrate sequence analysis, whereas the increase in Al Redhill (2378 Da) was close to that expected (2392 Da) for an Arg-albumin with a disialylated N-linked biantennary oligosaccharide and an Ala→Thr mutation. The circulating proalbumins, Christchurch and Blenheim, had mass increases of 748 and 756 Da, respectively, over Al A; in excellent agreement with theoretical values of 744 and 756. Clear shifts in mass were also detected for the point substitutions 177Cys→Phe (44 Da), 1Asp→Val (20 Da), and Arg-albumin (160 Da).
Time-dependent changes of serum carboxy-terminal peptide of type I procollagen and carboxy-terminal telopeptide of type I collagen concentrations in patients with acute myocardial infarction after successful reperfusion: correlation with left ventricular volume indices
Authors: Murakami Takashi , Kusachi Shozo , Murakami Masahiro , Sano Issei , Uesugi Tadahisa , Murakami Masaaki , Hirami Ryouichi , Kajiyama Akio , Kondo Jun , Tsuji Takao
Published: January 2020

To test the hypothesis that in patients with acute myocardial infarction (AMI), changes in the concentrations of the serum carboxy-terminal peptide of type I procollagen (PICP) and the carboxy-terminal telopeptide of type I collagen (ICTP) reflect extracellular matrix reformation and degradation, respectively, in the infarct healing processes, we measured these serum concentrations by RIA and compared their values with left ventricular (LV) indices obtained by left ventriculography. We studied 13 consecutive patients with their first AMI who underwent successful reperfusion. Blood samples were taken the day of admission and on days 2, 3, 4, 5, 7, and 14. LV volume indices were determined at 1 month after AMI, when LV remodeling was almost completed. The serum concentrations of both PICP and ICTP changed in a time-dependent manner. The average serum PICP concentration was lower than 1 SD below the mean control values on days 2 and 3 and increased thereafter, returning to the lower end of the control range at day 14. The area under the curve (AUC) for PICP was significantly correlated with the LV end systolic (ES) and end diastolic (ED) volume indices and LV ejection fraction for the first 14 days after AMI. The serum PICP on days 5–14 was inversely correlated or tended to be correlated with the LVES and LVED volume indices. The average serum ICTP concentrations on admission were within the control range, began to increase on day 2, and reached maximal concentrations on day 5, remaining at a plateau concentration until day 14. Although the AUC of ICTP for 14 days, the ICTP concentrations on days 1 and 14, and the minimal and maximal concentrations were significantly correlated with creatine kinase (CK) release and the period from AMI onset to the peak CK time, the concentrations were not significantly correlated with any LV indices except for the concentration on day 4, which was weakly correlated with the LVES volume index. The serum concentrations of PICP showed a significant time-dependent change that correlated with LV indices, indicating that PICP may provide additional information for evaluating the healing process because it affects LV remodeling after AMI. Although the serum concentration of ICTP changed in association with CK release, the ICTP concentration was found to be a poor indicator for LV indices.
Smoking, obesity, and hypertension alter the dose–response curve and test sensitivity of carbohydrate-deficient transferrin as a marker of alcohol intake
Authors: Whitfield John B , Fletcher Linda M , Murphy Theresa L , Powell Lawrie W , Halliday June , Heath Andrew C , Martin Nicholas G
Published: January 2020

Serum carbohydrate-deficient transferrin (CDT) is a specific and comparatively sensitive marker of excessive alcohol use; however, reports of its sensitivity vary according to the population or patient groups studied and their average alcohol intake. We have characterized the dose–response curve between alcohol intake and CDT concentrations in a study of 1400 men and women from a community-based twin registry. Our results show that mean CDT increases with increasing reported alcohol consumption even within the range of alcohol use considered to be nonhazardous. We found significant effects of sex, age, smoking, previous alcohol dependence, body mass index, and diastolic hypertension on the alcohol-CDT dose–response curve. These variables either affect test sensitivity or require adjustment of reference intervals. The results also provide insight into the physiological and biochemical factors that affect CDT concentration.
Urinary and plasma urodilatin measured by a direct RIA using a highly specific antiserum
Authors: Meyer Markus , Armbruster Franz P , Maier Ina , Schwing Jens , Haller Christlieb , Missbichler Albert , Adermann Knut , Forssmann Wolf-Georg , Woloszczuk Wolfgang
Published: January 2020

Urodilatin (95–126) (URO) appears to play a major physiologic role in fluid homeostasis and produces major changes when administered intravenously. Here we describe a monospecific, high-affinity antiserum against URO with no cross-reactivity (<0.01%) against the structural highly homologous atrial natriuretic peptide 99-126 (ANP-99-126), ANP analogs, and related peptides such as brain natriuretic peptide. A competitive RIA was developed, based on this antiserum. Urine samples with or without ethanol extraction and plasma samples without pretreatment were analyzed by the RIA, which had a detection limit of 10.5 ng/L, a linear measuring range between 10.5 and 1000 ng/L, and recoveries of 93–102% in urine and 90–104% in plasma. The intraassay CVs were 8.2% and 8.1% for urine samples with 269 and 669 ng/L URO; the interassay CV was 9.7% at 839 ng/L. Using this assay, we present URO data for urine from healthy volunteers receiving low and routine sodium diets and from clinical urine specimens; we also present pharmacokinetic data for URO in plasma from patients suffering from bronchial asthma and treated by URO infusion.
A family with high serum leucine aminopeptidase activity derived from a novel variant CD13
Authors: Kawai Makoto , Hara Yukichi , Kubota Tetsuo , Shiba Kiyoko , Hosaki Seijin
Published: January 2020

We investigated a family in which some individuals showed extremely high serum leucine aminopeptidase (LAP) (EC 3.4.11.2) activity, mainly derived from a variant CD13. The isoelectric points of the variant and normal CD13 were 3.3 and 4.1, respectively, and both points converged at 4.4 after treatment with neuraminidase, indicating that more sialic acids are bound to the variant CD13 than normal. The molecular masses of both CD13s were 144 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After treatment with neuraminidase, N-glycosidase, and O-glycosidase, apparent molecular masses of the variant and normal CD13 were 106 kDa and 100 kDa, respectively, suggesting that the variant CD13 contains a longer peptide than normal. This is the first case of familial high serum LAP activity in which the origin could be demonstrated by anti-CD13 monoclonal antibodies to be a variant CD13 inherited in an autosomal dominant mode. The isoelectric point of the LAP activity after neuraminidase treatment was different from that previously reported.
Simultaneous determination of methylenetetrahydrofolate reductase C677T and factor V G1691A genotypes by mutagenically separated PCR and multiple-injection capillary electrophoresis
Authors: Ulvik Arve , Ren Jicun , Refsum Helga , Ueland Per Magne
Published: January 2020

The C677T mutation of the methylenetetrahydrofolate reductase gene and the G1691A (Leiden) mutation of the factor V gene are established risk factors for thromboembolic disease. We here present an assay for the simultaneous genotyping of these common genetic variants. The assay involves a strategy based on multiplex mutagenically separated PCR performed in a single tube containing six primers. Separation of the resulting four PCR products (197, 207, 233, and 246 bp) was performed by capillary electrophoresis coupled with laser-induced fluorescence detection. The time for the automated electrophoresis was reduced to 2.5 min per sample by performing the capillary electrophoresis analysis in a multiple-injection mode.
Therapeutic drug monitoring in special populations
Authors: Walson Philip D
Published: January 2020

Therapeutic drug monitoring (TDM) is commonly used to maintain “therapeutic” drug concentrations. Even in compliant patients, with “average” drug kinetics, TDM is useful to identify the causes of unwanted or unexpected responses, prevent unnecessary diagnostic testing, improve clinical outcomes, and even save lives. TDM has greatest promise in certain special populations who are: (a) prone to under- or overrespond to usual dosing regimens, (b) least able to tolerate, recognize, or communicate drug effects, or who are (c) intentionally or accidentally misdosed. TDM is especially useful in patients at the extremes of age, in adolescents, and in patients who are either taking multiple drugs or expressing unusual pharmacokinetics as a result of physiological, environmental, or genetic causes. Less-well-appreciated uses of TDM include prevention of dangerousunderdosing of patients, investigation of adverse drug reactions, and identification of serious medication errors, even for a number of drugs that are not traditionally monitored. TDM can be useful for some drugs in any patient and for most drugs in some special populations.
Inhibition of DNA methylation in malignant MOLT F4 lymphoblasts by 6-mercaptopurine
Authors: Lambooy Lambert H J , Leegwater Peter A J , van den Heuvel Lambert P , Bökkerink Jos P , De Abreu Ronney A
Published: January 2020

Treatment of MOLT F4 lymphoblasts with 6-mercaptopurine (6-MP) resulted in a decrease of ATP and a depletion of S-adenosylmethionine (AdoMet). To investigate whether this might affect the methylation of DNA, we treated MOLT F4 lymphoblasts with increasing concentrations of 6-MP, followed by labeling with [methyl-14C]methionine and [methyl-3H]thymidine. After DNA isolation, we measured the incorporated radioactivity and determined the14C/3H ratio as a measure for the methylation of newly formed DNA. The 14C/3H ratio was decreased by 17% with 1 μmol/L 6-MP; treatment with increasing concentrations of 6-MP up to 10 μmol/L showed a further decrease to 70%, in comparison with untreated cells. To demonstrate that the methylation of deoxycytidine residues in DNA was reduced, we quantified hydrolyzed DNA by HPLC. The 14C/3H ratio showed a decrease with increasing 6-MP concentrations, indicating that treatment with 6-MP resulted in hypomethylation of DNA.
A new method to measure iron absorption from the enrichment of 57Fe and 58Fe in young erythroid cells
Authors: van den Heuvel Ellen G H M , Muys Theo , Pellegrom Hillie , Bruyntjes Joost P , van Dokkum Wim , Spanhaak Steven , Schaafsma Gertjan
Published: January 2020

Iron absorption can be measured by the incorporation of stable iron isotopes into erythrocytes, 14 days after isotope administration. The disadvantage of this method is the high dose of isotopes needed to obtain a sufficient enrichment. Therefore, in this study cell fractions rich in young erythroid cells were prepared by using a density separation method. From 10 women blood was taken 4, 5, and 7 days after oral and intravenous administration of 57Fe and58Fe. In these cell fractions and in whole blood taken 14 days after isotope administration, isotope enrichment was measured and absorption calculated. Absorption calculated from the isotope enrichment in the reticulocyte-rich cell fractions (12.2 ± SEM 3.7%) was not significantly different from absorption based on whole-blood values (13.0 ± 3.3%). Because a threefold higher isotope enrichment was found in the cell fractions, the required dose of stable isotopes can be reduced to one-third of the dose used in the traditional method without loss of sensitivity.
Detection and classification of paraproteins by capillary immunofixation/subtraction
Authors: Bossuyt Xavier , Bogaerts Ann , Schiettekatte Gilberte , Blanckaert Norbert
Published: January 2020

A selection of 58 specimens with a monoclonal component identified by immunoelectrophoresis and/or immunofixation was analyzed with the immunosubtraction procedure on the Paragon 2000 capillary electrophoresis system. The capillary system detected 93% of the paraproteins and, using immunosubtraction, correctly identified 91% of the paraproteins. Paraproteins that were detected by immunofixation and/or immunoelectrophoresis but not by capillary electrophoresis were also missed by agarose electrophoresis and cellulose acetate electrophoresis. Cellulose acetate electrophoresis was the least sensitive method for detection of paraproteins. Only 74% of the monoclonal components were detected by this technique, whereas 86% were revealed by agarose electrophoresis. In addition to monoclonal paraproteins, we also studied biclonal paraproteins and oligoclonal banding. Capillary electrophoresis and immunosubtraction correctly detected and identified three specimens containing biclonal paraproteins. In one specimen, capillary zone electrophoresis detected only one band, whereas agarose gel electrophoresis detected two bands. The sensitivity for detection and identification of oligoclonal banding by capillary electrophoresis was inferior to immunofixation.
Zinc protoporphyrin as screening test in female blood donors
Authors: Harthoorn-Lasthuizen Else J , Lindemans Jan , Langenhuijsen Mart M A C
Published: January 2020

Erythrocyte zinc protoporphyrin (ZPP) was measured in 102 women blood donors to evaluate its usefulness in screening for evolving iron deficiency anemia, a reason for the deferral of donors. The results were compared with serum ferritin determinations. Five women were deferred before their first donation and eight women were deferred after one or two donations. Women with increased ZPP values all had low serum ferritin concentrations, indicating iron-deficient erythropoiesis that was caused by iron depletion. The positive predictive value of an increased ZPP in predicting deferral of the donor after one or two donations was 75%, whereas a serum ferritin concentration ≤12 μg/L predicted deferral in 26% of the donors. The results indicate that the ZPP test can be recommended as a feasible and inexpensive predonation test to determine a subset of donors with iron-deficient erythropoiesis at risk of developing iron deficiency anemia.
Evaluation of an acidic deproteinization for the measurement of ascorbate and dehydroascorbate in plasma samples
Authors: Koshiishi Ichiro , Mamura Yoshie , Liu Ji , Imanari Toshio
Published: January 2020

The most popular pretreatment method of plasma samples for the measurement of ascorbate (AsA) and dehydroascorbate (DHA) has been an acidic deproteinization via metaphosphoric acid or trichloroacetic acid. In general, DHA is absent in plasma samples prepared from human blood in a conventional manner. However, when these plasma samples were subjected to acidic deproteinization, DHA was detected in the acidified sample solutions. In the present study, we demonstrate that the oxidation of AsA to DHA in the solutions was promoted by at least two mechanisms, one involving catalysis by ferric ion released from transferrin, and the other involving catalysis by plasma hemoglobin. In the acidified transferrin solution by trichloroacetic acid, an oxidation of AsA to DHA proceeded with standing time, whereas the oxidation was not observed in that by metaphosphoric acid. This oxidation appeared to be catalyzed by ferric ion released from transferrin. In contrast, plasma hemoglobin functioned as a catalyst for AsA oxidation in both metaphosphoric acid and trichloroacetic acid solutions. Therefore, DHA content in the trichloroacetic acid-treated plasma sample was markedly higher than that in the metaphosphoric acid-treated one. These results suggest that DHA detected in acidified plasma samples is an artifact resulting from AsA oxidation.
Optimizing frequency and number of controls for automatic multichannel analyzers
Authors: Neubauer Aljoscha , Wolter Christian , Falkner Christine , Neumeier Dieter
Published: January 2020

Sampling strategy fundamentally influences the effectiveness of quality control with control charts. This study shows a simple approach for optimizing the control strategy for automatic multichannel analyzers that takes into account cost-efficiency considerations. Our main focus is on the frequency of controls necessary. The methods used are based on a field study (on a Hitachi/BM 747), the views of experts, and computer simulations of customary cost-models together with a survey of the literature. We found that industrial cost-models are applicable only with distinct limitations, but–unlike the test-yield model–they offer consistent solutions. On the basis of the field study and the opinions of experts, we adjusted the control strategy to account for inadequacies in the theoretical models. The combined result is that, for effective operation, the number of samples between controls may reach values up to 100 and should not require controls more often than every 30 samples on comparable multichannel analyzers. For adequate statistical performance, a simple 3-SD Shewhart chart usually requires not more than two controls of the same material at each time.
Accurate measurement of cholecystokinin in plasma
Authors: Rehfeld Jens F
Published: January 2020

Shortage of reliable plasma assays has hampered studies of cholecystokinin (CCK). The assay problems are low plasma concentrations, extensive molecular heterogeneity, and close homology of CCK to gastrin, which circulates in higher concentrations. To develop an accurate CCK RIA, antibodies were raised in rabbits, guinea pigs, and mice in titers from 200 to 4 000 000. The specificity of the antisera was tested with homologous peptides, and tissue and plasma extracts. Rabbit 92128 produced antibodies in high titer (≥500 000) with sufficient avidity (K \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(_{eff}^{{^\circ}}\) \end{document} ≥ 1012 mol−1) and the desired specificity. The antiserum binds the bioactive forms of CCK with equimolar potency and displays no reactivity with gastrin. CCK concentrations in plasma from healthy humans rose from 1.13 ± 0.10 pmol/L (mean ± SE, n = 26) to 4.92 ± 0.34 pmol/L after a mixed meal. Chromatography of human plasma revealed traces of CCK-58, a predominance of CCK-33 and CCK-22, and moderate amounts of CCK-8. The results show that it is possible to produce specific CCK-antisera using a sulfated CCK-12 analog.
Quantification of prostate-specific antigen mRNA by coamplification with a recombinant RNA internal standard and microtiterwell-based hybridization
Authors: Verhaegen Monique , Ioannou Pinelopi C , Christopoulos Theodore K
Published: January 2020

We report a quantitative analytical methodology for prostate-specific antigen (PSA) mRNA, which is based on the coamplification of the target with a recombinant RNA internal standard (IS) using reverse transcriptase-polymerase chain reaction. PSA mRNA and the RNA IS contain the same primer recognition sites and generate amplification products that have identical sizes but differ in a 24-bp sequence located in the center of the molecule. Amplified sequences are labeled with biotin using a biotinylated upstream primer. The products are captured on streptavidin-coated microtiter wells and hybridized to specific probes labeled with the hapten digoxigenin. The hybrids are determined using alkaline phosphatase-labeled anti-digoxigenin antibody and time-resolved fluorometry. The ratio of the fluorescence values obtained for the PSA mRNA and the RNA IS is a linear function of the amount of PSA mRNA present in the sample. Samples containing total RNA from PSA-expressing cells (LNCaP cells) in addition to 1 μg of RNA from healthy cells give fluorescence ratios related linearly to the number of cells in the range of 4 to 3000 cells.
Urokinase plasminogen activator: a prognostic marker in breast cancer including patients with axillary node-negative disease
Authors: Duffy Michael J , Duggan Catherine , Mulcahy Hugh E , McDermott Enda W , O’Higgins Niall J
Published: January 2020

Urokinase plasminogen activator (uPA) is a serine protease causally involved in cancer invasion and metastasis. In this study, high concentrations of uPA in primary breast cancers were independently associated with both a shortened disease-free interval and overall survival. For the disease-free interval as endpoint, uPA was a stronger indicator of outcome than lymph node status, whereas for overall survival, nodal status was stronger than uPA. In patients without metastasis to axillary nodes, uPA was also an independent prognostic marker, using both the disease-free interval and overall survival as end points. In contrast to uPA, neither tumor size nor estrogen receptor status was prognostic in the node-negative patients. Measurement of uPA concentrations might thus be of value in selecting the more aggressive subpopulation of node-negative breast cancer patients that could benefit from adjuvant therapy.
Detection and identification of monoclonal gammopathies by capillary electrophoresis
Authors: Henskens Yvonne , de Winter John , Pekelharing Maurits , Ponjee Gabrielle
Published: January 2020

Capillary electrophoresis (CE) and immunosubtraction capillary electrophoresis (IS-CE) were compared with the conventional methods agarose gel electrophoresis (AGE) and immunofixation electrophoresis (IFE) for detection and identification of paraproteins. In total, 74 paraproteins out of 468 serum samples were detected by both methods. Seventy-three monoclonal bands with concentrations ranging from 0.6 to 50.9 g/L were detected by the routine method. With CE, 70 paraproteins were detected and quantified on the electropherogram. Four paraproteins were not detected by CE; three of these were IgG (0.6, 1.1, and 2.2 g/L, respectively), and one was a IgM paraprotein (20.3 g/L) that could be visualized by minor changes in the running conditions. In comparison with IFE, 69 paraproteins were typed identically using IS-CE; only one paraprotein (IgMκ, 14.9 g/L) could not be identified. On the other hand, a monoclonal IgA band that had not been detected by AGE was identified by CE and IS-CE. We conclude that, in general, CE could be a useful method for detection of paraproteins and that IS-CE is a good alternative to IFE. Additional studies are required to investigate the ionic strength and pH of the running buffer, because these prove to be the most crucial factors for routine CE separation of paraproteins.
Comparison of the Axis %CDT TIA and the CDTect method as laboratory tests of alcohol abuse
Authors: Viitala Katja , Lähdesmäki Kaija , Niemelä Onni
Published: January 2020

Carbohydrate-deficient transferrin (CDT) has been suggested as a specific marker of alcohol abuse. We designed this study to compare the conventional CDTect method (Pharmacia & Upjohn) and the new semiautomated Axis %CDT turbidimetric immunoassay (%CDT TIA) for their diagnostic performance to identify problem drinking. The sensitivities of the %CDT TIA and CDTect for correctly classifying heavy drinkers (n = 90) were 29% and 59% with the thresholds currently recommended by the manufacturers, respectively. In the control group (n = 114), which included hospitalized patients with abnormal serum transferrin concentrations, the CDTect assay gave 21 false-positive values (18%), whereas the %CDT TIA showed 100% specificity. With the cutoff limits based on the present healthy control group (mean + 2 SD), the sensitivities of the %CDT TIA and CDTect were 61% and 86%, respectively. For men, the ROC plot area of the CDTect results in comparisons of alcohol abusers and healthy controls was significantly (P <0.05) higher than that of the %CDT TIA results, whereas for women, there was no significant difference in this respect. The slope and intercept (with 95% confidence intervals) for linear regression between CDTect and %CDT TIA were 0.13 (0.12–0.15) and 1.16 (0.73–1.59), respectively (Sy‖x = 1.51, r = 0.744). CDTect results correlated positively with serum transferrin (r = 0.224, P <0.001), whereas the %CDT TIA results showed a slight inverse correlation with serum transferrin (r = −0.132, P = 0.07). The data suggest that CDTect is more sensitive than %CDT TIA in detecting drinking problems. However, the %CDT TIA method yields more specificity when analyzing samples from patients with high serum transferrin concentrations.
Novel immunoassay for the measurement of complexed prostate-specific antigen in serum
Authors: Allard W Jeffrey , Zhou Zeqi , Yeung Kwok K
Published: January 2020

Serum prostate-specific antigen (PSA) is an effective diagnostic tool for detection of prostate cancer (CaP) at an early and potentially curable stage, but specificity is low. Studies have shown that the proportion of serum PSA complexed with α-1-antichymotrypsin (ACT) is higher in men with CaP than in men with benign prostate disease. We developed a novel immunoassay for complexed PSA based on the unique binding properties of a monoclonal antibody that fails to bind free PSA in the presence of antibodies specific for free PSA. The assay measured mixtures of free and complexed PSA accurately, and the measured values of free + complexed PSA in artificial mixtures and in patient sera were equivalent to the measured value of total PSA. Both the serum concentration and the proportion of complexed PSA was substantially higher in patients with CaP compared with patients with benign prostate disease. The cPSA assay may have utility in improving specificity in screening for prostate cancer.
Determination of monoethylglycinexylidide by fluorescence polarization immunoassay in highly icteric serum samples: modified precipitation procedure and HPLC compared
Authors: Schütz Ekkehard , Shipkova Maria , Niedmann Paul Dieter , Wieland Eberhard , Armstrong Victor William , Oellerich Michael
Published: January 2020

Hyperbilirubinemia, which frequently occurs in severe liver disease, interferes with the fluorescence polarization immunoassay (FPIA) monoethylglycinexylidide (MEGX) assay manufactured by Abbott Diagnostics. Because the MEGX test is particularly helpful in this clinical situation, strategies have been developed to overcome this problem. Precipitation of serum with the Abbott Digoxin II precipitation reagent eliminates bilirubin. Therefore, we compared FPIA results after precipitation of 81 icteric samples from 27 MEGX tests to results obtained using a validated HPLC method. The precipitation did not substantially alter the performance characteristics of FPIA: detection limit, 8 μg/L; between-days imprecision, 5.3–6.2%; recovery, 102–104% (50–200μg/L). This pretreatment of serum did not eliminate all interference, and only a poor correlation was observed between serum MEGX concentrations measured with HPLC or modified FPIA (r2 = 0.46; Sy‖x = 20.0 μg/L). In contrast, MEGX formation values calculated by subtraction of the prelidocaine MEGX concentration were in close agreement (r2 = 0.98; Sy‖x = 2.3 μg/L). Because only MEGX formation is clinically relevant, this modified FPIA procedure offers a simple and rapid alternative to HPLC.
Validation of the Point-EXACCT method in non-small cell lung carcinomas
Authors: Somers Veerle A M C , Leimbach Darcy A , Theunissen Paul H M H , Murtagh James J , Holloway Brian , Ambergen Anton W , Thunnissen Frederik B J M
Published: January 2020

K-ras point mutations are often detected in part of the lung carcinomas. For the validation of a highly sensitive and rapid assay for known point mutations, Point-EXACCT (Biochim Biophys Acta 1998; 1379:42–52), we analyzed 89 non-small cell lung carcinomas and compared the results with two sequencing methods. No point mutations were found with double-stranded sequencing. Single-stranded sequencing detected six patients positive for K-ras codon 12. When Point-EXACCT was used, K-ras codon 12 mutations were detected in 8 of 52 patients with squamous cell carcinomas, 10 of 29 patients with adenocarcinomas, and 3 of 8 patients with large cell carcinomas. The finding of K-ras mutations in squamous cell carcinomas is explained by the high sensitivity of the method. Therefore, Point-EXACCT may be applicable to detection of those alterations occurring at a low frequency among an excess of cells with wild-type DNA.
Comparison of seven immunoassays for the quantification of CA 125 antigen in serum
Authors: Davelaar Elvira M , van Kamp Gerard J , Verstraeten Rob A , Kenemans Peter
Published: January 2020

Seven CA 125 immunoassays were compared for their clinical performance. CA 125 concentrations were determined in 289 serum samples obtained from women with benign pelvic tumors (samples from 98 patients) and patients with various cancers (samples from 111 patients). In the range of 0–1000 kilounits/L, all assays tested were linearly correlated, with correlation coefficients ranging from 0.89 to 0.99. In relation to the original Centocor CA 125 assay, there was an overall tendency to measure higher absolute values in the lower CA 125 value range. This was not seen in relation to the Centocor CA 125 II assay. ROC curves (benign vs pretreatment ovarian cancer patients) were nearly identical for all assays, and the areas under the ROC curves were not markedly different. We conclude that the CA 125 assays tested are strongly related to each other and are clinically reliable for the quantification of serum CA 125 and that none of the assays offers higher diagnostic accuracy or better discrimination between patient groups, especially not in the lower ranges.
Human cholesteryl ester transfer protein measured by enzyme-linked immunosorbent assay with two monoclonal antibodies against rabbit cholesteryl ester transfer protein: plasma cholesteryl ester transfer protein and lipoproteins among Japanese hypercholesterolemic patients
Authors: Sasai Kanna , Okumura-Noji Kuniko , Hibino Takeshi , Ikeuchi Reiko , Sakuma Nagahiko , Fujinami Takao , Yokoyama Shinji
Published: January 2020

Plasma cholesteryl ester transfer protein (CETP) concentrations were measured in Japanese subjects by an ELISA with two different monoclonal antibodies that were raised against rabbit CETP and cross-reacted against human CETP. Among 63 patients who consecutively underwent coronary angiography, the plasma CETP of 37 patients with luminal stenosis ≥50% in their coronary arteries was not significantly different from that of the 26 patients with luminal stenosis <50%. No other lipoprotein-related measurement except HDL-cholesterol differentiated the two groups. Among 40 hypercholesterolemic patients, no lipoprotein-related measurement other than LDL-cholesterol was found to positive correlate with the CETP. Before and after the treatment of 23 patients with simvastatin 5 mg a day for 4 weeks, plasma CETP markedly decreased in those whose pretreatment CETP was ≥3 mg/L; no change was observed for those with lower pretreatment CETP. In the former group, negative correlation between CETP and HDL-cholesterol was demonstrated only in the posttreatment plasma.
Radioreceptor assay of an endothelin A receptor antagonist in plasma and urine
Authors: Cernacek Peter , Franchi Luigi , Dupuis Jocelyn , Rouleau Jean-Lucien , Levy Mortimer
Published: January 2020

Orally active nonpeptide antagonists of endothelin (ET) receptors may prove beneficial in the treatment of cardiovascular and renal disease. The pharmacodynamics and pharmacokinetics of these drugs are not sufficiently known, and practical methods for their analysis have not been developed. We describe a simple, sensitive, and reproducible radioreceptor assay (RRA) for LU135252, a selective antagonist of the ETA receptor, using porcine aortic smooth muscle membranes as the acceptor and 125I-endothelin-1 as the ligand. With methanol extraction of plasma and urine samples, recovery of LU135252 ranged from 79% to 91% at 60–1000 nmol/L. The logit-log transformed calibration curves constructed with LU135252 added to plasma or to urine were linear (r = 0.993 ± 0.005, n = 11) in the range from 18.7 to 2400 nmol/L. The detection limit with plasma- and urine-based calibration curves was 19 nmol/L. The interassay coefficient of variation was 12.6% at 70 nmol/L (n = 9) and 6.5% at 590 nmol/L (n = 9). Endothelin-1 did not interfere in the RRA at pathophysiologically and clinically relevant concentrations [up to 15 pmol/L (40 pg/mL)]. When LU135252 was added to plasma, the signal was completely stable after storage for 1 week at 4 °C, although there was a modest loss of the signal after 24 h at room temperature. The practical performance of this RRA was then tested in plasma samples obtained from (a) rats after a single oral administration of LU135252, (b) from coronary-ligated rats chronically treated with LU135252, and (c) in plasma and urine samples obtained from dogs during intrarenal infusion of LU135252.
Endothelium and acute coronary syndromes
Authors: Liao James K
Published: January 2020

The vascular endothelium is a dynamic endocrine organ that regulates contractile, secretory, and mitogenic activities in the vessel wall and hemostatic processes within the vascular lumen. Risk factors for atherosclerosis such as cigarette smoking, hypertension, and increased serum lipid concentrations impair endothelial function and lead to the development of atherosclerotic vessels, which cause acute coronary syndromes. Atherosclerotic vessels progress from scattered foam cells to complex lesions with a lipid core and fibrous cap. Factors that weaken and cause the rupture of the fibrous cap will expose circulating blood products to the procoagulant materials in the lipid core. Thrombosis and subsequent remodeling of the unstable plaque may be catastrophic or clinically silent depending on the degree of vascular occlusion and availability of collateral blood flow. Evidence is presented that supports the view that endothelial dysfunction is an early marker of atherosclerosis and an important contributor to the atherogenic process.
Cardiac marker point-of-care testing in the Emergency Department and Cardiac Care Unit
Authors: Brogan Gerard X , Bock Jay L
Published: January 2020

There has been much interest in improving the accuracy and speed with which chest pain patients presenting to the emergency department are diagnosed and treated. Recently, attention has been directed toward alternative site or point-of-care testing for biochemical markers of myocardial cell necrosis in addition to traditional diagnostic methodologies. The various point-of-care cardiac marker devices available and their potential applications are discussed. Regulatory and quality management issues related of point-of-care testing are reviewed.
Different intracellular compartmentations of cardiac troponins and myosin heavy chains: a causal connection to their different early release after myocardial damage
Authors: Bleier Jürgen , Vorderwinkler Karl-Paul , Falkensammer Jürgen , Mair Peter , Dapunt Otto , Puschendorf Bernd , Mair Johannes
Published: January 2020

We investigated the net myocardial release of creatine kinase isoenzyme MB (CKMB), myoglobin, cardiac troponin T (cTnT), cardiac troponin I (cTnI), and cardiac β-type myosin heavy chain (β-MHC) into the coronary circulation after cardioplegic cardiac arrest in humans. Cardiac markers were measured in paired arterial, central venous, and coronary sinus blood in 19 patients undergoing elective coronary artery bypass grafting (CABG) before aortic cross-clamping and 1, 5, 10, and 20 min after aortic declamping. cTnT and cTnI were released into the coronary sinus in parallel to each other and almost simultaneously to myoglobin and CKMB within 20 min of reperfusion. In contrast, no β-MHC was released in the same patients during the study period. The average soluble cTnT and cTnI pools in right atrial appendages of 11 patients with right atrial and right ventricular pressures within reference values were comparable and were ∼8% of total myocardial troponin content. The soluble β-MHC pool was <0.1% in all patients. Our results demonstrate the impact of the different intracellular compartmention of regulatory and contractile proteins on their early release from damaged myocardium.
Measurement of specific immunoglobulin E: intermethod comparison and standardization
Authors: Plebani Mario , Bernardi Daniela , Basso Daniela , Borghesan Franco , Faggian Diego
Published: January 2020

Recently introduced “second-generation” techniques for specific IgE measurement have produced some analytical improvement, offering better clinical sensitivity than previous techniques. The aims of our study were to compare the analytical and clinical performances of four second-generation techniques for allergen-specific IgE measurement in serum and to ascertain whether the new system for reporting quantitative results contributes to greater clinical agreement between findings using the techniques considered. Allergen-specific IgE was measured using the CAP® System, CARLA®, ENEA®, and AlaSTAT®, and the findings were compared. A significant disagreement was found between CAP and ENEA for all allergens and between CAP and CARLA for D1 and G5. However, the clinical discrepancies were reduced by selecting method-specific thresholds using ROC analysis. Second-generation techniques enable us to obtain better standardization of results; however, the identification of a specific threshold appears to be a prerequisite for the appropriate clinical interpretation of the test findings.
Thermal Instability of the Trimeric Structure of the N-terminal Propeptide of Human Procollagen Type I in Relation to Assay Technology
Authors: Brandt Jette , Krogh Thomas N , Jensen Charlotte H , Frederiksen Jette K , Teisner Børge
Published: January 2020

The N-terminal propeptide of procollagen type I (PINP) appeared in two peaks after size chromatography. The high-molecular weight form was transformed to the low-molecular weight form during incubation at 37 °C, whereas the low-molecular weight form remained unchanged. The PINP concentrations in amniotic fluid and sera remained unchanged during 37 °C incubation when measured using an ELISA; however, concentrations decreased by 89–93% when measured using an RIA. The ELISA:RIA ratio varied from 1.1 to 2.9 in these fluids because of different size distributions and the inability of the RIA to measure the low-molecular weight form. Thermal transition of the high-molecular weight form caused a change in its elution volume but did not change its migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectrometry revealed identical results for both forms. We reached the following conclusions: (a) the trimeric structure of PINP is unstable at 37 °C; (b) the two molecular forms represent intact α1 chains in trimeric and monomeric forms; (c) thermal transition is an ongoing in vivo process; and (d) this is important in the choice of assay technology.
Whole Blood Capcellia CD4/CD8 Immunoassay for Enumeration of CD4+ and CD8+ Peripheral T Lymphocytes
Authors: Carrière Dominique , Vendrell Jean Pierre , Fontaine Claude , Jansen Aline , Reynes Jacques , Pagès Isabelle , Holzmann Catherine , Laprade Michel , Pau Bernard
Published: January 2020

We evaluated the Whole Blood Capcellia® CD4/CD8, an immunoenzymatic method that provides absolute counts of CD4+ and CD8+ T cells in peripheral blood. The assay is based on the separation of T cells by use of an anti-CD2 magnetic bead suspension, followed by reaction of the CD4 or CD8 molecules with the corresponding monoclonal antibody coupled to peroxidase. CD4-positive monocytes were excluded from the assay. Freeze-dried magnetic bead-T-cell complexes were used as calibrators. Capcellia counts from HIV-1-infected patients were compared with those obtained by flow cytometry as the comparison method. The results by Capcellia correlated well with those by flow cytometric analysis: r2 = 0.95; P <0.001; (y = 0.96x − 22.1); Sy|x = 64 for CD4; r2 = 0.81; P <0.001; (y = 1.26x − 76.4); Sy|x = 139 for CD8; n = 76. The correlation between CD4+ T-cell counts determined by two trained experimenters was significant (r2 = 0.96). Our results indicate that this new ELISA technique for lymphocyte immunophenotyping is an efficient alternative to flow cytometry.
Sensitive Assay for Mitochondrial DNA Polymerase γ
Authors: Naviaux Robert K , Markusic David , Barshop Bruce A , Nyhan William L , Haas Richard H
Published: January 2020

Background: The mitochondrial DNA polymerase γ is the principal polymerase required for mitochondrial DNA replication. Primary or secondary deficiencies in the activity of DNA polymerase γ may lead to mitochondrial DNA depletion. We describe a sensitive and robust clinical assay for this enzyme.

Methods: The assay was performed on mitochondria isolated from skeletal muscle biopsies. High-molecular weight polynucleotide reaction products were captured on ion-exchange paper, examined qualitatively by autoradiography, and quantified by scintillation counting.

Results: Kinetic analysis of DNA polymerase γ by this method showed a Km for dTTP of 1.43 μmol/L and a Ki for azidothymidine triphosphate of 0.861 μmol/L. The assay was linear from 0.1 to 2 μg of mitochondrial protein. The detection limit was 30 units (30 fmol dTMP incorporated in 30 min). The linear dynamic range was three orders of magnitude; 30–30 000 units. Imprecision (CV) was 6.4% within day and 12% between days. Application of this assay to a mixed population of 38 patients referred for evaluation of mitochondrial disease revealed a distribution with a range of 0–2506 U/μg, reflecting extensive biologic variation among patients with neuromuscular disease.

Conclusion: This assay provides a useful adjunct to current laboratory methods for the evaluation of patients with suspected mitochondrial DNA depletion syndromes.
Human Glandular Kallikrein in Breast Milk, Amniotic Fluid, and Breast Cyst Fluid
Authors: Magklara Angeliki , Scorilas Andreas , López-Otín Carlos , Vizoso Francisco , Ruibal Alvaro , Diamandis Eleftherios P
Published: January 2020

Background: Human glandular kallikrein (hK2) belongs to the serine protease family of enzymes and has high sequence homology with prostate-specific antigen (PSA). The physiological role of hK2 has not as yet been determined, but there is evidence that it can regulate the proteolytic activity of PSA through processing and activating pro-PSA, an inactive precursor. Thus, it is conceivable that these two secreted proteins may coexist in biological fluids. Currently, hK2 is considered an androgen-regulated and prostate-specific protein. Recently, it has been demonstrated that hK2 is expressed in the breast cancer cell line T-47D after stimulation by steroid hormones, and we reported that hK2 can be detected in a subset of breast tumor extracts. These data suggest that hK2 may be expressed in tissues other than the prostate, such as those in which PSA has already been detected. Because hK2 is a secreted protein, it may be present in various biological fluids.

Methods: We analyzed milk samples from lactating women, amniotic fluid from pregnant women, and breast cyst fluid from patients with gross breast cystic disease, using a highly sensitive and specific immunoassay for hK2.

Results: hK2 was present in all three biological fluids. We suggest that the female breast may produce hK2 and provide evidence that hK2 may have value as an additional marker for the discrimination between type I and type II breast cysts.

Conclusions: The female breast produces hK2 in addition to PSA. More studies are necessary to establish the role of this kallikrein in nondiseased breast, gross breast cystic disease, and breast cancer.