bluuebunny/crossref_metadata_2025 huggingface dataset:
New tools for laboratory design and management
Authors: Truchaud Alain , Le Neel Tanguy , Brochard Hugues , Malvaux Sophie , Moyon Marine , Cazaubiel Murielle
Published: January 2020

The clinical laboratory is changing from a place of activity based on sample analysis to an in vitro diagnostic network. To convince our team, partners, and administrators, we need new comprehensive tools to define a strategy with limited risk of failure or conflicts. Specific quality goals should be established before choosing automated tools for sample handling, analytical systems, laboratory information systems, communication systems, or advanced technologies. A system approach maps and simplifies the process, based more on a functional study than on classical disciplines. A customer–supplier approach establishes the requirements between partners either inside or outside the laboratory. The quality system must be a management tool, linking samples, tasks, information, and documents. Quantitative simulation modeling explores different automation alternatives and their impact on laboratory workflow. Finally, integration of results in interactive semirealistic simulation tools for laboratory design or reengineering can be used as communications tools to involve laboratory professionals in the change of their practice.
ProC® Global: the first functional screening assay for the complete protein C pathway
Authors: Dati Francesco , Hafner Gerd , Erbes Hilde , Prellwitz Winfried , Kraus Michael , Niemann Frank , Noah Michael , Wagner Carola
Published: January 2020

In clinical practice, venous thromboembolic complications are much more frequent than bleeding disorders. In fact, disturbances within the protein C pathway due to coagulation factor V (FV) Leiden mutation and deficiency of protein C or protein S are the most frequent abnormalities in hereditary thrombophilia. Furthermore, acquired dysfunctions of the protein C system may predispose the single individual to an increased thrombotic risk. A routine-suited screening assay that would allow the monitoring of the proper interplay of factors in the protein C pathway could add an important factor to the basic coagulation profile. This consists of the prothrombin time and of the activated partial thromboplastin time, which currently allow only a screening for increased risk for bleeding but not for venous thromboembolism. A new functional screening test for the protein C system such as the presented ProC® Global should therefore facilitate detection of FV Leiden as well as deficiency of protein C and protein S. The results of the present evaluation indicate that ProC Global is highly sensitive to activated protein C resistance/FV Leiden (100%) and protein C deficiency (90%) and sensitive to protein S deficiency (63%). Furthermore, the assay gives a quantitative measure of the net potential of the protein C pathway in relation to the intrinsic procoagulant system. The use of this assay for a prospective assessment of thromboembolic risk is the subject of current studies.
In vitro effects of a novel hemoglobin-based oxygen carrier on routine chemistry, therapeutic drug, coagulation, hematology, and blood bank assays
Authors: Callas Demetra D , Clark Terri L , Moreira Paulo L , Lansden Cara , Gawryl Maria S , Kahn Stephen , Bermes Edward W
Published: January 2020

Red blood cell (RBC) replacement solutions are being developed as alternatives to allogeneic RBC use in blood transfusions in the treatment of massive trauma, to achieve hemodynamic stability during elective surgery, and to increase oxygen-carrying capacity in anemia. Hemoglobin-based oxygen carrier (HBOC)-201 (Biopure Corp.) is a purified, sterile, isosmotic glutaraldehyde-polymerized bovine hemoglobin. Because this product is acellular, blood components containing this substance appear hemolyzed. This study reports on the interferences produced by the presence of HBOC-201 in a variety of clinical assays. This product was added in vitro at concentrations up to 60 g/L (6.0 g/dL) to normal human serum, plasma, or whole blood before testing for serum chemistries, coagulation profiles, and hematology and blood bank assays. In addition, a set of normal human sera containing HBOC-201 was supplemented with various therapeutic drugs and assayed for these agents. The results of these studies demonstrate that the presence of HBOC-201 in blood components does not result in significant analytical interference that would be of concern with many clinical assays at HBOC-201 concentrations encountered during routine clinical use of this RBC replacement solution in patients.
A laboratorian’s perspective on evaluation and implementation of new laboratory tests
Authors: O’Kane Dennis J , Ebert Thomas A , Hallaway Brenda J , Roberts Steven G , Bhuiyan Abul K M J , Tenner Kathleen S
Published: January 2020

New assay development should be directed toward answering fundamental clinical questions. Caveats that must be considered before initiating assay development projects are: New assays should allow the clinician to interact with and treat a patient more effectively, thereby improving medical outcome; and new assays should facilitate recapture of system resources, enabling cost savings or reinvestment of resources. Defining the clinical questions and consideration of the caveats permit a means of prioritizing assay development activities. Laboratorians are faced with evaluating several types of development activities that lead to assay implementation in routine clinical testing. Assays can be prioritized for up-grading to newer cost-effective technologies, provided the changes maintain or improve analytical and clinical performance. Predicting which research assay will have future value is difficult when clinical performance is not fully validated. However, such assay development has the greatest potential for changing the delivery of healthcare by a clinician.
Validation by isoelectric focusing of the anion-exchange isotransferrin fractionation step involved in determination of carbohydrate-deficient transferrin by the CDTect assay
Authors: Arndt Torsten , Hackler Rolf , Kleine Tilman O , Gressner Axel M
Published: January 2020

Serum concentration of carbohydrate-deficient transferrin (CDT) is used for laboratory diagnosis of chronic alcohol abuse. Using isoelectric focusing for validation of the initial isotransferrin fractionation step involved in the determination of CDT by the CDTect assay, we found a complete in vitro iron saturation of transferrin and sufficient stability of the transferrin iron load during column passage; effective separation of non-CDT-isotransferrins and CDT-isotransferrins at the microcolumns; partial coelution of trisialo-Fe2-transferrin, which did not significantly affect CDT measurement; partial retention of CDT-isotransferrins, especially disialo-Fe2-transferrin, which may cause falsely negative results for CDT at the upper reference limits; good precision of the isotransferrin fractionation step; and no significant effects of low concentrations of serum protein and transferrin. We strongly urge standardization of CDT analysis and suggest isoelectric focusing for validation of CDT analysis methods and verification of odd results.
Chemical mismatch cleavage combined with capillary electrophoresis: detection of mutations in exon 8 of the cystathionine β-synthase gene
Authors: Ren Jicun , Ulvik Arve , Refsum Helga , Ueland Per Magne
Published: January 2020

Mutation detection by chemical mismatch cleavage (CMC) is based on the chemical modification and cleavage at the site of mismatched C or T in heteroduplexes, using hydroxylamine or osmium tetroxide (OsO4) as chemical probes. In the present study, we evaluated CMC in combination with capillary electrophoresis (CE) by determining the common T833C and G919A mutations in exon 8 of the cystathionine β-synthase gene in heterozygous and homozygous samples. A 186-bp fragment encompassing exon 8 was amplified by PCR with both primers labeled with 5′-fluorescein. Labeled single strands of 40 and 61 nucleotides (nt) were formed from the coding strand of the T833C sample and non-coding strand from the G919A sample, respectively. These single-stranded DNA (ssDNA) products were analyzed under denaturing conditions by CE with short-chain linear polyacrylamide as the sieving matrix and were detected by laser-induced fluorescence (LIF), using a sensitive, one-channel sheath-flow detector. The CE-LIF format afforded relatively high resolution of ssDNA (down to 1 nt), precise size assessment of CMC products, sensitive detection with small sample requirements, and fast analysis. Thus, CMC combined with CE-LIF is suitable for screening of known mutations, giving expected CMC products, but will also detect unknown mutations, the locations of which are indicated by the fragment sizes.
Analytical and clinical performance characteristics of Tandem-MP Ostase, a new immunoassay for serum bone alkaline phosphatase
Authors: Broyles Dennis L , Nielsen Randall G , Bussett Elizabeth M , Lu W Douglas , Mizrahi Isaac A , Nunnelly Patricia A , Ngo Tram A , Noell Julia , Christenson Robert H , Kress Barry C
Published: January 2020

The performance characteristics of the Tandem®-MP Ostase® assay, a new microplate immunoassay for bone-specific alkaline phosphatase (bone ALP; EC 3.1.3.1) in human sera, are described. Bone ALP is bound to streptavidin-coated microwells by a single biotinylated anti-bone ALP monoclonal antibody. Antigen is detected by the addition of p-nitrophenyl phosphate. The assay is performed at room temperature in <90 min. Imprecision was 2.3–6.1% with a detection limit of 0.6 μg/L. Method comparison of bone ALP measurements with the Tandem-MP Ostase assay and the mass-based Tandem-R Ostase assay (n = 285) indicated regression statistics of Tandem-MP Ostase = 1.03 Tandem-R Ostase + 0.22 μg/L, Sy‖x = 4.0 μg/L, r = 0.97. Serum bone ALP values in apparently healthy men and in pre- and postmenopausal women were also similar between the two Ostase assay formats. Liver ALP reactivity determined using the slope and heat inactivation methods was similar in both Ostase assays. Liver ALP reactivity ranged from 3 μg/L (heat inactivation) to 6 μg/L (slope method) per 100 U/L of liver ALP activity, whereas bone ALP reactivity was 37 μg/L per 100 U/L of bone ALP activity, indicating a liver ALP relative reactivity of 8.1–16.2%. Similar results were obtained with the Alkphase-B bone ALP immunoassay. The Tandem-MP Ostase bone ALP assay demonstrated increased concentrations of serum bone ALP in conditions where bone metabolism is increased and showed a rapid, temporal decrease in serum bone ALP in Paget disease patients on bisphosphonate therapy. In conclusion, the Tandem-MP Ostase assay for serum bone ALP is a rapid, simple, robust nonisotopic alternative to the Tandem-R Ostase immunoradiometric assay that provides an accurate and sensitive assessment of bone turnover.
CO-Oximetry interference by perflubron emulsion: comparison of hemolyzing and nonhemolyzing instruments
Authors: Shepherd A P , Steinke J M
Published: January 2020

Perflubron emulsion is expected to be in clinical use soon as a non-hemoglobin blood substitute. A preliminary report indicates that this new oxygen-carrying fluorocarbon interferes with the measurements of CO-oximeters. Therefore, we have quantified the interference that perflubron causes in the measurements of eight widely used oximeters and CO-oximeters. The AVL Omni 6, CC270, IL482, IL682, and OSM3 are conventional CO-oximeters that hemolyze blood samples before analyzing them. In contrast, the AVOXimeters 1000 and 4000 and the IL Synthesis 35 make their measurements without hemolyzing the samples. Because perflubron is expected to be used most frequently on surgical patients in a hemodiluted state, we conducted all tests on human erythrocytes suspended in plasma at a hemoglobin concentration standardized to 70 g/L (7 g/dL) and with oxyhemoglobin saturation set at 97%. When perflubron was added to the blood samples, the nonhemolyzing CO-oximeters were not seriously affected by perflubron concentrations in and above the therapeutic range. In contrast, some of the hemolyzing CO-oximeters experienced concentration-dependent interference in their measurements of all analytes except total hemoglobin concentration. Thus, we conclude that the nonhemolyzing CO-oximeters provide an effective means for determining whether a hemolyzing CO-oximeter is experiencing clinically important interference in blood from patients receiving perflubron.
Direct reverse transcription-PCR on oligo(dT)-immobilized polypropylene microplates after capturing total mRNA from crude cell lysates
Authors: Hamaguchi Yohei , Aso Yoshimasa , Shimada Hiroshi , Mitsuhashi Masato
Published: January 2020

To simplify gene expression analysis, oligo(dT)-immobilized polypropylene microplates were used serially to capture mRNA, synthesize cDNA, and amplify specific genes. The amounts of immobilized oligonucleotide, hybridized mRNA, and synthesized cDNA were quantified fluorometrically using either Yoyo-1 or AttoPhos. The immobilized oligonucleotides captured ∼40–55% of mRNA directly from crude cell lysates. Hybridized mRNA was then amplified by one-step reverse transcription (RT)-PCR with rTth polymerase or two-step PCR with initial cDNA synthesis followed by PCR, where the latter exhibited more sensitivity. In two-step RT-PCR, microplates can be reused for multiple PCRs with the same or different primer sets because synthesized cDNA was covalently attached to the plates at its 5′ end. We believe this microplate may be acceptable as a platform for various mRNA expression analyses, including basic research, drug screening, and molecular toxicology, as well as for molecular pathological diagnostics.
Detection of intracellular antigens by flow cytometry: comparison of two chemical methods and microwave heating
Authors: Millard Isabelle , Degrave Etienne , Philippe Marianne , Gala Jean-Luc
Published: January 2020

Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. This study compares three fixation/permeabilization techniques: two commercial chemical reagents, the ORTHOPermeaFixTM (OPF) and the FIX&PERM Cell Permeabilization Kit® (F&P), and a novel method based on microwave heating (MWH). They have been applied to the detection of two nuclear (p53 and rb/p105) and two cytoplasmic (bcl-2 and mdr-1/gp-170) antigens, using positive- and negative-control cell lines and peripheral blood mononuclear cells. Western blotting was performed as a control of protein expression. For the four antigens assessed, cellular morphology, discrimination between intact cells and debris, percentage of positive cells, and mean fluorescence intensity were examined. For this last parameter, the assessment of the MWH technique was performed using SD and a graphical approach inspired by the concepts described by Bland and Altman (Lancet 1986;346:1085–7) as well as Petersen et al. (Clin Chem 1997;43:2039–46). The statistical analysis shows that MWH is comparable to the commercial methods and that its reproducibility is also equivalent to OPF and F&P. As assessed for some of the most clinically relevant intracytoplasmic and intranuclear antigens, the MWH method appears to be a valuable and inexpensive alternative. It is worth noting that, unlike commercial reagents, MWH altered surface antigens. Interestingly, this feature, which would prevent cell selection on the basis of combined membrane and intracellular epitopes, is associated with a decrease of nonspecific background fluorescence.
Clinical outcome and economic impact of aminoglycoside peak concentrations in febrile immunocompromised patients with hematologic malignancies
Authors: Binder Lutz , Schiel Xaver , Binder Claudia , Menke Cristina Fernandes Almeida , Schüttrumpf Silke , Armstrong Victor W , Unterhalt Michael , Erichsen Norbert , Hiddemann Wolfgang , Oellerich Michael
Published: January 2020

The aim of this study was to investigate the clinical and economic significance of aminoglycoside peak concentrations in febrile neutropenic patients with hematologic malignancies. Sixty-one patients were treated according to protocol II of the Paul-Ehrlich-Gesellschaft: initial application of gentamicin or tobramycin in combination with a cephalosporin or ureidopenicillin and, after 3 days, a potential change of antibiosis to be decided in case of nonresponse. At the same time, samples were collected by an independent controller. We found a significant dependence of clinical outcome on aminoglycoside peak concentrations (P = 0.004). Twelve of 17 patients with peak concentrations >4.8 mg/L, but only 13 of 44 patients with concentrations ≤4.8 mg/L, responded to initial therapy. Average infection-related costs per patient with peak values >4.8 mg/L were US$1429, $1790, and $1701 for nursing, diagnostics, and therapeutics, respectively (total $4920). Expenses for patients with peak concentrations ≤4.8 mg/L were ∼1.8-fold higher (average total $8718). If all 61 patients had achieved peaks >4.8 mg/L, the potential savings would have totalled $167 112. We conclude that neutropenic patients form a target group for successful pharmacokinetic intervention and cost saving.
Development of new cancer chemoprevention agents: role of pharmacokinetic/pharmacodynamic and intermediate endpoint biomarker monitoring
Authors: Lieberman Ronald , Crowell James A , Hawk Ernest T , Boone Charles W , Sigman Caroline C , Kelloff Gary J
Published: January 2020

Recently, several promising strategies have been advanced for improving the efficiency of new agent development. These include pharmacokinetic/pharmacodynamic (PK/PD) and intermediate endpoint bio-marker (IEB) monitoring. Here, we review their essential role as practical tools for guiding the evaluation of agents for cancer chemoprevention (CP) and provide examples of CP agents that utilize these approaches. Several important categories of IEBs are delineated, including histologically based (intraepithelial neoplasias and nuclear morphometry). The use of select IEBs combined with a Bayesian method for clinical trial monitoring for rapid identification of ineffective or promising agents is discussed. The similarities between IEB and TDM are described. Finally, we present future tools for enhanced monitoring of CP agents that will impact on laboratory medicine and are also applicable to many other drug classes, e.g., laser capture microdissection and cDNA chip microarrays that assess gene expression patterns of precancerous and cancerous lesions.
Isolation and identification of a C39 demethylated metabolite of rapamycin from pig liver microsomes and evaluation of its immunosuppressive activity
Authors: Nickmilder Marc J M , Latinne Dominique , De Houx Jean-Paul , Verbeeck Roger K , Lhoëst Georges J J
Published: January 2020

We studied in vitro metabolism of rapamycin using pig liver microsomes. After extraction of the metabolites from the incubation medium, the crude metabolite extract was submitted to normal and subsequently to reversed-phase HPLC chromatography. We describe in the current study a metabolite of retention time 23.2 min collected from reversed-phase HPLC and identified by fast atom bombardment mass spectrometry (MS) and electrospray MS-MS as a C39 demethylated rapamycin metabolite. In vitro immunosuppressive activity of this metabolite, determined by the mixed lymphocyte reaction, was negligible compared with that of the parent compound. The decrease of in vitro immunosuppressive activity compared with the parent compound is likely to be attributed to important structural modifications of the rapamycin binding region to the FK-506 binding protein.
Strontium absorption and excretion in normocalciuric subjects: relation tocalcium metabolism
Authors: Vezzoli Giuseppe , Baragetti Ivano , Zerbi Simona , Caumo Andrea , Soldati Laura , Bellinzoni Piera , Centemero Antonella , Rubinacci Alessandro , Moro GianLuigi , Bianchi Giuseppe
Published: January 2020

The relationships of Sr intestinal absorption and renal excretion with biohumoral factors regulating Ca metabolism were studied in 47 normocalciuric subjects with Ca kidney stones. Sr concentrations were measured in serum and urine after an oral load of stable Sr (30.2 μmol/kg body wt). Enteral absorption of the ion (9.77 ± 0.438 mmol · L−1 · min, 240 min after Sr administration), expressed as the area under the plasma concentration–time curve (AUC), and renal clearance (CRE) in these subjects during the test (2.80 ± 0.336 mL/min) were not different from values for 27 controls. CRE was not correlated with AUCs. Plasma concentrations of parathyroid hormone (PTH) negatively correlated with AUCs (P <0.01) and correlated with CRE after one outlier was excluded (P <0.05). Plasma concentrations of 1,25-dihydroxyvitamin D correlated positively with AUCs (P <0.01) when normalized to the plasma concentration of PTH. Multiple stepwise regression showed that PTH and phosphatemia were significantly related to AUC values at 240 min (P <0.01). These findings suggest that Sr absorption and excretion reflect the regulation of Ca metabolism, but some differences in renal handling of the two ions may exist.
Intracellular and extracellular blood magnesium fractions in hemodialysis patients; is the ionized fraction a measure of magnesium excess?
Authors: Huijgen Henk J , Sanders Renata , van Olden Rudolf W , Klous Marjolein G , Gaffar Faryal R , Sanders Gerard T B
Published: January 2020

To establish the best measure for determining magnesium overload, we measured ionized and total magnesium in serum and mononuclear blood cells and total magnesium in erythrocytes in blood of 23 hemodialysis patients, known for their disturbed magnesium homeostasis. When comparing the mean magnesium values obtained in the patient population with those of a control population, all of these magnesium markers, including the biologically active fractions, were significantly (P <0.05) increased. Because serum total magnesium was not increased in all dialysis patients studied, the population was divided into two groups, according to total serum magnesium >1.0 mmol/L or less than that. Results in these two populations showed that ionized serum magnesium and ionized magnesium in mononuclear blood cells might give a better indication about the magnesium status of the tested patients than the currently used total serum magnesium data. However, neither of the two markers, especially ionized serum magnesium, was able to discriminate fully between normal magnesium homeostasis and magnesium excess. We therefore conclude that the two ionized magnesium markers offer minimal advantage for this discrimination, and that the total magnesium concentration in serum remains the measurement of choice.
Multicenter evaluation of the Bayer Immuno 1™ CA 15-3™ assay
Authors: Cheli Carol D , Morris Deborah L , Kish Linda , Goldblatt Joan , Neaman Irene , Allard W Jeffrey , Yeung Kwok K , Wu Alan H B , Moore Robert , Chan Daniel W , Fritsche Herbert A , Schwartz Morton K , Very Donald L
Published: January 2020

We conducted a multicenter evaluation of the analytical and clinical features of the automated Bayer Immuno 1™ CA 15-3™ assay and compared assay performance to two manual tests. Results of the 10-day imprecision study of the Bayer Immuno 1 assay pooled across four evaluation sites and three lots of reagent produced total CV ≤4%. Lot-to-lot reproducibility for 26 different lots of reagents and calibrators manufactured over a 2-year period was demonstrated (CV, 1.1%). Results for the Bayer Immuno 1 assay correlated well with the Biomira TRUQUANT® BR™ 27.29 and Centocor® CA 15-3 RIAs (r ≥0.94). The upper limit of the reference interval for the Bayer Immuno 1 assay was 35.9 kilounits/L (35.9 units/mL); values were similar for all methods. Longitudinal monitoring of healthy women yielded assay values with an average CV of 11% and 21% for the Bayer Immuno 1 and Biomira assays, respectively. The Bayer Immuno 1 assay demonstrated the analytical features, intermethod correlation, and long-term performance characteristics that are essential for longitudinal monitoring of breast cancer patients.
Clinical laboratory test menu changes in the Pacific Northwest: 1994 to 1996
Authors: LaBeau Kathleen M , Simon Marianne , Steindel Steven J
Published: January 2020

Laboratory testing services are presently undergoing dynamic changes in response to a wide range of external factors. Government regulations, reimbursement, and managed care are only a few of the influences affecting the availability of testing services and on-site testing capabilities in hospital, independent, and physician office laboratories. Medical practice changes, marketplace influences, test technologies, and costs also play a role in determining where testing is being performed. To better understand the factors influencing clinical laboratory test volumes and menus and to identify on-site testing deemed essential in physician office laboratories, we gathered information from a network of clinical laboratories in the Pacific Northwest. Questionnaires were sent to 257 Laboratory Medicine Sentinel Monitoring Network participants in March 1996. In the past 2 years, changes in on-site test volumes and test menus have been primarily due to medical practice changes and marketplace influences. When laboratories had a decrease in test volumes or test menu choices, the size of the patient workload and the volumes of test orders have had the greatest impact. Laboratory regulations and managed care contracts have played a role in shifting on-site testing to outside sources; however, these factors did not appear to be primary influences. Only 5% of physician office laboratories identified tests that they believed were essential for optimal patient care but did not perform on-site.
Immunoradiometric assay for the N-terminal fragment of proatrial natriuretic peptidein human plasma
Authors: Numata Yoshito , Dohi Keiji , Furukawa Ayako , Kikuoka Shino , Asada Hidehisa , Fukunaga Takahiro , Taniguchi Yasushi , Sasakura Kazuyuki , Tsuji Tetsuo , Inouye Ken , Yoshimura Michihiro , Itoh Hiroshi , Mukoyama Masashi , Yasue Hirofumi , Nakao Kazuwa
Published: January 2020

Recently, the N-terminal fragment of proatrial natriuretic peptide (N-terminal proANP) has been proposed as a marker of chronic congestive heart failure. In this study, we established a two-step immunoradiometric assay using monoclonal antibodies and synthetic N-terminal proANP (1–67) as a standard. It allows us to measure plasma N-terminal proANP in only 4 h without prior extraction. The detection limit of this assay was 15 pmol/L for a 100 μL sample of plasma. Within-run CVs ranged from 1.7% to 2.9% and between-run CVs ranged from 4.2% to 5.1%. The dilution curves of plasma samples showed good linearity and analytical recovery was 89–104%. The mean (±SD) N-terminal proANP in plasma of 33 healthy subjects was 188 (±71) pmol/L and 1030 (±411) pmol/L in 25 patients with heart failure. Our immunoradiometric assay is rapid and precise enough for routine determination of N-terminal proANP in human plasma.
Flow cytometry with a monoclonal antibody to the low density lipoprotein receptor compared with gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia
Authors: Raungaard Bent , Heath Finn , Brorholt-Petersen Jens Uffe , Jensen Henrik Kjærulf , Faergeman Ole
Published: January 2020

We used a fluorescence flow cytometry assay with a monoclonal low density lipoprotein (LDL) receptor-specific antibody to detect LDL receptor expression on blood T lymphocytes and monocytes. We prepared peripheral blood mononuclear cells from patients with genetically verified LDL receptor-defective (Trp66-Gly mutation, n = 17) or receptor-negative (Trp23-stop mutation, n = 17) heterozygous familial hypercholesterolemia (FH) and from healthy individuals (n = 24). The cells were stimulated to express the maximum amount of LDL receptor by preincubation in lipoprotein-deficient medium. A dual-labeling technique allowed flow cytometric analysis of LDL receptor expression on cells identified by fluorescently conjugated surface marker antibodies. Knowing the LDL receptor gene mutation of the FH patients allowed us to compare the diagnostic capability of this functional assay with the DNA diagnosis and to validate the assay with molecular genetics instead of clinical indices of heterozygous FH. T lymphocytes expressed more LDL receptors and gave better diagnostic results than monocytes, and cells from patients with either the Trp66-Gly or the Trp23-stop mutation had variable but significantly reduced LDL receptor expression. The data indicate that this fluorescence flow cytometry assay is unsuitable for diagnosis of individual cases of heterozygous FH but that it may be useful for functionally characterizing mutations in the LDL receptor gene.
Marked gender differences in ambulatory morning growth hormone values in young adults
Authors: Engström Britt Edén , Karlsson F Anders , Wide Leif
Published: January 2020

The influence of gender on serum concentrations of growth hormone (GH) and 12 other endocrine analytes was investigated in sera drawn from 291 healthy medical students in the ambulatory state in the morning, after fasting overnight. GH was measured with a sensitive noncompetitive fluoroimmunoassay. The median GH value was 80-fold higher in women 21–26 years old than in age-matched men (14.4 vs 0.18 mIU/L), compared with a female/male ratio of 2.2 for 17β-estradiol and a male/female ratio of 14 for testosterone. Furthermore, the values for sex hormone-binding globulin, follicle-stimulating hormone, luteinizing hormone, prolactin, and insulin-like growth factor 1 (IGF-1) were higher, whereas the values for free thyroxine, triiodothyronine, thyroid-stimulating hormone, and parathyroid hormone were lower in the women. The median GH value was 68-fold higher in women 27–43 years old than in age-matched men (10.9 vs 0.16 mIU/L). Women taking contraceptives with ethinyl estradiol and desogestrel or levonorgestrel had higher GH values, and the desogestrel group had lower IGF-1 values than women not taking contraceptives. The median GH values in these groups were 125- and 117-fold higher, respectively, than in men 21–26 years old. The results suggest that routine morning activity produces a marked GH response in >90% of young women but in very few age-matched men. The effect on GH was even more pronounced in women taking oral contraceptives, suggesting that the intake of ethinyl estradiol contributes to higher GH concentrations in these women.
Simultaneous determination of mycophenolic acid and its glucuronide in human plasma using a simple high-performance liquid chromatography procedure
Authors: Shipkova Maria , Niedmann Paul Dieter , Armstrong Victor William , Schütz Ekkehard , Wieland Eberhard , Shaw Leslie M , Oellerich Michael
Published: January 2020

We describe a reversed-phase HPLC method for determination of total mycophenolic acid (MPA), its free concentration (MPAf), and the glucuronide metabolite (MPAG), based on simple sample preparation and gradient elution chromatography. The compounds were quantified in parallel by absorbance at 254 nm and 215 nm in the internal standard mode. Linearity was verified up to 50 mg/L for MPA and up to 500 mg/L for MPAG (r >0.999). Detection limits at 215 and 254 nm were, respectively, 0.01 and 0.03 mg/L for MPA, and 0.03 and 0.1 mg/L for MPAG. The recovery of MPA was 95–106%;recovery of MPAG was 96–106%. The imprecision (CV) for MPA (0.2–25 mg/L) was <8.4% (254 nm) and <4.4% (215 nm) within day (n = 12) and <9.2% (254 nm) and <6.2% (215 nm) between days (n = 12). The imprecision for MPAG (10–250 mg/L) was <4.9% (254 nm) and <3.4% (215 nm) within day, and <6.1% (254 nm) and <5.9% (215 nm) between days. For quantification of MPAf, 100 μL of ultrafiltrate was applied directly to the column. The detection limit was 0.005 mg/L at 215 nm and 0.015 mg/L at 254 nm. In the range between 18–210 μg/L, the within-day CVs were <11.8% (n = 12) and the between-day CVs were <15.8% (n = 12).
Genetic testing for Gilbert’s syndrome: how useful is it in determining the cause of jaundice?
Authors: Rudenski Aram S , Halsall David J
Published: January 2020

Gilbert’s syndrome is a benign condition causing hyperbilirubinemia, which is also a symptom of liver or hemolytic disease. A genetic test may be possible for Gilbert’s syndrome because an associated gene defect has been isolated. Here we present a mathematical analysis of the use of this test in excluding harmful causes of hyperbilirubinemia. The effectiveness of the test depends on a low prevalence and high penetrance of the gene defect and a low prevalence of harmful hyperbilirubinemia. If the gene defect has a 12% prevalence and 10% penetrance, then the prevalence of harmful hyperbilirubinemia needs to be <1.5% for a positive test to reduce this risk fivefold. Estimates of the prevalence of harmful hyperbilirubinemia and of the prevalence and penetrance of the Gilbert genotype are required to assess the value of the genetic test in determining a cause of hyperbilirubinemia. Until these values for prevalence and penetrance are known, genetic testing for Gilbert’s syndrome cannot be recommended on a routine basis.
Identification and haplotype analysis of apolipoprotein B-100 Arg3500→Trp mutation in hyperlipidemic Chinese
Authors: Tai Der-Yan , Pan Ju-Pin , Lee-Chen Guey-Jen
Published: January 2020

DNA screening for apolipoprotein (apo) B-100 mutations was performed in hyperlipidemic Chinese. The apo B-100 gene segment surrounding previously identified familial defective apo B-100 (FDB) mutations was amplified by PCR and subjected to single-strand conformation polymorphism (SSCP) analysis. One subject’s aberrant SSCP band was cloned and sequenced to study the molecular lesions. A recurrent ArgCGG-to-TrpTGG mutation (R3500W) in the codon 3500 of the apo B-100 gene was identified. The C-to-T transition creates a NlaIII site and permits rapid restriction analysis of the mutation. A total of 373 hyperlipidemic patients and 309 controls were screened for R3500W. Nine unrelated subjects were shown to be heterozygous for the mutation, and no R3500W carriers were found in the control group (P = 0.004). Six polymorphic markers, including five restriction fragment length polymorphisms and one hypervariable repeat region, were used for haplotype analysis on the mutant allele. In two families, the R3500W mutation could be unambiguously assigned to a unique haplotype XbaI−/MaeI+/MspI+/EcoRI+/Eco57I+/34 3′HVR repeats; in the other seven unrelated heterozygotes, this finding was consistent when an unequivocal haplotype was deduced. The results suggest that all R3500W alleles are identical by descent in our population. The fact that the same mutant allele was identified in other Asians with FDB indicates a common Asian origin for the R3500W mutations.
Biochemical markers of the acute coronary syndromes
Authors: Christenson Robert H , Azzazy Hassan M E
Published: January 2020

The acute coronary syndromes represent a continuum of myocardial ischemia ranging from angina, reversible tissue injury → unstable angina, frequently associated with minor myocardial damage → myocardial infarction and extensive tissue necrosis. Historically, coronary artery disease assessment has been mainly binary, using WHO criteria of symptoms, electrocardiography, and biochemical markers. The creatine kinase-MB isoenzyme (CK-MB) has been a benchmark for markers, but it is not specific for myocardium. Cardiac-specific isoforms of troponin T and I have emerged as sensitive myocardial infarction (MI) indicators and, importantly, for risk stratification of acute coronary syndrome patients. In addition to markers of myocardial cell necrosis, markers of plaque disruption (C-reactive protein and serum amyloid A), “angry” platelets (P-selectin), ischemia (glycogen phosphorylase-BB isoenzyme), and the procoagulant state and thrombosis (soluble fibrin) have potential use. Also, CK-MB and myoglobin have been combined with clinical indicators for monitoring reperfusion after thrombolytic therapy. Biochemical markers will continue to be an important clinical adjunct for MI diagnosis, risk assessment, and reperfusion monitoring in the future.
Coronary atherosclerosis and its effect on cardiac structure and function: evaluation by electron beam computed tomography
Authors: Stanford William , Thompson Brad H
Published: January 2020

Coronary artery disease affects millions of Americans annually. In evaluating coronary artery disease, it is important to develop diagnostic methodology that can screen patients before the onset of symptoms or cardiac events and, in addition, evaluate the functional aspects of coronary artery disease, including any residual effects on the heart after events have occurred. Electron beam computed tomography allows the identification of coronary calcium, which is a marker for coronary atherosclerotic disease, and also allows the quantification of cardiac function, which may be altered from coronary atherosclerosis or the occurrence of a cardiac event. Thus, electron beam computed tomographic imaging is having a major impact on the diagnosis and follow-up on coronary artery disease.
Evaluation of the tacrolimus II microparticle enzyme immunoassay (MEIA II) in liver and renal transplant recipients
Authors: Cogill Jan L , Taylor Paul J , Westley Ian S , Morris Raymond G , Lynch Stephen V , Johnson Anthony G
Published: January 2020

We evaluated the MEIA II with blood samples with added tacrolimus (3.0, 5.0, 11.0, and 22.0 μg/L). The assay had acceptable recoveries (99–103%) and intraday imprecision (<16.0%) across the range of concentrations studied, except for the recoveries at 3.0 μg/L (86.3%) and 5.0 μg/L (80.7%). Comparison of liver (n = 116) and renal (n = 113) patient samples measured by MEIA II against HPLC-tandem mass spectrometry (HPLC-MS/MS) found a mean overestimation of 15.6%. From these comparison data it can be calculated that at values of 5 and 20 μg/L in liver or renal transplant patient samples, measured by HPLC-MS/MS, MEIA II will have the corresponding range estimates of 3.6–7.9 μg/L and 20.9–25.4 μg/L, respectively. No clinically significant difference in results, in terms of overestimation or correlation, was observed between the two transplant groups studied. The MEIA II is an improvement on the previous MEIA I and is suitable for the therapeutic drug monitoring of tacrolimus where HPLC-MS/MS is unavailable.
Serum digoxin in the presence of Digibind: determination of digoxin by the Abbott AxSYM and Baxter Stratus II immunoassays by direct analysis without pretreatment of serum samples
Authors: Ocal Idris T , Green Terrence R
Published: January 2020

We have reevaluated the feasibility of using direct immunochemical methods to track free digoxin in patients receiving Digibind®. We report here that results obtained by the Stratus II and AxSYM immunoassays on patients receiving digoxin (without Digibind), digoxin-fortified serum samples supplemented with Digibind, and a digitoxic patient treated with Digibind, show no clinically significant biases. We conclude that useful free digoxin concentrations may be obtained for Digibind-treated patients using either the AxSYM or Stratus immunoassays without subjecting samples to ultrafiltration before analysis.
Luminometric single step urea assay using ATP-hydrolyzing urease
Authors: Näslund Birgitta , Ståhle Lars , Lundin Arne , Anderstam Björn , Arner Peter , Bergström Jonas
Published: January 2020

An automatic enzyme kinetic luminometric method for determination of small quantities of urea in biological fluids and in microdialysates is presented. The method is based on the ATP-hydrolyzing urease reaction [urea amidohydrolase (ATP-hydrolyzing); EC 3.5.1.45], monitored by a luciferin-luciferase ATP reaction. The assay range is 100 pmol to 50 nmol with a detection limit of 5 μmol/L in the sample, compared with detection limits of 0.1 mmol/L in earlier spectrophotometric methods. To reduce the non-urea-dependent ATPase activity (vblank) and to increase the urea-dependent activity, 1,2-propanediol was included. Assay conditions were optimized by multivariate analysis. Recoveries of urea added to blood dialysate and plasma were 96–103%. No analytical interference of common metabolites, drugs, or other additives was observed. The total CVs (6 days and six concentrations, 1.2–21.8 mmol/L) were 3.6–8.5%. The results obtained with the present assay were highly correlated for dialysate (r = 0.979) and for plasma (r = 0.978) with those obtained by a spectrophotometric kit method with slopes of 1.02–1.03 and intercepts of 0.08–0.23 mmol/L.
Evaluation of Interferences in Rate and Fixed-Time Nephelometric Assays of Specific Serum Proteins
Authors: Bossuyt Xavier , Blanckaert Norbert
Published: January 2020

We performed interference studies for IgG, IgA, IgM, haptoglobin, and α1-antitrypsin assayed in serum, using either fixed-time nephelometry on the BN 100 from Behring or rate nephelometry on two analyzers from Beckman Instruments. For clear serum samples, results for IgG, IgA, IgM, and haptoglobin obtained with the three nephelometers showed good agreement. Values for α1-antitrypsin in clear sera were lower with the BN 100 than with the Array 360 or Immage. In lipemic samples, the BN 100 gave higher values than the Array 360 or Immage for all analytes except IgG. Addition of Intralipid to serum produced atypical reactions with the BN 100 (fixed-time nephelometry) but not with the Array 360 or Immage (rate nephelometry). The interference of lipemia on the BN 100 was also seen when the Beckman antibody was used, indicating that the effect was reagent-independent. For hemolyzed samples, the BN 100 gave higher values than the Array 360 or Immage for haptoglobin but not for the other analytes. Addition of increasing amounts of a hemolysate to serum revealed a negative interference in all assay systems. This effect was more pronounced with the Beckman reagent than with the Behring reagent in all three nephelometers and was independent of the type of instrument (fixed-time vs rate nephelometry).
Real-Time Quantitative PCR for the Measurement of MYCN Amplification in Human Neuroblastoma with the TaqMan Detection System
Authors: Raggi Claudia Casini , Bagnoni Maria Letizia , Tonini Gian Paolo , Maggi Mario , Vona Giovanna , Pinzani Pamela , Mazzocco Katia , De Bernardi Bruno , Pazzagli Mario , Orlando Claudio
Published: January 2020

Background: Neuroblastoma is the most common extracranial malignant solid tumor in children under 5 years and is characterized by a wide clinical and biological heterogeneity, from spontaneously regressive forms to cancers with a rapid and fatal progression. MYCN oncogene amplification is considered the most important prognostic factor to evaluate survival and therapeutic choices in these patients.

Methods: Here we present a new assay for rapid and accurate measurement of MYCN amplification, based on real-time quantitative PCR with the TaqManTM reaction. The degree of MYCN amplification was derived from the ratio of the MYCN oncogene and the single-copy reference gene, β-actin. The absolute abundance of these two genes in tumor sample DNA was obtained by extrapolation on external calibration curves generated with reference DNA.

Results: We found a variable degree of MYCN amplification, from 2 to 29, in 26 of 49 (53%) neuroblastomas. These results were well correlated to those obtained with a competitive PCR assay in the same samples (r = 0.987). MYCN amplification was associated mainly with advanced cancer stages, and the analysis of overall survival confirmed that the measurement of MYCN amplification is a predictor of patient outcome in neuroblastoma. Patients without MYCN amplification had a cumulative survival significantly higher than patients with low (<9; P = 0.02) and high (≥9; P = 0.03) oncogene amplification.

Conclusion: The assay is rapid and reproducible and does not require any post-PCR analytical procedure.
Dynamic Reaction in a Homogeneous HDL-Cholesterol Assay Visualized by Electron Microscopy
Authors: Kondo Akira , Muranaka Yoshinori , Ohta Isao , Kanno Takashi
Published: January 2020

Background: Measurement of HDL-cholesterol (HDL-C) by homogeneous assays with automated analyzers is replacing precipitation methods. However, in this reaction-type assay, interactions between the reagents and lipoproteins remain unknown.

Methods: Electron microscopy was used to investigate the reactions in a homogeneous HDL-C assay. Negative staining with 10 g/L uranyl acetate was performed for lipoprotein visualization by electron microscopy. Observations of the interactions between lipoproteins and the reagents of a polyanion-polymer/detergent assay were achieved by cooling the reaction mixture in ice water. This treatment also allowed observation of the time course of the reaction.

Results: In the first-reagent reaction (polyanion-polymer), every lipoprotein aggregated almost completely. In the second-reagent reaction (enzymes and detergent), only HDL in the lipoprotein aggregates was selectively resolved and reacted enzymatically. Reagent 1 contains two important substances: polyanion and synthetic polymer. Using x-ray microanalysis, we confirmed that aggregation of lipoproteins in the first reaction occurred through interaction with the phosphotungstate of the polyanion.

Conclusion: Electron microscopy morphologically revealed the dynamic reaction in a homogeneous HDL-C assay.
Freedom from Drug Interference in New Immunoassays for Urinary Catecholamines and Metanephrines
Authors: Wassell Julie , Reed Paul , Kane John , Weinkove Cyril
Published: January 2020

Background: Determination of urinary free catecholamine and total (i.e., free plus conjugated) metanephrine excretion is considered the most clinically sensitive biochemical test for pheochromocytoma. In this study, we evaluated new immunoassay methods for the measurement of these analytes for potential drug-based interference.

Methods: Urine samples collected from patients on a variety of medications were grouped by specific drug type. The significance of any difference in the free catecholamine or total metanephrine concentrations in the different groups was assessed by one-way ANOVA. A group of patients receiving no medication was included as a control (no analytical interference). Additionally, analytical accuracy, detection limit, and precision were determined.

Results: No significant differences were found in the concentrations of free catecholamines or total metanephrines in urine from patients taking the medications investigated and the control group: P = 0.649 (fE), 0.221 (fNE), 0.149 (tM), and 0.170 (tNM). For free catecholamines, intraassay CVs were 4.6–18%; interassay CVs were 10–25%. For total metanephrines, intraassay CVs were 9.6–27%; interassay CVs were 5.8–22%. Detection limits were 0.009 and 0.027 μmol/L for fE and fNE and 0.119 and 0.346 μmol/L for tM and tNM, respectively.

Conclusions: None of the drugs examined in this study interfered in the measurement of free catecholamines or total metanephrines by these immunoassays. The technique is easier to use, requires less equipment, and is more accessible than HPLC. In combination, these assays are suitable as initial screening tests for pheochromocytoma.