bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in serum apolipoprotein and lipoprotein profile induced by chronic alcohol consumption and withdrawal: determinant effect on heart disease?
Authors: Lecomte E , Herbeth B , Paille F , Steinmetz J , Artur Y , Siest G
Published: January 2020

The effects of alcohol consumption on serum concentrations of apolipoproteins (apo) A-I, C-III, B, and E and of lipoproteins (Lp) A-I, A-I:A-II, C-III, C-III:B, and (a) were studied in 132 healthy subjects, including 55 low drinkers of alcohol (<20 g/day), 36 moderate drinkers (20-50 g/day), and 41 heavy drinkers (>50 g/day), and in 97 hospitalized alcoholic patients (> 100 g/day) without severe liver disease (especially functional insufficiency), before and after 21 days of withdrawal treatment. Serum concentrations of apo A-I, LpA-I, LpA-I:A-II, apo C-III, and LpC-III significantly (P </= 0.01) increased with alcohol intake (mean +/- SE in low drinkers vs in alcoholics)--1.45 +/- 0.03 vs 1.78 +/- 0.05 g/L; 0.45 +/- 0.02 vs 0.56 +/- 0.02 g/L; 0.99 +/- 0.02 vs 1.22 +/- 0.04 g/L; 27.6 +/- 1.5 vs 39.7 +/- 1.7 mg/L; and 8.4 +/- 0.9 vs 24.7 +/- 1.7 mg/L, respectively-whereas apo B and LpC-III:B concentrations tended to decrease--1.20 +/- 0.04 vs 1.06 +/- 0.04 g/L and 19.3 +/- 1.2 vs 14.9 +/- 1.0 mg/L, respectively. No significant difference between these four types of alcohol consumption was noticed for cholesterol, triglycerides, apo E, and Lp(a). After withdrawal, the concentrations of serum apo A-I, apo C-III, LpA-I, LpA-I:A-II, and LpC-III decreased significantly (P </= 0.01), reaching values comparable with those in low drinkers; concentrations of triglycerides, apo B, apo E, and Lp(a) rose; and cholesterol concentration was unaffected. In multiple regression analysis, after adjustment for serum concentrations of albumin, aspartate aminotransferase, and gamma-glutamyltransferase and for the Quetelet index, alcohol consumption remained positively correlated to apo A-I, LpA-I:A-II, apo C-III, and LpC-III concentrations. Study of other determinants of serum apo and lipoprotein concentrations suggests that alcohol-related variations in some of them, especially apo A-I, might depend on the metabolic ability of the liver to synthesize proteins and on induction phenomena. Finally, although the increase of antiatherogenic apo- and lipoproteins and the decrease of those known to be atherogenic were generally marked in alcoholics, alcohol-related modifications of these markers were very limited in our sample of French healthy men. We conclude, therefore, that moderate alcohol consumption (20-50 g/day) is unlikely to protect against ischemic heart disease through an effect on the proteins measured in this study.
Design and assessment of average of normals (AON) patient data algorithms to maximize run lengths for automatic process control
Authors: Westgard J O , Smith F A , Mountain P J , Boss S
Published: January 2020

Achieving high quality and high productivity with automated testing processes will require process control systems that are optimized for the necessary error detection, minimum false rejection, and maximum run length. This study investigates whether run length could be monitored by average of normals (AON) algorithms that truncate the patient test distribution and estimate the average of a suitable number of patient results. The design of AON algorithms for individual analytes is facilitated by computer-simulated power curves that consider the ratio of the population biological variation (Spop) to the test method variation (Smeas), represent a range of Spop/Smeas ratios from 2 to 15, and include numbers of patient test results from 10 to 600. The potential applications of AON algorithms are assessed for 38 tests whose quality requirements represent the total error criteria from the Ontario Medical Association Laboratory Proficiency Testing Program, Spop/Smeas ratios from 0 to 32, critical systematic shifts from 0.02 to 10.85 Smeas, and test workloads representative of a regional reference laboratory. Approximately half of these tests provide high potential for applying AON algorithms to monitor run length.
Competitive RT-PCR to quantify CFTR mRNA in human endometrium
Authors: Mularoni A , Adessi G L , Arbez-Gindre F , Agnani G , Nicollier M
Published: January 2020

Because the down-regulation by progesterone of cystic fibrosis transmembrane conductance regulator (CFTR) expression could be a useful specific marker to define the state of implant receptivity in endometrium, a competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed for quantifying the CFTR mRNA concentration in human endometrial samples. A competitor RNA was constructed with the same sequence as the CFTR sequence except for a 20-nucleotide insertion in the middle. The amplified products were separated by polyacrylamide gel electrophoresis. The ratio of CFTR band areas to competitor band areas provided the basis of quantification. Using this competitive RT-PCR, we measured CFTR mRNA in human endometrial samples taken at different periods of the menstrual cycle, in endometriosis, and in hyperplasia. Results show that the method is suitable for measuring the concentration of CFTR mRNA.
Chemiluminescence immunoassay for vascular endothelial growth factor (vascular permeability factor) in tumor-tissue homogenates
Authors: Schlaeppi J M , Eppenberger U , Martiny-Baron G , Küng W
Published: January 2020

We developed a two-site chemiluminescence immunoassay for human vascular endothelial growth factor (VEGF). The assay recognized both VEGF121 and VEGF165 isoforms, but had no detectable cross-reactivity with platelet-derived growth factor or placenta growth factor. The range of detection was between 30 ng/L and 30 micrograms/L VEGF. Inter- and intraassay variations were 8.2-8.3% and 7.2-7.6%, respectively. VEGF concentrations were measured in the cytosolic extracts of 45 ovarian and 142 primary breast tumors. The amount of VEGF in the ovarian tumors (median = 0.46 ng/mg total protein, range 0-15.8 ng/mg) was significantly (P = 0.03) higher compared with the breast tumors (median = 0.24 ng/mg total protein, range 0-12.3 ng/mg). In 32 and 7 extracts of normal breast tissues adjacent and distant to the tumors, respectively, VEGF concentrations were significantly much lower (P < 0.0001). The detection of substantial amounts of VEGF in two invasive tumors (compared with normal tissues) suggests that the assay should be a useful tool for investigating the prognostic value of VEGF in breast and ovarian carcinomas and for selecting patients for future anti-VEGF therapy.
Prostate-specific antigen concentrations in serum in acute illnesses
Authors: Honda S A , Goldstein A P , Morita T , Sugiyama C , Cody L , Rios C N , Bhagavan N V
Published: January 2020

In light of recent studies showing that prostate-specific antigen (PSA) complexes with certain blood proteins, we studied the effects of acute-phase reactants and alpha 2-macroglobulin (A2MG) on serum concentrations of PSA. Serum samples were obtained from 419 men admitted to an acute-care facility. Various acute-phase reactants-including C-reactive protein, alpha 1-acid glycoprotein, alpha 1-antitrypsin, and alpha 1-antichymotrypsin-and A2MG were measured with a Beckman Array analyzer in parallel with determinations of PSA concentrations by two methods, the Hybritech Tandem RIA and the Abbott PSA IMx. Evaluation by Spearman rank correlation revealed a significant negative correlation of A2MG with PSA values (P < 0.01) and (as expected) a positive correlation of age with PSA values (P < 0.001). The former correlation suggests the possibility that patients with high serum concentrations of A2MG may give falsely decreased results for PSA concentrations in serum.
Growth hormone assays: early to latest test generations compared
Authors: L'Hermite-Balériaux M , Copinschi G , Van Cauter E
Published: January 2020

We compared the data from four growth hormone (GH) immunoassays for analyzing 24-h GH profiles in four apparently normal subjects and four obese subjects (508 serum samples). The detection limit was 0.02 microgram/L for one immunochemiluminometric assay (ICMA), 0.1 microgram/L for two IRMAs, and 0.4 microgram/L for one RIA. All GH pulses with a peak ICMA value > 1 microgram/L were detected by each of the other methods. Overall, the correlation coefficient between the values obtained with all four assays exceeded 0.90. However, for GH concentrations < or = 0.25 microgram/L, acceptable concordance (r2 > or = 0.80) was reached only between the ICMA and one IRMA; between the ICMA and the RIA, concordance was acceptable only for GH concentrations > or = 10 micrograms/L. In the normal subjects, the percentage of undetectable values was 0% with the ICMA but 29% with one of the IRMAs; in obese subjects, the corresponding values were 12% and 38%.
Comparison of total and bone-specific alkaline phosphatase in patients with nonskeletal disorder or metabolic bone diseases
Authors: Woitge H W , Seibel M J , Ziegler R
Published: January 2020

To evaluate the diagnostic validity of new assays for bone-specific alkaline phosphatase (BAP), we compared measurements of total alkaline phosphatase (TAP) in serum with results for three different assays of serum BAP in healthy adults (n = 119), patients with chronic nonskeletal disorders (n = 123), and patients with metabolic bone diseases (n = 113). Serum TAP was determined by a standard colorimetric assay, BAP by the methods of lectin precipitation (L-BAP), enzyme immunoassay (E-BAP), and immunoradiometric assay (I-BAP). Impairment of liver function resulted in significant increases of all alkaline phosphatase (AP) measurements, with the smallest changes being exhibited by E-BAP. Compared with the results by TAP, diagnostic sensitivity (i.e., of values exceeding the reference interval) was not improved by BAP, but receiver-operating characteristic (ROC) curve analyses revealed improved discrimination for primary hyperparathyroidism by E-BAP. These results indicate that, in the presence of liver disease, the specificity of AP measurements is improved by measuring BAP. In most other clinical situations, serum TAP appears to provide sufficient clinical information; however, the cross-sectional study design used here allows no statement about the usefulness of BAP in serial measurements.
Microtiter plate immunoenzymometric assay for estrogen receptor
Authors: Delage V , Teulon J M , Bellanger L , Seguin P , Descotes F , Saez S
Published: January 2020

The estrogen receptor (ER) status of breast cancer is used both as a prognostic factor and as a predictor of response to endocrine therapy. An immunoenzymometric assay for ER was developed on 96-well microtiter plates (EIA96). This technique involves two monoclonal antibodies directed against different epitopes in the B domain of ER. The two-step protocol (16-18 h and 3 h at 4 degrees C) requires 100 microL of cytosol. This assay showed a detection limit of 0.58 pmol/L. Intra- and interassay CVs of clinical specimens were < or = 5% except for the least concentrated sample (6.5 pmol/L, CV = 6.7%). In a comparison study involving cytosols of breast adenocarcinoma tissue biopsies, we compared the EIA96 with the radioligand assay (RLA) and the Abbott ER-EIA, widely used techniques for determining ER concentration in cytosols of breast cancer tumors. The two EIAs showed excellent agreement; however, two samples showed discrepant results by EIA96 and RLA.
Epitope mapping of prostate-specific antigen with monoclonal antibodies
Authors: Jette D C , Kreutz F T , Malcolm B A , Wishart D S , Noujaim A A , Suresh M R
Published: January 2020

Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. We identified and characterized the epitopes of two anti-PSA monoclonal antibodies (mAbs) designated B80 and B87. The epitopes were initially mapped as nonoverlapping by developing a sandwich immunoassay to measure PSA with the two anti-PSA mAbs. The two antibodies do not cross-react with homologous pancreatic kallikrein, but recognize epitopes unique to PSA. B80 and B87 can recognize both free and complexed PSA and hence measure total PSA. Epitope scanning and bacteriophage peptide library affinity selection procedures were used to identify and locate an epitope on PSA. A possible epitope for B80 was identified as being located on or near PSA amino acid residues 50-58 (-GRH-SLFHP-). The epitope for B87 was likely on an exposed nonlinear conformational determinant, unique to PSA, and not masked by the binding of B80 or alpha 1-antichymotrypsin.
Radioimmunoprecipitation assay for glutamic acid decarboxylase antibodies evaluated clinically with sera from patients with insulin-dependent diabetes mellitus
Authors: Ohta M , Obayashi H , Takahashi K , Kitagawa Y , Nakano K , Matsuo S , Nishimura M , Itoh N , Ohta K
Published: January 2020

We evaluated a new, commercially developed radioimmunoprecipitation assay for measuring glutamic acid decarboxylase (GAD) antibodies by using recombinant human GAD65. The intra- and interassay CVs were 8.0% (n = 20) and 8.6% (n = 15), respectively. We found GAD antibodies in 74% (23 of 31; 95% confidence interval 55-88%), 70% (14 of 20; 46-88%), and 65% (28 of 43; 49-79%) of patients at, respectively, < or = 1 year, 1-2 years, and 2-4 years after the onset of insulin-dependent diabetes mellitus (IDDM) and in 30% (30 of 99; 21-40%) of patients with long-term diabetes (4-22 years). We also detected GAD antibodies in 8% (9 of 106; 4-16%) of patients with non-insulin-dependent diabetes mellitus (NIDDM). The frequency of GAD antibodies in the NIDDM group was markedly higher in the insulin-deficient patients [67% (6 of 9; 30-93%)], who initially were nonketotic and non-insulin-dependent for > or = 6 months but later became insulin dependent, than in the non-insulin-deficient patients [3% (3 of 97; 1-9%)]. This new commercial assay is easy to use and provides a specific and sensitive method for evaluating GAD antibodies in IDDM.
Pre beta 1-high-density lipoprotein increases in coronary artery disease
Authors: Miida T , Nakamura Y , Inano K , Matsuto T , Yamaguchi T , Tsuda T , Okada M
Published: January 2020

Pre beta 1-HDL promotes cholesterol efflux from cell membranes. Its plasma concentration is regulated by lecithin: cholesterol acyltransferase (LCAT). To clarify whether the concentration of pre beta 1-HDL changes in coronary artery disease (CAD), we determined the distribution of apolipoprotein A-I (apoA-I) among HDL subfractions in 20 CAD patients and 20 healthy controls, using nondenaturing two-dimensional gel electrophoresis. We found that CAD patients had significantly higher concentrations of pre beta 1-HDL than the controls [7.6% +/- 3.4% vs 4.6% +/- 2.3% of apoA-I (P < 0.01)]. Even after correcting for apoA-I concentrations, this increase remained significant [87 +/- 37 vs 63 +/- 28 mg/L apoA-I (P < 0.05)]. The mean LCAT concentration was significantly lower (P < 0.05) in CAD patients than in controls. These findings, that pre beta 1-HDL concentrations increase in CAD, strongly suggest that the process of reverse cholesterol transport could be altered in CAD.
Rapid determination of fecal fat by Fourier transform infrared analysis (FTIR) with partial least-squares regression and an attenuated total reflectance accessory
Authors: Franck P , Sallerin J L , Schroeder H , Gelot M A , Nabet P
Published: January 2020

Fecal lipid content is usually determined by titrimetric or gravimetric methods, but these methods are time consuming and involve dangerous solvents. We have developed a new method of measuring fecal lipids by Fourier transform infrared spectrometry (FTIR) with an attenuated total reflectance accessory that is fast and requires no solvents. The spectra of stools from 4000 to 750 cm-1 were analyzed, and the lipid concentrations were measured by using a calibration curve prepared by partial least-squares analysis of data from 34 stools. The linearity of the method was tested by mixing low-lipid stools with lipid-overloaded stools to give a range of 0.5-15% lipid. The prediction residual values were -0.49-0.78% for calibrators, and -2.55-2.34% for unknown samples. There was good agreement between the fecal lipids measured by gravimetric (x) and FTIR(y) methods: y = 0.87x + 5.5. The standard error of prediction was 1.07%.
Immunosensors: technology and opportunities in laboratory medicine
Authors: Morgan C L , Newman D J , Price C P
Published: January 2020

An immunosensor is a device comprising an antigen or antibody species coupled to a signal transducer, which detects the binding of the complementary species. An indirect immunosensor uses a separate labeled species that is detected after binding by, e.g., fluorescence or luminescence (i.e., a heterogeneous immunoassay). A direct device detects the binding by a change in potential difference, current, resistance, mass, heat, or optical properties (i.e., a homogeneous immunoassay). Although indirect sensors may encounter fewer problems due to nonspecific binding effects, the direct sensors are capable of real-time monitoring of the antigen-antibody reaction. A wide range of molecules can be detected with detection limits ranging between 10(-9) and 10(-13) mol/L. However, there are only a few successful commercial applications of direct immunosensors, these being of the optical type. This review describes the principles underlying the technologies, their merits, limitations, and applications.
Urine sulfatides and the diagnosis of metachromatic leukodystrophy
Authors: Natowicz M R , Prence E M , Chaturvedi P , Newburg D S
Published: January 2020

A deficiency of the lysosomal enzyme arylsulfatase A (ASA) causes the lysosomal storage disorder metachromatic leukodystrophy (MLD). The diagnosis of MLD is straightforward in cases with deficient leukocyte or fibroblast ASA activity and a typical clinical history. However, several atypical and late-onset forms of MLD have been described. The diagnosis is also complicated by the high frequency of presumably benign polymorphisms at the ASA gene locus that are associated with markedly diminished in vitro ASA activity. Additional diagnostic tools are needed in the clinically and (or) enzymatically atypical cases. Although analyses of urinary sulfatides have been reported to be helpful in the diagnosis of MLD, previously described methods are complex and incompletely characterized and validated. We developed an improved method for determining urinary sulfatides and applied it to a cohort of individuals with MLD. The sulfatides are extracted from urine, separated from glycerol-based lipids by alkaline hydrolysis, isolated by ion-exchange chromatography, and hydrolyzed to galactosylceramide, which is then perbenzoylated and quantified by HPLC. This assay provides excellent resolution of sulfatides from other lipids and good analytical precision. In addition, the urinary sulfatide concentrations of healthy controls (mean +offSD: 0.16 +/- 0.07 nmol/mg creatinine; range: 0.07-0.34; n = 18) are clearly distinguished from those of individuals with MLD (7.6 +/- 6.1 nmol/mg creatine; 1.2-24.2; n = 20).
Ammonium persulfate: a safe alternative oxidizing reagent for measuring urinary iodine
Authors: Pino S , Fang S L , Braverman L E
Published: January 2020

The chloric acid method is most commonly used to obtain accurate and reproducible measurements of iodine and remove interfering substances. Unfortunately chloric acid is a potential hazard, requiring an explosion-proof hood, among other precautions. We have developed a simple, convenient, and economic method for measuring urinary iodine by using 1 mol/L ammonium persulfate, a nonexplosive, nonhazardous chemical, as the oxidizing reagent. The oxidation procedure can be completed in 30 min at a temperature of 91-95 degrees C. The iodine in the urine is then measured by a modification of the traditional colorimetric method of Sandell and Kolthoff. Urine samples (110) collected from a mixed population of healthy males and females, ranging in age from 6 to 79 years and living in the US, were analyzed for urine iodine content by two methods: the proposed ammonium persulfate method and the chloric acid method. The ammonium persulfate method has an intraassay CV of 9.1% at 0.42 +/- 0.04 micromol/L (mean +/- SD), 7.8% at 1.46 +/- 0.11 micromol/L, and 4.0% at 3.54 +/- 0.14 micromol/L. The interassay CV is 10.2% at 0.46 +/- 0.05 micromol/L, and 7.9% at 3.27 +/- 0.26 micromol/L. Recovery of iodine added to urine in vitro was 107%, 94%, and 97% for 0.42 micromol/L, 0.77 micromol/L and 3.64 micromol/L, respectively. The lower limit of detectability was 0.0034 microgram of iodine. Values for iodine in 110 urines measured by the reference chloric acid method ranged from 0.06 to 8.03 micromol/L and by the ammonium persulfate method from 0.05 to 7.4 micromol/L. The persulfate method (y) correlated extremely closely with the reference chloric acid method (x) by the Pearson correlation (y = 0.923x + 0.810 micromol/L, and r = 0.994, Sy/x = 1.841).
Highly sensitive immunoradiometric assay for serum thyroglobulin with minimal interference from autoantibodies
Authors: Marquet P Y , Daver A , Sapin R , Bridgi B , Muratet J P , Hartmann D J , Paolucci F , Pau B
Published: January 2020

Five monoclonal antibodies (MAbs) directed against antigenic domains on thyroglobulin (Tg) not recognized by most anti-Tg human autoantibodies (aAbs) have been used to develop an improved IRMA for serum Tg with a limit of detection of 0.2 micrograms/L. Samples are incubated for 3 h in tubes coated with four anti-Tg MAbs. After washing, the tubes are incubated with the tracer MAb for 20 h at room temperature. Dilution and reproducibility tests demonstrated assay reliability. Tests performed on samples with (n = 361) or without (n = 283) aAbs showed that the TG IRMA Pasteur is largely independent of the marked interference generally caused by aAbs. These results were confirmed with an extended population of 2759 samples. For a cutoff of 1 micrograms/L, sensitivity and specificity were 0.97 and 1, respectively, in a follow-up of differentiated thyroid carcinoma in patients treated by total thyroidectomy.
DELISA: sensitive nonisotopic assay for GAD65 autoantibodies, a key risk-assessment marker for insulin-dependent diabetes mellitus
Authors: Mehta H B , Vold B S , Minkin S , Ullman E F
Published: January 2020

Nonisotopic assays for the measurement of autoantibodies to 65-kDa glutamic acid decarboxylase (GAD65) have not previously achieved performance equivalent to radiobinding assays (RBA). We have developed a modified ELISA protocol, DELISA, for measuring autoantibodies to GAD65 in serum. The method overcomes the problems of poor sensitivity and specificity associated with conventional ELISAs. Serum containing GAD65 autoantibodies is incubated with biotinylated GAD65 (bGAD65). Sufficient soluble Protein A-dextran conjugate is added to bind the immunoglobulins in the sample, including GAD65 autoantibodies to which GAD65 is bound. After incubation, the mixture is transferred to a streptavidin-p4ated microtiter well, which binds free bGAD65 but not bGAD65 bound to autoantibodies. Streptavidin-bound bGAD65 is detected by means of a peroxidase-GAS65MAb conjugate. The method appears to have comparable sensitivity and specificity to those of RBAs. Reaction of the antibodies with soluble antigen to increase the binding rate and the use of high serum concentrations and very low antigen concentrations to increase sensitivity are critical elements of the method.
Human placenta radioreceptor assay with digoxin and ouabain to detect endogenous digitalis-like factor(s) in human plasma and urine
Authors: Paci A , Ciarimboli G , Biver P
Published: January 2020

We describe the optimization and validation of a clinically feasible radioreceptor assay to detect endogenous digitalis-like factor(s) (EDLF) in human plasma and urine. The assay is based on the competitive replacement of 125I-labeled digoxin on human placenta membranes by ligands present in sample extracts. Digoxin and ouabain were used as calibrators. We also describe simple and effective methods for extraction and enrichment of EDLF from human plasma and urine. Assay sensitivity and precision were enhanced by using a sequential saturation technique with appropriate concentrations of tracer and receptors. Filtration was used to separate bound from free ligand. A two-step solid-state extraction with acetonitrile allowed the separation of two EDLFs with different polarity (EDLF-1 and EDLF-2) from the same plasma sample. A one-step solid-state extraction with methanol was suitable for urine. EDLF-1 and EDLF-2 in healthy adults were respectively 204 +/- 155 and 207 +/- 423 pmol/L ouabain equivalents, or 312 +/- 241 and 302 +/- 581 pmol/L digoxin equivalents. Plasma concentrations of EDLFs in newborns and pregnant women were higher than in healthy adults, and the concentrations in urine were higher than in plasma. Several cross-reactivity experiments showed that physiological concentrations of endogenous steroids and lipids did not inhibit binding, and supported the hypothesis that EDLFs are endogenous compounds other than the steroids and lipids also investigated.
Enzymatic measurement of creatine in erythrocytes
Authors: Beyer C , Alting I H
Published: January 2020

We describe an automated enzymatic procedure for quantifying creatine in erythrocytes. In this assay, after sample clean-up, creatine kinase (CK; EC 2.7.3.2) and pyruvate kinase (EC 2.1.7.40) are used as auxiliary enzymes; lactate dehydrogenase (EC 1.1.1.27) is used in the indicator reaction. CK is also used as the starting reagent. Data obtained with the present method (y) correlated as follows with those from the comparison method (x) for creatine measurement in erythrocytes (colorimetric, diacetyl-alpha-naphthol): y = 1.017x - 27.5 (S(y/x) = 11.9, r = 0.997). Precision data were obtained by analyzing three different samples of erythrocytes nine times per day for 25 days with both methods. The total CVs determined by our method and the comparison method were respectively 3.0-4.8% (measured creatine range, 171-463 micromol/L) and 5.1-7.1% (creatine, 185-485 micromol/L). We established the reference interval (mean +/- 2SD) for creatine in erythrocytes from healthy individuals as 194-538 micromol/L for men (n = 71) and 241-677 micromol/L for women (n = 100). Using erythrocytes from patients with various hematological abnormalities and chronic renal failure, who were being treated with continuous ambulatory peritoneal dialysis or hemodialysis, we performed a clinical evaluation and obtained results in agreement with existing data on biochemical markers for the mean age of erythrocytes.
Detection of prostate-specific antigen mRNA and protein in breast tumors
Authors: Zarghami N , Diamandis E P
Published: January 2020

We have developed reverse transcription-polymerase chain reaction (RT- PCR) methods for detecting prostate-specific antigen (PSA) mRNA. Using these methods, and a highly sensitive immunofluorometric assay for measuring PSA protein, we have assessed the concentrations of PSA mRNA and PSA protein in 30 primary breast tumors and a few other control tissues. We found good agreement between presence of PSA protein and PSA mRNA in breast tumors. We thus propose that, in women, detection of PSA protein or PSA mRNA in tissues and tumors offers equivalent information. Because PSA protein is present in male blood and thus could contaminate extracts from tumors and tissues from men, we propose that the RT-PCR methods we describe be used to assess nonprostatic expression of the PSA gene in men.
Matrix effects and the performance and selection of quality-control procedures to monitor PO2 measurements
Authors: Olafsdottir E , Westgard J O , Ehrmeyer S S , Fallon K D
Published: January 2020

We have assessed how variation in the matrix of control materials would affect error detection and false-rejection characteristics of quality-control (QC) procedures used to monitor PO2 in blood gas measurements. To determine the expected QC performance, we generated power curves for S(mat)/S(meas) ratios of 0.0-4.0. These curves were used to estimate the probabilities of rejecting analytical runs having medically important errors, calculated from the quality required by the CLIA '88 proficiency testing criterion and the precision and accuracy expected for a typical analytical system. When S(mat)/S(meas) ratios are low, the effects of matrix on QC performance are not serious, permitting selections of QC procedures based on simple power curves for a single component of variation. As S(mat)/S(meas) ratios increase, single-rule procedures generally show a loss in error detection, whereas multirule procedures, including the 3(1)s control rule, show an increase in false rejections. An optimized QC design is presented.
Clinical trials of a novel centrifugation method: axial separation
Authors: Estey C A , Felder R A
Published: January 2020

Specimen centrifugation is one of the most time-consuming tasks associated with preanalytical specimen processing. We report the first clinical use and impact study of the novel centrifugation method, axial separation, using the Axial Separation Module (ASM). The ASM centrifuges medical specimens one at a time (in 1 min 9 s) in proprietary Axial Process Containers (APC). Here we report tests of the ASM in an outpatient clinical laboratory. Use of the ASM in the outpatient clinic laboratory led to an average total improvement (decrease in total bench time) of 8 min 9 s per specimen compared with conventional centrifugation. Laboratory analysis revealed no statistical difference between plasma obtained from the APC vs that from a Becton Dickinson Vacutainer Tube. We conclude the ASM offers a cost-effective alternative to conventional centrifugation. Further, the use of axial separation may allow specimen separation to become an integral part of the phlebotomy process.
Homogeneous assay for direct determination of high-density lipoprotein cholesterol evaluated
Authors: Nauck M , März W , Haas B , Wieland H
Published: January 2020

We evaluated a new homogeneous assay for quantifying high-density lipoprotein cholesterol (HDL-C). The assay included four reagents: polyethylene glycol for "wrapping" chylomicrons, very-low-density lipoproteins (VLDL), and low-density lipoproteins (LDL); antibodies specific for apolipoprotein (apo) B and apo C-III to produce aggregates of chylomicrons, VLDL, and LDL; enzymes for the enzymatic cholesterol determination of the noncomplexed lipoproteins with 4-aminoantipyrine as the color reagent; and guanidine salt to stop the enzymatic reaction and to solubilize the complexes of apo B-containing lipoproteins, which would otherwise interfere with the reading of absorbance. The total CVs of the new method ranged between 2.4% and 8.4%. The HDL-C values (y) were in good agreement with those by a comparison phosphotungstic acid/MgCl2 method (x): y= 0.987x + 17.2 mg/L (68th percentile of the residuals on the regression line= 21.49, r= 0.970). At triglyceride concentrations of 20 g/L (Intralipid) the homogeneous HDL-C concentrations increased by 2%. Hemoglobin markedly increased the results, whereas bilirubin reduced them. The homogeneous HDL-C assay was easy to handle and allows full automation. This test should considerably facilitate the screening of individuals at an increased risk of cardiovascular disease.
Interference in glucose and other clinical chemistry assays by thiocyanate and cyanide in a patient treated with nitroprusside
Authors: Randell E W , St Louis P
Published: January 2020

A 3-year-old patient treated with nitroprusside for congestive heart failure had 6.5 mmol/L thiocyanate (toxic, >1.5 mmol/L) and 110 micromol/L cyanide (toxic, >5 micromol/L) present in her blood. At this time a whole-blood glucose concentration assayed on the Nova Stat Profile 5 Plus (Stat Profile) was 25.1 mmol/L. Plasma from that specimen analyzed on a Kodak Ektachem 700 analyzer (E700) indicated 5.2 mmol/L glucose. We investigated the potential interference of dissolved thiocyanate or cyanide on glucose and other routine assays. Toxic concentrations of thiocyanate increased Stat Profile glucose values and E700 total calcium, chloride, and creatinine values. Stat Profile ionized calcium values were decreased by toxic concentrations of thiocyanate. Cyanide (100 micromol/L) decreased alanine aminotransferase activity measured on the E700. Interference with the Stat Profile glucose assay may have been caused by thiocyanate oxidation at the glucose electrode.
Reference intervals for plasma apolipoprotein B determined with a standardized commercial immunoturbidimetric assay: results from the Framingham Offspring Study
Authors: Contois J H , McNamara J R , Lammi-Keefe C J , Wilson P W , Massov T , Schaefer E J
Published: January 2020

We evaluated a commercially available, standardized immunoturbidimetric assay for apolipoprotein (apo) B, the protein constituent of low-density lipoprotein (LDL), to establish reference ranges for men and women, and to determine the concentrations associated with increased risk of coronary heart disease (CHD). The between-run CV for assay of a normal-concentration control for this assay was 6.60%. The mean (+/-SD) apo B concentration was 1.03 +/- 0.24 g/L in 1880 men, significantly higher than the mean for 1944 women (0.96 +/- 0.26 g/L) participating in cycle 4 of the Framingham Offspring Study (P<0.001). An apo B value of 1.20 g/L corresponded roughly to the 75th percentile in men, similar to an LDL cholesterol concentration of 1.60 g/L, and subjects with concentrations greater than this were significantly more likely to have CHD than subjects with apo B concentrations less than 1.00 g/L, the approximate 50th percentile (P<0.05 in men and P<0.001 in women).
Four frequently used test systems for serum cholesterol evaluated by isotope dilution gas chromatography-mass spectrometry candidate reference method
Authors: Thienpont L M , Van Landuyt K G , Stöckl D , De Leenheer A P
Published: January 2020

We evaluated the performance of four frequently used cholesterol test systems, using split-sample measurements with a panel of 79 patients' specimens and isotope dilution gas chromatography-mass spectrometry (ID GS-MS) as a comparison method. The test systems were from Beckman, a Boehringer Mannheim, Merck, and Johnson & Johnson Clinical Diagnostics, performed on the Synchron CX7, Hitachi 717, Mega, and Ektachem 250 analyzers, respectively. The liner regression data for the method comparison [ID GS-MS as independent variable (x)] were for Beckman: slope = 1.012, intercept = 0.0243 mmol/L, dispersion (S(y/x)) = 0.1303 mmol/l, and correlation coefficient (r) = 0.9867; for Boehringer Mannheim: slope = 1.002, intercept = 0.114 mmol/L, Sy/x = 0.0759 mmol/L, r = 0.9954; for Merck: slope = 1.034, intercept = -0.0613 mmol/L, Sy/x = 0.0886 mmol/L, r = 0.9941; and for Johnson & Johnson Clinical Diagnostics: slope = 1.007, intercept = 0.01 mmol/L, Sy/x = 0.15 mmol/L, and r = 0.9811. These data demonstrate excellent state-of-the-art cholesterol measurement for some of the most widely used test systems.
Nephelometric determination of carbohydrate deficient transferrin
Authors: Schellenberg F , Martin M , Cacès E , Bénard J Y , Weill J
Published: January 2020

We describe a technique for measuring carbohydrate-deficient transferrin (CDT) in serum. Serum transferrin fractions are separated by anion-exchange chromatography on microcolumns. Sialic acid-deficient transferrin fractions are collected in the eluate, and transferrin is then quantified by a rate-nephelometric technique. Imprecision (CV) was 4-5% within-run and 7-9% between runs (n = 15). Comparison with an isoelectric focusing-immunofixation method for transferrin index (x) yielded y = 761x + 7, Sy/x = 39 mg/L. Assay of sera from 90 abstainers or moderate consumers of alcohol showed that 81 (90%) had CDT concentrations between 30 and 70 mg/L. Among 74 alcoholics admitted to an alcohol treatment center, 54 (73%) had CDT > 70 mg/L, i.e., the diagnostic sensitivity was 73% at a specificity of 90% (area under receiver-operator characteristic curve = 0.891).
Quantification of circulating 1,25-dihydroxyvitamin D by radioimmunoassay with 125I-labeled tracer
Authors: Hollis B W , Kamerud J Q , Kurkowski A , Beaulieu J , Napoli J L
Published: January 2020

We report here the first RIA for 1,25-dihydroxyvitamin D utilizing a radioiodinated (125I) tracer. This is also the first validated RIA for 1,25-dihydroxyvitamin D [1,25(OH)2D] that does not require sample prepurification by HPLC before the binding assay. The assay involves acetonitrile extraction, treatment of the crude extract supernate with sodium periodate, extraction and purification of endogenous 1,25(OH)2D by solid-phase chromatography, and finally, quantification by RIA. Calibrators were prepared in stripped human serum and processed exactly the same as samples, eliminating the need for internal control for procedural losses of endogenous 1,25(OH)2D. The assay consists of a 2-h room temperature incubation with the primary antibody, a 20-min incubation with a second antibody, and separation of bound from free by centrifugation. Assay results can be in hand with 5 h. The detection limit of the assay is 2.4 ng/L 1,25-dihydroxyvitamin D3. Results compare well with those from an accepted radioreceptor assay. Sample pretreatment with sodium periodate is absolutely essential before quantification by RIA; otherwise, concentrations of endogenous 1,25(OH)2D may be greatly overestimated.
Influence of specimen carryover on sensitive thyrotropin (TSH) assays: is there a problem?
Authors: Sadler W A , Murray L M , Turner J G
Published: January 2020

A relatively slow transition has occurred from so-called 1st-generation thyrotropin (TSH) assays (e.g., RIAs) through 2nd-generation assays (e.g., IRMAs) to 3rd-generation assays (e.g., immunochemiluminometric assays). Analysis of data from a modified internal quality-control design, followed up by a computer simulation, showed that specimen carryover has minimal effect on 2nd-generation TSH assays. However, extension of the simulation to a 3rd-generation assay showed the possibility of substantial effects in the subnormal region. Carryover of 1:1250 (0.08%), for example, may reduce the theoretical 10-fold precision improvement claimed for 3rd-generation assays to nearer fourfold. Simulation results suggest maximum allowable specimen carryover of approximately 1:10,000 (approximately 0.01%) for 3rd-generation TSH assays. We suggest that when automated specimen handling is used in a TSH assay, a well-designed carryover experiment should become a routine part of reports that claim 3rd-generation (or better) performance characteristics.
Denaturing gradient gel electrophoresis to diagnose multiple endocrine neoplasia type 2
Authors: Blank R D , Sklar C A , Martin M L
Published: January 2020

Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant cancer syndrome caused by mutations in the RET protooncogene. Others have already demonstrated the value of genetic testing in known MEN 2 kindreds. Previously described approaches to DNA-level diagnosis, particularly of index cases, are tedious. We developed appropriate denaturing gradient gel electrophoresis (DGGE) conditions for analysis of exons 10, 11, and 16 of this gene, where many of the pathogenic mutations map. We screened 16 members of a three-generation MEN 2 kindred by DGGE and found five affected but still asymptomatic patients, ranging in age from 5 to 67 years. We used DGGE to localize the pathogenic mutations and screen at-risk individuals in several other kindreds. DGGE--which requires no radioactive, fluorescent, or chemiluminescent labeling--is ideally suited to the diagnosis of MEN 2 because of the syndrome's dominant genetics and the rarity of clinically silent variants in the RET gene.
Use of neural networks to diagnose acute myocardial infarction. I. Methodology
Authors: Jørgensen J S , Pedersen J B , Pedersen S M
Published: January 2020

We investigated several aspects of using neural networks as a diagnostic tool: the design of an optimal network, the amount of patients' data needed to train the network, the question of training the network optimally while avoiding overfitting, and the influence of redundant variables. The specific clinical problem chosen for illustration was the diagnosis of acute myocardial infarction, given only the electrocardiogram and the concentration of potassium in serum at the time of admission. We found that, in contrast to usual practice, the termination of the training process should be based on the generalization performance and not on the training performance. We also found that a principal component analysis can be used to eliminate redundant variables, thereby reducing the data space. The diagnostic performance of the neural network we used was 78%--superior to that of linear discriminant function analysis but similar to that of quadratic discriminant function analysis.
Laboratory testing for recent alcohol consumption: comparison of ethanol, methanol, and 5-hydroxytryptophol
Authors: Helander A , Beck O , Jones A W
Published: January 2020

The ratio of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid (5HTOL/5HIAA) in urine was compared with concentrations of ethanol and methanol as a way to monitor recent alcohol consumption. During detoxification of alcohol-dependent subjects, ethanol persisted longer in urine than in breath or plasma. Blood and urinary methanol remained increased for 2-6 h after blood ethanol had returned to background concentrations, whereas 5HTOL/5HIAA remained increased for 6-15 h. In healthy volunteers who had ingested alcohol (range 3-98 g) the previous afternoon or evening, 87% (for men) and 59% (for women) of all drinking occasions exceeding 7 g of alcohol were identified by an increased 5HTOL/5HIAA in the first morning urine void. This compared with 32% and 12%, respectively, identified by analysis of ethanol (>200 micromol/L). No gender difference in the excretion pattern of 5HTOL/5HIAA was seen. The results demonstrate that 5HTOL/5HIAA provides a specific and more sensitive method to detect recent alcohol consumption than does ethanol or methanol.
Circulating total and ionized magnesium after ethanol ingestion
Authors: Wu C , Kenny M A
Published: January 2020

Hypomagnesemia is associated with alcoholism (Lim P, Jacob E. Metabolism 1972;21:1045-51). Here we assess two measurements of blood magnesium in emergency care patients with confirmed ethanol ingestion. Serum total and ionized magnesium (tMg, iMg) were measured in 88 patients with ethanol concentrations of 6-128 mmol/L and in sera of 97 hospitalized patients (control group). iMg was measured by an ion-selective electrode method; tMg was measured spectrophotometrically. iMg was significantly lower for the ethanol-containing specimens (0.35 +/- 0.12 mmol/L, mean +/- SD) than for the control group (0.46 +/- 0.15 mmol/L), with P <0.0001. The tMg for the test group (0.87 +/- 0.20 mmol/L) was not significantly different from the controls (0.88 +/- 0.33 mmol/L), with P = 0.5987. tMg was not well-correlated with iMg in the ethanol-positive specimens. Most ethanol-positive patients had abnormally low serum iMg (87 of 88 had iMg <0.53 mmol/L).
Automated HPLC screening of newborns for sickle cell anemia and other hemoglobinopathies
Authors: Eastman J W , Wong R , Liao C L , Morales D R
Published: January 2020

Automated HPLC is used to test dried blood-spot specimens from newborns for hemoglobins (Hb) F, A, S, C, E, and D. We present the method and report on its performance determined during >4 years of testing 2.5 x 10(6) newborns. The method features automated derivation of presumptive phenotypes; quantitative quality control and proficiency testing; throughput of one specimen per minute; small sample volume; hemoglobin concentrations quantified with an interlaboratory CV of 14-18%; retention times with interlaboratory CV of <2% and matching, within +/- 0.03 min, of laboratories and reagent lots; control of peak resolution; 0.5% detection limit for Hb S and C, and 1.0% for Hb F, A, E, and D; few interferences; and negligible background and carryover. Shortcomings of the method are the absence of microplate barcode identification and the need for manually pipetting the sample eluate into the microplate.
Laboratory tests of iron status: correlation or common sense?
Authors: Hastka J , Lasserre J J , Schwarzbeck A , Reiter A , Hehlmann R
Published: January 2020

We demonstrate that simple correlation between the various tests of iron status is not sufficient for examining their value in diagnosing iron deficiency (ID). Three degrees of ID are recognized: Iron depletion (ID grade I) is defined by decreased total body iron and normal iron support to erythropoiesis, as diagnosed by decreased storage iron, decreased ferritin, normal sideroblast count, normal zinc protoporphyrin (ZPP), and transferrin saturation >15%. When the iron supply to erythropoiesis becomes insufficient, as diagnosed by transferrin saturation < or = 15%, increased ZPP, and decreased sideroblast count, iron-deficient erythropoiesis (ID grade II) occurs. When finally hemoglobin is below its normal range, iron-deficiency anemia (ID grade III) results. The various tests for ID cannot be compared without taking into account the severity of the deficiency. Depending on the grade of ID examined, the correlation of markers seen in our patients' data varied considerably. We conclude that a "best" marker of ID does not exist. However, the different tests efficiently complement each other by detecting different stages and individually show the clinical extent of ID. Ferritin reflects the iron stores. ZPP indicates whether the ID in a given patient is clinically relevant or not. Finally, the extent of a clinically relevant ID can be assessed by the measured ZPP, hemoglobin concentration, and red cell indices.
HPLC method for measurement of purine nucleotide degradation products in cerebrospinal fluid
Authors: Kuracka L , Kalnovicová T , Líska B , Turcáni P
Published: January 2020

We describe a convenient method for the separation and quantification of xanthine, hypoxanthine, and uric acid in 20 microL of cerebrospinal fluid (CSF) with use of HPLC and ultraviolet detection. The analysis is performed on a Sepharon SGX C18 column and the elution system consists of potassium phosphate buffer, pH 5.1, with 20 mL/L methanol. The lower limit of detection was 4 pmol for hypoxanthine and xanthine and 6 pmol for uric acid. Analytical recoveries of purine metabolites ranged from 98.6% to 102.9%. The intra- and interassay CVs were <3%. The applicability of the method is illustrated with the determination of micromolar concentrations of xanthine, hypoxanthine, and uric acid in CSF samples obtained from 113 patients with various neurological disorders.
HPLC micromethod for amrinone and metabolites in patients receiving concurrent cephalosporin therapy
Authors: Pappas J B , Allen E M , Ross M , Banner W
Published: January 2020

Amrinone (AMR), a bipyridine derivative, is receiving increasing use in postoperative cardiac patients as an inotrope and vasodilator. The hemodynamic response to amrinone in adults is linearly related to AMR concentrations, warranting therapeutic drug monitoring. We report a rapid microsample HPLC method for monitoring AMR and its principal metabolites, N-acetyl (N-ac) and N-glycolyl (N-gly) AMR. Serum was precipitated with acetonitrile, and the supernatant fluid was then injected into a C18 narrow-bore column. The mobile phase consisted of a 0.1 mol/L sodium phosphate buffer (pH 6) with a gradient of acetonitrile going from 50 to 100 mL/L of eluent. Detection with a diode-array detector (DAD) concurrently monitored the absorbances at 320 and 345 nm. Monitoring 320 nm allows optimal quantification of AMR, N-gly, and N-ac. Patients often receive concurrent cephalosporin therapy, which is detectable at 320 nm but not 345 nm. Because cephalosporins coelute with AMR or metabolites, monitoring at 345 nm allows separation of these antibiotics from AMR and metabolites while retaining a detection limit of 0.5 mg/L.