bluuebunny/crossref_metadata_2025 huggingface dataset:
Preliminary Treatment of Urinary Proteins Improves Electrophoretic Analysis and Immunodetection
Authors: Verdier J M , Dussol B , Dupuy P , Berland Y , Dagorn J C
Published: January 2020

Analysis of urinary protein composition is an important tool in studies on renal physiology and physiopathology. Urine is, however, a complex mixture containing, besides protein, a variety of compounds such as salts, peptides, oligosaccharides, and glycosaminoglycans. Some of these compounds interfere with the electrophoretic migration of protein in sodium dodecyl sulfate-polyacrylamide gels and prevent correct analysis of the protein pattern. We describe a simple method for extracting urinary proteins that considerably improves their electrophoretic migration and subsequent immunodetection. This treatment involves ammonium sulfate fractionations (for precipitating proteins), EDTA (for inhibiting protein aggregation), and HCl hydrolysis (for removing glycosylaminoglycans). Recovery during extraction was found to be almost quantitative for total protein and three representative proteins: albumin, alpha 1-glycoprotein acid, and beta 2-microglobulin.
Intralaboratory Performance Requirements Necessary to Pass Proficiency Testing: CAP-1990 vs CLIA-1967 (March 14, 1990) Formats Compared
Authors: Laessig R H , Ehrmeyer S S , Leinweber J E
Published: January 2020

The pre-1990 College of American Pathologists' (CAP) Proficiency Testing (PT) program used a two samples per analyte/four challenges per year format with performance or pass-fail grading criteria determined by the program. On Jan. 1, 1991, the Clinical Laboratory Improvement Act of 1967 (CLIA-67) final rules (March 14, 1990) mandated a revised PT format of five samples per analyte/four challenges per year, with the regulations specifying minimum performance criteria. Extending our previous analysis, we compare the maximum permissible intralaboratory imprecision at low bias compatible with passing external PT in the former CAP and current CLIA-67 formats. If a laboratory is able to reduce its internal coefficient of variation (CV) to less than 44% of the PT criterion for each analyte, its overall chance of adverse action for any of the 27 routine chemistry analytes specified in CLIA-67 will be less than 1% in a two-year (eight PT challenges or events) period. Consideration of actual interlaboratory CVs from CAP surveys suggest that a reduction of this magnitude may be difficult for the analytes total cholesterol and blood urea nitrogen, where intralaboratory imprecision comparable with the group standard deviation (SD) from 1990 CAP surveys would yield individual adverse action (PT failure) rates of 5% and 1%, respectively. Five other analytes have CLIA-67 performance limits dangerously close to CAP interlaboratory CVs.
Failure of Common Glycation Assays to Detect Glycation by Fructose
Authors: Ahmed N , Furth A J
Published: January 2020

Serum albumin was modified by in vitro glycation with either fructose or glucose, to see whether the common clinical assays for glycation were able to detect both fructose- and glucose-induced changes in protein structure in diabetes. Although fluorescence measurements showed that fructose causes far more protein damage than glucose, neither serum fructosamine (SFA) nor phenylboronate affinity (PBA) glycation assays reflected these changes. The SFA method implied that fructose causes only about 5% of the glycation induced by glucose; with PBA the proportion was 25%. The thiobarbituric acid- and periodate-based assays also greatly underestimated the true extent of fructation. We discuss these discrepancies with respect to the underlying chemistry, emphasizing the difference between aldehydic and ketonic Amadori products (exemplified by fructose and glucose derivatives, respectively). The implications for detecting fructose-induced secondary diabetic complications are also discussed.
Effects of Serum Bilirubin on Determination of Uric Acid by the Uricase-Peroxidase Coupled Reaction
Authors: Aoki Y , Ihara H , Nakamura H , Aoki T , Yoshida M
Published: January 2020

We examined the possible effect of different bilirubin species, including unconjugated (Bu), sugar-conjugated (Bc), and authentic delta bilirubin (Bd) isolated from serum, on the direct enzymatic measurement of uric acid by using the uricase (EC 1.7.3.3) and peroxidase (EC 1.11.1.7) coupled reaction. Bc, isolated from human bile, produced the most interference with uric acid determination. Although the presence of human serum albumin reduced interference by Bu and Bc, albumin-bound Bc complex still produced greater interference than Bu. Interference with the peroxidase reaction by Bc covalently bound to serum albumin (Bd) was less than that by Bu. We examined this phenomenon by using serum-isolated Bd obtained by ammonium sulfate precipitation and ultrafiltration and by using commercially available ditaurobilirubin (DTB) and chemically synthesized Bd (via Woodward's reagent, WBd) as surrogates for bile-isolated Bc and natural Bd, respectively. DTB produced the same amount of interference as natural Bc, but the interference produced by DTB in the presence of serum albumin was not the same as that produced by natural Bc with albumin. Our synthetic Bd appears to be a reliable surrogate for natural Bd in testing for bilirubin interference.
Multiple Occurrence of Macro Creatine Kinase in One Family
Authors: Bayer P M , Kraus S
Published: January 2020

We measured creatine kinase (CK, EC 2.7.3.2), CK-MB isoenzyme activity and mass concentration, and distribution of CK isoenzymes (by electrophoresis) in serum from five members of one family. The mother and two young children showed CK-IgG complexes in their sera. The concentration of the CK-IgG complexes in the children decreased over time, suggesting that the complex involved maternal IgG and had been transferred across the placenta from the mother to her children.
Robotic Automation of Cyclosporine Analysis in Whole Blood
Authors: Holman J W , Felder R A
Published: January 2020

Cyclosporin A (CsA) is currently the most selective immunosuppressant used clinically to prevent organ rejection after transplantation. We used a reprogrammable robot, the Benchmate (Zymark Inc., Hopkinton, MA), to automate much of the sample preparation involved in solid-phase extraction of CsA. Lysed whole-blood specimens containing previously added internal standard were placed on the Benchmate in the specimen-holding area, and a C18 Bond-Elut column was placed in the top of the sample tube. Specimen extraction from this point was handled automatically by the Benchmate, after which we manually injected the sample into the HPLC system for quantification. Analytical recovery of CsA in two concentrations of calibrator was similar for the manual and Benchmate methods. Analytical imprecision for the Benchmate was less than for manual extraction: within-run imprecision (CV) was less than 8% at either concentration. Between-run imprecision, determined by having the Benchmate extract CsA from two specimens each day for 20 days, was less than 9%. Patients' specimens were extracted manually (x) or by using the Benchmate robot (y); the results, compared by linear regression, agreed well: (y = 0.99 (SD 0.02)x + 2.6 (SD 2.8) micrograms/L (Sy[x = 7.59 micrograms/L, n = 27). We conclude that the Benchmate usefully reduces the manual steps necessary to extract specimens before HPLC analysis for CsA.
Characterization of Hemoglobin Lepore Variants by Advanced Mass-Spectrometric Procedures
Authors: De Caterina M , Esposito P , Grimaldi E , Di Maro G , Scopacasa F , Ferranti P , Parlapiano A , Malorni A , Pucci P , Marino G
Published: January 2020

We describe an analytical protocol for characterizing the molecular structure of hemoglobin (Hb) Lepore variants by using two different mass-spectrometric approaches. The first method consists of direct examination of the chromatographically separated hybrid globins by electro-spray mass spectrometry; the variant Lepore globin is identified through the accurate determination of its molecular mass. Alternatively, the anomalous globins are digested with trypsin and their structures are determined by fast atom bombardment mass-spectrometric analysis of the peptide mixture. The application of this procedure to the identification of Hb Lepore Boston and Hb Lepore Baltimore is described.
Electrophoretic Fractionation of Pancreatic Lipase
Authors: Panteghini M
Published: January 2020

Human pancreatic isolipases were separated by electrophoresis on cellulose acetate membrane. Band visualization by fluorescence detection of NADH produced by beta-oxidation cyclic reaction of fatty acid liberated from 1,2-dilinoleoylglycerol by lipase was achieved by making a two-plate "sandwich" of an electrophoresis plate and a substrate-bearing plate. Two true lipase forms, named L1 and L2 to reflect their electrophoretic migration, were resolved with this method and quantified by densitometry. The procedure was not only simple and rapid but also precise (between-run CV less than 13.5%) and sensitive (detected lipase fractions of less than 2 U/L). L1 (mean 57% of total lipase, SD 19.3%) and L2 (mean 43%, SD 19.3%) lipase forms were found in all normal persons studied. In a preliminary clinical study of 27 emergency room patients, evaluation of the lipase electrophoretic pattern in serum did not separate patients with acute pancreatitis from those with other acute abdominal disorders.
Direct Double Monoclonal Immunoradiometric Assay of Urinary Human Growth Hormone
Authors: Porquet D , Rigal O , Brion D E , Valade F , Leger J , Czernichow P
Published: January 2020

Several reports indicate that urinary growth hormone (GH) excretion might reflect central release of the hormone, and that measurement of urinary GH shows promise in the investigation of physiological and pathological GH secretion. We have developed and evaluated a direct immunoradiometric assay (IRMA) in which two monoclonal antibodies are used to measure GH in the urine of children. The detection limit is approximately 0.018 pmol/L for a sample volume of 2 mL. Within- and between-run variations (CVs) were 5.6% and 14.2%, respectively. Analytical recovery and dilution experiments showed the specificity of the method for GH. In normal-stature prepubertal children ages 3-12 years, 24-h urinary GH excretion was 0.189 (SD 0.100) pmol and correlated well with the amount of GH in the first morning miction, which showed wide day-to-day variations. Like others, we found a strong correlation between GH concentrations in serum and urine during stimulation tests with GH-releasing hormone (somatoliberin) and (or) during physiological nocturnal secretion, confirming that urinary GH measurement may be of help in investigating patients (particularly young children) with diseases in which GH secretion is impaired.
Sensitive and Selective Liquid-Chromatographic Assay of Fluoxetine and Norfluoxetine in Plasma with Fluorescence Detection After Precolumn Derivatization
Authors: Suckow R F , Zhang M F , Cooper T B
Published: January 2020

We determined fluoxetine (Prozac) and its major metabolite norfluoxetine in plasma by liquid chromatography with fluorescence detection. After liquid-liquid extraction from 1 mL of plasma, the extract was derivatized at room temperature with dansyl chloride, and the highly fluorescent derivatives were chromatographed with a reversed-phase C18 column and a mobile phase of phosphate buffer and acetonitrile. Dansylated fluoxetine, norfluoxetine, and the internal standard were eluted in less than 14 min with no interference from endogenous material. The calibration curve was linear over the concentration range 25-800 micrograms/L with inter- and intra-assay imprecision (CV) of less than 10%. Validity of the assay was checked by comparing results for 110 patients' samples with those by a liquid-chromatographic method with ultraviolet detection (r = 0.993 for fluoxetine, 0.957 for norfluoxetine). The identity of the dansylated derivatives was verified by positive chemical ionization mass spectroscopy. The lower limit of detection was approximately 3 micrograms/L. Because no major antidepressant, neuroleptic, or respective drug metabolites interfere with the quantification of fluoxetine and norfluoxetine, this is a useful procedure for pharmacokinetic studies and in clinical settings.
Presence of Angiotensin Peptides in Human Urine
Authors: Hermann K , Rittweger R , Phillips M I , Ring J
Published: January 2020

Immunoreactive angiotensin I and angiotensin II were found in human urine that was purified on octadecasilylsilica cartridges. The daily excretion of angiotensin I and II in healthy volunteers was 189.00 (SE 38.36) and 17.54 (SE 3.07) pmol/24 h or 148.09 (SE 32.22) and 12.82 (SE 2.34) pmol/L, respectively (n = 12). No circadian rhythm was observed in the excretion patterns of angiotensin I and II. In vitro degradation of angiotensin I or II could not be detected in acidified urine samples. A marked increase in the excretion of angiotensin I and II could be demonstrated in patients with anaphylactoid reactions to drugs and food additives after oral challenge. Immunoreactive angiotensin I and II could be characterized by HPLC as Ile5-angiotensin I, Ile5-angiotensin II, and angiotensin II metabolites.
Detection of Testosterone Administration by Increased Ratio Between Serum Concentrations of Testosterone and 17 Alpha-Hydroxyprogesterone
Authors: Carlström K , Palonek E , Garle M , Oftebro H , Stanghelle J , Björkhem I
Published: January 2020

An increased ratio between urinary testosterone (T) and epitestosterone (epiT) has been accepted by the International Olympic Committee as a marker for T doping. However, in a few subjects, we and others have observed constantly above-normal urinary T/epiT ratios that are unlikely to be related to exogenous T administration. To find a better test for T doping, we studied several serum and urinary androgens and androgen precursors, estrogens, and luteinizing hormone (LH) in seven healthy volunteers for 35 days after an intramuscular injection of 250 mg of testosterone enanthate. Among urinary analyses, only the T/epiT ratio was a suitable marker of T doping; of the serum assays, 17 alpha-hydroxyprogesterone (17OHP), T/17OHP ratio, LH, and T/LH ratio were fair to good markers of T doping. The serum T/17OHP ratio was the best marker of those tested, with all seven subjects having above-normal values for this in the first 3 days of the observation period. No other marker showed abnormal values in all subjects at any time. Moreover, the T/17OHP ratio was affected by neither diurnal variation nor physical stress. The value of this marker for T doping was further supported by the finding of normal T/17OHP ratios in a subject with increased urinary T/epiT ratios caused by an abnormally low testicular epiT production, probably related to genetic factors.
Simple Method for Quantifying Alpha-Tocopherol in Low-Density+Very-Low-Density Lipoproteins and in High-Density Lipoproteins
Authors: Carcelain G , David F , Lepage S , Bonnefont-Rousselot D , Delattre J , Legrand A , Peynet J , Troupel S
Published: January 2020

We assessed the distribution of alpha-tocopherol in serum lipoprotein samples after separating the lipoprotein fractions by either sequential ultracentrifugation or selective precipitation with sodium phosphotungstate-magnesium chloride reagent. alpha-Tocopherol concentrations were determined by reversed-phase high-performance liquid chromatography. After ultracentrifugation, we found that in men, low- and very-low-density serum lipoproteins (LDL-VLDL) contained 53.6% of alpha-tocopherol vs 46.4% in high-density lipoproteins (HDL). In women, serum LDL-VLDL contained 45.6% alpha-tocopherol after ultracentrifugation vs 54.4% in HDL. After selective precipitation, the proportions of alpha-tocopherol in men were 56.1% in LDL-VLDL vs 43.9% in HDL, and in women, 45.4% in LDL-VLDL vs 54.6% in HDL. After selective precipitation, alpha-tocopherol recovery from whole lipoprotein fractions was 97% to 100% vs 80% after ultracentrifugation, thus allowing more accurate alpha-tocopherol quantification than after separation by ultracentrifugation.
Detection of Antithyroglobulin Autoantibodies with Defined Epitopic Specificity by a Microparticle-Enhanced Nephelometric Immunoassay
Authors: Harchali A A , Montagne P , Cuillière M L , Bouanani M , Pau B , Duheille J
Published: January 2020

To hydrophilic, polyfunctional spherical microparticles of predetermined diameter, produced by copolymerization of acrylic monomers, we covalently bound human thyroglobulin. The thyroglobulin-microsphere conjugate was agglutinated, in the presence of antimouse immunoglobulins antiserum, by four monoclonal antibodies, each recognizing a different antigenic domain on the thyroglobulin molecule. These agglutinations were quantified by measuring with a specially designed nephelometer the light scattered by clusters of the conjugates. Agglutination with the monoclonal antibody recognizing antigenic domain II of the thyroglobulin molecule was specifically inhibited by some human sera that contained antithyroglobulin autoantibodies. This allowed us to develop a microparticle-enhanced nephelometric immunoassay for these autoantibodies with defined epitopic specificity. Using this assay, we detected and quantified antithyroglobulin autoantibodies in serum samples from all eight patients examined with Hashimoto disease and from most (75%) patients with untreated Graves disease.
Quantification of beta 2-microglobulin and albumin in plasma and peritoneal dialysis fluid by a noncompetitive immunoenzymometric assay
Authors: Kandoussi A , Cachera C , Pagniez D , Saile R , Tacquet A
Published: January 2020

An enzyme-linked immunosorbent assay (ELISA) was developed for measuring beta 2-microglobulin (beta 2m) and albumin in continuous ambulatory peritoneal dialysis (CAPD) fluid. Plasma concentrations of beta 2m were twofold greater in hemodialysis patients (41.3 +/- 13.3 mg/L) than in CAPD patients (23.6 +/- 5.5 mg/L) matched for duration of treatment. Measurement of beta 2m in CAPD fluid showed a substantial loss of this protein, approximately 31% of total body beta 2m, compared with a 5% loss of a protein of middle molecular mass (albumin). Because of the molecular sieving effects of the peritoneal membrane, peritoneal clearance of beta 2m was sixfold greater than that of albumin. Whether beta 2m losses prevent or delay the incidence of dialysis-induced amyloidosis in these patients remains to be established.
Competitive assay to improve the specificity of detection of single-point mutations in alpha 1-antitrypsin deficiency
Authors: Gunneberg A , Scobie G , Hayes K , Kalsheker N
Published: January 2020

Allele-specific oligonucleotides are used widely for the detection of single point mutations in genes. A modification of this assay based on competition has been developed for detection of the Z mutation of alpha 1-antitrypsin (alpha 1-AT). The normal alpha 1-AT allele is referred to as M, and the Z mutation arises from a single base substitution. Amplified DNA products corresponding to homozygous M, heterozygous MZ, and homozygous Z obtained by the polymerase chain reaction were incubated with a twofold molar excess of unlabeled oligonucleotide prior to hybridization with a radiolabeled oligonucleotide. Thus, initial incubation with unlabeled M-specific oligonucleotide was followed by hybridization with radiolabeled Z-specific oligonucleotide, and vice versa. This assay increased the specificity of single-point mutation detection three- to four-fold. Furthermore, specific hybridization was obtained at a lower temperature as a consequence of improving the signal-to-noise ratio.
Simultaneous determination of vitamins A and E and carotenoids in plasma by reversed-phase HPLC in elderly and younger subjects
Authors: Zaman Z , Fielden P , Frost P G
Published: January 2020

A reversed-phase high-performance liquid-chromatographic method for the simultaneous determination of retinol, alpha-tocopherol, alpha-carotene, beta-carotene, cryptoxanthin, lutein/zeaxanthin, and lycopene is described. This method was applied to plasma measurements in healthy young and elderly subjects. The plasma, deproteinized with ethanol, is extracted twice with n-hexane. After evaporation, the residue is dissolved in 50 microL of tetrahydrofuran and made up to 200 microL with ethanol. Samples (50 microL) are injected onto a 250 x 4.6 mm column of 5-microns-particle Spherisorb ODS1 (Phase Separations) that had been equilibrated with solvent mixture A:B (90:10 by vol) [A = 100 mmol/L ammonium acetate in methanol: acetonitrile (80:20 by vol) and B = 100 mmol/L ammonium acetate in water] at 2 mL/min. The analytes are eluted by running a 12-min linear gradient to 100% A; solvent A is then maintained for 10 min. Intrabatch CVs were 2.3%, 3.3%, 2.8%, 3.6%, 3.6%, and 3.0% for retinol, alpha-tocopherol, lutein/zeaxanthin, cryptoxanthin, lycopene, and beta-carotene, respectively. The corresponding interbatch CVs were 4.9%, 5.8%, 12.3%, 6.5%, 8.0%, and 3.4%.
Evaluation of EMIT Cyclosporine Assay for use with whole blood
Authors: Beresini M H , Davalian D , Alexander S , Toton-Quinn R , Barnett B , Cerelli M J , Hu M W , Berger D E , Blohm W P , Jaklitsch A
Published: January 2020

We evaluated the EMIT Cyclosporine Assay, designed for specific quantification of cyclosporine (CsA) in whole blood. The assay was run on the Roche Cobas Mira or Cobas Mira S analyzer. Analytical recovery from both normal donor whole blood and transplant patients' samples was within 17% of the standards. Measurement of diluted out-of-range samples also yielded excellent recovery (101-105%). Within-run, between-run, and total CVs were < or = 8.1%, 10.9%, and 12.1%, respectively. The detection limit was < 32 micrograms/L. The assay was linear from 0 to 500 micrograms/L. No significant cross-reactivity was observed for CsA metabolites AM1, AM19, and AM4N. Slight cross-reactivity occurred with metabolite AM9; 670 micrograms/L AM9 increased the measured CsA concentration by 49 micrograms/L. High concentrations of bilirubin, uric acid, triglycerides, and cholesterol, as well as 54 commonly coadministered drugs did not interfere with CsA quantification. Similarly, neither extreme values of hematocrit nor choice of anticoagulant affected CsA recovery. Sample extract stability was > 4 h, and assay calibration was stable for at least 2 weeks. Patients' samples analyzed by the EMIT assay showed strong correlation with both HPLC and 125I-RIA (r > 0.97). We conclude that the EMIT Cyclosporine Assay provides a convenient, accurate, and precise method for specific quantification of CsA in whole blood.
HPLC assay of melatonin in plasma with fluorescence detection
Authors: Peniston-Bird J F , Di W L , Street C A , Kadva A , Stalteri M A , Silman R E
Published: January 2020

We report an HPLC assay for melatonin that incorporates automated injection, methanol/water mobile phase, and fluorescence detection. Plasma samples were extracted by solid and liquid phases. Recovery was > 70% for 1-10 mL of plasma extracted, approximately 40 pg-250 ng of melatonin. Samples were dried and reconstituted in 100 mL/L methanol. Injections were 25 microL or 150 microL, depending on sample concentration, and the melatonin peak was eluted in 380 mL/L methanol. The detection limit of the assay was 6 pg on the column, allowing a practical sensitivity in plasma of 11 pmol/L for 8-mL samples and 34 pmol/L for 2-mL samples. More than 100 plasma samples from volunteers and patients were assayed and the results compared with an established RIA. The mean daytime concentration of melatonin was 20.7 pmol/L (SEM = 1.2) and 18.5 pmol/L (SEM = 1.6) for HPLC and RIA, respectively, and the mean nighttime concentration was 82.4 pmol/L (SEM = 6.5) and 82.2 (SEM = 7.3), respectively.
Colorimetric assay of glycoprotein glycation free of interference from glycosylation residues
Authors: Kennedy D M , Skillen A W , Self C H
Published: January 2020

We have developed a colorimetric assay for determining the degree of glycation of serum proteins that is unaffected by glycosylation residues. This was accomplished by reducing the proteins with sodium borohydride prior to periodate oxidation. Previous periodate-based methods, which offer several advantages over other glycation assays, cannot determine glycoprotein glycation because interference from sialic residues in the glycan chain can lead to overestimation of the amount of glycation products. Without reduction, glycation of fetuin was double that of asialofetuin glycated under identical conditions. We found that borohydride reduction before periodate oxidation increases the amount of formaldehyde released in proportion to the extent of glycation, irrespective of the degree of glycosylation. Using two glycoproteins and an unglycosylated protein, we showed how measurement of the formaldehyde increase enables the extent of glycoprotein glycation to be determined without removal of interfering sugars.
Nonradioactive, colorimetric microplate hybridization assay for detecting amplified human immunodeficiency virus DNA
Authors: Rapier J M , Villamarzo Y , Schochetman G , Ou C Y , Brakel C L , Donegan J , Maltzman W , Lee S , Kirtikar D , Gatica D
Published: January 2020

A nonradioactive, colorimetric microplate hybridization procedure was used to assay human immunodeficiency virus (HIV) DNA, amplified by the polymerase chain reaction (PCR). Under the PCR conditions used, four proviral copies per 150,000 cells were detected by amplifying a series of DNA mixtures that contained various copy numbers of HIV. Assays of PCR-amplified DNA from peripheral blood mononuclear cells of seronegative individuals yielded negative results (104 of 104), whereas samples from seropositive individuals yielded > 99% positive results (141 of 142). Similar results were obtained in a chemiluminescent assay with an acridinium ester-labeled probe and in a solution hybridization assay in which a 32P-labeled probe was used.
Hapten-heterologous conjugates evaluated for application to free thyroxine immunoassays
Authors: Khosravi M J , Papanastasiou-Diamandi A
Published: January 2020

We evaluated the effect of hapten heterology on free thyroxine (FT4) immunoassays involving the biotin-streptavidin system and time-resolved fluorometry. We compared protein derivatives of thyroxine (T4) and triiodothyronine (T3) as solid-phase antigen or biotinylated protein-tracer conjugate for competitive (or sequential) binding to a mouse anti-T4 monoclonal antibody. In both one- and two-step assays, the heterologous combination of the antibody and T3 conjugates showed superior standard curve sensitivity but up to eightfold lower zero standard signal (B(o)) when the same amounts of antibody and conjugates were used. The improved sensitivity was not altered when the amount of coupled T3 was increased to obtain a B(o) value similar to that of the homologous combination of antibody and T4 conjugates. In the two-step format, the sensitivity of the homologous assay was insufficient for routine use, consistent with displacement of bound T4 during the antibody back-titration step (demonstrated in the T4 displacement experiment with excess conjugate). Results from the one-step (labeled antibody) heterologous assay for approximately 85 clinical samples correlated well with those from an immunofluorometric assay and a two-step radioimmunoassay. The assay was not affected by a wide variation in endogenous serum concentrations of T4-binding globulin and albumin.
Selective removal of beta 2-microglobulin from plasma specimens of long-term hemodialysis patients by high-performance immunoaffinity chromatography
Authors: Mogi M , Harada M , Kojima K , Adachi T , Nakamura S
Published: January 2020

We examined the suitability of high-performance immunoaffinity chromatography (HPIAC) for removing beta 2-microglobulin (beta 2-MG) from plasma of patients (n = 26) undergoing long-term hemodialysis. We used immobilized antibody to beta 2-MG to prepare an immunoaffinity column with a 0.5-mL bed volume. Plasma samples of 0.5 mL were chromatographed at a flow rate of 0.5 mL/min and pressures < 7 kg/cm2. Although only a small amount of beta 2-MG can be removed with a conventional dialyzer, 92.1% +/- 0.4% of the globulin could be removed by HPIAC. The column could be reused after acid elution. After 50 cycles the efficiency of beta 2-MG trapping was 73% of the original efficiency.
Time-resolved immunofluorometric assay of complement C3: application to cerebrospinal fluid
Authors: Gaillard O , Meillet D , Diemert M C , Musset L , Delattre J , Schuller E , Galli J
Published: January 2020

Complement components have a role in various neurological disorders. Complement C3 can be measured by immunochemical methods, but only radioimmunoassays and electroimmunodiffusion assays (EIDs) are sufficiently sensitive to be applied to biological fluids in which the C3 concentration is low, especially cerebrospinal fluid (CSF). We report a sandwich-type time-resolved immunofluorometric assay (TR-IFMA) for C3 in CSF. The linearity (0.7-3650 micrograms/L) and intra- (CV < 4.8%) and inter-assay (CV < 10.9%) precision were satisfactory and the results agreed with those of EID. The assay is extremely sensitive (< 1 microgram/L) and its analytical range is large and well suited to clinical applications. This simple TR-IFMA is thus a nonisotopic alternative to radioimmunoassay for the quantification of complement C3 in CSF.
Clinical measurement of serum amiodarone and desethylamiodarone by using solid-phase extraction followed by HPLC with a high-carbon reversed-phase column
Authors: Jandreski M A , Vanderslice W E
Published: January 2020

We describe a rapid, simple HPLC method routinely used in our clinical laboratory for determining amiodarone and its metabolite desethylamiodarone. These compounds are released from serum proteins by pretreatment with an acidic solution and then extracted onto a C2 reversed-phase clean-up column. After elution from the extraction column, the compounds are separated and quantified by HPLC with a C18 reversed-phase column and spectrophotometric detection. The standard curves for the drug and metabolite are linear up to 20.0 mg/L, with a lower limit of detection of 0.16 mg/L. The CVs for intra-assay precision were 5.0% at 0.58 mg/L and 2.9% at 5.96 mg/L; for inter-assay precision, they were 9.6% at 0.52 mg/L and 6.1% at 2.09 mg/L. Lipemia, hemoglobin, and bilirubin up to 300 mg/L do not interfere with this assay. None of > 550 cardiac patients' samples tested contained a compound that interferes with this assay.
Rapid determination of erythrocyte pyruvate kinase activity
Authors: Mosca A , Tagarelli A , Paleari R , Scarpelli P , Brancati C
Published: January 2020

We report a new potentiometric method for determining pyruvate kinase (PK). Enzymatic activity is measured by monitoring the change in pH produced in the reaction buffer under International Committee for Standardization in Haematology (ICSH) standardized assay conditions, and the lactate dehydrogenase reaction is automatically subtracted in each measuring cycle. The analysis, performed at 37 degrees C, requires a 10-microL sample (isolated erythrocytes or whole blood) and is completed in 2.5 min. The intra-assay CV is < 4% (PK between 3 and 35 U/g Hb); the interassay CV is 4.0% (PK 15 U/g Hb); results are linear from 3 to 30 U/g Hb. A good correlation with the ICSH reference method (x) was found: y = 1.011x - 0.05; n = 32; r = 0.9939; Sylx = 0.75 (units: U/g Hb). The reference intervals of the PK activity in isolated erythrocytes (RBC-PK) were estimated in 89 normal subjects. We found that women possess a higher RBC-PK than do men (P < 0.0001) and that the biological variability (CVb) of RBC-PK is 13.5%. Applications of the proposed method to the hematological routine are reported.
Increased urinary excretion of pyridinium cross-links in cancer patients
Authors: Lipton A , Demers L , Daniloff Y , Curley E , Hamilton C , Harvey H , Witters L , Seaman J , Van der Giessen R , Seyedin S
Published: January 2020

Pyridinoline (PYD) and deoxypyridinoline (DPD), two collagen-based cross-links found in bone, were measured by high-performance liquid chromatography in urine samples from 65 control subjects and 97 patients with either untreated or progressive cancer. Patients with cancer had significantly (P < 0.001) higher urine concentrations of PYD and DPD than did control subjects. Both cross-links were increased in cancer patients with and without clinically detectable bone metastases, although patients with bone and liver involvement had higher mean concentrations. The mean concentrations of both cross-links were also significantly higher in the urine samples of inpatients than in an outpatient ambulatory population. These findings suggest that the measurement of PYD and DPD in urine may be useful in assessing bone metastases and bone resorption in cancer patients.
Measurement of heroin and its metabolites by isotope-dilution electron-impact mass spectrometry
Authors: Goldberger B A , Darwin W D , Grant T M , Allen A C , Caplan Y H , Cone E J
Published: January 2020

A solid-phase extraction procedure was developed for the isolation of heroin, 6-acetylmorphine, and morphine from blood, plasma, saliva, and urine with subsequent assay by gas chromatography/mass spectrometry. Aprotic solvents, mild elution conditions, and an enzyme inhibitor were used to ensure maximum analyte stability. Samples were extracted and the extract was divided into two equal portions. One portion was assayed directly for heroin; detector response was linear over a concentration range of 1.0 to 250 micrograms/L. The second part of the extract was reacted with N-methyl-bis-trifluoroacetamide and assayed for the trifluoroacetyl derivatives of 6-acetylmorphine and morphine; detector response was linear over a concentration range of 1.0 to 500 micrograms/L. The limit of sensitivity was 1.0 microgram/L for each analyte. Hydrolysis of heroin to 6-acetylmorphine during extraction and analysis was < 5%. The method can be used to corroborate heroin use and to study the pharmacological effects of heroin and its metabolites.
A quantitative measure of nonlinearity
Authors: Emancipator K , Kroll M H
Published: January 2020

Quantitative measures of the nonlinearity of an analytical method are defined as follows: the "(dimensional) nonlinearity" of a method is the square root of the mean of the square of the deviation of the response curve from a straight line, where the straight line is chosen to minimize the nonlinearity. The "relative nonlinearity" is defined as the dimensional nonlinearity divided by the difference between the maximum and minimum assayed values. These definitions may be used to develop practical criteria for linearity that are still objective. Calculation of the nonlinearity requires a method of curve-fitting. In this article, we use polynomial regression to demonstrate calculations, but the definition of nonlinearity also accommodates alternative nonlinear regression procedures.
Glycated calmodulin from platelets as an index of glycemic control
Authors: Muruganandam A , Romsa G J , Thibert R J , Cheung R M , Draisey T F , Mutus B
Published: January 2020

In an effort to test whether a significant fraction of calmodulin would become glycated within the life span of the platelet (10-14 days), we monitored the kinetics of calmodulin glycation in vitro. Under the conditions we used, the fraction of glycated calmodulin reached a maximum (approximately 21%) within 10 days. We then extended the studies to human subjects. The intraplatelet concentrations of calmodulin and glycated calmodulin from age-matched type I diabetic subjects were monitored by a combination of m-aminophenylboronate affinity chromatography and enzyme-linked immunosorbent assay. The results indicate that the concentrations of total intraplatelet calmodulin (nonglycated plus glycated) were not dependent on the glycemic state of the subjects. Data from control and diabetic subjects showed a poor correlation between the concentrations of glycohemoglobin and of glycated calmodulin. However, a better correlation was obtained when glycated calmodulin concentrations were compared with those of serum fructosamine. The fraction of glycated calmodulin in the control population (7.71% +/- 0.75%) was significantly (P < 0.05) different from that of the diabetic population (21.6% +/- 1.26%). Given that the clinical role of the fructosamine assay remains controversial, estimation of glycated calmodulin in platelets might be useful as a short time-window index of glycemic control.
Reference intervals for 21 clinical chemistry analytes in arterial and venous umbilical cord blood
Authors: Perkins S L , Livesey J F , Belcher J
Published: January 2020

Reference intervals were determined for 21 clinical chemistry analytes in umbilical cord arterial and venous blood from healthy term infants. Nonparametric analysis (rank number) was used to determine the central 95% reference interval. No significant differences were observed between male and female infants. Reference intervals for glucose, urea, creatinine, urate, phosphate, calcium, albumin, total protein, cholesterol, triglycerides, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, creatine kinase, lactate dehydrogenase, gamma-glutamyltransferase, and magnesium all were significantly different from adult values.
Comparison of cyclosporin G (Nva2-cyclosporin) concentrations measured in whole blood by monoclonal fluorescence polarization immunoassay, monoclonal radioimmunoassay, and HPLC
Authors: Annesley T M , Coombs R C , Orsulak P J
Published: January 2020

Two commercial monoclonal immunoassays for monitoring cyclosporin A were used to measure whole-blood concentrations of the immunosuppressant cyclosporin G (CsG) in renal transplant patients. We performed a three-way comparison of these two immunoassays and HPLC. Although the two immunoassays agreed favorably, both the monoclonal fluorescence polarization immunoassay and the monoclonal RIA yielded higher CsG results for patients' specimens than did the liquid-chromatographic assay. The experimental data indicate that the observed differences are most likely due to the cross-reactivity of CsG metabolites in the immunoassays.
Inaccuracy of osmometry for measurement of plasma water in acute diabetes mellitus
Authors: Davidson D F
Published: January 2020

Osmometry is reportedly a relatively imprecise technique for the measurement of plasma water. However, reconsideration of the theoretical background of the test reveals that it is also likely to be inaccurate and subject to both positive errors as well as viscosity-dependent negative errors simultaneously, when applied to samples from patients admitted in acute diabetes mellitus. This was confirmed by comparison with Waugh's method of results for 59 specimens from 14 cases. Plasma water was shown to be relatively underestimated at high protein concentrations and overestimated at lower ones.
Simultaneous Determination of Free Testosterone and Testosterone Bound to Non-Sex-Hormone-Binding Globulin by Equilibrium Dialysis
Authors: Barini Angela , Liberale Italiana , Menini Edoardo
Published: January 2020

We describe a procedure based on equilibrium dialysis that allows the simultaneous determination of free testosterone and testorerone bound to non-sex-hormone-binding globulin (non-SHBG) in plasma. After saturating SHBG with 5α-dihydrotestosterone (DHT) according to a technique recently described, the percentage of free testosterone in the treated and the untreated samples is measured by equilibrium dialysis with use of a semiautomated instrument that allows rigorous standardization of the experimental conditions. The present method is simpler and faster than the previously described technique in which, after the saturation of SHBG with DHT, the unbound fractions were measured by centrifugal ultrafiltration dialysis. The method is also reproducible and suited for the analysis of a large number of samples. The technique has been applied to the determination of the fractional distribution of testosterone in plasma pools from normally menstruating, pregnant, and postmenopausal women and from normal men.
Transferrin allelic variants may cause false positives in the detection of cerebrospinal fluid fistulae
Authors: Sloman A J , Kelly R H
Published: January 2020

Variations in either the polypeptide sequence or the carbohydrate moieties of transferrin may result in altered electrophoretic mobility of this molecule. We report a case of an allelic (polypeptide) variant of transferrin with mobility similar to that of the beta 2 (sialic acid-depleted) transferrin found in cerebrospinal fluid (CSF) and a few other body fluids. Allelic variation and other transferrin anomalies may be mistaken for the CSF isoform, resulting in false diagnoses of CSF fistulae.
The history of evaluation criteria for CAP surveys
Authors: Hamlin W B
Published: January 2020

I summarize the evolution of target-value assignment methods and laboratory performance assessment (evaluation) within the College of American Pathologists Interlaboratory Survey Programs and review the importance of, and longstanding interest in, medical relevance criteria in setting analytical goals.
Profiles of very-long-chain fatty acids in plasma, fibroblasts, and blood cells in Zellweger syndrome, X-linked adrenoleukodystrophy, and rhizomelic chondrodysplasia punctata
Authors: Schutgens R B , Bouman I W , Nijenhuis A A , Wanders R J , Frumau M E
Published: January 2020

Profiles of saturated very-long-chain (> C22) fatty acids were studied in plasma, fibroblasts, erythrocytes, platelets, and leukocytes of patients affected by peroxisomal disorders such as Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD), and classic rhizomelic chondrodysplasia punctata (RCDP) and in controls. In Zellweger patients, the concentration of hexacosanoic acid (C26:0) and the C26:0/C22:0 ratio are greatly increased in plasma and fibroblasts. However, the plasma concentration of docosanoic acid (C22:0) is greatly decreased. Also in platelets, leukocytes, and to a lesser extent erythrocytes, the C26:0 concentrations and both the C26:0/C22:0 and C24:0/C22:0 ratios are greatly increased. The C24:0/C22:0 ratio is significantly increased in plasma, platelets, and leukocytes, but not in erythrocytes. In X-ALD, the C26:0 concentration and the C26:0/C22:0 and C24:0/C22:0 ratios are significantly increased in plasma, fibroblasts, platelets, and leukocytes, but the erythrocytes show substantial overlap in the 5-90% ranges between controls and patients. In RCDP, slightly increased C26:0 and C26:0/C22:0 ratios are found in erythrocytes, platelets, and leukocytes, but not in plasma and fibroblasts. We conclude that plasma and fibroblasts are the specimens of choice for biochemical diagnosis of Zellweger syndrome and X-ALD, respectively. The slight increase in C26:0 in blood cells of RCDP patients suggests a decreased flux of very-long-chain fatty acids through the peroxisomal beta-oxidation pathway in liver in this genetic disorder.
Evaluation of evacuated blood-collection tubes: effects of three types of polymeric separators on therapeutic drug-monitoring specimens
Authors: Landt M , Smith C H , Hortin G L
Published: January 2020

The potential of three types of separator materials found in conventional blood-collection tubes for interference in therapeutic drug measurements was assessed. None of the separators (based on acrylic, silicone, or polyester polymers) had any significant effect on the concentrations of seven drugs (theophylline, digoxin, phenytoin, phenobarbital, gentamicin, ethanol, and cyclosporine) in blood specimens that were processed and analyzed promptly. Storage of specimens for 24 h resulted in an average 2.4% increase in theophylline values in specimens collected in tubes with the acrylic separator (P = 0.024); an average 8.1% decrease in phenytoin in specimens collected in tubes with the polyester-based separator (P < 0.001); and an average 4.2% decrease in phenobarbital in specimens collected in tubes with the polyester-based separator (P = 0.02). All other drug concentrations were not significantly affected. A small decrease in phenytoin (7.9%; P < 0.01) was seen when the specimen volume in 7-mL tubes containing polyester-based separator was reduced to 1.0 mL; all other drug concentrations were unaffected by partial filling of tubes. Paired blood specimens from pediatric patients, when collected in plain tubes and tubes containing acrylic separator, yielded no significant differences for theophylline, digoxin, tobramycin, phenytoin, or phenobarbital concentrations. The three commercially available separators had only small effects on therapeutic drug concentrations, and a newly developed separator based on an acrylic resin was suitably inert.
Substantial urinary concentrations of material resembling beta-core fragment of chorionic gonadotropin beta-subunit in mid-menstrual cycle
Authors: Neven P , Iles R K , Howes I , Sharma K , Shepherd J H , Edwards R , Collins W P , Chard T
Published: January 2020

We measured the day-to-day variations in concentrations of beta-core, luteinizing hormone (LH), and alpha-subunit in urine during the menstrual cycle. The alpha-subunit concentrations showed a pattern similar to that of the LH concentrations. beta-Core-like material was increased during and up to 3 to 4 days after the surge in urine LH. The urine LH concentration was associated with the presence of beta-core immunoreactivity during the urine LH peak. Chromatography showed that, at the peak LH concentration and at 2 days after the LH peak, beta-core immunoreactivity could be accounted for by the presence of a peptide of low molecular mass similar to the beta-core molecule of hCG, but probably originating from the degradation of LH. The prolonged excretion of gonadotropin metabolites in the midcycle must be considered when beta-core is being assessed as a tumor marker.
Applications of a microfabricated device for evaluating sperm function
Authors: Kricka L J , Nozaki O , Heyner S , Garside W T , Wilding P
Published: January 2020

Mesoscale structures (microns dimensions, nL-pL volumes) have been designed and fabricated in silicon for use in various analytical tasks. We studied sperm motility and performed sperm selection in channels (80 microns wide x 20 microns deep), branching structures (40 microns wide x 20 microns deep, eight bifurcations), and channels containing barriers (7 microns feature size). Sperm-cervical mucus and sperm-hyaluronic acid interactions were assessed by using appropriate microchannel-chamber structures filled with either cervical mucus or hyaluronic acid. Simultaneous assessment of the potency of different spermicides (e.g., nonoxynol-9, C13G) and spermicide concentrations was achieved with structures comprising chambers containing spermicide connected via channels to a central chamber into which semen was introduced. Semen was also tested for the presence of sperm-specific antibodies by using microchannels filled with human anti-IgG antibody-coated microbeads.