bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct measurement of high-density lipoprotein cholesterol in serum with polyethylene glycol-modified enzymes and sulfated alpha-cyclodextrin
Authors: Sugiuchi H , Uji Y , Okabe H , Irie T , Uekama K , Kayahara N , Miyauchi K
Published: January 2020

We have developed an automated method for measuring high-density lipoprotein (HDL)-cholesterol in serum without prior separation, using polyethylene glycol (PEG)-modified enzymes and sulfated alpha-cyclodextrin. When cholesterol esterase and cholesterol oxidase enzymes were modified with PEG, they showed selective catalytic activities towards lipoprotein fractions, with the reactivity increasing in the order: low-density lipoprotein < very-low-density lipoprotein approximately chylomicron < HDL. In the presence of magnesium ions, alpha-cyclodextrin sulfate reduced the reactivity of cholesterol, especially in chylomicrons and very-low-density lipoprotein, without the need for precipitation of those lipoprotein fractions. The combination of PEG-modified enzymes with alpha-cyclodextrin sulfate provided selectivity for the determination of HDL-cholesterol in serum in the presence of a small amount of dextran sulfate without the need for precipitation of lipoprotein aggregates. The results of the HDL-cholesterol assayed in serum by this direct method correlated well with those obtained by precipitation-based methods and also that by an ultracentrifugation method.
Measuring outcomes: why now?
Authors: Witte D L
Published: January 2020

Intense scrutiny of the American healthcare paradigm will alter the activities of patients, providers, and payors. Government reform and marketplace-driven managed care programs create uncertainty. Quality, access, and cost concerns also drive change. Quality is conformity to requirements, and specification of requirements creates policy debate. Variability in utilization creates an accountability chasm between payors and providers that cannot be bridged without understanding the uncertainties in "appropriateness" research. Quality and access appear secondary to cost. Cost discussions must differentiate cost and charge. Inappropriate charge benefit analyses may temporarily benefit a specific organization but are unlikely to create long-term societal benefit. Multiple transitions have begun, including a shift from disease care to healthcare, provider mentality to consumer mentality, and provider autonomy to collaboration and accountability. Laboratories will be expected to provide outcomes, not tests; income will be related to covered lives, not volume, and profit will shift from "piecework and efficiency profit" to "prevention profit." Only good "outcomes" measurement can reduce uncertainty. The laboratory contribution to value in care processes remains unclear. What information is added by each result? How can results help prevent the need for future services? These are our challenges.
Diagnostic information as a commodity
Authors: Johnson H A
Published: January 2020

In estimating the cost-effectiveness of diagnostic procedures, it is helpful to treat diagnostic information as a commodity with a unit price. The amount of useful information provided by a test result can be measured in binary units (bits), and the unit price of the information produced by the test result can be expressed in dollars per bit in much the same way that the price of gold is given in dollars per ounce. This allows comparison of the unit prices of various diagnostic tests, examination of the effect of multiple testing, and calculation of the most cost-effective conditions for screening tests.
Conducting outcomes research: past experience and future directions
Authors: Boone D J , Steindel S J
Published: January 2020

The Centers for Disease Control and Prevention (CDC) was delegated authority to conduct the studies mandated in the Clinical Laboratory Improvement Amendments of 1988 (CLIA). Since that time, the CDC has been planning and implementing a research program, Evaluation of Quality of Laboratory Practices and Standards (EQLPS), aimed at demonstrating a clear linkage between patient outcome and laboratory practices and standards such as proficiency testing, quality assurance, and personnel standards. The goal of EQLPS is to improve the quality of laboratory medicine by providing a scientific and technical basis for laboratory practices and standards. In October 1995, the CDC will sponsor an Institute entitled "Frontiers in Laboratory Practice Research," during which strategies for conducting laboratory practice research will be discussed. The Institute should help identify research strategies that will eventually provide the information necessary to develop appropriate practice guidelines for laboratory medicine.
Structural analysis of CFTR gene in congenital bilateral absence of vas deferens
Authors: Jézéquel P , Dorval I , Fergelot P , Chauvel B , Le Treut A , Le Gall J Y , Le Lannou D , Blayau M
Published: January 2020

Congenital bilateral absence of the vas deferens (CBAVD) is found in most males with cystic fibrosis (CF), but this malformation can be observed without any pulmonary or digestive features. We have analyzed 13 exons of the CF gene in a cohort of 25 CBAVD patients. Among the 50 chromosomes studied, 24 mutations were identified: delta F508 (14 cases), R117H (7 cases), R1070W (2 cases), 621 + 1 G --> T (1 case), and A1067V (1 case). Except for delta F508, the most frequent mutations (R117H, R1070W) were not observed in the CF group (109 patients) studied in our laboratory. We discuss the significance of these results.
Prognostic role of antioxidant enzymes in sepsis: preliminary assessment
Authors: Warner A , Bencosme A , Healy D , Verme C
Published: January 2020

The prognostic potential of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) was evaluated in sepsis. Enzyme concentrations were determined in samples obtained from septic patients at time of diagnosis. Statistically significant increases in activities of total plasma SOD (P < 0.003, n = 32), erythrocyte (RBC) SOD (P < 0.007, n = 16), plasma CAT (P < 0.0001, n = 32), and RBC CAT (P < 0.005, n = 16) were found in septic patients when compared with healthy adult controls (n = 7). Further, within the group of septic patients, statistically significant differences were found for total plasma SOD (P < 0.05) and plasma CAT (P < 0.009) (but not for RBC determinations) when survivors (n = 15) were compared with nonsurvivors (n = 17). No significant differences were found for either plasma or RBC enzyme concentrations when patients who developed adult respiratory distress syndrome were compared with those who did not. The most striking finding was that plasma total SOD values of > 10 kU/L were found in 7 of 21 (30%) patients who did not survive their sepsis and that these values did not overlap with any surviving patients or controls. However, while high total plasma SOD activity appears to have some potential as a prognostic indicator, lower values (0.0-8.8 kU/L) do not. For plasma CAT, despite finding statistically significant differences between survivors and nonsurvivors, the substantial overlap in the values obtained for the two groups limits the practical prognostic potential of this enzyme.
Nitrite and nitrate determinations in plasma: a critical evaluation
Authors: Moshage H , Kok B , Huizenga J R , Jansen P L
Published: January 2020

Plasma nitrite and nitrate determinations are increasingly being used in clinical chemistry as markers for the activity of nitric oxide synthase and the production of nitric oxide radicals. However, a systematic evaluation of the determination of nitrite and nitrate in plasma has not been performed. In this study the recovery and stability of nitrite and nitrate in whole blood and in plasma, the relation between nitrite and nitrate concentrations in plasma, and possible sources of artifacts were investigated. The main conclusions are: (a) Recovery of nitrite and nitrate from plasma is near-quantitative (87%) and reproducible; (b) nitrite and nitrate are stable in (frozen) plasma for at least 1 year; (c) nitrite in whole blood is very rapidly (> 95% in 1 h) oxidized to nitrate, and therefore plasma nitrite determination alone is meaningless; (d) the ranges of nitrite and nitrate concentrations in plasma samples of 26 healthy persons are 1.3-13 mumol/L (mean 4.2 mumol/L) and 4.0-45.3 mumol/L (mean 19.7 mumol/L), respectively; (e) plasma nitrite and nitrate concentrations were not correlated (nitrite as % of total nitrite + nitrate varied from 3.9% to 88% in plasma samples); and (f) plasma samples should be deproteinized, and background controls for each sample should be included in the assay, to avoid measuring artifactually high nitrite and nitrate concentrations in plasma.
Determination of total serum sulfite by HPLC with fluorescence detection
Authors: Ji A J , Savon S R , Jacobsen D W
Published: January 2020

An estimated 500,000 individuals in the US, mostly steroid-dependent asthmatics, suffer severe adverse reactions to sulfites in foods, beverages, and pharmaceutical products. In an attempt to understand the pathogenesis of sulfite hypersensitivity, we have developed an assay for the determination of total serum sulfite by utilizing: (a) reductive release of serum protein-bound sulfite; (b) derivatization of free sulfite with monobromobimane; (c) separation of sulfite-bimane from thiol-bimanes by reversed-phase HPLC; and (d) quantitation of sulfite-bimane by fluorescence detection. The detection limit of this assay was 0.44 mumol/L serum sulfite. The intra- and interassay CVs for total serum sulfite at 5.4 mumol/L were 8.1% and 22.0%, respectively. The standard addition method was used to determine total serum sulfite in normal subjects. More than 70 samples were prepared in 2-3 h, followed by automated overnight analysis. The mean concentrations (+/- SD) of total serum sulfite in female (n = 41) and male (n = 35) donors were 4.63 +/- 2.33 and 5.16 +/- 2.68 mumol/L, respectively (not statistically significant: P = 0.368). The combined mean concentration of total sulfite in both sexes was 4.87 +/- 2.49 mumol/L. There was no correlation between total serum sulfite and total serum cysteine, cysteinylglycine, homocysteine, subject age, serum cobalamin, or serum folic acid. The reference range (mean +/- 2 SD) for total serum sulfite in normal subjects is 0-9.85 mumol/L.
Accumulation of atherogenic remnants and lipoprotein(a) in the nephrotic syndrome: relation to remission of proteinuria
Authors: Joven J , Simó J M , Vilella E , Camps J , Espinel E , Villabona C
Published: January 2020

Although lipoprotein abnormalities of the nephrotic syndrome are assumed to be related to the presence of proteinuria, this topic has not been investigated extensively. We measured lipoproteins from 19 nonuremic patients during and after remission of the nephrotic syndrome in an effort to determine the extent of their putative atherogenicity. As expected, disturbances involved primarily the apoprotein B-containing lipoproteins. No patient showed serum lipoprotein(a) [Lp(a)] < 300 mg/L during the acute phase. Lp(a) concentrations correlated significantly with those of apoprotein B, and both values decreased dramatically with the remission of the nephrotic syndrome. Surprisingly, despite the resolution of proteinuria, concentrations of intermediate-density lipoproteins and Lp(a) remained above normal in hypertriglyceridemic patients, suggesting a residual effect of nephrosis in the overall lipoprotein transport. Accumulation of atherogenic remnants should be considered a characteristic of the hyperlipidemia of the nephrotic syndrome, and aggressive treatment to reduce proteinuria is mandatory.
Immunospecific scintillation proximity assay of cholesteryl ester transfer protein activity
Authors: Lagrost L , Loreau N , Gambert P , Lallemant C
Published: January 2020

We describe a novel, immunospecific scintillation proximity assay for determining cholesteryl ester transfer protein (CETP) activity in total human serum and in reconstituted experimental mixtures. The assay is based on the measurement of radiolabeled cholesteryl esters transferred from a tracer dose of biosynthetically labeled high-density lipoprotein subfraction 3 to unlabeled apolipoprotein (apo) B-containing lipoproteins. The radioactivity content of the apo B-containing lipoprotein fraction can be evaluated without separating the donor from the acceptor lipoprotein substrates, and is measured through the formation of ternary complexes involving the radiolabeled apo B-containing lipoproteins, specific anti-apo B antibodies from sheep, and anti-sheep antibody-labeled fluoromicrospheres. Good correspondences were observed between CETP activity values obtained either with the ultracentrifugation method or the immunospecific scintillation proximity assay (n = 70; r = 0.94; P = 0.0001), and between values obtained for either fresh or frozen serum samples (n = 70; r = 0.93; P = 0.0001). Because of its potential for automation, the immunospecific scintillation proximity assay may constitute a convenient tool to measure serum CETP activity in the clinical laboratory.
Effect of beta-glucuronidase on urinary benzodiazepine concentrations determined by fluorescence polarization immunoassay
Authors: Simonsson P , Lidén A , Lindberg S
Published: January 2020

In samples from patients treated with oxazepam, beta-glucuronidase increased the immunoreactivity of urinary benzodiazepines analyzed by fluorescence polarization immunoassay (FPIA). Increasing concentrations of beta-glucuronidase added to samples from drug-free controls did not influence the results. In the absence of beta-glucuronidase, 22 of 35 samples from patients undergoing detoxification gave positive results at a cutoff concentration of 200 micrograms/L. Pretreatment with beta-glucuronidase increased the number of drug-positive samples to 33. The drug-negative samples were obtained from two patients who had been oxazepam-free for at least 1 week. Thus, beta-glucuronidase can be used to increase the sensitivity of the urinary benzodiazepine FPIA without reducing the specificity of the method.
Vitamin E and vitamin A concentrations in plasma adjusted for cholesterol and triglycerides by multiple regression
Authors: Jordan P , Brubacher D , Moser U , Stähelin H B , Gey K F
Published: January 2020

The plasma concentration of vitamins A and E varies with the amount of concurrent lipids and thus requires lipid standardization. The present study compares a new multiple regression-based method for adjusting vitamins A and E concentrations for cholesterol and triglycerides with previous methods (adjustment for cholesterol only, and adjustment for the sum of cholesterol and triglycerides). The results show that the new method can reduce influence of the concurrent lipids.
Modified (desialylated) low-density lipoprotein measured in serum by lectin-sorbent assay
Authors: Tertov V V , Sobenin I A , Orekhov A N
Published: January 2020

Modified low-density lipoprotein (LDL) with a low sialic acid content was found in the blood of patients with coronary atherosclerosis. This desialylated lipoprotein causes lipid accumulation in arterial smooth-muscle cells and stimulates cell proliferation and production of the extracellular matrix, i.e., induces all atherogenic manifestations at the cellular level. We have developed a lectin-sorbent assay for the determination of desialylated LDL in sera. The assay is based on the binding of desialylated LDL by immobilized Ricinus communis agglutinin with subsequent measurement of lipoprotein through use of anti-apolipoprotein (apo) B antibody. The assay is sensitive to desialylated apo B concentrations as low as 5 micrograms/L. The intraassay and interassay CVs were 4.8% and 11.3%, respectively. Comparison between the lectin-sorbent assay and a lectin chromatographic technique showed a good correlation. This determination of modified desialylated LDL in human serum with high accuracy and reproducibility may help establish the diagnostic value of this lipoprotein as a risk factor of atherosclerosis.
Protocol for collection and HPLC analysis of volatile carbonyl compounds in breath
Authors: Lin Y , Dueker S R , Jones A D , Ebeler S E , Clifford A J
Published: January 2020

This noninvasive method for collection and analysis of a wide range of aldehydes and ketones in human breath may enable assessment of lipid peroxidation and metabolic status in vivo. Breath samples are drawn through silica cartridges impregnated with 2,4-dinitrophenylhydrazine, which traps carbonyls as their hydrazone derivatives. The hydrazone derivatives are eluted from the cartridges with acetonitrile, separated by reversed-phase HPLC, and quantified spectrophotometrically. Using this method, we have measured formaldehyde, acetaldehyde, acetone, propanal, 2-butanone, butanal, pentanal, and hexanal. Recoveries of carbonyls added to Douglas bags were > 90%, except for 2-butanone, which was 86.2%. The overall CVs for sampling plus analyzing duplicate aliquots of breath were < 11%. The results indicate that this protocol can be used to monitor changes of carbonyl production by analyzing expired air, which may, with further study, indicate physiological and pathological status.
Results of B-vitamin supplementation study used in a prediction model to define a reference range for plasma homocysteine
Authors: Ubbink J B , Becker P J , Vermaak W J , Delport R
Published: January 2020

Because high plasma concentrations of homocysteine constitute an enhanced risk for premature coronary heart disease, it is necessary to establish a reference range for normal concentrations of plasma homocysteine. The frequency distribution of plasma homocysteine concentrations tails to the right, and the nonparametric approach is unsatisfactory for defining a normal plasma homocysteine reference range. By using subjects' responses to appropriate vitamin supplementation, we developed a mathematical prediction model to calculate the plasma homocysteine concentration that could be expected for each individual treated with a vitamin supplement. With this model, we can predict that plasma homocysteine concentrations will approach a normal frequency distribution with a 95% reference range (mean +/- 2 SD) of 4.9-11.7 mumol/L, provided the vitamin status of the study population is improved.
Plasminogen activator activity and plasma-coagulum lysis measured by use of optimized fibrin gel structure preformed in microtiter plates
Authors: Sidelmann J , Jespersen J , Gram J
Published: January 2020

We introduce a new fibrin plate assay performed in microtiter plates. By means of spectroscopic studies we optimized the structure of the fibrin gel and then used the optimized fibrin gel to determine plasminogen activator activity. Plasminogen activator solutions were applied on top of the fibrin gel, and the absorbance of the gel was recorded at 405 nm. After incubation for 17 h at 25 degrees C, the absorbance was measured again. The difference in absorbance was proportional to the concentration of plasminogen activator, such that the dose-response curves were linear when the difference in absorbance was plotted as a function of the logarithmic concentration of plasminogen activator. We assayed both tissue-type and urokinase-type plasminogen activator activity. The intraassay CV was < 4.7% (n = 20); the interassay CV was < 3.1% (n = 15). Using the optimized procedure, we modified the assay for determination of plasma-coagulum lysis time in human plasma. We established a reference interval for lysis time in apparently healthy subjects of 75 to 201 ks. Patients with deep vein thrombosis showed significantly (P = 0.013) higher values.
Rapid, fully automated measurement of plasma homocyst(e)ine with the Abbott IMx analyzer
Authors: Shipchandler M T , Moore E G
Published: January 2020

We have developed a totally automated fluorescence polarization immunoassay for homocyst(e)ine with no pretreatment or chromatographic steps. Comparison with four well-established chromatographic methods yielded r values ranging from 0.980 to 0.997 and slopes from 1.030 to 1.493. Inter- and intraassay CVs ranged from 0.0% to 8.0% and from 0.0% to 6.4%, respectively. Imprecision (CV) of measuring six plasma samples on three instruments ranged from 6.3% to 10.2%. The assay was linear for plasma samples diluted with buffer from 0 to 8-fold. Mean recovery of homocysteine added to two plasma samples was 97.1% and 99.9%. The assay exhibited almost no cross-reactivity towards cysteine and methionine, and a batch of 20 samples can be processed in 60 min.
Apolipoprotein E: an important gene and protein to follow in laboratory medicine
Authors: Siest G , Pillot T , Régis-Bailly A , Leininger-Muller B , Steinmetz J , Galteau M M , Visvikis S
Published: January 2020

The human apolipoprotein (apo) E gene is polymorphic, with three common alleles (epsilon 2, epsilon 3, epsilon 4) coding for three isoforms (E2, E3, E4). The isoforms differ from each other by a single amino acid substitution, and also differ in their binding affinity for the four apo E receptors. Apo E polymorphism is an important determinant of risk for the development of cardiovascular and Alzheimer diseases, the prevalence of the epsilon 4 allele being increased in both kinds of patients compared with control subjects. Furthermore, the prevalence of the epsilon 4 allele differs among populations (range 5-40%, respectively, for Taiwanese and Papua New Guineans). Genotyping or phenotyping needs to be introduced in clinical laboratories. The choice of the method should be based on the types of patients who are examined. The apo E genotype is also a determinant of apo E plasma concentration. Standardization of apo E measurement is an important prerequisite before investigating the clinical interest of plasma apo E concentration. Determination of apo E genotype/phenotype and later the plasma concentration are expected to yield useful clinical laboratory information.
Increased urinary excretion of analogs of Krebs cycle metabolites and arabinose in two brothers with autistic features
Authors: Shaw W , Kassen E , Chaves E
Published: January 2020

A marked increase in analogs of Krebs cycle metabolites was found in the urine of two brothers with autistic features. These metabolites included citramalic, tartaric (3-OH-malic), and 3-oxoglutaric acids and compounds tentatively identified as a citric acid analog and partially identified as a phenylcarboxylic acid by the fragmentation pattern of the trimethylsilyl (TMS) derivatives of the compounds and mass shifts of the same compounds derivatized with perdeuterated N,O-bis(trimethylsilyl)trifluoroacetamide. The molecular mass of the TMS derivative of the tentatively identified citric acid analog was 596 Da, based on a finding of a significant M - 15 ion at m/z 581. The citric acid analog was excreted in quantities as high as 137 mmol/mol creatinine, based on the response factor of citric acid as a surrogate calibrator. A carbohydrate with a retention time and mass spectrum identical to arabinose was also found in high concentrations in the urine of these brothers.
Inborn errors of metabolism diagnosed in sudden death cases by acylcarnitine analysis of postmortem bile
Authors: Rashed M S , Ozand P T , Bennett M J , Barnard J J , Govindaraju D R , Rinaldo P
Published: January 2020

Fatty acid oxidation (FAO) disorders represent a frequently misdiagnosed group of inborn errors of metabolism. Some patients die at the first episode of fasting intolerance and, if appropriate investigations are not undertaken, often meet the criteria of sudden infant death syndrome (SIDS). To expand existing protocols for the postmortem diagnosis of FAO and other metabolic disorders, we tested the hypothesis that analysis for acylcarnitine in bile, a specimen readily available at autopsy, may be utilized for diagnostic purposes. Using electrospray/tandem mass spectrometry, we analyzed for acylcarnitine postmortem bile specimens from two infants with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, one infant with glutaryl-CoA dehydrogenase deficiency, and 17 uninformative SIDS cases as controls. The affected cases, and none of the controls, showed marked accumulation of C10-C18 acylcarnitines or glutarylcarnitine (acyl/free carnitine ratio: 5.2, 2.7, and 1.9, respectively; controls 0.2 +/- 0.1). In one patient, all other diagnostic methods were uninformative, suggesting that bile acylcarnitine profiling could lead to identification of previously overlooked cases.
Amylase, lipase, pancreatic isoamylase, and phospholipase A in diagnosis of acute pancreatitis
Authors: Clavé P , Guillaumes S , Blanco I , Nabau N , Mercé J , Farré A , Marruecos L , Lluís F
Published: January 2020

To determine the utility of serum amylase (AMY), lipase (Lp), pancreatic isoamylase (isoA), phospholipase A (PLA), and urine AMY in the diagnosis of acute pancreatitis, samples of serum and urine were obtained on admission and every day thereafter for 5 days from 384 patients with acute abdominal pain. Diagnostic accuracy, determined as the area under the receiver operating characteristic curve, was > 0.975 for serum AMY, Lp, isoA, and urine AMY. For each of these enzymes, a threshold value (twice to sixfold the upper limit of the reference values) offering diagnostic efficiency > 95% could be determined. In contrast, accuracy and efficiency of serum PLA were low. The profiles of these enzymes in acute pancreatitis decreased in a parallel fashion over 5 days except for PLA. We conclude that diagnostic utilities are similar for serum AMY, Lp, isoA, and urine AMY for acute pancreatitis, provided that an appropriate threshold is established.
Sensitive method for analysis of strontium in human and animal plasma by graphite furnace atomic absorption spectrophotometry
Authors: Barto R , Sips A J , van der Vijgh W J , Netelenbos J C
Published: January 2020

For long-term pharmacokinetic studies in humans as well as in experimental animals, an analysis of strontium (Sr) with a low detection limit and high sensitivity is necessary. The data presented here describe the optimization of the furnace program and sample handling for measuring Sr in plasma by using graphite furnace atomic absorption spectrophotometry. The method was validated and applied to studies on the pharmacokinetics of SrCl2 in humans and rats. Calibration curves were linear up to 57.1 nmol/L. The limit of detection and lower limit of quantification were 0.21 and 0.57 nmol/L, respectively. Reciprocal sensitivity was 0.53 nmol/L Sr at A = 0.0044. The intraassay precision was 2.2%, 1.5%, and 1.1% (n = 6) at 4.57, 22.7, and 49.2 nmol/L Sr, respectively. At these concentrations, the interassay precision and recovery were 0.7%, 1.5%, and 1.8% and 100.4%, 99.1%, and 100.6% (n = 12), respectively. The endogenous Sr concentration in human plasma samples was 0.27 +/- 0.07 mumol/L (n = 18). Pharmacokinetic studies in a human volunteer and in rats demonstrated that this procedure was suited for measuring low Sr concentrations and was applicable to small sample volumes.
Spectrophotometric determination of ascorbic acid and dehydroascorbic acid
Authors: Moeslinger T , Brunner M , Volf I , Spieckermann P G
Published: January 2020

We present a method for measuring ascorbic acid in methanol/trichloroacetic acid extracts prepared from human plasma after enzymatic oxidation of ascorbic acid to dehydroascorbic acid by ascorbate oxidase. Samples were assayed by spectrophotometrically monitoring the kinetics of the concentration-dependent absorbance changes of dehydroascorbic acid with phosphate-citrate-methanol buffers. Ascorbic acid was determined as the difference between dehydroascorbic acid and total ascorbic acid content. The detection limit was < 0.5 mumol/L. The calibration curve was linear (r > 0.995) over the range 0-1000 mumol/L. Analytical recovery of ascorbic acid added to plasma was 93-105%. The between-day variance was < 7%. Comparison of the spectrophotometric determination (y) with a chromatographic procedure (x) gave y = 1.02x - 0.653 (Sylx = 3.61) over the range of physiologically relevant concentrations. Total analysis time is < 10 min per sample and allows the simultaneous analysis of multiple samples.
Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays
Authors: Chen Z , Prestigiacomo A , Stamey T A
Published: January 2020

We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.
Electrophoresis-tutor: an image-based personal computer program that teaches clinical interpretation of protein electrophoresis patterns of serum, urine, and cerebrospinal fluid
Authors: Astion M L , Rank J , Wener M H , Torvik P , Schneider J B , Killingsworth L M
Published: January 2020

High-resolution protein electrophoresis of serum, urine, and cerebrospinal fluid (CSF) can aid in the diagnosis of multiple myeloma, amyloidosis, macroglobulinemia, multiple sclerosis, and other diseases. Electrophoresis-Tutor is a personal computer program based on approximately 150 digital images that teaches the clinical interpretation of agarose gel electrophoretic patterns. The program is divided into the following sections: introduction, CSF, serum, urine, review of disease states, program navigator, and final exam. The CSF section describes normal and abnormal CSF findings with emphasis on oligoclonal banding, as seen in the CSF of patients with multiple sclerosis. The serum section emphasizes monoclonal gammopathy patterns but also has detailed descriptions of inflammation, liver disease, protein-losing disorders, genetic deficiencies, and other patterns. Monoclonal gammopathy is described in the context of specific associated clinical conditions (e.g., myeloma, amyloidosis). For each monoclonal gammopathy example, results of standard electrophoresis, densitometry, and immunofixation are presented. The review of disease states uses animation to illustrate the development and remission of a variety of pathological patterns. The program navigator allows the user to jump quickly to any place in the program. The optional exam contains 20 questions, and detailed feedback is given after each question. Electrophoresis-Tutor can be used as a stand-alone teaching tool, a companion to traditional instruction, or a reference source.
Novel nonseparation sandwich-type electrochemical enzyme immunoassay system for detecting marker proteins in undiluted blood
Authors: Meyerhoff M E , Duan C , Meusel M
Published: January 2020

A novel nonseparation electrochemical enzyme immunoassay (NEEIA) for detecting marker proteins in undiluted blood is described. The approach is based on preferential electrochemical measurement of surface-bound enzyme-labeled reporter antibody (E-Ab), relative to an excess of this reagent in the sample solution. NEEIAs are carried out on microporous membranes coated with a thin, circular area of gold. The gold serves simultaneously as a working electrode and solid phase for immobilized capture anti-protein antibodies. In the assay, analyte protein is incubated concurrently with the Ab-coated gold surface and excess E-Ab conjugate. Detection of bound E-Ab is achieved by introducing the substrate for the enzyme through the back side of the membrane. The product of bound E-Ab is detected immediately by oxidation or reduction at the gold electrode, and the resulting current is proportional to the concentration of protein in the sample. The feasibility of the NEEIA approach is demonstrated via the detection of prostate-specific antigen in undiluted plasma samples (n = 64), with alkaline phosphatase as the label. Use of multiple gold films deposited on the same porous membrane to perform simultaneous NEEIAs is also described.
Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer
Authors: Mathis G
Published: January 2020

A homogeneous assay method based on the long-lived fluorescence of rare earth cryptates and amplification by nonradiative energy transfer has been developed for immunoassays. The principles of the assay allow a double discrimination of the emitted signal through spectral and temporal selectivity. The cage structure of the complex, ion pairing around europium, as well as double-wavelength detection, fully shield the assay from perturbations of media. Events based on short-range interactions involving biomolecules are of tremendous importance in many domains of biology, either for analytical purposes or for molecular mechanism studies. Therefore, the principles and the reagents used to devise this homogeneous assay were adapted to various models representative of molecular and cellular processes and were chosen from the signaling pathways involved in cellular communication and expression: epidermal growth factor (EGF) receptor-ligand interaction, EGF receptor kinase activity, Jun/Fos protein-protein interaction, and DNA hybridization. Evaluation of the homogeneous assays yielded results compatible with those from comparison assays and demonstrates the versatility and wide range of applicability of this methodology.
Evaluation of thyroid status in patients with thyrotoxicosis
Authors: Braverman L E
Published: January 2020

The generic term thyrotoxicosis defines the clinical syndrome of hypermetabolism associated with excess amounts of circulating free thyroxine (T4) and (or) triiodothyronine (T3) concentrations, irrespective of the source of the excess hormones. The term hyperthyroidism is reserved for those patients with thyrotoxicosis caused by increased synthesis and secretion of thyroid hormones from the gland due either to thyroid stimulators in the blood or to autonomously functioning thyroid nodules and is almost always associated with an increased radioactive iodine uptake (RAIU) by the thyroid. Another major cause of thyrotoxicosis is increased release of thyroid hormone from the gland, not associated with increased synthesis, caused by inflammatory changes, and always associated with a low thyroid RAIU. The most common miscellaneous cause of thyrotoxicosis is the exogenous ingestion of excess thyroid hormone, associated with a low thyroid RAIU. The serum concentration of thyrotropin (TSH) is low in all causes of thyrotoxicosis, except for TSH-secreting pituitary tumors and selective pituitary resistance to thyroid hormones. Anti-thyroglobulin and anti-thyroid peroxidase antibodies are present in patients with autoimmune thyroid disease, and serum thyroglobulin is increased in all patients with thyrotoxicosis except those with thyrotoxicosis facticia. A decreased serum TSH and normal concentrations of serum free T4 and T3 define the syndrome of subclinical thyrotoxicosis.
Optimal laboratory testing for diagnosis and monitoring of thyroid nodules, goiter, and thyroid cancer
Authors: Ladenson P W
Published: January 2020

Optimal use of laboratory tests to diagnose and monitor patients with goiter, thyroid nodules, or thyroid cancer requires an appreciation of the pathophysiologic factors implicated in thyroid hyperplasia and neoplasia: growth factors (especially thyrotropin, TSH), growth-stimulating immunoglobulins, activating mutations of the TSH receptor, and other oncogenic transformations. In patients with diffuse goiter and thyroid nodules, serum TSH measurement in a highly sensitive assay excludes both primary hypothyroidism and common causes of thyrotoxicosis. In selected patients, screening for anti-thyroid peroxidase with or without anti-thyroglobulin antibodies can confirm the diagnosis of autoimmune thyroiditis. Serum calcitonin measurement is appropriate only when medullary thyroid carcinoma (MTC) is clinically suspected. Laboratory testing is essential in management of thyroid carcinoma patients after primary surgical therapy. Serum TSH measurement is vital to ensure that thyroxine replacement and TSH suppression are adequate in treatment of epithelial cancers. Serial monitoring of serum thyroglobulin (Tg) can detect tumor recurrence and quantify tumor burden. Interpretation of serum Tg results requires an appreciation of certain technical considerations (e.g., anti-Tg antibody interference) and the patient's concurrent TSH status. Periodic serum Tg measurements and 131I scans are complementary monitoring techniques. Serum calcitonin measurement and screening for ret protooncogene mutations are both valuable for identifying individuals with MTC.
Guidelines for diagnosis and monitoring of thyroid disease: nonthyroidal illness
Authors: Stockigt J R
Published: January 2020

On the basis of low specificity, poor positive predictive value, and cost, there is at present no basis for routine assessment of thyroid function in acutely hospitalized patients, unless clinical features suggest the possibility of thyroid dysfunction, or a patient's background increases the likelihood of thyroid dysfunction. When used in severely ill patients, estimates of both thyroxine (T4) and thyrotropin (TSH) show a high prevalence of abnormal results, but lack specificity and have poor positive predictive value for true thyroid disease. When thyroid function is tested in the critically ill, the positive predictive value for true thyroid disease of both free T4 and TSH measurements could be improved by using wider reference intervals than for unselected populations. The knowledge of nonspecific disease-related abnormalities of triiodothyronine, T4, and TSH is not currently likely to yield useful prognostic information or to alter management for individual patients. Thyroid testing should be readily available for any acutely ill patient with any clinical features that suggest thyroid dysfunction, and for groups at increased risk of thyroid dysfunction. An initial abnormal result for either TSH or free T4 estimate should be followed by combined analysis of free T4 and TSH with the best available methodology. Diagnosis of thyroid dysfunction should be based on the T4-TSH relation rather than either value alone. Persistence of an apparent diagnostic abnormality should be confirmed before therapy is commenced.
Multicenter evaluation of the Abbott IMx CA 15-3 assay
Authors: van Kamp G J , Bon G G , Verstraeten R A , Lynch D , Krikau M , Flückiger J , Ruibal A , Kenemans P
Published: January 2020

We describe a new fully automated procedure for the quantitative measurement of CA 15-3: the microparticle enzyme immunoassay (MEIA) technology developed by Abbott Labs. for the IMx automated immunoassay analyzer. The new IMx CA 15-3 test uses two mouse monoclonal antibodies, 115D8 and DF3. The test has a dynamic range to 250 kilounits/L and a minimal detectable dose of CA 15-3 < 0.2 kilounits/L. On dilution, linearity is excellent, with recoveries ranging from 94% to 101%. Studies were conducted at four sites to evaluate the performance characteristics of this assay. The intra- and interassay CVs were < 4.7% and < 5.6%, respectively, and showed a between-laboratory CV < 5.9%. Test results of the Abbott IMx CA 15-3 (y) were correlated with those obtained with the Centocor CA 15-3 RIA (x), a solid-phase heterologous RIA. Linear regression analysis on results for 1973 samples yielded: y = 0.97x - 2.09 (r = 0.9899, Sy/x = 22.2, range 1-4089 kilounits/L).
Evaluation of clinical assays for measuring high-dose methotrexate in plasma
Authors: Albertioni F , Rask C , Eksborg S , Poulsen J H , Pettersson B , Beck O , Schroeder H , Peterson C
Published: January 2020

Four routine assays commonly used for monitoring plasma methotrexate (MTX) during high-dose therapy were validated by HPLC as the comparison method. MTX and its main metabolite, 7-hydroxymethotrexate (7-OHMTX), were analyzed by HPLC with postcolumn derivatization and fluorometric detection. About 200 clinical plasma samples from 13 children with acute lymphoblastic leukemia who received 5-8 g/m2 MTX as 24-h infusions were analyzed. The fraction of measured concentrations of MTX that were within 75-125% of the values obtained by HPLC were 64.5% for enzyme inhibition assay, 56.4% for fluorescence polarization immunoassay with polyclonal antibodies (FPIA1; Abbott), 58.9% for FPIA2 (with monoclonal antibodies; Abbott), and 46.4% for enzyme-multiplied immunoassay (Emit; Syva). All nonchromatographic procedures were subject to interferences from MTX plasma metabolites or endogenous substances. The interference from 7-OHMTX was, however, somewhat less pronounced for FPIA2 (monoclonal) than for FPIA1 (polyclonal).
Evaluation of a fully automated end-tidal carbon monoxide instrument for breath analysis
Authors: Vreman H J , Baxter L M , Stone R T , Stevenson D K
Published: January 2020

We evaluated a novel, portable breath sampler/CO-quantifying instrument [Baby's Breath Carbon Monoxide Analyzer (BB); Natus Medical], developed for use at the bedside or with gas samples collected into bags. Bench tests demonstrated that the CO measurements were linear, accurate, and precise when compared with gas chromatography (GC) results. In vivo tests (n = 30) performed with adults showed excellent correlation between end-tidal breath CO measurements (ETCO) corrected for inhaled CO (ETCOc) as determined by BB and GC. Breath sampling efficiency was 96%. ETCOc measurements and blood carboxyhemoglobin fractions (% of total hemoglobin) corrected for inhaled CO (COHbc) correlated strongly: COHbc = 0.25 ETCOc--0.01 microL/L CO (r2 = 0.97, Sy/x = 0.47, SE slope = 0.01, n = 30). The imprecision, assessed by the mean of the population's CV for triplicate determinations, was 11%. Measurements with healthy and hemolytic term newborns showed that ETCOc values of > 3 microL/L correlated with known hemolytic conditions. We conclude that this instrument is clinically reliable and can be used to noninvasively measure ETCO in neonates and adults.
Rapid and simultaneous quantification of rhamnose, mannitol, and lactulose in urine by HPLC for estimating intestinal permeability in pediatric practice
Authors: Miki K , Butler R , Moore D , Davidson G
Published: January 2020

Determination of intestinal permeability by measuring nonmetabolized sugars has been used to assess the integrity of intestinal mucosa. We have developed and validated a modified HPLC method for determining the concentration of L-rhamnose, mannitol, and lactulose in urine, using an amine-modified silica column and refractive index detection. Probe sugars are simultaneously resolved within 18 min. The calibration curve for each sugar is linear to 20 mmol/L. The minimum detectable concentration of lactulose is 0.05 mmol/L. Recovery of probe sugars is between 99.3% and 105.1%. Overall precision (CV) of estimation of probe sugars ranges from 4.2% to 6.5%. In 14 urine samples from healthy children who ingested the test solution containing 1 g of L-rhamnose, 1 g of mannitol, and 5 g of lactulose, the 5-h urinary excretion ratios of lactulose/rhamnose and lactulose/mannitol were 0.047 +/- 0.018 and 0.021 +/- 0.010 (mean +/- SD), respectively. This method presents a rapid, convenient, and practical technique for determining intestinal permeability in clinical pediatric practice.
Biological and clinical significance of haptoglobin polymorphism in humans
Authors: Langlois M R , Delanghe J R
Published: January 2020

Haptoglobin is a hemoglobin-binding protein expressed by a genetic polymorphism as three major phenotypes: 1-1, 2-1, and 2-2. Most attention has been paid to determining haptoglobin phenotype as a genetic fingerprint used in forensic medicine. More recently, several functional differences between haptoglobin phenotypes have been demonstrated that appear to have important biological and clinical consequences. Haptoglobin polymorphism is associated with the prevalence and clinical evolution of many inflammatory diseases, including infections, atherosclerosis, and autoimmune disorders. These effects are explained by a phenotype-dependent modulation of oxidative stress and prostaglandin synthesis. Recent evidence is growing that haptoglobin is involved in the immune response as well. The strong genetic pressure favoring the 2-2 phenotype suggests an important role of haptoglobin in human pathology.
Nonisotopic method for accurate detection of (CAG)n repeats causing Huntington disease
Authors: Muglia M , Leone O , Annesi G , Gabriele A L , Imbrogno E , Grandinetti C , Conforti F L , Naso F , Brancati C
Published: January 2020

Huntington disease (HD) is a neurodegenerative disorder caused by an expanded trinucleotide repeat (CAG)n located at the 5' end of the novel IT15 gene. Discovery of this expansion allows the molecular diagnosis of HD by measuring repeat length. We applied a simple nonisotopic method to detect (CAG)n repeats, avoiding both radioactive and Southern transfer analysis. The assay is based on direct visualization of electrophoresed PCR products, after silver nitrate gel staining. Its accurate sizing of HD alleles allows presymptomatic diagnosis of at-risk persons. By avoiding isotopic manipulations, the method is safe and accurate, with no radioactive background bands. Furthermore, because it permits direct allele visualization after gel staining, the method is simple and rapid, allowing allele sizing within hours rather than days.
Free thyroxine concentrations in serum measured by equilibrium dialysis in chronic renal failure
Authors: Okabayashi T , Takeda K , Kawada M , Kubo Y , Nakamura S , Chikamori K , Terao N , Hashimoto K
Published: January 2020

The serum concentration of free thyroxine (FT4) is often low in patients with chronic renal failure (CRF) with low serum concentrations of triiodothyronine (T3). We evaluated the serum FT4 concentration by using both an equilibrium dialysis RIA kit (D-FT4) and a labeled-antibody kit (M-FT4) in two different groups of CRF patients, undergoing chronic hemodialysis (HD, n = 145) or not (non-HD, n = 30), and in a group of normal healthy subjects (n = 58). Thyroid peroxidase antibodies and thyroglobulin antibodies were not detected in any patient. Serum FT4 concentrations (mean +/- SD, pmol/L) by the D- and M-FT4 assays were, respectively, 21.5 +/- 4.6 and 16.6 +/- 2.0 in the healthy subjects, 17.8 +/- 4.3 and 13.9 +/- 3.6 in the non-HD patients, and 16.9 +/- 4.9 and 10.7 +/- 1.9 in the HD patients. By the D-FT4 assay, results for both CRF groups were significantly different from those for the healthy group (P <0.01), as were the results for each pair of groups by the M-FT4 assay (P <0.01). FT4 values were reported as being within the healthy reference range by D-FT4 in 73 of 113 HD subjects who had low T3 and low M-FT4 values. Serum FT4 concentrations measured by both assay kits showed a significant inverse correlation with the serum concentration of creatinine (P <0.01), but the serum concentrations of sex-hormone-binding globulin did not differ significantly among the three groups. Our results indicate that the low FT4 concentration measured by D-FT4 in patients with CRF, particularly those on HD, probably reflects the actual, mild nonthyroidal illness of renal failure.