bluuebunny/crossref_metadata_2025 huggingface dataset:
Measuring c-erbB-2 oncogene amplification in fresh and paraffin-embedded tumors by competitive polymerase chain reaction
Authors: Sestini R , Orlando C , Zentilin L , Gelmini S , Pinzani P , Bianchi S , Selli C , Giacca M , Pazzagli M
Published: January 2020

We present an original application of competitive polymerase chain reaction (PCR) for measuring oncogene amplification in DNA from human tumors by simultaneous PCR amplification of genomic DNA with fixed amounts of an internal standard (competitor DNA). Competitors share the same sequence as the target genes but contain an additional 15- to 20-base-pair insert, which allows resolution of the amplified products after polyacrylamide gel electrophoresis and ethidium bromide staining. The gene copy number is derived from the ratio between the intensities of the bands corresponding to the amplified products. Using this procedure, we measured c-erbB-2 amplification in breast and bladder carcinomas in both fresh tumor tissues and paraffin-embedded tissue samples and assessed the precision, sensitivity, and accuracy of the assay. Competitive PCR is a simple, reliable, and accurate method for the evaluation of c-erbB-2 amplification and is potentially suitable for use in the clinical laboratory.
Fast-type electrophoretic variant of lactate dehydrogenase M(A) and comparison with other missense mutations in lactate dehydrogenase M(A) and H(B) genes
Authors: Maekawa M , Sudo K , Kobayashi A , Sugiyama E , Li S S , Kanno T
Published: January 2020

An electrophoretic variant of lactate dehydrogenase (LD) M(A) subunit was discovered in a female patient with chest pain. Her LD activity in serum was within the normal reference interval, and analysis of her LD isoenzyme pattern showed an abnormal migration indicating a fast-type LD-M(A) subunit variant. DNA analysis of the mutant LD-M gene detected a single base substitution, an A to G transition at codon 220 (AAA-->GAA). This mutation resulted in the replacement of a lysine by a glutamic acid (mutation K220E) and produced a subunit variant (electrophoretic fast type). This missense mutation was also observed in the patient's son, and genotypes of mother and son were consistent with their biochemical phenotypes, as evaluated by LD isoenzyme analysis.
Lowering the federally mandated cannabinoid immunoassay cutoff increases true-positive results
Authors: Huestis M A , Mitchell J M , Cone E J
Published: January 2020

Proposed changes to the Health and Human Services Guidelines for forensic urine drug testing will lower the required cannabinoid immunoassay cutoff concentration from 100 to 50 micrograms/L. We investigated the effect of this change on the sensitivity, specificity, and efficiency of eight cannabinoid immunoassays: Syva Emit d.a.u. 100; Syva Emit II 100; Syva Emit d.a.u. 50; Syva Emit II 50; Roche Abuscreen Online; Roche Abuscreen radioimmunoassay; Diagnostic Reagents; and Abbott ADx. All specimens also were assayed by gas chromatography/mass spectrometry. Lowering the cutoff concentration from 100 to 50 micrograms/L increased efficiencies and sensitivities for all immunoassays, with minor decreases in specificity (1.0-2.6%). There was a 23.2-53.6% increase in the number of true-positive specimens identified. Thus, lowering the cannabinoid immunoassay cutoff concentration from 100 to 50 micrograms/L resulted in detection of a substantial number of additional true-positive specimens, with an accompanying small increase in unconfirmed positive results.
Immunoradiometric assay of human intact proinsulin applied to patients with type 2 diabetes, impaired glucose tolerance, and hyperandrogenism
Authors: Chevenne D , Ruiz J , Lohmann L , Laudat A , Leblanc H , Gray I P , Passa P , Porquet D
Published: January 2020

We describe an immunoradiometric assay for human intact proinsulin in serum. In this method, one monoclonal antibody, coated onto polyacrylamide beads, cross-reacts with proinsulins and insulin. A sandwich is formed with intact proinsulin, split (65-66) proinsulin, and des (64-65) proinsulin binding with an 125I-labeled monoclonal antibody specific for an epitope at the intact B-C junction of proinsulin. Because split (65-66) and des (64-65) proinsulin concentrations are very low in serum, this assay essentially measures intact proinsulin. When we used 1-mL serum samples, the mean detection limit was 0.4 pmol/L. Mean proinsulin concentrations (pmol/L) were 3.4 (range 1-9.1) in healthy fasting subjects, 28.5 (9.7-101) in patients with type 2 diabetes (treated with metformin and sulfonylureas), 5.0 (1.6-9.3) in women with hyperandrogenism and normal insulinemia, 10.3 (2.6-36) in women with hyperandrogenism and hyperinsulinemia, and 8.5 (4.8-21.3) in patients with impaired glucose tolerance.
Development of indices for determining extracellular fluid sodium and water status in acute diabetic ketoacidosis: possible tools for clinical audit
Authors: Davidson D F , Williamson J , Boag D E , Millar T
Published: January 2020

The limitation of plasma sodium concentration as an indicator of extracellular hydration status in cases of acute diabetes is well recognized and could lead to individually inappropriate fluid therapy. However, in view of the small analytical and biological variations exhibited by plasma concentrations of protein, water, and sodium in health, we have developed simple laboratory indices that may better describe the extracellular environment. Preliminary data presented here for 20 patients with acute diabetic ketoacidosis admitted as emergencies to Crosshouse Hospital suggest that the type of approach we describe has the potential to supply meaningful therapeutic data to the managing physician and, therefore, merits further study in a clinical setting.
Iron-deficiency anemia is associated with high concentrations of transferrin receptor in serum
Authors: Punnonen K , Irjala K , Rajamäki A
Published: January 2020

We evaluated the use of transferrin receptor (TfR) in serum as an index of iron deficiency in 19 patients diagnosed as having iron-deficiency anemia, in 17 patients with anemia of chronic disease, and in a control group of 19 nonanemic patients who underwent elective ocular or nasopharyngeal surgery. The assessment of iron status of the anemic patients was based on the presence of stainable iron on bone marrow examination. In the patients with iron-deficiency anemia, the serum TfR concentration was 5.3 +/- 1.8 mg/L (mean +/- SD), significantly higher than in the control group (1.7 +/- 0.5 mg/L) or in the patients with anemia of chronic disease (1.6 +/- 0.4 mg/L). This study suggests that serum TfR measurement is a reliable index of iron depletion and potentially of importance in the diagnosis of iron-deficiency anemia.
Lithium determinations evaluated in eight analyzers
Authors: Sampson M , Ruddel M , Elin R J
Published: January 2020

We compared five ion-selective electrodes (ISEs; AVL 985S, Baxter Lytening 2Z, Beckman EL-ISE, Ciba-Corning 654 Na/K/Li, and Nova CRT 11) and a colorimetric method (Ektachem) for determination of lithium with flame atomic absorption and atomic emission spectroscopy. We evaluated precision, recovery, interference by drugs (procainamide, N-acetylprocainamide, quinidine, lidocaine, carbamazepine, and valproic acid) and inorganic analytes (Na, K, Ca, Mg, and Br), and performance with sera from patients receiving lithium. Imprecision was < 5% (CV) for all analyzers except Ektachem and Beckman. Analytical recovery was 100% +/- 5% for all analyzers except Ektachem and Baxter. Some drug interference was seen with all analyzers except AVL and Corning. Calcium caused interference with AVL, Corning, and Ektachem analyzers, and sodium and potassium interfered with the Ektachem analyzer. The results with the Baxter, Beckman, and Corning analyzers were closest to those by flame atomic emission (mean differences, 0.01 to 0.02 mmol/L); the AVL and Nova analyzers gave lower results (mean differences, -0.11 to -0.13 mmol/L) and the Ektachem gave higher results (mean difference, 0.08 mmol/L).
SR1 assays of alpha-fetoprotein, carcinoembryonic antigen, and prostate-specific antigen compared with corresponding established commercial assays
Authors: Jolley N L , Bacarese-Hamilton T
Published: January 2020

We compared results obtained with the newly developed alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate-specific antigen (PSA) assays for the fully-automated Serono SR1 analyzer with those obtained with the major, established methods for these analytes: Serono Serozyme and Bridge, Abbott IMx, Kodak Amerlite, Boehringer Mannheim ES600, Pharmacia Delfia, Hybritech Tandem-E and Tandem-R, Ciba Corning ACS180, and DPC IRMA-Count. The correlations were good for all methods studied (r > or = 0.94). For AFP, numerical agreement was good, with linear regression slopes of 0.88 to 1.15; for CEA, correlation slopes of 1.03 to 1.16 were observed. The SR1 PSA assay agreed well with five of the seven methods studied (slopes of 0.98 to 1.22), but the Ciba Corning ACS180 PSA assay gave results higher than all other methods studied (slope 0.54 vs SR1 at low doses) and the Abbott IMx PSA assay gave results lower than all other methods studied (slope 1.47 vs SR1).
Cardiac troponin-T immunoassay for diagnosis of acute myocardial infarction
Authors: Wu A H , Valdes R , Apple F S , Gornet T , Stone M A , Mayfield-Stokes S , Ingersoll-Stroubos A M , Wiler B
Published: January 2020

We evaluated the analytical and clinical performance of an immunoassay for cardiac troponin T (cTnT). Within-run and total imprecision ranged from 1.6 to 11.3%. The sensitivity and linear range was 0.015 and 13 micrograms/L, respectively. Frozen samples were stable for at least 8 weeks. No interferences were seen with lipids or bilirubin (total and conjugated). Hemoglobin caused a negative bias at concentrations > 4 g/L. Heparinized plasma showed a 6% negative bias compared with serum. The clinical utility of cTnT was compared with that of creatine kinase (CK)-MB (mass assay). The sensitivity of cTnT measurements from 63 patients with acute myocardial infarction (AMI) (cTnT cutoff 0.1 microgram/L) was 60% at 0-3 h, 59% at 3-6 h, 94% at 6-9 h, 90% at 9-12 h, 99% at 12-24 h, 92% at 24-48 h, 83% at 48-72 h, and 100% at 72-96 h. Corresponding results for CK-MB (cutoff 5.0 micrograms/L and 2.5% relative index) were 45%, 64%, 82%, 97%, 87%, 81%, 54%, and 59%. The specificity of the markers from 49 non-AMI patients was 46% and 79% for cTnT and CK-MB, respectively. We show that CK-MB is more specific for diagnosis of AMI, and propose that cTnT is more sensitive to myocardial injury.
HPLC analysis of oligosaccharides in urine from oligosaccharidosis patients
Authors: Peelen G O , de Jong J G , Wevers R A
Published: January 2020

Analysis of urinary oligosaccharides by thin-layer chromatography (TLC) is used as screening procedure for 10 different lysosomal diseases. We tested the usefulness of HPLC in screening, using a CarboPac PA1 column (Dionex), pulsed amperometric detection (PAD), and post-column derivatization (PCD). Patterns from six types of oligosaccharidoses were compared with normal urinary patterns and with the TLC patterns. PAD appeared to be nonspecific and therefore is applicable only to desalted urine samples. PCD was more specific and applicable to nondesalted urine samples, albeit with a lower resolving power. Peaks in urines from oligosaccharidoses patients were identified on the basis of retention times of commercially available oligosaccharides or TLC bands after isolation and HPLC of the corresponding oligosaccharides. Abnormal oligosaccharide peaks were seen in urines from patients with alpha-mannosidosis, GM1-gangliosidosis (juvenile), GM2-gangliosidosis (Sandhoff disease), Pompe disease, and beta-mannosidosis. HPLC detected no abnormal oligosaccharides in urine from patients with fucosidosis. Although TLC is a simple and reliable screening procedure for detecting classical lysosomal diseases with oligosaccharide excretion, HPLC, by its higher resolution and possibility of quantification, can more generally be used for recognition of abnormal oligosaccharides or detection of increased excretion or content for known oligosaccharides in urine, other body fluids, and cells.
New international reference preparation for proteins in human serum (RPPHS)
Authors: Whicher J T , Ritchie R F , Johnson A M , Baudner S , Bienvenu J , Blirup-Jensen S , Carlstrom A , Dati F , Ward A M , Svendsen P J
Published: January 2020

Quality-control surveys in recent years, in various parts of the world, have shown poor between-laboratory agreement for measurements of plasma proteins. Despite the existence of international reference materials distributed by the World Health Organization, standards produced by diagnostics manufacturers and professional organizations differ significantly in their ascribed values. The reasons for this are complex but include poor availability of the primary materials, confusion about their use, and the fact that their turbidity on reconstitution precludes their use in modern optical immunoassays. This unfortunate situation led to an important initiative to produce sufficient quantities of a widely available, optically clear secondary reference material for plasma proteins that could be used worldwide by manufacturers, professional organizations, and laboratories. Here we present an overview on how the laboratory community, including manufacturers, clinical laboratories, professional societies, and regulators, has reached what we consider is a successful conclusion to a difficult problem.
European specifications for imprecision and inaccuracy compared with operating specifications that assure the quality required by US CLIA proficiency-testing criteria
Authors: Westgard J O , Seehafer J J , Barry P L
Published: January 2020

Proposed and interim European quality specifications for imprecision and inaccuracy have been compared with the US CLIA total error criteria for proficiency testing (PT). To assess the relative demands of separate imprecision and inaccuracy specifications vs total error criteria, we derived the imprecision and inaccuracy that would be allowable if a testing process were to provide 90% assurance of achieving the analytical quality required by CLIA PT criteria. Charts of operating specifications (OPSpecs charts) were prepared for commonly used single-rule and multi-rule quality control procedures with 2 and 4 control measurements per run. Of the 23 tests studied, the proposed European specifications for imprecision and inaccuracy were more demanding than the CLIA requirements for 12 tests (albumin, alkaline phosphatase, amylase, calcium, chloride, creatinine, lactate dehydrogenase, lithium, magnesium, total protein, sodium, and thyroxine). The CLIA total error criteria were more demanding than the proposed European specifications for nine tests (alanine aminotransferase, aspartate aminotransferase, total bilirubin, cholesterol, creatine kinase, iron, triglycerides, uric acid, and urea nitrogen). Two tests (glucose, potassium) showed different requirements at different decision levels. Manufacturers and laboratory analysts need to compare these different quality specifications on a test-by-test basis to guide the development, selection, evaluation, and control of laboratory measurement procedures.
Stable expression of enzymatically active human pancreatic lipase in V79 cells: purification and characterization of the recombinant enzyme
Authors: Canalias F , Visvikis A , Thioudellet C , Siest G
Published: January 2020

The serum activity of human pancreatic lipase (HPL; EC 3.1.1.3), the main lipolytic enzyme secreted by the pancreas, is a valuable marker of pancreatic disorders. However, determining lipase activity in human serum is difficult because the substrates used vary in their lipase specificity. A lipase reference material is therefore needed. We describe the production of recombinant HPL (rHPL) in V79 Chinese hamster lung cells. A cDNA encoding the sequence of HPL was subcloned into the pcDNAI eukaryotic expression vector, which was then used to transfect V79 cells. The 50-kDa purified recombinant enzyme is fully active, is glycosylated, and has a pI of 7.0. The catalytic properties of rHPL, determined by using triolein as the substrate, were similar to those of the native enzyme. The V79rHPL cell line we developed might be useful for the production of rHPL suitable for the preparation of a reference material.
Alkaline phosphatase isoforms in serum after liver allograft surgery
Authors: Randolph-Habecker J , Lott J A , Tesi R J
Published: January 2020

Orthotopic liver transplantation (OLT) is now the only available treatment for end-stage liver disease; the major postoperative complications of OLT are rejection and infection. Fractionation of alkaline phosphatase (ALP) isoforms in serum by isoelectric focusing can be used to identify patients with complications. Reference ranges for liver-function tests (LFT) and liver ALP isoforms were established for post-OLT patients with stable postoperative courses and compared with those of patients with complications. We found canalicular, hepatocyte, and high-molecular-mass ALP to be statistically higher in nearly all patients with complications as compared with patients who had a stable postoperative course; these tests may identify patients requiring a liver biopsy. When used in conjunction with LFT and other clinical findings, ALP isoforms could aid in the monitoring of complications and treatment and in the adjustment of immunosuppressive therapy in stable OLT cases.
Relevance of macro creatine kinase type 1 and type 2 isoenzymes to laboratory and clinical data
Authors: Lee K N , Csako G , Bernhardt P , Elin R J
Published: January 2020

From 8322 patients for whom creatine kinase (CK; EC 2.7.3.2) isoenzyme analysis was ordered, we identified 136 patients with macro CK isoenzyme in their serum. There were 36 cases with type 1 (prevalence: 0.43%) and 100 cases with type 2 isoenzyme (prevalence: 1.20%). About three-fourths of the patients were ambulatory at the time of testing, and approximately 90% of the first 68 patients identified survived at least 1 year after macro CK was found in their serum. Age and gender did not differ significantly between the two groups. The serum total CK was significantly higher (P < 0.0005), and an increased CK-MB proportion (> 0.05 of total CK) was also significantly more common (P < 0.0005) in patients with macro CK type 1 than in those with type 2. On average, macro CK type 2 accounted for approximately 25% and macro CK type 1 for approximately 10% of the serum total CK activity. Patients with macro CK type 1 most often had myositis, whereas those with macro CK type 2 most commonly had a malignancy. We conclude that the presence of macro CK isoenzymes has a low prognostic value for impending death, but may support the diagnosis of an autoimmune process (type 1) or malignant cell proliferation (type 2).
Rapid automated enzyme immunoassay of serum amyloid A
Authors: Wilkins J , Gallimore J R , Tennent G A , Hawkins P N , Limburg P C , van Rijswijk M H , Moore E G , Pepys M B
Published: January 2020

Serum amyloid A (SAA), a sensitive acute-phase protein, is the precursor of AA fibrils in reactive amyloidosis. However, SAA is poorly immunogenic, and development and standardization of immunoassays of this protein have been difficult. We established an automated polyclonal/monoclonal microparticle capture enzyme immunoassay, standardized with the World Health Organization prospective reference standard for SAA. A stabilized concentrate of SAA was used for controls and calibrators. The assay range was 1-750 mg/L with CVs < 7% throughout. Plasma and serum gave identical results and no interferences were observed. Linear regression against radial immunodiffusion assay gave a slope of 1.04 (95% confidence interval 0.99-1.10), intercept of -9 mg/L (95% confidence interval -14-3), and residual SD (SEE) of 20 for samples containing < or = 200 mg/L (n = 173). In 105 apparently healthy adults the mean (SD) SAA concentration was 3.7 (3.6) mg/L, the median was 3.0 mg/L, and the range, 0.7-26.4 mg/L. In clinical acute-phase sera, values up to 2200 mg/L were seen. This method will facilitate measurement and investigation of SAA in clinical practice generally and in AA amyloidosis.
Automated HPLC assay of fluoxetine and norfluoxetine in serum
Authors: Nichols J H , Charlson J R , Lawson G M
Published: January 2020

This automated assay determines the concentration of the antidepressant fluoxetine (Prozac) and its active metabolite norfluoxetine in serum by reversed-phase HPLC with spectrophotometric detection. Extraction, injection, and quantification are performed by the Gilson Aspec automated sample handler. The patient's specimen, with added protriptyline as internal standard, is extracted with solid-phase and liquid-liquid methods. Separation is conducted isocratically on a 5-microns (particle size) Supelcosil LC-8-DB reversed-phase column with a triethylamine acetate:acetonitrile mobile phase. The detection limit is 10 micrograms/L and absorbance varies linearly with concentration between 20 and 1,000 micrograms/L for both compounds. Mean recovery was 62% for fluoxetine and 70% for norfluoxetine over linear limits. Within-run and day-to-day imprecision (CV), evaluated at three concentrations (50, 200, and 500 micrograms/L), ranged from 2% to 7%. An extensive interference study of 108 drugs was conducted. Results (n = 58) by the automated method (y) correlated well with those by a manual HPLC method (x): y = 0.96x + 10.20 (r = 0.951, Sylx = 42.9) for fluoxetine, and y = 0.95x - 1.37 (r = 0.917, Sylx = 47.2) for norfluoxetine.
Changes in biliary (high-molecular-mass) and liver isoforms of alkaline phosphatase after baboon-to-human liver transplantation
Authors: Mercer D , Tang M , Marino I R , Demetris A , Fung J , Starzl T , Warty V
Published: January 2020

We report a case of hyperphosphatasemia in a 35-year-old patient with hepatitis B who underwent an orthotopic xenogeneic liver transplant. Marked increases in total alkaline phosphatase (ALP; EC 3.1.3.1) activity began 5 days posttransplantation (six times human normal) and increased to approximately 17 times normal at day 11. Increased ALP persisted for > 40 days and steadily increased to 75 times normal in the patient's last 30 days. Gel electrophoresis detected both liver (LALP) and biliary (high-molecular-mass, BALP) isoforms. LALP measured with ion-exchange columns revealed an activity time course pattern similar to that of total ALP. Results for BALP activity also obtained with ion-exchange columns exhibited broad variability, ranging from 2 to 428 times normal.
Aspartate aminotransferase macroenzyme complex in serum identified and characterized
Authors: Stasia M J , Surla A , Renversez J C , Pene F , Morel-Femelez A , Morel F
Published: January 2020

Macromolecular aspartate aminotransferase was found in the serum of an apparently healthy patient. This complex was composed of aspartate aminotransferase (AST; EC 2.6.1.1) and immunoglobulin. Electrophoresis of the patient's serum showed an abnormal band migrating between mitochondrial (m) and cytosolic (s) AST. The macromolecular complex was purified by gel filtration on Sephacryl S300. The molecular mass of the complex was estimated to be 250 kDa, suggesting that the complex probably consists of one immunoglobulin molecule associated with one AST molecule. By immunoelectrophoresis, the immunoglobulin was found to be an IgG with kappa-lambda type light chain. When we used polyclonal antibodies against human mAST or sAST, the sAST antibodies strongly inhibited the AST activity of the macrocomplex, whereas the mAST antibodies had no effect. Thus the AST molecule of the macrocomplex is an sAST type.
Critical evaluation of current concepts in exposure assessment
Authors: Heinzow B G , McLean A
Published: January 2020

The exploitation of natural resources and the improper use and disposal of thousands of chemicals have resulted in environmental pollution and a potential threat to human health on a global scale. Increasing public concern about environmental exposure to and consequent ill health from contaminants demands informed answers based on valid risk assessment. By assessing internal exposure to pollutants, human biomonitoring focuses on early markers of potential risks to prevent serious adverse effects. Exposure assessment may provide a rational basis for risk assessment, with knowledge of the adequacy of limit values; it may also uncover long-term changes in body burdens and thus help identify the sources and transfer pathways of environmental pollutants. The techniques of biological exposure assessment should be incorporated into epidemiological studies if suitable specimens are available, such as exhaled air, blood, urine, breast milk, or adipose or keratinous tissue. Special precautions must be taken in sampling, storage, and analysis if the findings are to be interpreted correctly and reliable conclusions drawn.
Validity of lead exposure markers in diagnosis and surveillance
Authors: Graziano J H
Published: January 2020

Extensive research has been devoted to the development of biomarkers of environmental and occupational exposure to lead (Pb). This body of work can serve as a paradigm for biomarker development for other chemical exposures. Early efforts focused on indirect measurements of exposure by analyzing precursors and enzymes of a biosynthetic pathway (heme) in blood and urine. However, the direct measurement of Pb in blood has become increasingly simple and reliable and is now widely accepted for pediatric surveillance programs, in part because of known associations of Pb with adverse health outcomes. Other markers of exposure include measurements of Pb in important compartments: bone Pb, tooth Pb, and chelatable Pb. In addition, the technique of stable isotope dilution is available, since Pb exists in numerous nonradioactive isotopic forms. The strengths and weaknesses of all Pb biomarkers for confirming a diagnosis or for epidemiologic research vary widely depending upon the hypothesis under investigation.
Blood concentrations of volatile organic compounds in a nonoccupationally exposed US population and in groups with suspected exposure
Authors: Ashley D L , Bonin M A , Cardinali F L , McCraw J M , Wooten J V
Published: January 2020

Exposure to certain volatile organic compounds (VOCs) commonly occurs in industrialized countries. We developed a method for measuring 32 VOCs in 10 mL of whole blood at low concentration. We used this method to determine the internal dose of these compounds in 600 or more people in the US who participated in the Third National Health and Nutrition Examination Survey. From our study results, we established a reference range for these VOCs in the general population of the US. We found detectable concentrations of 1,1,1-trichloroethane, 1,4-dichlorobenzene, 2-butanone, acetone, benzene, chloroform, ethylbenzene, m,p-xylene, styrene, tetrachloroethane, and toluene in most of the blood samples of nonoccupationally exposed persons. The accuracy of VOC evaluations depends on the ability of investigators to make sensitive and reproducible measurements of low concentrations of VOCs and to eliminate all sources of interference and contamination.
Determining acceptable risks: experimental and epidemiological issues
Authors: Kimbrough R D
Published: January 2020

The extent to which results of experimental toxicology studies can be used to assess risk for humans varies. Administering high doses of chemicals to experimental animals may produce indirect adverse metabolic and nutritional effects, and metabolic pathways may differ at high and low doses. The rate of metabolism at high and low doses and the metabolic pathways of chemicals in different species are important for establishing the reasons for differences in species responses. Animal species best suited to serve as a surrogate for humans vary for different chemicals. For realistic risk assessments, more detailed information on the toxicokinetics of chemicals in humans is needed. As data are developed to link early biochemical changes with future disease, it is important to determine their predictive value. Our present expectations of using various biomarkers to predict future health outcomes may be unrealistic. More baseline data are needed to determine which biomarkers are expressions of exposure and which are predictors of future disease. It is also unclear how lifestyle affects most biomarkers.
Enzymatic determination of sodium, potassium, and chloride in abnormal (hemolyzed, icteric, lipemic, paraproteinemic, or uremic) serum samples compared with indirect determination with ion-selective electrodes
Authors: Hübl W , Wejbora R , Shafti-Keramat I , Haider A , Hajdusich P , Bayer P M
Published: January 2020

We evaluated the effect of hemolysis, icteric discoloration, lipemia, paraproteinemia, and uremia on enzymatic methods for determining sodium, potassium, and chloride, according to the National Committee for Clinical Laboratory Standards EP7-P proposals for testing interference from endogenous substances. The sodium, potassium, and chloride assays (reagent kits supplied by Boehringer Mannheim) were based on electrolyte-dependent beta-galactosidase, pyruvate kinase, and alpha-amylase, respectively. The results were compared with those obtained by indirect ion-selective electrodes (ISE), which in turn had been validated by flame photometry. We analyzed the samples with Hitachi 717, 737, and 911 chemistry analyzers and with an IL943 flame photometer. The enzymatic results were in good agreement with those by ISE, the interference-related differences generally being without clinical significance; however, none of the enzymatic methods could analyze grossly lipemic samples.
Erythrocyte Na+,K(+)-ATPase activity does not predict therapeutic response to calcium antagonists in essential hypertension
Authors: Tanyalçin T , Kutay F , Soydan I , Erlaçin S
Published: January 2020

We investigated whether pre- and posttreatment analysis of erythrocyte membrane Na+,K(+)-ATPase (EC 3.6.1.37) activity would be a useful marker for screening hypertensive patients to determine who might benefit from treatment with calcium antagonists. Erythrocyte Na+,K(+)-ATPase activity and sodium and potassium (ENa, EK) contents were determined in controls and in patients with untreated essential hypertension before and after 4 weeks of treatment with nitrendipine. Na+,K(+)-ATPase activity was significantly (P < 0.0001) less in untreated hypertensive patients (n = 15; 104.60 +/- 29.37 nmol of phosphate produced per milligram of protein per hour) than in controls (n = 15, 171.87 +/- 34.42). After 4 weeks of nitrendipine treatment Na+,K(+)-ATPase activity was greater than in the pretreatment group: 158.13 +/- 26.80 (P < 0.001). Pretreatment ENa contents (22.34 +/- 4.77 mmol/L) were significantly (P < 0.0001) higher than in the normotensive group (13.14 +/- 3.32 mmol/L), but there was no significant difference between the controls and the posttreatment group (14.84 +/- 3.49 mmol/L). The control and pretreatment groups showed negative correlations between enzyme activity and systolic/diastolic blood pressure (P < 0.0001). The control and the posttreatment groups showed an inverse correlation between enzyme activity and ENa contents: r = -0.608 (P < 0.05) and r = -0.724 (P < 0.001), respectively. Although Na+,K(+)-ATPase is restored in hypertensive patients receiving nitrendipine treatment, relative changes in enzyme activity in relation to relative reduction in blood pressure response to treatment were not correlated.
Contribution of dialysis to endogenous oxalate production in patients with chronic renal failure
Authors: France N C , Holland P T , Wallace M R
Published: January 2020

We tested the possibility that the buffering agents in dialysis bath fluid might contribute to increased endogenous oxalate production in dialyzed patients. Using stable isotope dilution mass spectrometry, we obtained oxalate production rates and pool sizes directly for 10 patients in chronic renal failure, 5 of whom were undergoing continuous ambulatory peritoneal dialysis (lactate-buffered fluid). All peritoneal dialysis patients had either increased oxalate production rates or expanded oxalate pools when compared with undialyzed patients in renal failure. From a further four patients receiving maintenance hemodialysis we took blood samples immediately before and after three consecutive dialysis sessions in which the bath-fluid buffering agent (bicarbonate or acetate) was alternated; we analyzed these samples for oxalate and key precursors by capillary gas chromatography. Plasma glycine and serine concentrations remained within the physiological range. Glycolate and oxalate concentrations decreased, but the oxalate remained above normal after dialysis. All changes were independent of the bath-fluid buffering agent. We suggest that dialysis might stimulate the formation of oxalate by removing product inhibition of a late catabolic step.
HPLC determination of polyethylene glycol 400 in urine: oligomeric profile in healthy and celiac disease subjects
Authors: Oliva A , Armas H , Fariña J B
Published: January 2020

The absorption of orally administered polyethylene glycol (PEG) has been used to assess intestinal permeability. We describe a simple HPLC technique to determine the oligomeric profile of PEG excreted in urine. We measured the total (%) PEG excreted in 6 h and the ratio of the four smallest oligomers to the three largest oligomers (expressed as mean percentages). The proposed method differentiates distinct groups of subjects with varying degrees of intestinal permeability detected by intestinal biopsy. The percent of PEG excreted and the oligomer ratio values for healthy subjects were, respectively, 30.1 +/- 3.87 and 0.35 +/- 0.03; for celiac patients on a gluten-free diet, 24.5 +/- 6.65 and 0.45 +/- 0.16; and for celiac patients, 15.0 +/- 5.93 and 1.12 +/- 0.55.
Excess serum osmolality gap after ingestion of methanol: a methodology-associated phenomenon?
Authors: Demedts P , Theunis L , Wauters A , Franck F , Daelemans R , Neels H
Published: January 2020

A patient intentionally ingested an unknown amount of methanol and was admitted to the hospital 6 h later. On admission, the methanol concentration in blood was estimated as approximately of 134 mmol/L, based on the calculation of the osmolal gap. Intravenous ethanol administration and hemodialysis were promptly started. During hemodialysis, several blood samples were collected for determination of methanol and ethanol concentrations. Initially, we used gas chromatography with split-mode injection of pretreated serum samples; however, methanol concentrations turned out to be significantly lower than expected, based on calculated osmolal gap values. Because no explanation for the excess serum osmolal gap was apparent, we reanalyzed samples, using head-space gas chromatography. The methanol concentrations measured were significantly higher and osmolal gap values were no longer excessive.
The fiscal consequences of central vs distributed testing of glucose
Authors: Winkelman J W , Wybenga D R , Tanasijevic M J
Published: January 2020

We have compared the operational, medical, and fiscal consequences of performing urgent glucose testing at the bedside vs in the central laboratory. The turnaround time (TAT) with bedside testing was only 1-2 min shorter than that from the central laboratory, to which specimens are sent by pneumatic tube and from which results are automatically broadcast by computer to the originating site. No significant adverse medical outcomes were associated with this difference in TAT. The cost of bedside testing is approximately twice that of central laboratory testing.
Is glycohemoglobin testing useful in diabetes mellitus? Lessons from the diabetes control and complications trial
Authors: Goldstein D E , Little R R , Wiedmeyer H M , England J D , Rohlfing C L , Wilke A L
Published: January 2020

To address the question, Do laboratory tests cost money or save money? we have used as a model for discussion a common chronic disease, diabetes mellitus, and a widely used laboratory test, that for glycohemoglobin, a measure of long-term glycemia used to manage diabetic patients. Diabetes mellitus is serious, highly prevalent, and costly. In 1992, $1 of every $7 spent on health in the US was for diabetes, predominantly for treatment of the chronic complications of the disease. The recently completed Diabetes Control and Complications Trial (DCCT) demonstrated that development and progression of the chronic complications of diabetes are related to the degree of altered glycemia as quantified by determinations of glycohemoglobin. Thus, use of glycohemoglobin testing for routine diabetes care provides an objective measure of a patient's risk for developing diabetic complications. Results of this test can alert patients and health providers to the need for change in the treatment plan. Optimal use of glycohemoglobin testing for diabetes care will require standardization of test results.
Diagnostic utility of laboratory tests in alcoholic liver disease
Authors: Rosman A S , Lieber C S
Published: January 2020

Alcoholic liver disease is an important health problem in the US. A public health approach is proposed, using laboratory tests to identify patients with early fibrosis of the liver. A variety of serological markers of liver fibrosis based on collagen-related products (e.g., amino-terminal propeptides of type III procollagen) have been investigated. Further studies are needed to determine the optimal combination of tests for discriminating between steatosis and early fibrosis. Laboratory tests are also useful in excluding nonalcoholic liver diseases such as viral hepatitis, hemochromatosis, and Wilson disease. The monitoring of sobriety in patients with alcoholic liver disease by currently available tests is far from ideal. A new marker of excessive alcohol consumption, carbohydrate-deficient transferrin, is not usually affected by liver disease and thus shows promise as a marker of relapse in alcoholic patients. The development of reliable screening markers of fibrosis and sobriety could potentially reduce the health costs and suffering associated with the complications of alcoholic cirrhosis.
Japanese healthcare system: in vitro diagnostic tests and reimbursements
Authors: Shimauchi A
Published: January 2020

In 1961, a new and mandatory National Health Insurance plan was enacted in Japan. This healthcare system has succeeded in providing universal coverage while also containing the growth of national medical expenditures (NME) to the rate of growth of the gross national product (GNP), namely, approximately 4-5% annually, for several decades. All Japanese medical procedures, including dental procedures, prescription drugs, and diagnostic tests, are reimbursed by a fee schedule set by the Ministry of Health and Welfare. The combination of strict fee control and low administration costs has kept the Japanese NME growth below that of the GNP. In 1990, NME was 20.6 trillion yen ($187 billion), total diagnostic testing expenditures (DTE) were 2.3 trillion yen, representing 11.2% of national medical expenditures (NME). Of this amount, in vitro diagnostic testing accounted for 1.4 trillion yen, representing 61% of DTE and 6.8% of NME. Annually, 1.8 billion in vitro diagnostic tests are performed.
Understanding the sodium pump and its relevance to disease
Authors: Rose A M , Valdes R
Published: January 2020

Na,K-ATPase (sodium pump; EC 3.6.1.37) is present in the membrane of most eukaryotic cells and controls directly or indirectly many essential cellular functions. Regulation of this enzyme (ion transporter) and its individual isoforms is believed to play a key role in the etiology of some pathological processes. The sodium pump is the only known receptor for the cardiac glycosides. However, endogenous ligands structurally similar to digoxin or ouabain may control the activity of this important molecular complex. Here we review the structure and function of Na,K-ATPase, its expression and distribution in tissues, and its interaction with known ligands such as the cardiac glycosides and other suspected endogenous regulators. Also reviewed are various disorders, including cardiovascular, neurological, renal, and metabolic diseases, purported to involve dysfunction of Na,K-ATPase activity. The escalation in knowledge at the molecular level concerning sodium pump function foreshadows application of this knowledge in the clinical laboratory to identify individuals at risk for Na,K-ATPase-associated diseases.
Graphite furnace atomic absorption spectroscopic measurement of blood lead in matrix-matched standards
Authors: Bannon D I , Murashchik C , Zapf C R , Farfel M R , Chisolm J J
Published: January 2020

Now that the level of concern for a toxic blood lead concentration is 0.482 mumol/L (10 micrograms/dL), laboratories must meet new requirements to shorten analysis times and increase accuracy and precision of blood lead determinations. We used a matrix-matching method to estimate the lead concentration in blood by graphite furnace atomic absorption spectroscopy (GFAAS). For CDC proficiency samples and the NIST-Certified Blood Reference standard, the performance of this method compared favorably with that of previously published GFAAS methods and of the anodic stripping voltammetric method routinely used in our laboratory. At lead concentrations of 0.242 mumol/L (5.01 micrograms/dL) and 1.478 mumol/L (30.63 micrograms/dL), within-run CVs were 2.78% and 0.68%, respectively; between-run CVs were 4.9% and 1.35%. In 52 study samples with lead content ranging from 0.097 to 3.812 mumol/L (2 to 79 micrograms/dL), 87% of results by the matrix-modified method were within 0.048 mumol/L (1 microgram/dL) of consensus values.
Tamm-Horsfall glycoprotein: role in inhibition and promotion of renal calcium oxalate stone formation studied with Fourier-transform infrared spectroscopy
Authors: Knörle R , Schnierle P , Koch A , Buchholz N P , Hering F , Seiler H , Ackermann T , Rutishauser G
Published: January 2020

Tamm-Horsfall glycoprotein (THP) from healthy probands inhibits the precipitation of calcium oxalate, whereas THP from individuals who repeatedly develop calcium oxalate stones has no effect or even promotes precipitation. Using Fourier-transform infrared spectroscopy, we found a structural differentiation between these functionally different THPs: a decisive difference in sialic acid content. Quantitative analysis for sialic acid showed the same results. THP from healthy probands had a high sialic acid content (51 +/- 9 g/kg), whereas THP from recurrent stone formers had a decreased sialic acid content (21 +/- 4 g/kg). This explains the dual role of THP in the precipitation of calcium oxalate and the formation of renal stones and shows the importance of glycosylation in the function of this glycoprotein.
Sensitive time-resolved fluoroimmunoassay of salmon calcitonin
Authors: Rong H , Tørring O , Sääf M , Sjöstedt U , Sjöberg H E , Bucht E
Published: January 2020

A two-site assay was developed by use of the "dissociation and enhancement lanthanide fluoroimmunoassay" (DELFIA) technique for determination of salmon calcitonin (SCT) in serum after administration to osteoporotic patients. Polyclonal antibodies were produced in rabbits immunized with SCT coupled to ovalbumin. After affinity purification, the antibodies were used both as immobilized capture antibodies and as Eu-chelate-labeled signal antibodies. A sensitive assay with a detection limit of 1.1 pmol/L was achieved, and no cross-reaction with human calcitonin was observed. The intra- and interassay CVs were < 12% (n = 10) and < 15% (n = 4), respectively. Analytical recovery of SCT added to serum was 91% +/- 3% (mean +/- SD, n = 4). SCT was measurable in all the samples from eight osteoporotic patients after subcutaneous SCT administration. We conclude that this new sensitive and specific two-site DELFIA can reliably measure SCT in serum.
Miniature field-flow fractionation system for analysis of blood cells
Authors: Yue V , Kowal R , Neargarder L , Bond L , Muetterties A , Parsons R
Published: January 2020

Field-flow fractionation is an analytical tool that has been historically used to separate species, ranging from molecules to particles or cells several micrometers in size. This technology can effect separation by size, density, charge, or other physical properties, depending on the configuration of the field-flow system. We have developed a miniature field-flow system to analyze cell populations in a small, 125-microns-deep channel 19 cm long. Gravity is used as the primary field to effect separation, and cell analysis is performed in < 20 min. Erythrocytes elute as a single peak when diluted blood is fractionated in this system. Analysis of blood samples from several donors (normal controls and patients with sickle cell anemia) yields erythrocyte peaks with slightly different mobilities (elution times). Peak mobility does not directly correlate with mean cell volume or other standard erythrocyte parameters. Cell density appears to be a key factor in determining cell mobility with this system.
Starburst dendrimers: enhanced performance and flexibility for immunoassays
Authors: Singh P , Moll F , Lin S H , Ferzli C , Yu K S , Koski R K , Saul R G , Cronin P
Published: January 2020

Starburst dendrimers are novel, water-soluble polymeric materials, with a well-defined composition and structure. In our application, we used dendrimers composed of poly(amidoamine) groups to which we coupled several specific antibodies, to investigate potential formats based on radial partition immunoassay. The coupled antibodies have retained their stability and immunological binding after coupling, both in solution and when immobilized onto a solid support. On the basis of our feasibility studies with model systems, we conclude that immunoassays can be developed with performance equivalent to or better than that in many established systems. By application of a mixture of the dendrimer-coupled antibody and the analyte of interest to the solid phase, we have investigated the performance characteristics of solution-phase immunoassays. Our experiments demonstrate enhanced sensitivity for creatine kinase MB isoenzyme (CKMB), thyrotropin, and myoglobin assays and reduced instrumental analysis time for the CKMB assay.
Total porphyrin and coproporphyrin and uroporphyrin fractions in urine measured by second-derivative spectroscopy
Authors: Buttery J E , Chamberlain B R , Gee D , Pannall P R
Published: January 2020

A second-derivative scan of an acidified urine sample allows the amplitude of deflection (delta A) and the minimum wavelength of the trough (lambda min) to determine the correct porphyrin concentration and the coproporphyrin:uroporphyrin (copro:uro) ratio, respectively, from a nomogram constructed from calibrator solutions. We measured 24 urine samples for total porphyrin as coproporphyrin equivalents and adjusted the results with factors from the nomogram. The adjusted results (x) (mean +/- SE, 501 +/- 57 nmol/L) compared favorably with the expected results (y) (514 +/- 57). The regression equation and correlation coefficient were: y = 0.993x - 8.9 (r = 0.998, S(y/x) = 16.2). Results of the copro:uro ratio derived by second-derivative spectroscopy and HPLC showed no significant difference (chi 2-test) from samples with various copro:uro ratios. Recovery studies on four urine samples supplemented with known proportions of coproporphyrins and uroporphyrins gave good agreement between the measured and the expected porphyrin ratios. The overall imprecision (CV) of the assay ranged from 3.6% to 6.0% for coproporphyrin and from 3.2% to 9.1% for uroporphyrin.