bluuebunny/crossref_metadata_2025 huggingface dataset:
Biomarkers in epidemiology
Authors: Grandjean P
Published: January 2020

A biomarker is a measurable event occurring in a biological system, such as the human body. In environmental epidemiology, a biomarker represents a subclinical and reversible change; it is not a diagnostic test, but an indicator that an early change has occurred that could later lead to clinical disease. Although some biomarkers may belong to more than one class, they are often separated into biomarkers of exposure, biomarkers of effect, and biomarkers of susceptibility. Biomarkers can be used to classify and quantify environmental exposures and their related effects, and many methods may be applicable in toxicological experiments as well as in epidemiology. Accordingly, biomarker epidemiology is undergoing rapid development and expansion and is becoming one of the most promising areas of environmental research. Although expanded applications should be encouraged, many biomarkers are poorly characterized, and attention should be paid to defining their properties in detail.
Biomarkers as tools in human health risk assessment
Authors: Greim H , Csanády G , Filser J G , Kreuzer P , Schwarz L , Wolff T , Werner S
Published: January 2020

Evaluation of occupational or environmental risk due to exposure to chemicals requires sufficient information on the toxic profiles, mechanisms of action, toxicokinetics, dose-response relation, exposure, and the target dose. Usually exposure is estimated by measuring concentrations of the agent in air, food, water, soil, dust, or other media with which a population or an individual is in contact. However, this external exposure is only a rough estimate for the internal exposure (agent dose or its metabolite at the critical target in the organism). Factors of influence are bioavailability of the chemicals, variations in concentrations and routes of exposure, physical activity, and individual variation in rates of metabolism, distribution, and excretion. All these affect the concentration of the toxic agent at the critical target, which is the most precise information for risk assessment. Thus, internal exposure is best measured by determining the concentration of the toxicant or its ultimate metabolite at the critical site in the target organ or by determining adducts with cellular macromolecules such as proteins, amino acids, DNA, or its bases. The latter are easily available in experimental toxicology from animal experiments but only occasionally from humans. For health surveillance such data usually are not available, because they require invasive procedures such as biopsies. Therefore, more accessible body fluids or tissue are used, such as blood, urine, or adipose tissue, or adducts with macromolecules such as albumin or hemoglobin in the blood, DNA adducts in peripheral lymphocytes, or altered DNA bases in urine such as 8-hydroxyguanine. All of these are indicators for exposure, whereas risk can only be estimated if the correlation between their deviations from normal and the dose-response at the critical target is known.
Macromolecular adducts caused by environmental chemicals
Authors: Neumann H G , Albrecht O , van Dorp C , Zwirner-Baier I
Published: January 2020

We describe three biomonitoring studies in which hemoglobin (Hb) adducts were used as biochemical markers to assess indirectly the target dose of genotoxic chemicals. We monitored the exposure to 1,3-butadiene in occupationally exposed workers and in two control groups by analyzing the adducts formed by the reaction of the first activation product, butadiene monoepoxide, with the terminal valine of Hb; we also measured hydrolyzable adducts formed by the reaction of metabolically formed nitroso derivatives with Hb from five selected nitropolycyclic aromatic hydrocarbons (1-nitropyrene; 2-nitrofluorene, 3-nitrofluoranthrene, 6-nitrochrysene, and 9-nitrophenanthrene) in coke oven workers of different job categories and control workers of the same geographical area. We detected hydrolyzable adducts from monocyclic nitroarenes in blood from individuals living in a contaminated area where explosives had been produced and from controls. The contaminants considered were 2,4,6-trinitrotoluene; 2,4- and 2,6-dinitrotoluene; and 1,3-dinitrobenzene. Differences between groups were significant, but interindividual variation was great and back-ground exposures must be considered.
DNA repair and epidemiology of basal cell carcinoma
Authors: Grossman L , Wei Q
Published: January 2020

In a molecular epidemiological study of DNA repair, host reactivation assay was used to measure the DNA repair capacity of cryopreserved lymphocytes from 88 primary basal cell carcinoma (BCC) patients and 135 cancer-free controls. In this study population, reduced repair of ultraviolet radiation-induced DNA damage contributed to the risk of sunlight-induced BCC. A family history of BCC is associated with low DNA repair. Repair of ultraviolet radiation-damaged DNA declines at a rate of approximately 1%/year in noncancerous controls. Reduced DNA repair is more likely seen in young BCC patients, indicating that BCC is a premature aging disease of the skin. The persistence of photochemical damage because of reduced repair results in point mutations in the p53 gene and allelic loss of the nevoid BCC gene located on chromosome 9q. Xeroderma pigmentosum appears to be a valid paradigm for the role of DNA repair in BCC in the general population.
Polymorphisms in drug-metabolizing enzymes as modifiers of cancer risk
Authors: Spurr N K , Gough A C , Chinegwundoh F I , Smith C A
Published: January 2020

The identification of low-penetrance genes, the polymorphisms of which increase an individual's risk of developing cancer, are likely to be extremely important in the general population. In this report we analyzed two genes involved in detoxification. In a number of loci, we identified polymorphic variation correlating with the expression of the gene product. We analyzed two such loci, the cytochrome P-450 gene CYP2D6 and the N-acetyltransferase 2 (NAT2) genes, in patients with bladder and colon cancer, respectively. We observed no statistically significant associations between the control and cancer populations; however, there was a small increase in heterozygote number in bladder cancer.
Do environmental estrogens contribute to the decline in male reproductive health?
Authors: Jensen T K , Toppari J , Keiding N , Skakkebaek N E
Published: January 2020

Several observations suggest that male reproductive health has been declining since World War II in many countries. The incidence of testicular cancer, hypospadias, and cryptorchidism has been increasing and semen quality has been decreasing, and these may have a common etiology. Treatment of several million pregnant women with the synthetic estrogen diethylstilbestrol led to an increase in these conditions among the sons of these women. These abnormalities probably arise during fetal development. The similarity between these effects and the adverse change in male reproductive development and function raised the question of whether the adverse changes are attributable to altered exposures to estrogenic and other endocrine-disrupting agents during fetal development. We speculate that alteration in exposure to estrogen in the past half-century may have caused the changes in male reproductive health.
Improved PCR amplification/Hhal restriction for unambiguous determination of apolipoprotein E alleles
Authors: Appel E , Eisenberg S , Roitelman J
Published: January 2020

We present a modification to the polymerase chain reaction amplification/Hhal restriction isotyping method for human apolipoprotein (apo) E. This method includes a mutagenic forward primer and 5'-end labeling of both primers. These modifications of the original method described by Hixon and Vernier (J Lipid Res 1990;31:545-8) allow sensitive and unambiguous determination of apoE genotypes.
Analytical and clinical performance of improved Abbott IMx CA 125 assay: comparison with Abbott CA 125 RIA
Authors: Thomas C M , Massuger L F , Segers M F , Schijf C P , Doesburg W H , Wobbes T
Published: January 2020

We compared the improved Abbott IMx cancer antigen (CA) 125 assay (cat. no. 7A89) with the Abbott CA 125 RIA. Serum specimens were from healthy perimenopausal women (n = 124) and from patients with benign gynecologic and nongynecologic diseases (n = 124), ovarian carcinoma (n = 104), or other malignancies (n = 193). The IMx assay detected as little as 0.193 kAU/L CA 125 (AU = arbitrary Abbott unit), demonstrated up to 29% overestimation upon serum dilution, low within-assay (2.7-5.6%) and between-assay (4.8-8.2%) CVs, and no high-dose hook effect < or = 46,000 kAU/L nor influence from human anti-mouse antibodies in serum of women injected with OC 125 F(ab')2. Values by IMx were 20% lower than by RIA for healthy perimenopausal women (n = 100; IMx = 0.80 RIA - 2.5 kAU/L), and at least 50% higher for those with benign or malignant ovarian disorders at concentrations < 100 kAU/L. Receiver-operating characteristic (ROC) curve analysis of ovarian neoplasma vs perimenopausal controls indicated a gain of specificity and sensitivity with the improved IMx assay over the RIA, but ROC performance was the same with either assay if patients with benign ovarian disorders were used as controls.
Clinical utility of biochemical analysis of cerebrospinal fluid
Authors: Watson M A , Scott M G
Published: January 2020

In addition to microbial culture, cytology, and immunological studies, physicians rely on the clinical chemistry laboratory for biochemical analysis of patients' cerebrospinal fluid (CSF). However, apart from routine glucose and protein determinations, the clinical value of other CSF analytes is often unclear. Here, we review the literature pertaining to the use of CSF biochemical measurements in managing patients with infectious disease, neoplasia, stroke and trauma, and dementia. Although a small number of studies demonstrate potential usefulness of some markers, we conclude that, without further study, the data are insufficient to support the routine clinical use of most of the analytes examined.
Immunoprecipitation assay for autoantibodies to steroid 21-hydroxylase in autoimmune adrenal diseases
Authors: Colls J , Betterle C , Volpato M , Prentice L , Smith B R , Furmaniak J
Published: January 2020

Adrenal autoantibodies characteristic of autoimmune Addison disease are directed towards steroid 21-hydroxylase (21-OH; EC 1.14.99.10). We describe a new assay to measure 21-OH autoantibodies (21-OH Abs), based on immunoprecipitation by the antibodies of 35S-labeled human 21-OH. Using this immunoprecipitation assay (IPA), we detected 21-OH Abs in 42 of 64 (66%) patients with Addison disease and in 14 of 19 (74%) patients with autoimmune polyendocrine syndromes type I and type II. No 21-OH Abs were detected by the IPA in any patients with Addison disease attributable to tuberculosis (n = 9) or adrenoleukodystrophy (n = 9) or in patients with autoimmune thyroid disease (n = 28), systemic lupus erythematosus (n = 10), myasthenia gravis (n = 10), rheumatoid arthritis (n = 10), or insulin-dependent diabetes mellitus (n = 12). None of the 26 sera from healthy normal blood donors was positive for 21-OH Abs by the assay. We found good agreement between 21-OH Abs measured by IPA and by Western blotting (r = 0.83, n = 123, P < 0.001). The inter- and intraassay CVs for IPA were well < 10% at high, medium, and low concentrations of 21-OH Abs. Overall, our studies indicate that the IPA provides a specific, sensitive, and convenient system for measuring 21-OH Abs.
Direct enzyme-linked immunosorbent assay and a radioimmunoassay for melatonin compared
Authors: Chegini S , Ehrhart-Hofmann B , Kaider A , Waldhauser F
Published: January 2020

A novel commercially available ELISA for direct measurement of melatonin concentration in serum was evaluated and compared with an RIA routinely used in our laboratory. The direct ELISA is technically simpler, requires a smaller sample volume (0.1 mL), and compares well with RIA in terms of stability of the calibration curve and intra- and interassay CVs. Correlation with RIA measurements is, however, suboptimal (y = 0.39x + 56; r = 0.65, P < 0.001; n = 138), which may be due to a serum effect, as evidenced by dilution studies. Furthermore, the detection range of the ELISA does not cover the physiological daytime melatonin concentrations in humans. Adding an extraction and 10-fold concentration step shifted the detection range of the ELISA to include low physiological concentrations as well. Correlation with RIA measurements also improved significantly (y = 0.97x-23; r = 0.95, P < 0.001; n = 105), probably due to removal of the serum effect. Although extraction increases the required sample volume (1.5 mL), work load, and procedure time, this step is necessary for the ELISA to compete successfully with RIA.
Isotope dilution mass spectrometry as a candidate definitive method for determining total glycerides and triglycerides in serum
Authors: Ellerbe P , Sniegoski L T , Welch M J
Published: January 2020

A new isotope dilution mass spectrometric method for total glycerides and triglycerides in human serum is described. Total glycerides are defined as the sum of tri-, di-, and monoglycerides plus free glycerol; triglycerides are defined as the pure triglyceride species. In both determinations, serum samples are supplemented by addition of [13C3]tripalmitin, processed, derivatized, and the abundance ratios of selected ions are determined. Bias is investigated by quantifying the analyte in the same samples under different chromatographic and ionization conditions. The analytes were determined in two human serum pools. The CV for a single measurement ranged from 0.35% to 0.72%, and the relative SEM ranged from 0.10% to 0.34%; there was no significant bias in the measurements. The combination of high precision and absence of significant bias in the results qualify this method for consideration as a Definitive Method as defined by the National Committee for Clinical Laboratory Standards.
Significance of low serum alkaline phosphatase activity in a predominantly adult male population
Authors: Lum G
Published: January 2020

The causes for low serum alkaline phosphatase (ALP) activity (reference range 30-115 U/L) in a large Veterans Medical Center were reviewed. Of 69,864 ALP determinations made over a 4-year period, 130 were low (< 30 U/L, 0.19%), representing 88 individual patients. Of these, 83 (primarily men, 96%) patients' charts were reviewed and classified into two groups, those with and those without conditions previously reported to be associated with decreased serum ALP activity: 47% had conditions associated with low ALP activity, the most frequent being cardiac surgery and cardiopulmonary bypass (26.5%), malnutrition (12.0%), magnesium deficiency (4.8%), hypothyroidism (2.4%), and severe anemia (1.2%); 53% of patients did not have clinical conditions previously associated with low ALP activity. No case of clinically apparent hypophosphatasia, for which low ALP activity is the defining characteristic, was found in this population of veterans. A low serum ALP may be of significance in other patient populations such as children, where it is associated with achondroplasia and cretinism, or in postmenopausal women with osteoporosis taking estrogen replacement therapy. In the predominantly adult male population in this study, low ALP activity was rare; it was seen most frequently in cardiac surgery patients postoperatively, a clinical condition heretofore not commonly associated with low serum ALP activity.
Automated measurement of alpha-amylase isoenzymes with 6(3)-deoxymaltotriose as selective amylase inhibitor
Authors: Uchida R , Tokutake S , Motoyama Y , Hosoi K , Yamaji N
Published: January 2020

We developed an automated method for measurement of alpha-amylase isoenzymes in serum by a single kinetic assay (SKA) and a double kinetic assay (DKA) with 2-chloro-4-nitrophenyl-6(5)-azido-6(5)-deoxy-beta-maltopentaoside as a substrate and 6(3)-deoxymaltotriose (DOG3) as a novel selective amylase inhibitor. DOG3 showed a large difference in inhibitory activity between human pancreatic alpha-amylase (HPA; 86.9% inhibition) and salivary alpha-amylase (32.1% inhibition) at 0.33 mmol/L. Constant inhibition was obtained immediately after addition of DOG3. The inhibitory effect did not change with variation in concentrations of amylase up to approximately 3000 U/L. The results obtained by SKA correlated well with those obtained by three methods: monoclonal antibody (r = 0.988), wheat germ inhibitor (r = 0.989), and DKA (r = 0.995). The within-run and between-run CVs for HPA were 0.63-2.32% on SKA, 0.69-1.81% on DKA. No significant interferences by endogenous serum compounds were observed with the proposed methods.
Age-related reference values for urinary excretion of sialic acid and deoxysialic acid: application to diagnosis of storage disorders of free sialic acid
Authors: Romppanen J , Mononen I
Published: January 2020

We have established by HPLC age-related reference intervals for sialic acid urinary excretion in 364 control individuals to assist in evaluating the clinical significance of the free sialic acid concentration in urine. In addition, an HPLC method for quantitative analysis of free deoxysialic acid was developed, and age-related reference intervals for excretion of this compound in urine were established. In patients with storage disorders of free sialic acid (n = 11) the sialic acid excretion was increased 2- to 35-fold, compared with the mean value of the control subjects in the corresponding age group, and exceeded the interval in each case. The excretion of deoxysialic acid was within the reference interval in all of the patients, indicating that its metabolism was not affected in the disorders. The age-related reference values assist in evaluating the excretion of free sialic acid in the diagnosis of storage disorders of free sialic acid, especially in young children.
HLA class II genotyping: two assay systems compared
Authors: Thonnard J , Deldime F , Heusterspreute M , Delepaut B , Hanon F , De Bruyère M , Philippe M
Published: January 2020

In the last few years, a variety of DNA-based human leukocyte antigen (HLA) typing methods have emerged, revealing the extreme polymorphism of HLA genes. This polymorphism makes it difficult for a clinical laboratory to establish the best HLA typing strategy. In this study we have compared two techniques for performing HLA-DRB typing: a commercial rapid assay based on the polymerase chain reaction (PCR) followed by reverse dot-blot hybridization of the PCR products (the Inno-LiPA assay), and a method based on PCR followed by restriction fragment length polymorphism analysis. We found that both methods provide reliable results with a high rate of concordance (97%) and that Inno-LiPA is convenient for large-scale routine typing. However, if a high-resolution allelic typing is required, each method lacks accuracy but using them in association improves the accuracy of the results.
Serum concentrations of 10 acute-phase proteins in healthy term and preterm infants from birth to age 6 months
Authors: Kanakoudi F , Drossou V , Tzimouli V , Diamanti E , Konstantinidis T , Germenis A , Kremenopoulos G
Published: January 2020

Aiming to define the evolution pattern of 10 acute-phase proteins in early infancy, we measured nephelometrically the serum concentrations of albumin, prealbumin, retinol-binding protein, transferrin, ceruloplasmin, hemopexin, haptoglobin, alpha 1-acid glycoprotein, alpha 2-macroglobulin, and alpha 1-antitrypsin in 395 term and preterm infants (gestational ages 26-41 weeks). Measurements were performed within 24 h after birth and then at the end of 1 (n = 171), 3 (n = 155), and 6 (n = 90) months afterwards. Data obtained from 250 healthy adults were used as adult reference values. All proteins increased progressively with postnatal age, except for alpha 1-antitrypsin, which remained stable from birth to the 6th month. Concentrations of almost all measured proteins were significantly lower in preterm than in term infants in the first 3 months. Compared with adult values, alpha 2-macroglobulin and alpha 1-antitrypsin were higher in infants throughout the 6 months. The other proteins were significantly lower at birth than adult values but after 6 months, only albumin, prealbumin, retinol-binding protein, and alpha 1-acid glycoprotein still remained lower in infants. Thus both gestational and postnatal age should be considered when interpreting concentrations of these proteins in early infancy.
Immunoassay of a tartrate-resistant acid phosphatase in serum
Authors: Cheung C K , Panesar N S , Haines C , Masarei J , Swaminathan R
Published: January 2020

A tartrate-resistant acid phosphatase (TRACP) was purified from cord plasma by use of cation-exchange chromatography (carboxymethyl Sepharose), gel-filtration chromatography (Sephacryl S-200), and preparative isoelectric focusing. After raising polyclonal antibodies to the purified TRACP in rabbit, we used the antiserum to develop an ELISA. The antiserum cross-reacted with an extract of bone, but not with extracts of spleen, erythrocytes, platelets, or osteoblasts or with prostatic acid phosphatase. With this ELISA we determined the concentration of serum TRACP in healthy men to be 197 (61-301) micrograms/L (median and range). Serum TRACP concentrations were significantly higher in children and postmenopausal women and in patients with chronic renal failure, hyperparathyroidism, or hyperthyroidism. Estrogen replacement therapy of postmenopausal women for 3-6.5 months decreased their serum TRACP concentration by 70%.
Testing the accuracy of total cholesterol assays in an external quality-control program. Effect of adding sucrose to lyophilized control sera compared with use of fresh or frozen sera
Authors: Baadenhuijsen H , Demacker P N , Hessels M , Boerma G J , Penders T J , Weykamp C , Willems H L
Published: January 2020

We studied the suitability of various types of human serum preparations to test the accuracy of total cholesterol measurements in the External Quality Assessment scheme in The Netherlands, in which approximately 180 laboratories participate. Checked against the certified Abell/Kendall Reference Method, large reagent-dependent negative biases were observed with lyophilized serum that was insufficiently cryoprotected. The biases for the reagents of Du Pont, Roche, and Beckman averaged -16.7%, -9.2%, and -7.6% respectively; the least bias, -0.4%, was obtained with reagent from Boehringer Mannheim. The beneficial effect of cryoprotection with sucrose was demonstrated by the decrease in interreagent variation from 5.4% to 1.9%, the latter value being comparable with the values for fresh and once-frozen pooled serum (1.3% and 1.7%, respectively). We conclude that the detrimental effect of lyophilization on serum matrix can be minimized by suitable cryoprotection with 200 g/L sucrose.
Standardized method for high-resolution 1H-NMR of cerebrospinal fluid
Authors: Wevers R A , Engelke U , Wendel U , de Jong J G , Gabreëls F J , Heerschap A
Published: January 2020

This study describes a standardized method for recording single-pulse 1H-nuclear magnetic resonance (1H-NMR) spectra from cerebrospinal fluid (CSF). Quantitative data for alanine, valine, threonine, and lactic acid correlated well with data obtained with conventional techniques. The pH of the samples is important for the reproducibility of the chemical shift of resonances, and should be standardized to improve recognition and assignment of resonances. A database of resonances from various metabolites is presented. Fifty compounds could be identified in CSF, 15 of which had not been observed earlier in NMR studies of CSF. We describe for the first time in the literature, to our knowledge, 3-hydroxyisovaleric acid as a regular component of many CSF samples. As examples of the diagnostic power of the technique, spectra are shown of CSF from patients with three different inborn errors of metabolism. We found high concentrations of N-acetylaspartic acid, citric acid, and succinic acid in CSF from a patient with Canavan disease. This is indirect evidence for the existence of a carrier mechanism that is shared by these di- and tricarboxylic acids.
Nonisotopic competitive RT-PCR assay to measure MDR1 gene expression
Authors: Debuire B , Sol O , Lemoine A , May E
Published: January 2020

We report an original application of competitive reverse transcription-polymerase chain reaction (RT-PCR) for the quantification of MDR1 mRNA in clinical specimens by simultaneous reverse transcription and PCR amplification of cellular RNA with decreasing amounts of an internal standard. The competitor RNA shares the same MDR1 primer sequences as the cellular mRNA, but yields a different-sized PCR product. This allows resolution of the amplified cDNA fragments after agarose gel electrophoresis and ethidium bromide staining. The concentration of MDR1 mRNA is derived from the ratio between the intensities of the bands corresponding to the amplified products. We have used this assay to measure MDR1 expression in breast carcinomas and assessed the precision, sensitivity, and accuracy of the method. Competitive RT-PCR is a simple, highly specific, nonradioactive procedure for the quantification of MDR1 mRNA and is particularly suitable for use in the clinical laboratory.
Gene amplification for c-erbB-2, c-myc, epidermal growth factor receptor, int-2, and N-myc measured by quantitative PCR with a multiple competitor template
Authors: Sestini R , Orlando C , Zentilin L , Lami D , Gelmini S , Pinzani P , Giacca M , Pazzagli M
Published: January 2020

We recently proposed a quantitative PCR procedure for the absolute measurement of c-erbB-2 oncogene amplification, based on the simultaneous polymerase chain reaction (PCR) amplification of the target gene and of a competitor DNA molecule acting as internal standard. To increase the number of assayable oncogenes and the accuracy of the quantitative comparison of gene amplification degree within the same tumor, we have now constructed a single synthetic competitor for int-2, c-myc, N-myc epidermal growth factor receptor, and c-erbB-2 genes, and for the reference gene beta-globin. This competitor was constructed by a two-step recombinant PCR procedure and inserted as a 297-bp sequence in a plasmid. The order of primer insertion was designed to obtain competitors of comparable sizes to those of the respective genomic targets, but still easily recognizable from the latter ones by gel electrophoresis. The clone competitor was tested to evaluate the linearity range for each assay. The present application of quantitative PCR based on a multiple competitor represents the first approach for the achievement of a single reagent for the evaluation of a panel of genes potentially amplified in human tumors.
Nitrate determination in biological fluids by an enzymatic one-step assay with nitrate reductase
Authors: Bories P N , Bories C
Published: January 2020

We report a simple, enzymatic, end point method for determining nitrate in serum and urine with use of nitrate reductase from Aspergillus sp. (EC 1.6.6.2). The decrease in absorbance at 340 nm as a result of the oxidation of beta-NADPH is measured. We used FAD as a supplementary electron carrier and added an internal standard to avoid interference from possible inhibitors of the enzymatic reaction. The method was linear from 5 to 200 mumol/L nitrate in serum. Within-run (n = 12) and total (n = 14 days) imprecisions (CV) were 5.1-7.7% and 6.2-9.8%, respectively, at 20-90 mumol/L nitrate in serum. Recoveries of added nitrate were 80-106%. The median (range) concentration in serum of 20 healthy subjects was 16 (0-42) mumol/L.
Characteristics of creatine release during acute myocardial infarction, unstable angina, and cardiac surgery
Authors: Delanghe J R , De Buyzere M L , De Scheerder I K , Cluyse L P , Thierens H M
Published: January 2020

Creatine release was compared in various conditions of muscle damage: acute myocardial infarction (AMI), unstable angina, and cardiac surgery. After AMI, serum and urine creatine concentrations increased transiently. After cardiopulmonary resuscitation, serum creatine values were significantly higher because of impaired renal function, whereas urinary creatine concentrations were comparable. In 38 patients with unstable angina, no significant changes in serum and urine creatine concentrations were seen. In 37 of 92 AMI patients, secondary creatine peaks were observed 20.9 +/- 8.1 h after onset of symptoms. The magnitudes of the first and second peaks were correlated: Spearman r = 0.66. In 24 patients who underwent cardiac surgery, the changes in creatine concentration in serum during surgery were very small, despite the presence of muscle trauma.
Candidate reference method for determining serum creatinine by isocratic HPLC: validation with isotope dilution gas chromatography-mass spectrometry and application for accuracy assessment of routine test kits
Authors: Thienpont L M , Van Landuyt K G , Stöckl D , De Leenheer A P
Published: January 2020

We present a candidate Reference Method for determining creatinine in serum, based on isocratic HPLC. The chromatographic column, with alkaline-treated aluminum oxide as stationary phase, is eluted with an equivolume mixture of methanol/acetonitrile containing 70 mL/L aqueous NaOH (10 mmol/L), and ultraviolet absorbance is detected at 240 nm. We investigated the ruggedness of the method and validated its performance with isotope dilution gas chromatography-mass spectrometry (ID GC-MS) for a set of 22 patients' sera and 10 commercially available lyophilized control materials. The mean deviation from ID GC-MS was +0.1% (range, -2.2% to +2.2%). The between-day CV, calculated from six independent measurements performed on three different days, was 0.9% (range 0.2% to 1.9%); the within-run CV was 0.8%. The total error of the method was < 3%. These performance characteristics make the method suitable for target-setting of quality-control materials and accuracy assessment of routine test kits. For the latter application, done with split-sample measurements of a panel of 83 patients' specimens, we used the Boehringer Mannheim enzymatic creatinine PAP test on the Hitachi 911, the Kodak Ektachem single-slide enzymatic creatinine test on the Ektachem 700, the Merck Jaffé kinetic assay on the Mega analyzer, and the Roche Jaffé kinetic test on the Cobas Mira.
Detection of Charcot-Marie-Tooth type 1A duplication by the polymerase chain reaction
Authors: Blair I P , Kennerson M L , Nicholson G A
Published: January 2020

Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary peripheral neuropathy with a genetic locus on chromosome 17p11.2. The majority of patients carry a duplicated DNA segment that encompasses the gene PMP22, which encodes a peripheral myelin protein. PMP22 is the crucial gene involved in the pathogenesis of CMT1A. Molecular diagnosis of CMT1A requires detection of this duplicated segment. Existing methods for detection of the duplication are laborious and time consuming. We have developed a set of polymorphic (AC)n repeat markers (contained within the duplication) for use in the polymerase chain reaction, which give a high probability of detecting three unique alleles in affected individuals. This test detected 85% of a panel of 52 CMT1A patients in which the duplication had previously been demonstrated.
Fully automated assay for cobalamin-dependent methylmalonyl CoA mutase
Authors: Riedel B , Ueland P M , Svardal A M
Published: January 2020

We constructed a fully automated assay for the cobalamin-dependent enzyme methylmalonyl coenzyme A (CoA) mutase. The assay involves preincubation of the enzyme with adenosylcobalamin, incubation with substrate, termination of the reaction by adding trichloroacetic acid, filtration to remove precipitated protein, and finally analysis of the filtrate (containing methylmalonyl CoA and the product succinyl CoA) by HPLC. These steps were carried out by an inexpensive programmable autosampler equipped with thermostated sample racks and mobile disposable extraction column racks used here as a sample filtering device. A central element in the developmental work was to measure stability of reagents, enzyme, and product against the storage conditions during unattended analysis and the time table of the program. We evaluated the performance of the method by measuring methylmalonyl CoA mutase activity in rat liver, human fibroblasts, and human glioma cells. The within-run imprecisions (CV) were 2-10% for measuring enzyme activity in 20 replicate samples of a homogenate (test of the automated assay), and 7-12% for measuring enzyme activity in homogenates from 20 culture dishes (test of the total procedure). The method allows the unattended analysis of 56 samples per 24 h. This strategy for automation may be easily adapted for other enzyme assays.
Role of clinical practice guidelines and clinical profiling in facilitating optimal laboratory use
Authors: Kelly J T
Published: January 2020

Many studies have demonstrated the potential benefits of clinical practice guidelines in improving clinical practice. Moreover, several different kinds of clinical profiles have been developed to address issues related to quality, utilization, and outcomes of specific healthcare practices. With this expanded use of clinical practice guidelines and clinical profiles, clinical laboratory specialists have an opportunity to play an influential leadership role.
Equivalent early sensitivities of myoglobin, creatine kinase MB mass, creatine kinase isoform ratios, and cardiac troponins I and T for acute myocardial infarction
Authors: Mair J , Morandell D , Genser N , Lechleitner P , Dienstl F , Puschendorf B
Published: January 2020

Early sensitivities of creatine kinase (CK), CKMB (activity and mass), CKMM and CKMB isoform ratios, myoglobin, cardiac troponin I (cTnI), and cardiac troponin T (cTnT) were compared to find the most sensitive serum marker for acute myocardial infarction (AMI) during the first hours after onset of chest pain. In a prospective study we investigated 37 consecutive patients with AMI who were admitted to the coronary care unit within 4 h after onset of chest pain. Blood samples were drawn every hour for the first 10 h after admission. CKMB mass concentrations, CKMM and CKMB isoform ratios, myoglobin, cTnI, and cTnT increased significantly (P < or = 0.0067) earlier than CK and CKMB activity and were also significantly (P < or = 0.046) and markedly more sensitive on admission. Differences in early sensitivities of myoglobin, CKMB mass, CK isoform ratios, cTnI, and cTnT were small and not significant. Therefore, turnaround time and practicality for emergency determination of methods, specificities of markers, the required specificity in the individual patient, and costs mainly determine the choice among myoglobin, CKMB mass, CK isoforms, cTnI, and cTnT.
Tutorial software for clinical chemistry incorporating interactive multimedia clinical cases
Authors: Hooper J , O'Connor J , Cheesmar R , Price C P
Published: January 2020

We have developed computer-based clinical case histories incorporating multimedia elements to aid the learning of medicine in a problem-based manner. Topics have been developed in the specialty of Clinical Biochemistry but the approach used is suitable for any branch of clinical medicine. Each topic has material aimed at medical students and also postgraduate candidates for professional examinations. A browser program is also incorporated. Emphasis is made on interaction through the case and modeling of real-life decisions in diagnosis and treatment. Advantages of the program are self-paced learning, assessment of understanding, feedback, and emphasis on deep understanding of the basic physiological and biochemical processes underlying clinical problems.
Current status and performance goals for serum thyrotropin (TSH) assays
Authors: Spencer C A , Takeuchi M , Kazarosyan M
Published: January 2020

Current medical needs dictate that laboratories offer thyrotropin (TSH) assays that can reliably measure low TSH concentrations--a prerequisite for using the more cost-effective TSH-centered strategy currently recommended by the American Thyroid Association. This study reviews the functional performance of the TSH immunometric assay methods currently used in clinical practice. Methodological differences between methods, the rationale for using the 20% interassay CV as the functional sensitivity limit for patient reports, and both TSH-related and non-TSH-related specificity problems are reviewed. We recommend that manufacturers and clinical laboratories use a clinically relevant standard protocol for functional sensitivity assessments. In this protocol, human serum pools are analyzed in random order 10 or more times across a clinically realistic time span (which approximates to 6-8 weeks for TSH measurements used in an outpatient setting). Laboratories should independently establish and periodically check their functional sensitivity by the standard protocol and enlist the manufacturer's help to accomplish this if necessary. Manufacturers should (a) develop promotional material that realistically projects the assay's functional sensitivity expected in a clinical laboratory setting, (b) ensure that new clinical laboratory users achieve the projected functional sensitivity target when using the standard protocol, and (c) focus on the typical functional sensitivity obtained by clinical laboratory users rather than the assay's "generation" achieved under ideal conditions. If manufacturers and laboratories collaborate to solve the sensitivity and specificity problems discussed here, clinical laboratories should be better able to consistently deliver reliable serum TSH measurements across the full range of TSH concentrations encountered in clinical practice.
Monoclonal antibodies against a C-terminal peptide of human brain acetylcholinesterase distinguish between erythrocyte and brain acetylcholinesterases
Authors: Boschetti N , Brodbeck U , Jensen S P , Koch C , Nørgaard-Pedersen B
Published: January 2020

Monoclonal antibodies (mAbs) were raised against a peptide of the 10 C-terminal amino acids of human brain acetylcholinesterase (AChE): H-Tyr-Ser-Lys-Gln-Asp-Arg-Cys-Ser-Asp-Leu-OH. Two positive clones (mAbs 190-1 and 190-2) were selected and tested for their ability to distinguish between mammalian brain and erythrocyte AChEs. In a solid-phase enzyme antigen immunoassay as well as by Western- and dot-blot analysis, both antibodies showed clear binding to AChE from human and bovine brain but not to AChE from erythrocytes. MAbs 190-1 and 190-2 reacted with neither AChE from electric eel nor butyrylcholinesterase from human serum. Both antibodies were used in a quantitative assay for AChE in amniotic fluids, where AChE activity could be found only in samples from open neural tube-defect pregnancies, but not in fluids from normal pregnancies or in artificially blood-contaminated samples.
Fatty acid ethyl ester synthase, an enzyme for nonoxidative ethanol metabolism, is present in serum after liver and pancreatic injury
Authors: Aleryani S , Kabakibi A , Cluette-Brown J , Laposata M
Published: January 2020

Fatty acid ethyl esters (FAEE), esterification products of ethanol and fatty acids, have been implicated as mediators of ethanol-induced organ damage. Because cytosolic enzymes such as aspartate aminotransferase, lipase, and amylase appear in the blood after liver or pancreatic damage, we hypothesized that FAEE synthase, which is both cytosolic and membrane bound, is also released into the blood of patients with liver or pancreatic disease. We used a method involving thin-layer chromatography coupled with gas chromatography-mass spectrometry to reliably identify and quantify FAEE. In this study, we demonstrated that patients with liver or pancreatic disease release FAEE synthase into their plasma in amounts proportional to the amount of aspartate aminotransferase (r = 0.78), amylase (r = 0.65), and lipase (r = 0.63). These data indicate that liver and pancreatic damage results in release of FAEE synthase into the blood. The presence of FAEE synthase in plasma permits nonoxidative ethanol metabolism in the plasma.
Enzyme immunoassay validation for qualitative detection of cocaine in sweat
Authors: Spiehler V , Fay J , Fogerson R , Schoendorfer D , Niedbala R S
Published: January 2020

A solid-phase enzyme immunoassay (EIA) involving microtiter plates was modified for analysis of cocaine in sweat. Sweat was collected with the PharmChek sweat patch and drugs were eluted from the collection pad of the patch. The sweat contained primarily parent cocaine. The assay was determined to have cross-reactivity for cocaine of 102% relative to 100% for the benzoylecgonine (BE) calibrators and for cocaethylene of 148%. The optimum cutoff concentration for this modified assay, determined by receiver-operating characteristic curve analysis, was 10 micrograms/L cocaine or BE equivalents. At this concentration the assay had 94.5% sensitivity and 99.1% specificity vs gas chromatography-mass spectrometry (GC-MS) as an acceptable indicator of the true clinical state. The positive predictive value at a prevalence of 50% was 99%. Threshold analysis for positives suggested that the 95% confidence interval for a positive result by the EIA was between 12.5 and 15 micrograms/L and that quality-control samples at 5 and 15 micrograms/L could be run with each batch to certify the precision around the cutoff. All positive samples must be confirmed by GC-MS. The sensitivity and specificity of the overall analysis system (immunoassay screen and GC-MS confirmation) was 86% and 97%, with known cocaine dosing of volunteers as the acceptable indicator of the true clinical state.
Nighttime dosing assures postdistribution sampling for therapeutic drug monitoring of digoxin
Authors: Bernard D W , Bowman R L , Grimm F A , Wolf B A , Simson M B , Shaw L M
Published: January 2020

To study the appropriateness of phlebotomy for digoxin therapeutic drug monitoring (TDM) in outpatients, we conducted a retrospective chart review, a computer search of all previous TDM testing, and a questionnaire of all outpatients (n = 86) who had serum digoxin determinations between April 10 and April 28, 1992 (585 tests). In patients who took digoxin at the same time daily (40 patients, 300 tests), 52% of tests were performed on inappropriate samples drawn within 6 h of the last dose. No patient who took digoxin after 1700 had inappropriate tests. Phlebotomy for serum digoxin determinations before distribution of digoxin is complete is a common problem in outpatients, leading to clinically uninterpretable test results. Postdistribution sampling can be assured by nighttime dosing, and this recommendation has been implemented at our hospital.
Dynamic mutations pose unique challenges for the molecular diagnostics laboratory
Authors: McGlennen R C
Published: January 2020

Routine clinical molecular testing of diseases associated with unstable or dynamic trinucleotide repeat syndromes poses unique technical, medical, and ethical challenges to the laboratory. Although the pathophysiology of these disorders is to date still largely undefined, the uniformity of their genetics has led to the development of highly informative diagnostic tests. In general, amplification techniques, such as the polymerase chain reaction (PCR), are used to determine the size of alleles within the genes linked to these disorders. Technically, these assays require empirical optimization so that the PCR reactions are both robust and reproducible, and occasionally other methods must be used to confirm diagnoses. Beyond execution of the test, however, the molecular diagnostics laboratory needs also to be fundamentally involved in the process of interpreting these tests in the correct clinical context and in setting policy as to how these data are presented to patients.
GC-MS profiling of urinary organic acids evaluated as a quantitative method
Authors: Duez P , Kumps A , Mardens Y
Published: January 2020

We assessed the quantitative performances of a classical method for profiling urinary organic acids: ethyl acetate extraction/oxime-trimethylsilyl derivatization/GC-MS. Twenty-seven acids were quantified on the basis of specific ions in both scan and selected-ion monitoring modes. We found that the tuning of the mass detector severely affects the calibration factors, being critical to achieve quantitative results, and we propose a practical procedure for reproducible tuning. Of seven compounds tested, tropic acid was retained as the internal standard suitable for most of the acids of clinical interest; a second internal standard, 2-ketocaproic acid, was used in quantifying keto-acids. The within-day and total relative standard deviations (CVs), estimated from scan-mode analyses of urine, ranged from 2.6% to 12.7% and from 4.2% to 11.8%, respectively. Curvilinear relationships between analytical response and concentration were observed for most of the acids investigated.
Analyte and label binding assay read by flow cytometry
Authors: Utgaard J O , Frengen J , Stigbrand T , Ullen A , Schmid R , Lindmo T
Published: January 2020

A new immunometric two-site sandwich assay is introduced, in which a label-scavenging binding partner is added to the sample in addition to the analyte-binding partner. The scavenger binding partner binds excess label antibody, giving a signal proportional to the amount of excess label antibody in the sample solution. A set of two calibration curves is obtained from the two binding partners simultaneously, and a combination of the two signals gives an unambiguous determination of the analyte concentration, even for high analyte concentrations where the hook effect may occur. Two-particle immunofluorometric assays developed for placental alkaline phosphatase and human chorionic gonadotropin on the basis of this principle and yielding signals measured by flow cytometry gave rapid results (2 h) and had working ranges in excess of 5 and 6 orders of magnitude for the respective analytes.
Converging technologies and their impact on the clinical laboratory
Authors: Burtis C A
Published: January 2020

Rapid technological change is transforming the world in which we live. Because laboratory services are defined by available products and tools of technology, this "technological revolution" is having a significant and profound effect on how clinical laboratories are organized, staffed, equipped, and operated. In this review, I discuss individually and collectively the advances and developments being made in the general areas of measurement, digital, communication, and transportation technology and demonstrate how the convergence of the products evolving from these technologies is affecting the clinical laboratory.
Colorimetric ELISA measurement of specific mRNA on immobilized-oligonucleotide-coated microtiter plates by reverse transcription with biotinylated mononucleotides
Authors: Tominaga K , Miura Y , Arakawa T , Kobayashi K , Mitsuhashi M
Published: January 2020

In this study, alpha-globin mRNA was captured on plastic plates to which alpha-globin-specific oligonucleotides had been covalently immobilized; the captured mRNA was immediately reverse-transcribed into cDNA in the presence of biotinylated dUTP. Addition of alkaline phosphatase-conjugated streptavidin to react with the biotin was followed by addition of substrate (p-nitrophenyl phosphate), and the resulting colorimetric development was measured at 405 nm. Because multiple biotin molecules are incorporated into cDNA during reverse transcription, and because synthesized cDNA is more stable than mRNA, we could successfully quantify by conventional colorimetry the amount of alpha-globin mRNA on the plate within a linear range of 100 pg to 10 ng. This is more sensitive than conventional Northern blotting. Therefore, the present method may be applicable to measurements of specific mRNAs in various test materials.