bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ligase chain reaction assay for human mutations: the Sickle Cell by LCR assay
Authors: Reyes Antonio A , Carrera Paola , Cardillo Elena , Ugozzoli Luis , Lowery Jimmie D , Lin Ching-I P , Go Matthew , Ferrari Maurizio , Wallace R Bruce 
Published: January 2020

Abstract
We can detect the β-globin gene sickle cell mutation by using an assay based on the ligase chain reaction. The simultaneous amplification of the human growth hormone gene in the same reaction serves as a control for the amount of template DNA or amplification efficiency. Ligation products, which are biotinylated at one end and tagged with an arbitrary “tail” sequence at the other, are captured by hybridization to “tail”-complementary oligonucleotides immobilized on polystyrene microwells. The captured ligation products are detected colorimetrically by use of streptavidin–alkaline phosphatase conjugate. In a study of 24 subjects, the assay unequivocally discriminated among normal, carrier, and sickle cell genotypes.
Variability in the measurement of C-reactive protein in healthy subjects: implications for reference intervals and epidemiological applications
Authors: Macy Elizabeth M , Hayes Timothy E , Tracy Russell P 
Published: January 2020

Abstract
We developed a reproducible ELISA for C-reactive protein (CRP), calibrated with WHO Reference Material, for which intra- and interassay CVs were 3.0% and 6.0%, respectively. Analytical recovery was 97.9%. The distribution of CRP in a healthy blood donor population (n = 143) was nongaussian, with 2.5th, 50th, and 97.5th percentile values of 0.08, 0.64, and 3.11 mg/L, respectively. There was no sex-related difference, and the association with age was weak. In a study of variability [by the method of Fraser and Harris (Crit Rev Clin Lab Sci 1989;27:409–37)], the analytical variability was 5.2%; the within-subject variability, CVI, was 42.2%; and the between-subject variability, CVG, was 92.5%. The critical difference for sequential values significant at P ≤0.05 (i.e., the smallest percentage change unlikely to be due to analytical variability or CVI) was calculated as 118%, and the index of individuality, CVI/CVG, was 0.46. This suggests that CRP, like many clinical chemistry analytes, has limited usefulness in detecting early disease-associated changes when used in conjunction with a healthy reference interval. From a molecular epidemiological standpoint, the usefulness of CRP in longitudinal studies is suggested by the small index of individuality and by observations that (a) short-term fluctuations were infrequent, (b) all data stayed within the reference interval, and (c) relative rankings of the subjects over 6 months only moderately deteriorated.
Two-site immunofluorometric assay of intact salmon calcitonin with improved sensitivity
Authors: Rong Haiqin , Deftos Leonard J , Ji Hong , Bucht Elisabet 
Published: January 2020

AbstractWe recently developed a two-site immunofluorometric assay (IFMA) of salmon calcitonin (SCT) by DELFIA (dissociation enhancement lanthanide fluoroimmunoassay) technique using the same polyclonal antibodies both for “catching” the antigen and for signaling. In the present study we used a monoclonal antibody to SCT 1–11 as the capture antibody. This antibody was biotinylated before use in streptavidin-coated microtitration plates. The polyclonal antibody labeled with Eu chelate was used as a signaling marker. This combination of antibodies resulted in an assay that was three to four times more sensitive than the previous IFMA, with a detection limit of 0.3 pmol/L serum. Intact SCT 1–32 was detected by the assay (recoveries 94–96%), but not the fragments SCT 1–11 and SCT 10–32 or human calcitonin. Dilutions of plasma samples containing SCT were parallel to the calibration curve of SCT 1–32. Pharmacokinetic studies of SCT, 100 IU administered intramuscularly to 10 men, indicated peak serum concentrations of 32–128 pmol/L within 10–20 min with apparent half-life of 56 ± 18 min (mean ± SD). This new assay will allow study of the pharmacokinetics of new calcitonin preparations that do not require injection.
Adaptation of latex-enhanced assay for percent glycohemoglobin to a Dade Dimension® analyzer
Authors: Holownia Peter , Bishop Elizabeth , Newman David J , John W Gary , Price Christopher P 
Published: January 2020

Abstract
At present no method for glycohemoglobin (%HbA1c) is automated on a main-line analyzer to allow joint measurement with other indicators of diabetic control such as glucose and cholesterol. We describe an adaptation of a latex-enhanced competitive immunoassay for quantifying %HbA1c to the Dade International Dimension® analyzer. After a manual hemolysis step, HbA1c and total hemoglobin (Hb) are determined separately. The concentration of glycated β-subunit is obtained from the immunoassay, whereas Hb is assessed colorimetrically from a derivatized form. Both reactions were fully optimized for accuracy, precision, and specificity on the Dimension; stabilities of reagents and calibration were established; and potential interferences were assessed. The analyzer gave reliable results over the required clinical range of 1–15% HbA1c. Within-run and total assay variation were within 5% of the target CV limits, as determined by ANOVA with three representative sample pools across 20 days. Close agreement with an established HPLC procedure and a commercially available enzyme immunoassay was observed for 140 samples from clinically defined patient groups. Additional samples from patients with hemoglobinopathies (n = 20) demonstrated a more complex relationship between methods. We conclude that adaptation of the method for use with the Dimension analyzer is a valid method for quantifying %HbA1c.
Molecular diagnosis of B- and T-cell lymphomas: fundamental principles and clinical applications
Authors: Rezuke William N , Abernathy Evelyn C , Tsongalis Gregory J 
Published: January 2020

Abstract
Molecular diagnostic assays have become routine in the evaluation of lymphoid malignancies. Both Southern transfer and polymerase chain reaction (PCR) technologies are used to assess for B- and T-cell clonality, the presence of rearrangements involving protooncogenes such as bcl-1 and bcl-2, and the monitoring of minimal residual disease. We review the fundamentals of B- and T-cell ontogeny as well as the basic principles of the Southern transfer and PCR assays and their applications to the diagnosis of lymphoid malignancies.
Point On the meaning of “sensitivity”
Authors: Ekins Roger , Edwards Philip 
Published: January 2020

Abstract
The term “sensitivity” (as applied to an analytical method’s performance) has again become a subject of controversy. Certain authorities (e.g., IUPAC) define a system’s sensitivity as the response curve slope (or response/dose), others (e.g., IFCC) in terms of the detection limit. Many investigators have failed to perceive the contradiction between these concepts, wrongly assuming that maximizing “sensitivity” in the first sense maximizes it in the second (i.e., that they are inversely related). The existence of different meanings for this term (when used in the present context) is a source of confusion that has, among other things, led to erroneous ideas relating to immunoassay design. Such confusion should be terminated by adoption of one or the other of the definitions. However, the definitions are not of equal merit. We advance arguments against retention of the “slope” definition, which conflicts with the word’s common meaning and is meaningless as an indicator of the performance of a measuring system.
Rapid diagnosis of MCAD deficiency: quantitative analysis of octanoylcarnitine and other acylcarnitines in newborn blood spots by tandem mass spectrometry
Authors: Chace Donald H , Hillman Steven L , Van Hove Johan L K , Naylor Edwin W 
Published: January 2020

Abstract
We report the application of tandem mass spectrometry to prospective newborn screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. MCAD deficiency is diagnosed from dried blood spots on filter paper cards from newborns on the basis of the increase of medium chain length acylcarnitines identified by isotope dilution mass spectrometry methods. A robust and accurate semiautomated method for the analysis of medium chain length acylcarnitines as their butyl esters was developed and validated. Quantitative data from the analyses of 113 randomly collected filter paper blood spots from healthy newborns showed low concentrations of medium chain length acylcarnitines such as octanoylcarnitine. The maximum concentration of octanoylcarnitine was 0.22 μmol/L, with the majority being at or below the detection limit. In all 16 blood spots from newborns with confirmed MCAD deficiency, octanoylcarnitine was highly increased [median 8.4 μmol/L (range 3.1–28.3 μmol/L)], allowing easy detection. The concentration of octanoylcarnitine was significantly higher in these 16 newborns (&lt;3 days of age) than in 16 older patients (ages 8 days to 7 years) with MCAD deficiency (median 1.57 μmol/L, range 0.33–4.4). The combined experience of prospective newborn screening in Pennsylvania and North Carolina has shown a disease frequency for MCAD deficiency of 1 in 17 706. No false-positive and no known false-negative results have been found. A validated method now exists for prospective newborn screening for MCAD deficiency.
Effect of analytical run length on quality-control (QC) performance and the QC planning process
Authors: Parvin Curtis A , Gronowski Ann M 
Published: January 2020

Abstract
The performance measure traditionally used in the quality-control (QC) planning process is the probability of rejecting an analytical run when an out-of-control error condition exists. A shortcoming of this performance measure is that it doesn’t allow comparison of QC strategies that define analytical runs differently. Accommodating different analytical run definitions is straightforward if QC performance is measured in terms of the average number of patient samples to error detection, or the average number of patient samples containing an analytical error that exceeds total allowable error. By using these performance measures to investigate the impact of different analytical run definitions on QC performance demonstrates that during routine QC monitoring, the length of the interval between QC tests can have a major influence on the expected number of unacceptable results produced during the existence of an out-of-control error condition.
Disappearance of human chorionic gonadotropin and its α- and β-subunits after term pregnancy
Authors: Korhonen Juha , Alfthan Henrik , Ylöstalo Pekka , Veldhuis Johannes , Stenman Ulf-Håkan 
Published: January 2020

AbstractWe have used high-specificity and precision immunofluorometric assays to measure the elimination half-times of human chorionic gonadotropin (hCG), hCGα, and hCGβ in serum over 21 days after delivery in six women with term pregnancies. Baseline concentrations and half-times were calculated with the use of a curve-fitting algorithm for multiexponential decay. In contrast to the two-component model, a three-component exponential function with baseline provided a fit for which predicted values could not be distinguished from the observed values by analysis of variance. Median half-times were 3.6, 18.0, and 53.0 h for hCG; 1.0, 23.4, and 194 h for hCGβ; and 0.6, 6.2, and 21.9 h for hCGα. The mean ratio of hCGα to hCG decreased rapidly from 36.9% to 3.3% on day 3; thereafter it increased to 64.3% 21 days after delivery because of a higher baseline concentration of hCGα. hCGβ had the slowest total elimination rate, and the ratio of hCGβ to hCG in serum increased from 0.8% before delivery to 26.7% after 21 days. If the metabolism of hCG and hCGβ is similar in patients with trophoblastic disease, the ratio of hCGβ to hCG must be evaluated with caution in samples taken several days after initiating therapy. We conclude that the disappearance of hCGβ from plasma is slower than previously recognized and that the ratios of hCGβ or hCGα to intact hCG vary as a function of postpartum time. Such information may be important in clinical studies of pregnancy disorders.
Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR
Authors: Lay Marla J , Wittwer Carl T 
Published: January 2020

Abstract
A single-step method for factor V Leiden genotyping is presented that uses rapid-cycle PCR and simultaneous fluorescence analysis with resonance energy transfer probes. A fragment of the factor V gene containing the mutation is amplified asymmetrically through use of a primer labeled with Cy5TM in the presence of a 3′-fluorescein-labeled probe that covers the mutation site. When the fluorescein probe is annealed to the extension product of the Cy5-labeled primer, the fluorophores are brought into close enough contact for resonance energy transfer to occur. As the temperature increases, the probe melts from its target, decreasing the resonance energy transfer. When the probe is complementary to the product strand, it melts at 65 °C; if the single-base mutation is present, the probe melts at 57 °C. Concurrent amplification and analysis from genomic DNA takes 20–45 min and requires no sample manipulation after the fluorescence thermal cycler is loaded.
Quantification of parathyroid hormone-related protein mRNA by competitive PCR and time-resolved lanthanide fluorometry
Authors: Rong Haiqin , Ji Hong , Pernow Ylva , Sjöstedt Ulrika , Bucht Elisabet 
Published: January 2020

Abstract
Using dissociation and enhancement time-resolved lanthanide fluorometry, we have developed a quantitative competitive (QC)-PCR for measuring parathyroid hormone-related protein (PTHrP) mRNA after reverse transcription. A cloned PTHrP cDNA target was also modified by deletion of 10 bp and insertion of 21 bp in the midregion of the fragment and cloned for use as a competitor (i.e., internal standard). Two primers spanning 362 bp of target and 373 bp of competitor were designed and one of the primers was biotinylated. Two oligonucleotide probes, one recognizing the target and the other hybridizing to the competitor, were labeled with Eu chelate. Two equal aliquots of PCR products were assayed with each probe separately in streptavidin-coated wells. After 35 PCR cycles, the competitor signal decreased exponentially (y = e(3.74 −0.624x); r2 = 0.965) and the target signal increased exponentially (y = e(1.14 + 0.497x); r2 = 0.984) when 1000 copies/tube of the competitor and 0–100 000 copies/tube of the target DNA were added. Log-transformed data for the ratio of target to competitor signals (y) and the copies of the target DNA added (x) were used for plotting the linear calibration curve (y = 2.79+2.76x; r2 = 0.976). This QC-PCR enables analysis of multiple samples simultaneously and can be used to study PTHrP gene expression in malignancy and physiology.
Fully automated measurement of total iron-binding capacity in serum
Authors: Yamanishi Hachiro , Kimura Shigeki , Iyama Shigeru , Yamaguchi Yoshihisa , Yanagihara Takehiko 
Published: January 2020

Abstract
We established a method for fully automated measurement of total iron-binding capacity (TIBC) in serum without separation of the unbound excess iron after saturating serum transferrin. After saturation of serum transferrin with an excess amount of iron (first step), the unbound iron was eliminated by formation of a complex with ferrozine, which was used as a chromogenic reagent (second step). For the TIBC assay, iron dissociated from transferrin by shifting the pH to acidic was reacted with ferrozine, and the increase in the absorbance at 570 nm was measured (third step). Because the iron used as a calibrator, which was added to saturate transferrin, reacted completely with ferrozine in the second step (elimination of unbound iron), the change in the absorbance to generate a calibration factor could not be monitored in the third step. To solve this problem, we used N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid (HEDTA) to complex with the iron added to saturate transferrin in the second step. This made it possible to form an iron–ferrozine complex at acidic pH because iron was dissociated from HEDTA at acidic pH. The within-run CVs of this method were 0.66–2.43% at 17.7–77.0 μmol/L, and the day-to-day CVs were 1.06–1.57% at 29.9–60.4 μmol/L (n = 10). The correlation between the values obtained with this method (y) and those from the direct TIBC assay, which involved removal of unbound iron by ion-exchange resin (x), was: y = 0.963x + 0.29 μmol/L (r = 0.973, Sy|x = 2.83, n = 59), and with the TIBC values calculated from the serum iron concentrations and the unbound iron-binding capacities measured by a direct colorimetric method (x) was: y = 1.01x − 1.06 μmol/L (r = 0.994, Sy|x = 1.66, n = 51).
Pharmacogenetics: a laboratory tool for optimizing therapeutic efficiency
Authors: Linder Mark W , Prough Russell A , Valdes Roland 
Published: January 2020

AbstractPharmacogenetics is the study of the linkage between an individual’s genotype and that individual’s ability to metabolize a foreign compound. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Phenotypes exhibiting poor and ultraextensive metabolism result from genetic variance (polymorphism) of enzymes involved in metabolism. Thus, in pharmacogenetic studies one applies genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual’s drug metabolism phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic efficiency. More than 25 commonly prescribed medicines are metabolized by the cytochrome P-4502D6 (CYP2D6) isoenzyme, and polymorphism of the CYP2D6 gene affects the therapeutic management of up to 17% of individuals in some ethnic groups. In this review, we summarize and update information concerning drug-metabolizing genotypes with emphasis on CYP2D6 genotyping techniques that can be applied by the clinical laboratory for linking human genetics to therapeutic management.
Impact of CK-MB testing policies on hospital length of stay and laboratory costs for patients with myocardial infarction or chest pain
Authors: Wu Alan H B , Clive Jonathan M 
Published: January 2020

Abstract
We obtained data on hospital length of stay (LOS) and total laboratory charges for Medicare patients admitted to 82 hospitals in Massachusetts during 1994. Five Diagnosis Related Groups (DRGs) were selected: surviving acute myocardial infarction (AMI) with, and without, complications; AMI with death; angina pectoris; and chest pain. The hospitals were grouped according to their laboratory policies for testing CK-MB (e.g., frequency of assay runs; information obtained by telephone survey). The study was conducted to determine whether there was an association between turnaround times for results and LOS for cardiac DRGs. The mean LOS for AMIs with complication for 1513 patients admitted to 22 hospitals whose laboratories perform CK-MB testing once or twice daily was 8.4 days [95% confidence interval (CI): 8.2–8.7]. In contrast, the mean LOS for hospitals with CK-MB test policies of at least 3 runs daily or random-access stat was significantly (P &lt;0.05) lower, 7.7 days (CI: 7.4–8.0 and 7.5–7.9, respectively). Overall laboratory charges were lower in the hospitals with shorter LOS. With one exception, there was no significant difference in LOS between patients with DRGs of angina pectoris or chest pain or other AMI DRGs. For AMI, a CK-MB testing policy that produces shorter turnaround times may be justified because of an association with reductions in LOS and overall laboratory costs.
Population screening for the common G985 mutation causing medium-chain acyl-CoA dehydrogenase deficiency with Eu-labeled oligonucleotides and the DELFIA system
Authors: Seddon Helen R , Gray George , Pollitt Rodney J , Iitiä Antti , Green Anne 
Published: January 2020

Abstract
We have screened 10 171 neonatal blood spots from the Trent and West Midlands regions of the UK for the common G985 mutation to more accurately establish the incidence of medium-chain acyl coenzyme (Co)A dehydrogenase (MCAD) deficiency. We have used a technique involving PCR and Eu-labeled allele-specific oligonucleotides detected by using time-resolved fluorometry on the dissociation-enhanced fluorescence immunoassay (DELFIA) system for the detection of the G985 mutation. We have also evaluated the feasibility of neonatal screening with this technique. We identified 158 G985 heterozygotes and no G985 homozygotes. The calculated incidence of MCAD deficiency in the population studied (all mutations, assuming 90% of MCAD mutations are G985) is 1 in 13 426 (95% confidence limits 1 in 10 070–1 in 18 791). At the optimum cutoff criteria, the technique has a sensitivity of 97.5%, specificity of 99.6%, and positive predictive value of 80.2%. We conclude that this study confirms that MCAD deficiency is a common inherited metabolic disease and is a candidate for neonatal screening. The methodology used is robust and suitable for large-scale population studies such as this. The technique is also potentially suitable for screening.
Rapid, nonradioactive screening for mutations in exons 10, 11, and 16 of the RET protooncogene associated with inherited medullary thyroid carcinoma
Authors: Siegelman Mark , Mohabeer Ajay , Fahey Thomas J , Tomlinson Gail , Mayambala Chris , Jafari Sepideh , Noll Walter W , Thibodeau Stephen N , Dawson D Brian 
Published: January 2020

Abstract
Germline mutations in exons 10, 11, and 16 of the RET protooncogene are associated with the heritable cancer syndromes multiple endocrine neoplasia (MEN) type 2A, familial medullary thyroid carcinoma (FMTC), and MEN type 2B. Nonradioactive mutation analysis with nondenaturing Phastgels® and the Phast System™ was performed on DNA amplified by the polymerase chain reaction from exons 10, 11, and 16 of the RET protooncogene from patients with MEN 2A, MEN 2B, or FMTC. The analysis requires ∼45–90 min for electrophoresis and 35 min for staining. This assay detected 20 of 21 different mutations that represented ∼90% of all known mutations associated with these lesions. A rare silent polymorphism within exon 10 was also detected. This form of mutation analysis provides simple, rapid, and highly sensitive nonradioactive detection of mutations known to be associated with MEN 2A, FMTC, and MEN 2B.
Cardiac troponin T composition in normal and regenerating human skeletal muscle
Authors: Bodor Geza S , Survant Libby , Voss Ellen M , Smith Stephen , Porterfield Diane , Apple Fred S 
Published: January 2020

AbstractCardiac troponin T (cTnT), measurement of which has been recommended for diagnosing myocardial infarction, was initially believed to be specific for the heart. However, recent publications have reported cTnT in sera of patients without cardiac disease; therefore, we investigated whether cTnT could be found in human skeletal muscle tissues. Using immunohistochemistry, Western blot, and quantitative cTnT ELISA, we assayed human heart (n = 3), normal human skeletal muscle (n = 6), and diseased skeletal muscle samples from patients with polymyositis (PM, n = 13) and Duchenne muscular dystrophy (DMD, n = 6). All heart specimens contained cTnT, but the expression of cTnT in normal skeletal muscle samples varied widely, ranging from no expression (quadriceps femoris) to expression by up to 20% of the muscle fibers (diaphragm). Immunohistochemistry detected cTnT in skeletal muscle of 8 of the PM patients and all of the DMD patients. Mean myofibrillar cTnT concentrations (mg/g myofibrillar protein) were: cardiac = 10.0, normal skeletal = 0.8, PM skeletal = 0.7, and DMD skeletal = 4.37, confirming the results of immunohistochemistry. Western blot analysis also confirmed the expression of cTnT in muscle from DMD patients. These findings provide evidence that cTnT is not 100% cardiac-specific but also is expressed in regenerating (PM and DMD) as well as in normal (nonregenerating) skeletal muscle.