bluuebunny/crossref_metadata_2025 huggingface dataset:
Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR
Authors: Lay Marla J , Wittwer Carl T
Published: January 2020

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

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

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

Pharmacogenetics 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

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 <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

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

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

Cardiac 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.
Quantification of carbohydrate-deficient transferrin by ion-exchange chromatography with an enzymatically prepared calibrator
Authors: Renner Florian , Kanitz Rolf-Dieter
Published: January 2020

The current HPLC method for the determination of carbohydrate-deficient transferrin (CDT) yields ratios of CDT isoforms in relation to total transferrin, whereas the use of absolute concentrations obtainable in routine analysis by RIA and the reference ranges based hereupon is more convenient. We describe a modified HPLC method that likewise gives absolute CDT concentrations by using a calibrator prepared by treatment of transferrin with neuraminidase. Separation of isoforms could be improved and analysis time reduced to ∼2 h. Iron saturation proved stable during chromatography. In contrast to a commercial RIA, the cheaper and more time-saving HPLC method excludes erroneous results caused by aged samples or genetic transferrin variants and enables the determination of asialo- and disialotransferrin. Both methods showed comparable precision and correlated with each other (y = 1.76 + 0.27x; Sy|x = 5.38); for the HPLC method precision was 1.3–9.8% (within assay) and 6.2–10.6% (between assay). The clinical evaluation with a cutoff concentration of 80 mg/L resulted in a diagnostic specificity of 100% and a sensitivity of 82.5%.
The EWMA control chart: properties and comparison with other quality-control procedures by computer simulation
Authors: Neubauer Aljoscha Steffen
Published: January 2020

A quality-control chart based on exponentially weighted moving averages (EWMA) has, in the past few years, become a popular tool for controlling inaccuracy in industrial quality control. In this paper, I explain the principles of this technique, present some numerical examples, and by computer simulation compare EWMA with other control charts currently used in clinical chemistry. The EWMA chart offers a flexible instrument for visualizing imprecision and inaccuracy and is a good alternative to other charts for detecting inaccuracy, especially where small shifts are of interest. Detection of imprecision with EWMA charts, however, requires special modification.
A pragmatic approach to estimating total analytical error of immunoassays
Authors: Sadler William A , Smith Murray H , Murray Lynda M , Turner John G
Published: January 2020

Several groups have recently commented on the need for more realistic information on analytical performance of laboratory tests. The term “total analytical error” is sometimes used in this context. However, differing opinions have been expressed on how best to obtain estimates of clinical assay error, as it would be perceived by clinicians. We suggest a pragmatic definition of total analytical error for immunoassays and describe our attempts to estimate it by simple designs in the internal quality-control process. We use results over 29 months from a total serum thyroxine RIA. The most important error sources were those related to calibration materials and operator effects, errors not usually captured by short-term or snapshot experiments.
Development of a urodilatin-specific antibody and radioimmunoassay for urodilatin in human urine
Authors: Carstens Jan , Jensen Kaare T , Ivarsen Per , Rasmussen Lars M , Pedersen Erling B
Published: January 2020

Urodilatin (URO) (95–126) is a renal-derived natriuretic peptide that is isolated only from human urine. This study describes the development of a URO-specific antibody and a RIA for URO in urine. At present, there is no commonly available URO-specific antibody. We produced a URO-specific antibody without cross-reactivity with atrial natriuretic peptide (ANP) analogs by immunization of rabbits with the URO (95–126) peptide and subsequent purification of the resulting URO antiserum with affinity chromatography with CNBr-activated Sepharose 4B. The urine samples were ethanol-extracted before assay. The CVs were 6.7% (intraassay) and 14.1% (interassay). This study reports the circadian urinary excretion of URO in 24 healthy subjects with seven sampling periods per 24 h.
Development of enzyme immunoassay for endogenous ouabain-like compound in human plasma
Authors: Harwood Steven , Little John A , Gallacher Gerard , Perrett David , Edwards Raymond , Dawnay Anne
Published: January 2020

Widespread evidence supports the existence of an endogenous digitalis-like compound in mammals. We report here the development of a novel enzyme immunoassay for ouabain that, in conjunction with a detailed HPLC study, identifies a ouabain-like compound (OLC) in extracted human plasma. The assay is sensitive—minimum detection limit for OLC 37 pmol/L (11 pmol/L in plasma)—and has a working range (between-assay CV <10%) of 180–10 000 pmol/L (54–3000 pmol/L in plasma). Mean recoveries of ouabain added to plasma ranged from 90% to 100%, and plasma extracts diluted in parallel to the standard curve. Plasma OLC concentrations in 10 healthy volunteers averaged 92 pmol/L (range 55–168), assuming 100% cross-reactivity of OLC in the ouabain assay. HPLC analysis with two distinct chromatographic conditions demonstrated that endogenous human plasma OLC co-eluted with authentic ouabain. The enzyme immunoassay is rapid and easy to perform and will support further investigation of the nature of this controversial endogenous steroid.
Sweat testing for heroin and metabolites in a heroin maintenance program
Authors: Kintz Pascal , Brenneisen Rudolf , Bundeli Petra , Mangin Patrice
Published: January 2020

Recent advances in sensitive analytical techniques have enabled the analysis of drugs in unconventional biological materials such as sweat. In a study conducted during a heroin maintenance program, 14 subjects had sweat patches applied, then received intravenously two or three doses of heroin hydrochloride ranging from 80 to 1000 mg/day. The sweat patch was applied 10 min before the first dosage and removed ∼24 h later, minutes before the next dosage. Absorbent pads were stored at −20 °C in plastic tubes until analysis. The target drugs were extracted in 5 mL of acetonitrile in the presence of 100 ng each of heroin-d9, 6-acetylmorphine-d3, and morphine-d3. After agitation for 30 min, the acetonitrile solution was divided into two portions: 2 mL for heroin testing and the remainder for testing for the other compounds. After evaporation, the residue of the first portion was reconstituted in 35 μL of acetonitrile; the second was derivatized by silylation with 40 μL of N,O-bis(trimethylsilyl)trifluoroacetamide containing 10 mL/L trimethylchlorosilane. Drugs were analyzed by GC-MS in electron impact mode. Concentrations (nanograms per patch) ranged from 2.1 to 96.3 for heroin, 0 to 24.6 for 6-acetylmorphine, and 0 to 11.2 morphine. Except in one case, heroin was the major drug present in sweat, followed by 6-acetylmorphine and morphine. We observed no correlation between the doses of heroin administered and the concentrations of heroin measured in sweat.
Defining the best quality-control systems by design and inspection
Authors: Hinckley C Martin
Published: January 2020

Not all of the many approaches to quality control are equally effective. Nonconformities in laboratory testing are caused basically by excessive process variation and mistakes. Statistical quality control can effectively control process variation, but it cannot detect or prevent most mistakes. Because mistakes or blunders are frequently the dominant source of nonconformities, we conclude that statistical quality control by itself is not effective. I explore the 100% inspection methods essential for controlling mistakes. Unlike the inspection techniques that Deming described as ineffective, the new “source” inspection methods can detect mistakes and enable corrections before nonconformities are generated, achieving the highest degree of quality at a fraction of the cost of traditional methods. Key relationships between task complexity and nonconformity rates are also described, along with cultural changes that are essential for implementing the best quality-control practices.
Thoughts on quality-control systems: a laboratorian’s perspective
Authors: Cembrowski George S
Published: January 2020

State-of-the-art prospective quality-control systems entail the use of medically relevant, analyte-specific quality control limits. With analyte-specific limits broader than those generally used in the clinical laboratory, there will be fewer false rejections, fewer unnecessary reanalyses, and shorter delays in run reporting. If the analyte-specific limits are narrower than those used in the laboratory, more errors will be detected, but the user is at risk of identifying errors over which s/he and the manufacturer have little control. The use of various patient data quality-control algorithms is described. Conservatism is stressed in adopting manufacturers’ guidelines for surrogate, nondestructive quality-control testing. A simple, optimized approach is suggested for the systematic retrospective review of proficiency data. Finally, an approach is presented for converting from older, previously accepted quality control procedures to more efficient analyte-specific quality control.
Highly sensitive gas chromatographic analysis of ethanol in whole blood, serum, urine, and fecal supernatants by the direct injection method
Authors: Tangerman Albert
Published: January 2020

A highly sensitive, reproducible, and rapid gas chromatographic method for ethanol determination in various biological specimens (human whole blood, serum, urine, and fecal supernatants) was developed. The method involves direct injection of the biological specimen into the gas chromatograph, without any pretreatment. Contamination of the gas chromatographic column with nonvolatile material was prevented by the use of a glass liner in the injector. This liner, which acted as a precolumn, was partly filled with small glass beads. Injection was performed in between the glass beads. More than 50 injections of the various biological specimens could be done before the liner had to be replaced by a new one. This injection technique between glass beads allows direct injection of large sample volumes up to 10 μL without disturbing the gas chromatographic separation. Injection of these large sample volumes made the method very sensitive. The detection limit for ethanol amounted to 0.1 mg/L (2 μmol/L) when using an injection volume of 5 μL. Attention has also been paid to simultaneously monitoring ethanol, methanol, acetaldehyde, and acetone in blood and urine of control subjects.
Comparison of two assays for measuring LDL cholesterol
Authors: Maitra Anirban , Hirany Shaina V , Jialal Ishwarlal
Published: January 2020

The purpose of this study was to evaluate the LipiDirect assay (L-LDL) against the Direct LDL™ immunoseparation method (D-LDL) and beta quantification (BQ-LDL) for measurement of LDL cholesterol (LDL-C) in patients with normo- and hypertriglyceridemia. Samples from 156 patients [triglyceride (Tg) range 0.61–9.95 g/L] were assayed for LDL-C concentrations with the three methods. An additional seven patients with type III hyperlipidemia and 25 paired sera from fasting and nonfasting individuals also were analyzed by the three methods. Both assays displayed excellent precision. The mean LDL-C value from L-LDL was significantly higher than BQ-LDL and D-LDL in normo- and hypertriglyceridemic samples (P <0.001). The mean absolute bias of L-LDL vs BQ-LDL was 12.7% for Tg <4 g/L and 30.6% for Tg ≥4 g/L, compared with 6.2% and 12.5%, respectively, for D-LDL. L-LDL correctly classified only 68% of patients with LDL-C <1.30 g/L and 57% of patients with LDL-C between 1.30–1.59 g/L as compared with 98% and 93%, repectively, for D-LDL (P <0.001). In patients with type III hyperlipidemia, L-LDL had a 130% positive bias with BQ-LDL as compared with a 14% negative bias for D-LDL. With all three methods there were no significant differences between samples from fasting and nonfasting individuals. On the basis of these findings, the D-LDL assay appears to be superior to the L-LDL assay.
Denaturing gradient-gel electrophoresis screening of familial defective apolipoprotein B-100 in a mixed Asian cohort: two cases of arginine3500 → tryptophan mutation associated with a unique haplotype
Authors: Choong Meng-Ling , Koay Evelyn S C , Khoo Kah-Lin , Khaw Min-Cheh , Sethi Sunil K
Published: January 2020

The Arg-to-Trp substitution at codon 3500 in the apolipoprotein (apo) B-100 gene is established as a cause of familial defective apo B-100 (FDB), a functional mutation, resulting in reduced LDL receptor binding and manifest hypercholesterolemia. In a search for similar mutations in 163 Malaysians, we screened the putative receptor-binding region (codons 3456–3553) of the apo B-100 gene by PCR amplification and denaturing gradient-gel electrophoresis. Four single-base mutations were detected and confirmed by DNA sequencing. Two females, a Chinese and a Malay, had the same CGG3500 → TGG mutation, resulting in an Arg3500-to-Trp substitution. This is the second published report of such an independent mutation involving the same codon as the established Arg3500-to-Gln mutation. The two other mutations detected, CTT3517 → CTG and GCC3527 → GCT, resulted in degenerate codons with no amino acid substitutions. All four mutations were associated with a unique apo B haplotype, different from those found in Caucasian FDB patients but concurring with that previously reported for two other Asians with FDB.
Genetic mutations of butyrylcholine esterase identified from phenotypic abnormalities in Japan
Authors: Maekawa Masato , Sudo Kayoko , Dey Dilip Chandra , Ishikawa Jinko , Izumi Masakazu , Kotani Kazuo , Kanno Takashi
Published: January 2020

We have identified 12 kinds of genetic mutations of butyrylcholine esterase (BCHE) from phenotypic abnormalities, showing that BCHE activities were deficient or diminished in sera. These genetic mutations, detected by PCR–single-strand conformation polymorphism analysis and direct sequencing, consisted of one deletion (BCHE*FS4), nine missense (BCHE*24 M, *100S, *250P, *267R, *330I, *365R, *418S, *515C, *539T), and two nonsense mutations (BCHE*119STOP, *465STOP). All of the individuals deficient in serum BCHE activity were homozygous for silent genes (6 of 6). Fifty-eight percent of the individuals (31 of 53) with slightly reduced serum BCHE activity were heterozygous for silent genes. They also showed a higher frequency (47% as allele frequency) of the K-variant than the general population (17.5%). Finally, we confirmed low serum BCHE activity in 10 of 23 individuals heterozygous for silent genes.
Use of polymerase chain reaction to identify pneumococcal infection associated with hemorrhage and shock in two previously healthy young children
Authors: Messmer Trudy O , Whitney Cynthia G , Fields Barry S
Published: January 2020

A PCR assay was developed for detection of Streptococcus pneumoniae in clinical specimens including blood and paraffinized tissues. We were able to detect one organism of purified DNA or 4.5 colony-forming units in blood. The primers did not cross-react with other upper respiratory tract streptococci or with pathogens commonly found in clinical specimens. This assay was used in an investigation of an outbreak of severe illness characterized by septic shock and hemorrhage in previously healthy children. PCR detected S. pneumoniae in cerebrospinal fluid and autopsy tissues of the two infants who died. The findings from this assay indicated that PCR offers increased specificity and sensitivity over latex agglutination and counterimmunoelectrophoresis and should prove useful in the identification of additional cases of severe illness caused by S. pneumoniae.
Rapid and large-scale method to detect K-ras gene mutations in tumor samples
Authors: Lopez-Crapez Evelyne , Chypre Camille , Saavedra Jacqueline , Marchand Joseph , Grenier Jean
Published: January 2020

We have developed a rapid and large-scale method for the detection of K-ras gene mutations in tumors. First, DNA is amplified by an asymmetric PCR; second, the single-strand dinitrophenyl (DNP)-labeled amplified DNA is hybridized specifically to oligonucleotide probes affixed on a tube. Finally, perfectly matched duplexes are easily detected by a monoclonal anti-DNP antibody bearing125I. The usefulness of this technique is illustrated by analyzing K-ras codon 12 mutations in human colorectal samples. This reliable assay procedure can be applied to the rapid screening of virtually any genetic disease caused by previously described point mutations.
Misleading results from immunoassays of serum free thyroxine in the presence of rheumatoid factor
Authors: Norden Anthony G W , Jackson Rodwin A , Norden Lorraine E , Griffin A Jane , Barnes Margaret A , Little John A
Published: January 2020

A novel interference with measurements of serum free thyroxine (FT4) caused by rheumatoid factor (RhF) is described. We found misleading, sometimes gross, increases of FT4 results in 5 clinically euthyroid elderly female patients with high RhF concentrations. All 5 patients had high FT4 on Abbott AxSYM® or IMx® analyzers. “NETRIA” immunoassays gave misleading results in 4 of the 5 patients; Amerlex-MAB® in 2 of 4 patients; AutoDELFIA®in 2 of the 5; and Corning ACS-180® and Bayer Diagnostics Immuno 1® in 1 of the 5. BM-ES700® system results for FT4 in these women remained within the reference range. Results for serum T4, thyroid-stimulating hormone, free triiodothyronine, thyroid-hormone-binding globulin, and FT4 measured by equilibrium dialysis were normal in all 5 patients. Drugs, albumin-binding variants, and anti-thyroid-hormone antibodies were excluded as interferences. Addition to normal serum of the RhF isolated from each of the 5 patients increased the apparent FT4 (Abbott AxSYM). Screening of 83 unselected patients demonstrated a highly significant positive correlation between FT4 (Abbott AxSYM) and RhF concentrations. Discrepant, apparently increased FT4 with a normal result for thyroid-stimulating hormone should lead to measurement of the patient’s RhF concentration.
Detecting CFTR gene mutations by using primer oligo base extension and mass spectrometry
Authors: Braun Andreas , Little Daniel P , Köster Hubert
Published: January 2020

A new method for the reliable identification of localized variations in DNA by detection of associated diagnostic products with matrix-assisted laser desorption ionization time-of-flight mass spectrometry is described. The diagnostic products are generated by the primer oligo base extension (PROBE) reaction, which requires a single detection primer complementary to a region downstream of a target strand’s variable site. On addition of a polymerase, three dNTPs, and the fourth nucleotide in dideoxy form, the primer is extended through the mutation region until the first ddNTP is incorporated; the mass of the extension products determines the composition of the variable site. Tests for five cystic fibrosis mutations, including two exon 11 sites measured in a biplex reaction, and for differentiating between three common alleles of the poly(T) tract at the intron 8 splice acceptor site of the CFTR gene are presented. All experimental steps required for PROBE are amenable to the high degree of automation desirable for a high-throughput diagnostic setting. Furthermore, it requires no fluorescent, chemiluminescent, or radioactive labeling; the mass signals measured offer a far more analytically definitive signal, leading in all cases to high-quality unambiguous and easily interpreted results.
Preparation of lyophilized partial thromboplastin time reagent composed of synthetic phospholipids: usefulness for monitoring heparin therapy
Authors: van den Besselaar Anton M H P , Neuteboom Jacoline , Meeuwisse-Braun Joyce , Bertina Rogier M
Published: January 2020

To contribute to the development of a reference reagent for monitoring heparin therapy, a lyophilized partial thromboplastin time (PTT) reagent was prepared from synthetic dioleoylphosphatidylcholine, dioleoylphosphatidylserine, and dioleoylphosphatidylethanolamine, with colloidal silica as activator. The reagent, coded 91/558, was contained in sealed glass ampoules; it deteriorated in a heat degradation experiment, but its activity remained constant for at least 4 years when stored at −70 °C. Within- and between-run precision with this reagent complied with the requirements proposed by the International Committee for Standardization in Haematology (ICSH) Panel on PTT. The response of this reagent and of two other reagents to heparin added to pooled normal plasma was nonlinear. Citrated samples from 58 patients receiving intravenous heparin and from 24 apparently healthy volunteers were tested with reagent 91/558, with Automated APTT (Organon Teknika), with Manchester APTT reagent, with an anti-factor Xa assay, and with an anti-factor IIa assay. The correlation of APTT with anti-Xa and anti-IIa activity was poor. The best correlation was observed between reagent 91/558 and the Organon Teknika reagent. Correlations were improved when individual patients’ samples were replaced by pooled plasmas from heparinized patients, in whom the effect of oral anticoagulation was minimal. These results suggest that preparation of a lyophilized synthetic phospholipid reagent is feasible for use in monitoring heparin therapy.
Point Status of lipid and lipoprotein standardization
Authors: McNamara Judith R , Leary Elizabeth Teng , Ceriotti Ferruccio , Boersma-Cobbaert Christa M , Cole Thomas G , Hassemer David J , Nakamura Masakazu , Packard Christopher J , Seccombe David W , Kimberly Mary M , Myers Gary L , Cooper Gerald R
Published: January 2020

Cholesterol and triglyceride standardization procedures have been used extensively and continuously since the 1950s. Definitive and Reference Methods, as well as primary and secondary standards, have been developed and maintained as the basis for evaluating the accuracy of results by various methods in many laboratories. But, although standardization efforts for apolipoprotein A-I and B measurements have been reported in detail in the scientific literature, much less has been reported in the area of total and lipoprotein cholesterol and triglyceride standardization efforts. Standardized cholesterol and triglyceride concentrations, determined in multiple large epidemiological and clinical studies, have been instrumental to the National Cholesterol Education Program panels that have assessed the lipoprotein values associated with risk of coronary disease, and have determined the cutpoints that are now used extensively by physicians to guide diagnosis and treatment of individual patients.
Serum concentrations of myoglobin vs human heart-type cytoplasmic fatty acid-binding protein in early detection of acute myocardial infarction
Authors: Ishii Junnichi , Wang Jian-hua , Naruse Hiroyuki , Taga Shinn , Kinoshita Masatomo , Kurokawa Hiroshi , Iwase Masatsugu , Kondo Takeshi , Nomura Masanori , Nagamura Youichi , Watanabe Yoshihiko , Hishida Hitoshi , Tanaka Takao , Kawamura Keishiro
Published: January 2020

We compared the diagnostic utility of serum concentrations of human heart-type cytoplasmic fatty acid-binding protein (H-FABPc), myoglobin, and their ratio for the early diagnosis of acute myocardial infarction (AMI) in 104 healthy volunteers and 165 patients at admission within 6 h of the onset of chest pain. The ROC curves of the H-FABPc [0.946, 95% confidence interval (CI) = 0.913–0.979] and myoglobin (0.895, 95% CI = 0.846–0.944) between patients with AMI and healthy volunteers were significantly greater than the area under the ratio of myoglobin to H-FABPc (0.823, 95% CI = 0.765–0.881). In 165 patients, the sensitivity (81.8%, 95% CI = 74.2–89.4%), specificity (86.4%, 95% CI = 78.1–94.6%), and predictive accuracy (83.6%, 95% CI = 78.0–89.3%) of H-FABPc >12 μg/L in diagnosing AMI were significantly higher than those of myoglobin, and were similar to those of the combination of H-FABPc >12 μg/L and the ratio ≤14. We conclude that H-FABPc is a more sensitive and specific marker than myoglobin for the early diagnosis of AMI, and that their ratio cannot give a clear advantage over the measurement of H-FABPc alone.
Radioimmunoassay of cortisone in serum, urine, and saliva to assess the status of the cortisol–cortisone shuttle
Authors: Morineau Gilles , Boudi Ahmed , Barka Ahmed , Gourmelen Micheline , Degeilh Françoise , Hardy Noah , Al-Halnak Akram , Soliman Hany , Gosling James P , Julien René , Brerault Jean-Louis , Boudou Philippe , Aubert Philippe , Villette Jean-Marie , Pruna André , Galons Hervé , Fiet Jean
Published: January 2020

We have developed a new assay for cortisone (E) in serum, saliva, and urine involving Celite® chromatography followed by RIA with 125I-labeled E and scintillation proximity assay. The chromatography step separates cortisol (F) from E, and in combination with their RIAs, permits assessment of the status of the F–E shuttle. We report the results of basal, postcorticotropin (ACTH), and postdexamethasone E and F concentrations and their circadian fluctuations in the serum, saliva, and urine of healthy volunteers. The serum and urine F/E ratios were increased in patients with ectopic ACTH secretion, whereas in adrenal adenoma and Cushing disease only the urinary ratio was increased. In chronic renal insufficiency this ratio was increased in serum (23.5 ± 3.9) but diminished in saliva (0.38 ± 0.11), and in apparent mineralocorticoid excess the ratios were high in serum (44.3 ± 9.3) and urine (5.35 ± 0.85) compared with those of healthy subjects (serum 9.8 ± 3.5, urine 0.52 ± 0.29, saliva 0.52 ± 0.29).
Samples from Patients to Laboratories. Walter Guder, Sheshadri Narayanan, Hermann Wisser, Bernd Zawta. Darmstadt: Git Verlag, 1996, 101 pp., 48 DM. ISBN 3–928865-22-6.
Authors: Calam Roger R
Published: January 2020

An Analytical and Clinical Comparison of ACST BR with Two Other Breast Tumor Marker Assays Houser, S.M., Maimonis, P.J. Chiron Diagnostics, East Walpole, MA
Xenobiotic-induced hepatotoxicity: mechanisms of liver injury and methods of monitoring hepatic function
Authors: Sturgill Marc G , Lambert George H
Published: January 2020

Xenobiotic-induced liver injury is a clinically important etiology of hepatic disease that, if not recognized, can lead to hepatic failure. In this article we discuss the mechanisms of xenobiotic-induced liver injury, various factors that can alter the risk and severity of injury, the clinical and laboratory manifestations of injury, and the methods used to detect the presence of injury and (or) functioning liver mass.
Assessment of liver function: pre- and peritransplant evaluation
Authors: Shaked Abraham , Nunes Fredrick A , Olthoff Kim M , Lucey Michael R
Published: January 2020

Liver transplantation has been demonstrated to be a successful therapeutic modality for patients with end-stage liver disease. The high rate of survival for an otherwise terminal condition has resulted in significant expansion of the indications and diseases treated by this procedure, and is hampered only by the limited numbers of organs available for transplantation. Efforts in clinical and laboratory medicine should be directed to identify candidates who would benefit most from this procedure, to provide better means for accurate assessment of liver reserve and the appropriate timing for transplantation, to identify quality liver grafts that would have the potential to tolerate cold preservation and reperfusion injury, and to assist in accurate monitoring of graft function immediately after transplantation. The aim of this manuscript is to describe the existing pathways for clinical and laboratory assessment of pretransplant residual liver function, the donor liver graft, and immediate posttransplantation function.
Clinical chemistry and post-liver-transplant monitoring
Authors: Hickman Peter E , Potter Julia M , Pesce Amadeo J
Published: January 2020

Liver transplantation is an accepted therapy for end-stage liver disease. After allografting, a variety of clinical problems may require laboratory involvement for accurate and timely diagnosis and intervention. Critical factors in the choice of a laboratory test menu to support a transplant program include turnaround times that support clinical decisionmaking, real diagnostic value, and real value for money. Particular clinical problems, whose early presentation must be anticipated, include graft ischemia, primary nonfunction, and hepatic artery thrombosis. Acute rejection is common at 5–10 days posttransplantation, the principal target being the biliary tree. Longer-term problems are associated with the therapeutic drug measurement of cyclosporin A and, increasingly, tacrolimus (FK506); the side effects of immunosuppressant therapy also require monitoring. A successful liver transplant program can be adequately supported with a simple battery of automated tests that are cheap, fast, and available at all times.
Detecting familial defective apolipoprotein B-100: three molecular scanning methods compared
Authors: Henderson Bryony G , Wenham Philip R , Ashby J Peter , Blundell Gillian
Published: January 2020

Familial defective apolipoprotein (apo) B-100 (FDB), a condition that may give rise to hypercholesterolemia, is caused by mutations around codon 3500 of the apo B gene. We have compared the ability of three molecular-scanning techniques, heteroduplex analysis, single-strand conformation polymorphism (SSCP) analysis, and denaturing gradient gel electrophoresis (DGGE), to detect these mutations in a cohort of 432 hypercholesterolemic individuals. Heteroduplex analysis and DGGE detected 11 individuals with apo B mutations, 9 of whom were heterozygous for apo B R3500Q and 2 who were heterozygous for apo B R3531C. Whereas DGGE was able to distinguish between these two mutations, heteroduplex analysis was technically simpler and gave a higher sample throughput. In contrast, SSCP analysis detected only 7 of the R3500Q and none of the R3531C heterozygotes and was the most complex of the three techniques. We believe heteroduplex analysis to be the method of choice for screening large numbers of samples for FDB.
Monitoring of hemostasis in cardiac surgical patients: impact of point-of-care testing on blood loss and transfusion outcomes
Authors: Despotis George J , Joist J Heinrich , Goodnough Lawrence T
Published: January 2020

Patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) are at increased risk for excessive perioperative blood loss requiring transfusion of blood products. Strategies to optimize administration of heparin and protamine and the assessment of their effects on coagulation are evolving in cardiac surgical patients. Two recent evaluations have focused on the use of multiple point-of-care (POC) coagulation assays for patient-specific adjustment of heparin and protamine dosage. These studies indicate that blood loss and transfusion requirements in cardiac surgical patients may be reduced with more accurate control of heparin anticoagulation and its reversal. Blood component administration in patients with excessive post-CPB bleeding is generally empiric in part, related to turnaround times of laboratory-based tests. Methods are now available for rapid, POC assessment of coagulation to allow appropriate, targeted therapy for acquired hemostatic abnormalities. Recent studies indicate that a rapid evaluation of thrombocytopenia and coagulation factor deficiencies with POC tests can facilitate the optimal administration of pharmacologic and transfusion-based therapy in patients who exhibit excessive bleeding after CPB. POC tests that assess platelet function have been developed, and their use may facilitate identification of which patients at risk for excessive blood loss may respond to pharmacologic interventions such as desmopressin acetate or antifibrinolytic agents.
Cross-linking hybridization assay for direct detection of factor V Leiden mutation
Authors: Zehnder James , Van Atta Reuel , Jones Carol , Sussman Howard , Wood Michael
Published: January 2020

A nucleic acid photocross-linking technology was used in the development of a direct assay for factor V Leiden, a point mutation in the factor V gene (G1691A) that is the most common inherited risk factor for thrombosis. This cross-linking hybridization assay included two allele-specific capture probes and six signal-generating reporter probes; all were modified with a photoactivated cross-linking compound. By using two different capture probes complementary to a 16-base sequence at the factor V Leiden mutation site, but differing in the nucleotide opposite the mutation site (C vs T), wild-type and factor V Leiden alleles were differentiated in purified DNA specimens. The assay was also successfully applied to genomic DNA in leukocytes isolated from whole blood; the factor V status of 122 patients as determined by this method was in complete concordance with a standard PCR-based assay and clearly discriminated between healthy individuals and factor V Leiden heterozygotes.
Variability and determinants of total homocysteine concentrations in plasma in an elderly population
Authors: Clarke Robert , Woodhouse Peter , Ulvik Arve , Frost Chris , Sherliker Paul , Refsum Helga , Ueland Per M , Khaw Kay-Tee
Published: January 2020

The variability of plasma total homocysteine (tHcy) was examined in 96 individuals over a 1-yr period. Blood tHcy concentrations varied from 7.1 μmol/L in the bottom quintile to 14.5 μmol/L in the top quintile. The mean tHcy was 10.4 μmol/L, the between-person SD was 2.5 μmol/L, and the within-person SD was 0.93 μmol/L. There was little seasonal variation, and the reliability coefficient was 0.88. Mean tHcy concentrations were inversely related to mean plasma folate (r = −0.36) and vitamin B12 (r = −0.35) concentrations. Median tHcy concentrations were ∼1 μmol/L higher in men than in women and in older (70 to 74 years) than in younger (65 to 69 years) individuals and higher in those with the TT and CT genotypes for the methylenetetrahydrofolate reductase polymorphism than in those with the CC genotype (10.7 and 10.6 vs 9.6 μmol/L). Epidemiological studies based on single tHcy measurements may underestimate the magnitude of any risk associations with disease by 10–15%.
Measurement of soluble transferrin receptor in serum of healthy adults
Authors: Allen Jean , Backstrom Kristin R , Cooper Jeffrey A , Cooper MaryAnne C , Detwiler Thomas C , Essex David W , Fritz Rose P , Means Robert T , Meier Peter B , Pearlman Samuel R , Roitman-Johnson Beatrice , Seligman Paul A
Published: January 2020

The concentration of soluble transferrin receptor (sTfR) in serum is reported to be useful in the diagnosis of iron deficiency, especially for patients with concurrent chronic disease, where routine tests of iron status are compromised by the inflammatory condition. A new diagnostic assay for sTfR is calibrated against natural plasma sTfR, thus minimizing calibration discrepancies that result from differences between the analyte and the cellular transferrin receptor used in other assays. Use of the new assay to measure sTfR concentrations in 225 healthy, hematologically normal adults provided a reference interval against which pathological samples could be compared. There was no difference in the reference intervals for men and women and no correlation of [sTfR] with the age of the subject. Black subjects had significantly higher concentrations than nonblacks, and people living at high altitude had higher concentrations than those living closer to sea level. These differences were additive.