bluuebunny/crossref_metadata_2025 huggingface dataset:
Opportunistic Discovery of Occult Disease by Use of Test Panels in New, Symptomatic Primary Care Outpatients: Yield and Cost of Case Finding
Authors: Takemura Yuzuru , Ishida Haku , Inoue Yuji , Kobayashi Hiroyuki , Beck J Robert
Published: January 2020

Background: Diagnostic test panels have been advocated by the Japan Society of Clinical Pathology for evaluation of presenting complaints of new outpatients in primary care medicine. The tests have additional potential utility for opportunistic finding of asymptomatic diseases, but data are lacking on the number of new conditions identified by the test panels and on the cost per identified case.

Methods: We studied 540 new, symptomatic patients at the Comprehensive Medicine Clinics of National Defense Medical College during 1991–1997. All underwent testing with the “Essential Laboratory Tests” panel (2) [ELT(2) panel]. This panel includes hematologic tests, urinalysis, total protein, C-reactive protein, albumin, cholesterol, triglycerides, glucose, urea nitrogen, creatinine, uric acid, serum protein fractionation, six enzymes, and optional tests, including x-rays, electrocardiogram, and fecal occult blood.

Results: The ELT(2) panel uncovered 276 additional diagnoses of asymptomatic disease or abnormal health status. The most frequent occult condition was hyperlipidemia (100 cases) followed by liver dysfunction (53 cases). Clinical efficiency of the panel (occult diseases/patient) varied depending on the category of tentative initial diagnosis, with the highest efficiency in patients with cardiovascular disease. We created smaller panels by combining 11 basic tests [called the ELT(1) baseline panel] with one or more additional tests from the ELT(2) and analyzed their cost-effectiveness. Addition of four tests (total cholesterol, alanine aminotransferase, glucose, and uric acid) improved both clinical efficiency (0.41 occult disease/patient) and economic efficiency [¥2372 (∼$22.50 US)/occult disease] at a cost-effectiveness of ¥177 per incremental case of occult disease. Addition of further tests decreased cost-effectiveness.

Conclusions: Although the ELT(2) panel has supplemental utility for opportunistic screening of some significant, occult diseases and conditions, universal utilization of the full panel is not supported by the cost-effectiveness found in this study.
Quantification and Characterization of Pregnancy-associated Complexes of Angiotensinogen and the Proform of Eosinophil Major Basic Protein in Serum and Amniotic Fluid
Authors: Christiansen Michael , Jaliashvili Irakli , Overgaard Michael T , Ensinger Christian , Obrist Peter , Oxvig Claus
Published: January 2020

Background: The proform of eosinophil major basic protein (ProMBP) exists in serum from pregnant women complexed with a variable fraction of angiotensinogen (Ang). A subfraction further binds complement C3dg in a 2:2:2 complex. The function, physiology, and clinical importance of ProMBP complexes are unknown, and the specific quantification of these complexes has not been possible.

Methods: We developed an ELISA for the ProMBP/Ang complexes, using a monoclonal antibody against ProMBP for capture and a chicken anti-human Ang antiserum for detection. Calibrators were standardized with WHO IRP 78/610 for pregnancy proteins in the assay range 0.95–15.6 mIU/L.

Results: The concentrations of ProMBP/Ang complexes in serum of nonpregnant blood donors (n = 79) were log-normally distributed with a central 95th interval of 985-3655 mIU/L. In pregnancy, mean serum concentrations were increased from week 7, and the concentrations reached term concentrations in week 18. ProMBP/Ang complexes eluted in gel filtration as a broad peak with a molecular mass of ∼230 kDa. The concentration of ProMBP/Ang/C3dg increased during blood coagulation, suggesting that the ProMBP/Ang/C3dg complex may be a marker of complement activation.

Conclusions: ProMBP/Ang complexes are present in serum from nonpregnant persons as well as pregnant women, and the direct assays described here will make it possible to study the biochemistry and the clinical significance of different ProMBP complexes in pathological conditions and pregnancy.
Evidence-based Implementation of Free Phenytoin Therapeutic Drug Monitoring
Authors: Burt Martha , Anderson David C , Kloss Julie , Apple Fred S
Published: January 2020

Background: The majority of laboratories measure total phenytoin concentration for therapeutic drug monitoring. However, there are substantial interindividual variations in free phenytoin concentrations, the pharmacologically active component.

Methods: We describe the process and data used to implement monitoring of free phenytoin only in an urban medical center. Over a 6-week period, total and free phenytoin concentrations were measured, clinical charts reviewed, and indications for alterations in the percentage of free phenytoin fraction were determined.

Results: Of the 189 phenytoin requests from 139 patients, 136 data points were analyzed. Free phenytoin concentrations were 6.8–35.3%, with 50% outside the expected range of 8–12%. Clinical indications likely responsible for variations were hypoalbuminemia, drug interactions, uremia, pregnancy, and age. Overall, 30% of patients demonstrated a discrepancy between therapeutic, subtherapeutic, or supratherapeutic concentrations between free and total phenytoin concentrations. The largest discordance (53%) occurred in the patient group with free phenytoin <8% or >12%.

Conclusions: This study supports previous clinical findings that monitoring total phenytoin is not as reliable as free phenytoin as a clinical indicator for therapeutic and nontherapeutic concentrations. Thus, we recommend that therapeutic monitoring should use free phenytoin concentrations only.
Bone Lead Concentrations Assessed by in Vivo X-Ray Fluorescence
Authors: Ambrose Theresa M , Al-Lozi Muhammad , Scott Mitchell G
Published: January 2020

The assessment of past chronic lead exposure is difficult. Chronic lead burden is not always correctly assessed using laboratory-based tests that are useful for acute or recent exposures. We describe a case of suspected chronic lead exposure that illustrated the need for improved and possibly noninvasive methods to determine cumulative lead body burden. X-Ray fluorescence (XRF) is discussed as a method to obtain in vivo bone lead measurements. We discuss the potential of such measurements as accurate biomarkers of cumulative exposure and whether XRF can be used for retroactive exposure assessment or to predict risk of future health problems.
Understanding and Identifying Monoclonal Gammopathies
Authors: Attaelmannan Mohammed , Levinson Stanley S
Published: January 2020

Monoclonal gammopathies reflect conditions in which abnormal amounts of immunoglobulins are produced by a clone that developed from a single pro-B germ cell. The condition may reflect a disease process or be benign. The primary purpose of this review is to emphasize routine clinical laboratory techniques that currently are recommended for use in identifying monoclonal gammopathies from serum and urine. Selection of the preferred technique and correct interpretation often is dependent on an understanding of the immunological basis and clinical sequelae associated with these conditions. For this reason, we first briefly discuss the structure, production, and nature of immunoglobulins, and then describe important features of the associated diseases. Finally, we discuss strengths and weaknesses of the techniques and make reference to current recommendations to facilitate optimal testing. We discuss in detail high-resolution electrophoresis, methods for quantifying immunoglobulins, immunofixation electrophoresis, problems associated with analysis of urine immunoglobulins, and identification of cryoglobulins and immune complexes.
Multifaceted Approach to the Diagnosis and Classification of Acute Leukemias
Authors: McKenna Robert W
Published: January 2020

Until recently, the diagnosis and classification of acute myeloid (AML) and acute lymphoblastic (ALL) leukemias was based almost exclusively on well-defined morphologic criteria and cytochemical stains. Although most cases can be diagnosed by these methods, there is only modest correlation between morphologic categories and treatment responsiveness and prognosis. The expansion of therapeutic options and improvement in remission induction and disease-free survival for both AML and ALL have stimulated emphasis on defining good and poor treatment response groups. This is most effectively accomplished by a multifaceted approach to diagnosis and classification using immunophenotyping, cytogenetics, and molecular analysis in addition to the traditional methods. Immunophenotyping is important in characterizing morphologically poorly differentiated acute leukemias and in defining prognostic categories of ALL. Cytogenetic and molecular studies provide important prognostic information and are becoming vitally important in determining the appropriate treatment protocol. With optimal application of these techniques in the diagnosis of acute leukemias, treatment strategies can be more specifically directed and new therapeutic approaches can be evaluated more effectively.
Cardiac Troponin T and I in End-Stage Renal Failure
Authors: Wayand Diana , Baum Hannsjörg , Schätzle Gabriele , Schärf Julia , Neumeier Dieter
Published: January 2020

Background: In patients suffering from end-stage renal failure, cardiac troponin T (cTnT) and I (cTnI) may be increased in serum without other signs of acute myocardial damage. Whether these increases are specific to myocardial injury or nonspecific is not completely clear.

Methods: We investigated time courses of cTnT and cTnI over 1 year and the clinical outcome over 2 years in 59 patients with end-stage renal failure undergoing chronic hemodialysis. At the start of the study, we divided the patients into two groups, group 1, without history of cardiac failure, and group 2, with history of cardiac failure, and looked for differences between the groups in later adverse outcome. cTnT was measured using the Enzymun® troponin T assay on an ES 700 analyzer (Roche). cTnI was measured on a Stratus® II analyzer (Dade Behring). Creatinine and blood urea nitrogen were measured on a Vitros® 950 IRC (Ortho).

Results: Dialysis acutely increased cTnT (P <0.01) and decreased cTnI (P <0.001) regardless of the dialysis membrane used. Although statistically not significant, cTnT but not cTnI was increased more frequently in group 2 than in group 1, in some cases over the whole study period. Five patients (8.5%) died of cardiac complications within 2 years; all of them had mostly increased cTnT and, in one or more samples, increased cTnI.

Conclusions: Dialysis alters measured cTnT and cTnI concentrations in serum. In patients suffering from end-stage renal failure, sporadic or persistently increased cTnT and cTnI appear to predict cardiac complications. Because of the effects of the dialysis procedure on troponin values, we recommend that blood be collected before dialysis.
Determination of Total Interleukin-8 in Whole Blood after Cell Lysis
Authors: Reinsberg Jochen , Dembinski Jörg , Dorn Christoph , Behrendt Daniela , Bartmann Peter , van der Ven Hans
Published: January 2020

Background: It has been shown that a high percentage of interleukin-8 (IL-8) in blood is cell associated. Recently, a simple method for determination of cell-associated IL-8 in whole blood after cell lysis has been described. The purpose of this study was to evaluate this method, to examine the influence of preanalytic sample handling, and to establish the concentration range of total IL-8 and its relation to age and sex in healthy subjects.

Methods: Total IL-8 content of whole blood was determined after lysing blood cells with Milenia® cell lysis solution. IL-8 in the resulting blood lysate was measured with the IMMULITE® IL-8 immunoassay.

Results: When freshly drawn blood was stored up to 48 h on ice, no significant changes in total IL-8 were measured in the subsequently prepared lysate, whereas with storage at room temperature, total IL-8 increased after 3 h from 94 ± 13 ng/L to 114 ± 16 ng/L (n = 10). In lysate stored for 48 h at 4 °C, marginal changes of the IL-8 concentration were noted, with storage at room temperature, only 76% ± 5% (n = 12) of initial concentration was recovered. From lysate frozen at −20 and −80 °C, respectively, 84% ± 4% and 93% ± 2% of initial IL-8 was recovered after 70 days (n = 10). IL-8 was measured with comparable precision in plasma (CV, 3.2–4.2%) and blood lysate (CV, 3.7–4.1%). When plasma was diluted with cell lysis solution, a slightly overestimated recovery (125% ± 3%) was observed; for lysate specimens with a cell lysis solution content ≥75%, the recovery after dilution was 98% ± 2%. In lysate prepared from 12 blood samples with exogenous IL-8 added, IL-8 recovery was 104% ± 2% (recovery from plasma <35%). The median total IL-8 in blood lysates from 103 healthy subjects (22–61 years) was 83 ng/L of blood (2.5–97.5 percentile range, 49–202 ng/L of blood). In females but not in males, total IL-8 increased significantly with advancing age (P <0.002). We found grossly increased total IL-8 in six pregnant women with amniotic infection syndrome.

Conclusions: The evaluated method allows the assessment of total IL-8 in blood with good performance when appropriate conditions of sample pretreatment are considered. The values in healthy volunteers all were above the detection limit of the IL-8 assay; therefore, slight changes of total IL-8 could be noted. Thus, the present method is a suitable tool to study the diagnostic relevance of total IL-8 in blood.
Genetic Algorithm for Scheduling of Laboratory Personnel
Authors: Boyd James C , Savory John
Published: January 2020

Background: Staffing core laboratories with appropriate skilled workers requires a process to schedule these individuals so that all workstations are appropriately filled and all the skills of each worker are exercised periodically to maintain competence.

Methods: We applied a genetic algorithm to scheduling laboratory personnel. Our program, developed in Visual Basic 4.0, maximizes the value of a fitness function that measures how well a given scheduling of individuals and their skills matches a set of work tasks for a given work shift. The user provides in an Excel spreadsheet the work tasks, individuals available to work on any given date, and skills each individual possesses. The user also specifies the work shift to be scheduled, the range of dates to be scheduled, the number of days that an individual stays on a given workstation before rotating, and various parameters for the genetic algorithm if they differ from the default values.

Results: For >22 months, the program matched individuals to those tasks for which they were qualified and maintained personnel skills by rotating job duties. The schedules generated by the program allowed supervisory personnel to anticipate dates far in advance of when worker availability would be limited, so staffing could be adjusted. In addition, the program helped to identify skills for which too few individuals had been trained. This program has been well accepted by the staff in the clinical laboratories of a 670-bed university medical center, saving 37 h of labor per month, or approximately $11 000 per year, in time that supervisory personnel have spent developing work schedules.

Conclusions: The genetic algorithm approach appears to be useful for scheduling in highly technical work environments that employ multiskilled workers.
Comparison of HPLC and Small Column (CDTect) Methods for Disialotransferrin
Authors: Turpeinen Ursula , Methuen Taina , Alfthan Henrik , Laitinen Kalevi , Salaspuro Mikko , Stenman Ulf-Håkan
Published: January 2020

Background: Current methods for determination of carbohydrate-deficient transferrin (CDT) are based on separation of the CDT fraction by ion-exchange chromatography on minicolumns and quantification by immunoassay. Alternatively, the transferrin isoforms can be separated by HPLC anion-exchange chromatography and quantified by absorbance. This method has been reported to improve the validity of CDT as a marker of chronic alcohol abuse.

Methods: HPLC on either MonoQ or ResourceQ anion-exchange columns was used to separate and quantify isoforms of transferrin with detection at 460 nm. The result was expressed as the percentage of the disialo form (pI 5.7) of total transferrin (DST). The commercial CDTectTM assay was used as a comparison method. Serum samples from nondrinkers (n = 57), moderate drinkers (n = 77), and heavy drinkers (n = 139) were analyzed.

Results: In ROC analysis for differentiation between moderate and heavy drinkers, the area under the curve (AUC) for the HPLC method was 0.87 (95% confidence interval, 0.81–0.93), whereas that for CDTect was 0.72 (95% confidence interval, 0.64–0.80). At 90% specificity, the sensitivity of DST was 63% (95% confidence interval, 53–73%) compared with 33% (22–44%) for CDT. The reference interval of the HPLC method was 0.68–1.7%.

Conclusions: The HPLC anion-exchange method for quantification of CDT provides substantially better separation between moderate and heavy drinkers than the CDTect method.
Usefulness of Saliva for Measurement of 3,4-Methylenedioxymethamphetamine and Its Metabolites: Correlation with Plasma Drug Concentrations and Effect of Salivary pH
Authors: Navarro Mèonica , Pichini Simona , Farré Magí , Ortuño Jordi , Roset Pere N , Segura Jordi , de la Torre Rafael
Published: January 2020

Background: Saliva is an alternative biologic matrix for drugs-of-abuse testing that offers the advantages of noninvasive, rapid, and easy sampling. We studied the excretion profile of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolites in both saliva and plasma, as well the effect of the drug on salivary pH.

Methods: Saliva and plasma samples were obtained from eight healthy MDMA consumers after ingestion of a single 100-mg dose of the drug. Concentrations of MDMA and its main metabolites, 3,4-methylenedioxyamphetamine (MDA) and 4-hydroxy-3-methoxymethamphetamine (HMMA), in saliva and plasma were measured by gas chromatography-mass spectrometry. Apparent pharmacokinetic parameters for MDMA in saliva were estimated, and the saliva-to-plasma ratio at each time interval was calculated and correlated with salivary pH.

Results: MDMA, MDA, and HMMA were detected in saliva. Salivary concentrations of MDMA were 1728.9–6510.6 μg/L and peaked at 1.5 h after drug intake. This was followed by a progressive decrease, with a mean concentration of 126.2 μg/L at 24 h. The saliva-to-plasma ratio was 32.3–1.2, with a peak of 18.1 at 1.5 h after drug administration. Salivary pH seemed to be affected by MDMA administration; pH values decreased by 0.6 units (mean pH values of 6.9 and 6.8 at 1.5 and 4 h after drug administration vs predose pH of 7.4).

Conclusions: Measurement of MDMA in saliva is a valuable alternative to determination of plasma drug concentrations in both clinical and toxicologic studies. On-site testing is also facilitated by noninvasive and rapid collection of salivary specimens.
Reference Centiles for Serum Ferritin and Percentage of Transferrin Saturation, with Application to Mutations of the HFE Gene
Authors: Koziol James A , Ho Ngoc J , Felitti Vincent J , Beutler Ernest
Published: January 2020

Background: The gene that causes most cases of hereditary hemochromatosis is designated HFE. Individuals with mutations in the HFE gene may have increased serum iron, transferrin saturation, and ferritin concentrations relative to individuals with the wild-type genotype.

Methods: We generated reference centiles for percentage of transferrin saturation and serum ferritin concentrations in normal (wild-type), healthy Caucasian adults. We then examined transferrin and ferritin concentrations relative to these centiles in 81 individuals homozygous for the major hemochromatosis mutation C282Y and 438 individuals with the compound heterozygous HFE genotype C282Y/H63D.

Results: Serum ferritin concentrations, but not percentage of transferrin saturation, in normal, healthy women tended to increase sharply as they progressed through menopause. Transferrin and serum ferritin centiles for normal, healthy females were lower than the corresponding centiles in normal, healthy males. C282Y homozygotes had abnormally high transferrin saturation and serum ferritin values relative to the wild types. Compound heterozygotes appeared to be a mixture of individuals with unexceptional transferrin and ferritin values and those with abnormally large values similar to the homozygotes, with equal proportions of each.

Conclusions: There are age- and sex-related differences in reference centiles for the percentage of transferrin saturation and serum ferritin concentrations in normal, healthy adults. Individuals homozygous for the C282Y mutation in the HFE gene have abnormal transferrin saturation and serum ferritin values relative to the reference population; penetrance with the compound heterozygotes, as reflected by abnormal transferrin and ferritin values, is less than with the homozygotes.
Profiling of Tryptophan-related Plasma Indoles in Patients with Carcinoid Tumors by Automated, On-Line, Solid-Phase Extraction and HPLC with Fluorescence Detection
Authors: Kema Ido P , Meijer Wim G , Meiborg Gert , Ooms Bert , Willemse Pax H B , de Vries Elisabeth G E
Published: January 2020

Background: Profiling of the plasma indoles tryptophan, 5-hydroxytryptophan (5-HTP), serotonin, and 5-hydroxyindoleacetic acid (5-HIAA) is useful in the diagnosis and follow-up of patients with carcinoid tumors. We describe an automated method for the profiling of these indoles in protein-containing matrices as well as the plasma indole concentrations in healthy controls and patients with carcinoid tumors.

Methods: Plasma, cerebrospinal fluid, and tissue homogenates were prepurified by automated on-line solid-phase extraction (SPE) in Hysphere Resin SH SPE cartridges containing strong hydrophobic polystyrene resin. Analytes were eluted from the SPE cartridge by column switching. Subsequent separation and detection were performed by reversed-phase HPLC combined with fluorometric detection in a total cycle time of 20 min. We obtained samples from 14 healthy controls and 17 patients with metastasized midgut carcinoid tumors for plasma indole analysis. In the patient group, urinary excretion of 5-HIAA and serotonin was compared with concentrations of plasma indoles.

Results: Within- and between-series CVs for indoles in platelet-rich plasma were 0.6–6.2% and 3.7–12%, respectively. Results for platelet-rich plasma serotonin compared favorably with those obtained by single-component analysis. Plasma 5-HIAA, but not 5-HTP was detectable in 8 of 17 patients with carcinoid tumors. In the patient group, platelet-rich plasma total tryptophan correlated negatively with platelet-rich plasma serotonin (P = 0.021; r = −0.56), urinary 5-HIAA (P = 0.003; r = −0.68), and urinary serotonin (P <0.0001; r = −0.80).

Conclusions: The present chromatographic approach reduces analytical variation and time needed for analysis and gives more detailed information about metabolic deviations in indole metabolism than do manual, single-component analyses.
Tandem Mass Spectrometric Analysis for Amino, Organic, and Fatty Acid Disorders in Newborn Dried Blood Spots
Authors: Zytkovicz Thomas H , Fitzgerald Eileen F , Marsden Deborah , Larson Cecilia A , Shih Vivian E , Johnson Donna M , Strauss Arnold W , Comeau Anne Marie , Eaton Roger B , Grady George F
Published: January 2020

Background: Tandem mass spectrometry (MS/MS) is rapidly being adopted by newborn screening programs to screen dried blood spots for >20 markers of disease in a single assay. Limited information is available for setting the marker cutoffs and for the resulting positive predictive values.

Methods: We screened >160 000 newborns by MS/MS. The markers were extracted from blood spots into a methanol solution with deuterium-labeled internal standards and then were derivatized before analysis by MS/MS. Multiple reaction monitoring of each sample for the markers of interest was accomplished in ∼1.9 min. Cutoffs for each marker were set at 6–13 SD above the population mean.

Results: We identified 22 babies with amino acid disorders (7 phenylketonuria, 11 hyperphenylalaninemia, 1 maple syrup urine disease, 1 hypermethioninemia, 1 arginosuccinate lyase deficiency, and 1 argininemia) and 20 infants with fatty and organic acid disorders (10 medium-chain acyl-CoA dehydrogenase deficiencies, 5 presumptive short-chain acyl-CoA dehydrogenase deficiencies, 2 propionic acidemias, 1 carnitine palmitoyltransferase II deficiency, 1 methylcrotonyl-CoA carboxylase deficiency, and 1 presumptive very-long chain acyl-CoA dehydrogenase deficiency). Approximately 0.3% of all newborns screened were flagged for either amino acid or acylcarnitine markers; approximately one-half of all the flagged infants were from the 5% of newborns who required neonatal intensive care or had birth weights <1500 g.

Conclusions: In screening for 23 metabolic disorders by MS/MS, an mean positive predictive value of 8% can be achieved when using cutoffs for individual markers determined empirically on newborns.
Oligonucleotide Melting Temperatures under PCR Conditions: Nearest-Neighbor Corrections for Mg2+, Deoxynucleotide Triphosphate, and Dimethyl Sulfoxide Concentrations with Comparison to Alternative Empirical Formulas
Authors: von Ahsen Nicolas , Wittwer Carl T , Schütz Ekkehard
Published: January 2020

Background: Many techniques in molecular biology depend on the oligonucleotide melting temperature (Tm), and several formulas have been developed to estimate Tm. Nearest-neighbor (N-N) models provide the highest accuracy for Tm prediction, but it is not clear how to adjust these models for the effects of reagents commonly used in PCR, such as Mg2+, deoxynucleotide triphosphates (dNTPs), and dimethyl sulfoxide (DMSO).

Methods: The experimental Tms of 475 matched or mismatched target/probe duplexes were obtained in our laboratories or were compiled from the literature based on studies using the same real-time PCR platform. This data set was used to evaluate the contributions of [Mg2+], [dNTPs], and [DMSO] in N-N calculations. In addition, best-fit coefficients for common empirical formulas based on GC content, length, and the equivalent sodium ion concentration of cations [Na+eq] were obtained by multiple regression.

Results: When we used [Na+eq] = [Monovalent cations] + 120($\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\sqrt{{[}Mg^{2{+}}{]}\ {-}\ {[}dNTPs{]}}\) \end{document}$) (the concentrations in this formula are mmol/L) to correct ΔS0 and a DMSO term of 0.75 °C (%DMSO), the SE of the N-N Tm estimate was 1.76 °C for perfectly matched duplexes (n = 217). Alternatively, the empirical formula Tm (°C) = 77.1 °C + 11.7 × log[Na+eq] + 0.41(%GC) − 528/bp − 0.75 °C(%DMSO) gave a slightly higher SE of 1.87 °C. When all duplexes (matched and mismatched; n = 475) were included in N-N calculations, the SE was 2.06 °C.

Conclusions: This robust model, accounting for the effects of Mg2+, DMSO, and dNTPs on oligonucleotide Tm in PCR, gives reliable Tm predictions using thermodynamic N-N calculations or empirical formulas.
Creatine Kinase Gene Mutation in a Patient with Muscle Creatine Kinase Deficiency
Authors: Yamamichi Hiroshi , Kasakura Shinpei , Yamamori Shunzi , Iwasaki Ryu , Jikimoto Takumi , Kanagawa Sugayo , Ohkawa Jiro , Kumagai Shunichi , Koshiba Masahiro
Published: January 2020

Background: We describe a 56-year-old woman admitted to the hospital with a diagnosis of acute myocardial infarction without an increase of serum creatine kinase (CK) activity during her clinical course. She died on the 11th hospital day, and the diagnosis was confirmed by autopsy. The patient had had no previous muscular symptoms.

Methods: Expression of the CK-muscle (CK-M) protein in cardiac tissue was examined by immunoblotting and immunochemical staining. CK-M mRNA expression was estimated by semiquantitative reverse transcription–PCR. Gene structure of CK-M was determined by Southern blotting and direct sequencing of 2251 bp. Existence of a point mutation in the CK-M gene was examined by restriction fragment length polymorphism analysis of PCR products (PCR-RFLP) in the patient and in 108 controls.

Results: CK-M protein in the myocardial tissue of the patient was substantially lower (103 ± 7 ng/mg protein) than in control myocardial tissue (35 800 ± 2860 ng/mg protein). Immunoreactive CK-M in the patient tissue sample was 0.3% of the value for the control sample. CK-M mRNA was 53-fold less in the patient sample compared with the control. This very low expression of CK-M mRNA was considered to be the primary reason for CK-M deficiency. Direct sequencing revealed a point mutation at residue 54 in exon 2, which was specific for the patient. No other abnormalities were found in the CK-M gene of the patient.

Conclusions: This report identifies a molecular abnormality in human CK deficiency and discusses the physiologic relevance of CK-M.
Analysis of Dicarboxylic Acids by Tandem Mass Spectrometry. High-Throughput Quantitative Measurement of Methylmalonic Acid in Serum, Plasma, and Urine
Authors: Kushnir Mark M , Komaromy-Hiller Gabor , Shushan Bori , Urry Francis M , Roberts William L
Published: January 2020

Background: Methylmalonic acid (MMA) is a dicarboxylic acid whose concentration can be increased in blood and urine in patients with an inborn error of metabolism or vitamin B12 deficiency. We developed a method for the selective analysis of dicarboxylic acids that exploits the high specificity of tandem mass spectrometry (MS/MS) and the substantial difference in fragmentation patterns of the isomers methylmalonic (MMA) and succinic acid (SA).

Methods: Dicarboxylic acids were extracted from samples with methyl-tert-butyl ether and derivatized with butanolic HCl to form dibutyl esters. The derivative was injected into the liquid chromatography (LC)-MS/MS system using TurboIonSprayTM (nebulizer-assisted electrospray) ionization and quantified by the multiple reaction monitoring mode of MS/MS.

Results: The assay for MMA was linear up to 150 μmol/L. The total imprecision was ≤7.5% at both low and high concentrations. The limits of quantification and detection were 0.1 and 0.05 μmol/L, respectively. The degree of interference from SA could be predicted from the branching ratios of the major product ions.

Conclusions: The method is specific for dicarboxylic acids. The LC-MS/MS analysis for MMA requires minimal chromatographic separation and takes <60 s per sample. The entire analysis, including sample preparation, for a batch of 100 specimens can be performed in <4 h.
Fatty Acid Ethyl Esters in Hair as Markers of Alcohol Consumption. Segmental Hair Analysis of Alcoholics, Social Drinkers, and Teetotalers
Authors: Auwärter Volker , Sporkert Frank , Hartwig Sven , Pragst Fritz , Vater Hannelore , Diefenbacher Albert
Published: January 2020

Background: Fatty acid ethyl esters (FAEEs) are products of nonoxidative ethanol metabolism. After incorporation in hair, they should be suitable long-term markers of alcohol abuse.

Methods: Hair samples from 19 alcoholics in a treatment program, 10 fatalities with verified excessive alcohol consumption, 13 moderate social drinkers who consumed up to 20 g ethanol/day, and 5 strict teetotalers were analyzed in 1–12 segments for four FAEEs (ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate) by external degreasing with n-heptane, extraction with a dimethyl sulfoxide-n-heptane mixture, headspace solid-phase microextraction of the extracts, and gas chromatography-mass spectrometry with deuterated internal standards. The n-heptane washings were analyzed in the same way for FAEEs from the hair surface.

Results: The sum of the four ester concentrations in hair calculated for the proximal 0–6 cm segment was 2.5–13.5 ng/mg (mean, 6.8 ng/mg) for the fatalities, 0.92–11.6 ng/mg (mean, 4.0 ng/mg) for 17 of the alcoholics in treatment, 0.20–0.85 ng/mg (mean, 0.41 ng/mg) for the moderate social drinkers, and 0.06–0.37 ng/mg (mean, 0.16 ng/mg) for the teetotalers. In almost all cases the segmental concentrations increased from proximal to distal. There was no agreement between the self-reported drinking histories of the participants and the FAEE concentrations along the hair length. Ethyl oleate was the dominant ester in all samples.

Conclusions: FAEEs are deposited in hair mainly from sebum. Despite large individual differences, FAEE hair concentrations can be used as markers for excessive alcohol consumption with relatively high accuracy.
Effects of Hemoglobin Variants and Chemically Modified Derivatives on Assays for Glycohemoglobin
Authors: Bry Lynn , Chen Philip C , Sacks David B
Published: January 2020

Background: Glycohemoglobin (gHb), measured as hemoglobin (Hb) A1c or as total gHb, provides a common means for assessing long-term glycemic control in individuals with diabetes mellitus. Genetic variants and chemically modified derivatives of Hb can profoundly affect the accuracy of these measurements, although effects vary considerably among commercially available methods. The prevalence of genetic variants such as HbS, HbC, and HbE, and chemically modified derivatives such as carbamyl-Hb among patient populations undergoing testing is not insignificant. Clinical laboratories and sites responsible for point-of-care testing of gHb need to be aware of the interferences produced in assays by these Hbs.

Approach: We conducted a review of the literature describing the effects of variant Hbs on gHb assay methods commonly used in clinical laboratories.

Content: This review summarizes the documented effects of both common and uncommon Hb variants and derivatives on the measurement of gHb. Where known, we discuss mechanisms of interference on specific assays and methodologies. We specifically address effects of commonly encountered Hbs, such as carbamyl-Hb, HbS, HbC, HbE, and HbF, on assays that use cation-exchange chromatography, immunoassays, or boronate affinity methods for measuring gHb.

Summary: A variety of patient- and laboratory-related factors can adversely affect the measurement of gHb in patients harboring Hb variants or derivatives. Identification of the variant or derivative Hb before or during testing may allow accurate measurement of gHb by the selection of a method unaffected by the given variant or derivative. However, laboratories should make available alternative, non-Hb-based methods for assessing long-term glycemic control in individuals with HbCC, HbSS, or HbSC disease, or with other underlying disorders where the concentration of gHb does not accurately reflect long-term glycemic control.
Quantitative Real-Time Reverse Transcription-PCR Assay for Cyclin D1 Expression: Utility in the Diagnosis of Mantle Cell Lymphoma
Authors: Bijwaard Karen E , Aguilera Nadine S I , Monczak Yury , Trudel Michel , Taubenberger Jeffery K , Lichy Jack H
Published: January 2020

Background: The t(11;14)(q13;q32) translocation present in the majority of mantle cell lymphomas (MCLs) places the cyclin D1 gene under the control of immunoglobulin transcriptional regulatory elements, causing overexpression of cyclin D1. Quantification of cyclin D1 expression can distinguish MCL from other lymphomas.

Methods: A quantitative real-time reverse transcription (RT)-PCR assay was developed for cyclin D1 mRNA suitable for use with RNA extracted from fresh and formalin-fixed, paraffin-embedded tissues. Specimens were amplified in an Applied Biosystems Model 7700 Sequence Detection System in reactions containing primers and probes for cyclin D1 and a control gene, β2-microglobulin. Relative expression of the two genes was standardized against a control MCL cell line, M02058.

Results: The range of cyclin D1 expression among 20 MCLs was substantially higher than that in other lymphomas and reactive lymph nodes. By choosing an optimal cutoff point for assessing overexpression, the sensitivity and specificity of the assay for the diagnosis of MCL in lymph node specimens both approached 100%: Overexpression was detected in 20 of 20 MCLs, but in none of 21 non-mantle-cell lymphomas or 10 reactive lymph nodes.

Conclusions: Quantitative real-time RT-PCR for cyclin D1 overexpression provides a rapid diagnostic test with clinical utility in the diagnosis of MCL.
Macromolecular Chromogenic Substrates for Measuring Proteinase Activity
Authors: Hortin Glen L , Warshawsky Ilka , Laude-Sharp Maryline
Published: January 2020

Background: Proteinase activities are often measured using chromogenic substrates that are much smaller than physiological substrates.

Methods: The hydrodynamic size of macromolecular substrates (macrosubstrates) prepared by linking small chromogenic substrates to polyethylene glycol was determined by gel filtration. Efficiency of macrosubstrate cleavage by proteinases and α2-macroglobulin-proteinase complexes was monitored spectrophotometrically.

Results: Macrosubstrates had hydrodynamic radii of ∼20 Å, similar to proteins with a molecular weight of 18 000. Different macrosubstrates served as efficient substrates for chymotrypsin, trypsin, and thrombin. Linking small substrates to a polymer variably affected substrate efficiency, with the impact on activity ranging from a 60-fold decrease to a 30-fold increase. Proteinases complexed with α2-macroglobulin had ∼10-fold lower activity vs macrosubstrates than small substrates.

Conclusions: Macrosubstrates are efficient substrates that allow decreased measurement of sterically hindered proteinase molecules such as α2-macroglobulin-proteinase complexes. Thus, macrosubstrates may provide more accurate functional assays of proteinases such as coagulation factors.
Quantitative Determination of Vapor-Phase Compound A in Sevoflurane Anesthesia Using Gas Chromatography–Mass Spectrometry
Authors: Bouche Marie-Paule L A , Van Bocxlaer Jan F P , Rolly Georges , Versichelen Linda F M , Struys Michel M R F , Mortier Eric , De Leenheer André P
Published: January 2020

Background: During low-flow or closed-circuit anesthesia with the fluorinated inhalation anesthetic sevoflurane, compound A, an olefinic degradation product with known nephrotoxicity in rats, is generated on contact with alkaline CO2 adsorbents. To evaluate compound A formation and thus potential sevoflurane toxicity, a reliable and reproducible assay for quantitative vapor-phase compound A determination was developed.

Methods: Compound A concentrations were measured by fully automated capillary gas chromatography–mass spectrometry with cryofocusing. Calibrators of compound A in the vapor phase were prepared from liquid volumetric dilutions of stock solutions of compound A and sevoflurane in ethyl acetate. 1,1,1-Trifluoro-2-iodoethane was chosen as an internal standard. The resulting quantitative method was fully validated.

Results: A linear response over a clinically useful concentration interval (0.3–75 μL/L) was obtained. Specificity, sensitivity, and accuracy conformed with current analytical requirements. The CVs were 4.1–10%, the limit of detection was 0.1 μL/L, and the limit of quantification was 0.3 μL/L. Analytical recoveries were 100.6% ± 10.1%, 102.5% ± 7.3%, and 99.0% ± 4.1% at 0.5, 10, and 75 μL/L, respectively. The method described was used to determine compound A concentrations during simulated closed-circuit conditions. Some of the resulting data are included, illustrating the practical applicability of the proposed analytical approach.

Conclusions: A simple, fully automated, and reliable quantitative analytical method for determination of compound A in air was developed. A solution was established for sampling, calibration, and chromatographic separation of volatiles in an area complicated by limited availability of sample volume and low concentrations of the analyte.
Long-Term Evaluation of Electrospray Ionization Mass Spectrometric Analysis of Glycated Hemoglobin
Authors: Roberts Norman B , Amara Alieu B , Morris Michael , Green Brian N
Published: January 2020

Background: Electrospray ionization mass spectrometry (ESIMS) has been successfully applied to the identification of hemoglobin (Hb) variants and the presence of glucose adducts (mass difference of 162 Da) on the separate Hb α and β chains. To establish the potential of ESIMS as a routine and/or a reference method for the quantification of glycohemoglobin (HbA1c), we carried out a detailed evaluation over a 4-month period in a routine laboratory environment.

Methods: We optimized a procedure using ESIMS suitable for the routine quantitative analysis of HbA1c. We determined reliability and reproducibility over 4 months and assessed the potential for automated sample injection. We then compared values of 1022 blood samples from diabetic patients with a routine HPLC-based ion-exchange procedure (HA-8140; Menarini).

Results: Results of HbA1c measurement by ESIMS were available within 3 min. The analytical imprecision (CV) was 1.6–5.0% for both manual and automated injections. Data collection over the m/z 980-1400 range confirmed lower glycation of the α chain relative to the β chain (0.66:1). Only one glycation was observed per globin chain. The overall glycohemoglobin (i.e., the average of α- and β-chain glycations) measured by ESIMS (x) on 1022 blood samples was lower than by HPLC (y): y = 1.0432x + 0.4815. However, the β-chain glycation measured by ESIMS was up to 20% higher than the value measured by ion-exchange HPLC and showed a close conformity, particularly at 5–10% HbA1c, with the ion-exchange Diabetes Control and Complications Trial (DCCT)-corrected and the United Kingdom National External Quality Assessment Scheme DCCT mean return values.

Conclusions: ESIMS provides a precise measurement of HbA1c and, in particular, glycation of the β chain. The method is robust and could be proposed as a procedure to substantiate HbA1c measurement and/or calibration.
Evaluation of Blood Lead Proficiency Testing: Comparison of Open and Blind Paradigms
Authors: Parsons Patrick J , Reilly Andrew A , Esernio-Jenssen Debra , Werk Lloyd N , Mofenson Howard C , Stanton Noel V , Matte Thomas D
Published: January 2020

Background: Most proficiency testing (PT) programs operate with an open design in which clearly identified performance samples are distributed directly to participating laboratories on a shipping schedule announced in advance. In this study, we examine the effectiveness of assessing clinical laboratory performance for blood lead with an open PT by comparing its results with a double-blinded testing protocol.

Methods: Aliquots from up to 72 blood lead performance pools from the New York State Department of Health and the Wisconsin State Laboratory of Hygiene were disguised as routine patient specimens and submitted in two phases to up to 42 certified clinical laboratories for blood lead analysis. These 42 laboratories also received aliquots of the same performance samples for blood lead analysis directly from the “open” PT program provider.

Results: Data reported under blind and open strategies were scored against acceptable target ranges using the Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88) criteria established for blood lead, i.e., ± 0.19 μmol/L (± 4 μg/dL) or ± 10%, whichever is greater. Performance differences between the strategies were also assessed. We found that 17.7% of all blind PT results were classified as unacceptable compared with only 4.5% of open PT results (P <0.001). In phase 1, 13 of 22 laboratories (60%) exhibited a statistically significant difference (P <0.05) between their blind and open PT performances, although for 6 laboratories the poorer blind performance may not necessarily have led to unsuccessful PT participation under CLIA ’88 criteria. Seven (32%) laboratories had unsuccessful aggregate performance (<80%) under blind testing while maintaining successful performance in open testing. Of these seven, two had gross discrepancies motivating further investigation.

Conclusions: The data suggest that although ∼60% of clinical laboratories make special efforts to improve analytical performance on open PT samples relative to performance achieved for routine patient specimens, in most cases the differences are clinically insignificant and would not likely affect cumulative PT performance. Occasional use of blind PT may deter the inclination to treat performance samples more carefully.
High-Sensitivity C-Reactive Protein: A Novel and Promising Marker of Coronary Heart Disease
Authors: Rifai Nader , Ridker Paul M
Published: January 2020

Background: Coronary heart disease remains the leading cause of morbidity and mortality in the industrialized world. Clinical and laboratory studies have shown that inflammation plays a major role in the initiation, progression, and destabilization of atheromas. C-Reactive protein (CRP), an acute phase reactant that reflects low-grade systemic inflammation, has been studied in a variety of cardiovascular diseases.

Approach: Findings from prospective clinical trials were examined to determine the prognostic utility of CRP in acute coronary syndromes, and observations from epidemiological studies were reviewed to determine the ability of CRP to predict future first coronary events. The analytical considerations of CRP measurement in these clinical applications were also examined.

Content: In patients with established coronary disease, CRP has been shown to predict adverse clinical events. In addition, prospective studies have consistently shown that CRP is a strong predictor of future coronary events in apparently healthy men and women. The relative risk associated with CRP is independent of other cardiovascular disease risk factors. High-sensitivity CRP (hs-CRP) assays are needed for risk assessment of cardiovascular disease. Such assays are currently available but may require further standardization because patients’ results will be interpreted using population-based cutpoints. Preventive therapies to attenuate coronary risk in individuals with increased hs-CRP concentrations include aspirin and statin-type drugs.

Summary: hs-CRP has prognostic utility in patients with acute coronary syndromes and is a strong independent predictor of future coronary events in apparently healthy subjects.
Absence of Diurnal Variation of C-Reactive Protein Concentrations in Healthy Human Subjects
Authors: Meier-Ewert Hans K , Ridker Paul M , Rifai Nader , Price Nick , Dinges David F , Mullington Janet M
Published: January 2020

Background: The concentration of C-reactive protein (CRP) in otherwise healthy subjects has been shown to predict future risk of myocardial infarction and stroke. CRP is synthesized by the liver in response to interleukin-6, the serum concentration of which is subject to diurnal variation.

Methods: To examine the existence of a time-of-day effect for baseline CRP values, we determined CRP concentrations in hourly blood samples drawn from healthy subjects (10 males, 3 females; age range, 21–35 years) during a baseline day in a controlled environment (8 h of nighttime sleep).

Results: Overall CRP concentrations were low, with only three subjects having CRP concentrations >2 mg/L. Comparison of raw data showed stability of CRP concentrations throughout the 24 h studied. When compared with cutoff values of CRP quintile derived from population-based studies, misclassification of greater than one quintile did not occur as a result of diurnal variation in any of the subjects studied. Nonparametric ANOVA comparing different time points showed no significant differences for both raw and z-transformed data. Analysis for rhythmic diurnal variation using a method fitting a cosine curve to the group data was negative.

Conclusions: Our data show that baseline CRP concentrations are not subject to time-of-day variation and thus help to explain why CRP concentrations are a better predictor of vascular risk than interleukin-6. Determination of CRP for cardiovascular risk prediction may be performed without concern for diurnal variation.