bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of mutations in the apolipoprotein CII gene by denaturing gradient gel electrophoresis. Identification of the splice site variant apolipoprotein CII-Hamburg in a patient with severe hypertriglyceridemia
Authors: Nauck Markus S , Nissen Henrik , Hoffmann Michael M , Herwig Jürgen , Pullinger Clive R , Averna Maurizio , Geisel Jürgen , Wieland Heinrich , März Winfried
Published: January 2020

Familial apolipoprotein (apo) CII deficiency is a rare autosomal recessive inborn error of metabolism clinically resembling lipoprotein lipase deficiency. A number of mutations of the apo CII gene are known to date; they are located in the promoter region, the coding exons, or in the splice junctions. We present a simple assay based on PCR and denaturing gradient gel electrophoresis, which allows scanning of the promoter, the entire coding sequence, and the splice junctions of the apo CII gene for sequence variants. All gene fragments are amplified using a common PCR protocol and are examined for mutations on a single gradient gel. Using this method and direct sequencing, we identified homozygosity for a donor splice-site mutation in the second intron, previously designated apo CII-Hamburg, as the genetic cause of apo CII deficiency in a 9-year-old boy presenting with chylomicronemia, eruptive xanthoma, and pancreatitis. In addition, the method allowed us to detect all of six different other known mutations of the apo CII gene. We conclude, therefore, that our assay is highly sensitive; in addition, it is easy to perform and may facilitate the differential diagnosis of disorders of lipoprotein metabolism at the genetic level.
New immunoseparation-based homogeneous assay for HDL-cholesterol compared with three homogeneous and two heterogeneous methods for HDL-cholesterol
Authors: Nauck Matthias , März Winfried , Wieland Heinrich
Published: January 2020

We evaluated four new commercial methods for HDL-cholesterol determination. The three completely homogeneous assays were an immunoseparation-based (IS) method from Wako, a polyethylene glycol-modified enzyme (PEG) method from Boehringer Mannheim, and a synthetic polymer-based (SP) method from Genzyme. The fourth method was a new heterogeneous method in which lipoproteins are removed using dextran sulfate-coated magnetic beads and Mg2+ (MB, Reference Diagnostics). We compared these methods with the conventional phosphotungstic acid/MgCl2 precipitation (PTA) procedure. The homogeneous assays had good intraassay imprecision with total CVs <2.3%, whereas the CVs of the MB assay were <5.9%. Adding HDL to serum to achieve HDL-cholesterol (HDL-C) concentrations up to 1000 mg/L revealed nearly complete recoveries in the IS, PEG, and MB assays, whereas the SP assay showed a lower recovery (∼70%). The SP HDL-C apparently increased at increasing LDL-cholesterol and VLDL-triglyceride concentrations, whereas the IS, PEG, and MB methods were not influenced by LDL-cholesterol up to 6 000 mg/L (MB, 5 000 mg/L) and VLDL-triglycerides up to 9 000 mg/L. Free fatty acids above ∼2 mmol/L produced falsely high HDL-C in the IS and SP assays, the error amounting to as much as 50% in some samples. An intermethod comparison in 291 fresh serum samples yielded correlation coefficients of at least r = 0.95 for all assays, when compared with the PTA procedure. The slopes and intercepts of the regression lines were 1.05 and 57 (IS), 1.12 and 9.9 (PEG), 1.00 and 39 (SP), and 1.0 and 38 mg/L (MB), respectively. The new assays are precise and simplify the determination of HDL-C, but in part they lack specificity or are susceptible to interferences, resulting in discrepancies when compared with the established PTA procedure.
Assessment of interlaboratory performance in external proficiency testing programs with a direct HDL-cholesterol assay
Authors: Rifai Nader , Cole Thomas G , Iannotti Elizabeth , Law Terence , Macke Michael , Miller Richard , Dowd Dennis , Wiebe Donald A
Published: January 2020

Direct assays for the determination of HDL-cholesterol (HDL-C) have recently become available. The methods are precise, require small sample volume, and appear to be less affected by increased triglycerides than traditional precipitation methods. In this study, we describe the inter- and intralaboratory variability of the Boehringer Mannheim Corporation direct HDL-C assay and its performance in external proficiency testing surveys. A comparison study among three laboratories, using different analyzers and 85 serum specimens, showed a correlation coefficient (r) of 0.99. The direct HDL-C assay also showed good agreement with the ultracentrifugation-dextran sulfate-Mg2+ method (r = 0.98) and the Cholesterol Reference Method Laboratory Network-Designated Comparison Method (a = 0.98x + 4.75 mg/L, r = 0.98). Total error at medical decision levels ranged from −0.8% to +11.1%. Furthermore, this assay performed adequately in the College of American Pathologists and the ALERT® surveys as well as the CDC Lipid Standardization Program and met all performance criteria of regulatory agencies.
Water, fat, nitrogen, and sugar content in feces: reference intervals in children
Authors: Rivero-Marcotegui Adriana , Olivera-Olmedo José Emilio , Valverde-Visus Félix Sánchez , Palacios-Sarrasqueta Mercedes , Grijalba-Uche Ana , García-Merlo Sergio
Published: January 2020

Malabsorption-maldigestion syndromes are commonly found in several gastrointestinal diseases. Quantitative measures of fecal nutrients are important tools for the detection and diagnosis of these syndromes. Adequate food intake is important in the nutrition of children, especially during the first year of life. We have analyzed 180 stools of healthy children, divided into four age groups, to obtain the reference intervals of the major nutrients such as water, fat, nitrogen, sugar, and starch. Quantification of the nutrients was done by means of a near-infrared analyzer (Fenir 8820). Results show that this instrument exhibits a low coefficient of variation for all the nutrients except for starch. Fecal water, fat, nitrogen, and sugar concentrations ranged from 68.7 to 96.1 g/100 g, 0 to 14.5 g/100 g, 1.3 to 2.3 g/100 g, and 0.7 to 3.8 g/100 g, respectively. The results for the starch analyses were not acceptable because of instrument limitations. Near-infrared reflectance spectroscopy appears to be an alternative to standard chemical methods.
Plasma folic acid cutoff value, derived from its relationship with homocyst(e)ine
Authors: Brouwer D A Janneke , Welten H T M Erik , Reijngoud Dirk-Jan , van Doormaal Jasper J , Muskiet Frits A J
Published: January 2020

We established the cutoff value for plasma folic acid, using plasma homocyst(e)ine as the functional marker. To do this, we investigated the relationship of the plasma folic acid of 103 apparently healthy adults with their fasting plasma homocyst(e)ine and with their plasma homocyst(e)ine 6 h after oral methionine challenge (100 mg/kg). We also studied the relationship of their plasma folic acid with the decline of fasting plasma homocyst(e)ine after 7 days of folic acid supplementation (5 mg/day). The three approaches suggested a cutoff value of 10 nmol/L. The chances of individuals to significantly (P <0.05) lower their plasma homocyst(e)ine after folic acid supplementation proved significantly higher at plasma folic acid concentrations ≤10 nmol/L, as compared with folic acid concentrations above this value (odds ratio, 5.02; 95% confidence interval, 1.87–13.73). We suggest adopting a 10 nmol/L plasma folic acid cutoff value on functional grounds.
Microparticle-enhanced nephelometric immunoassay of lysozyme in milk and other human body fluids
Authors: Montagne Paul , Cuillière Marie Louise , Molé Claire , Béné Marie Christine , Faure Gilbert
Published: January 2020

Quantitation of lysozyme in human milk was performed by a microparticle-enhanced nephelometric immunoassay based on the measurement of the light scattered during the competitive immunoagglutination of a microparticle–lysozyme conjugate with an anti-lysozyme antiserum. This immunoassay has a detection limit of 8 μg/L of reaction mixture and can be performed using diluted milk (1:6000, in reaction mixture), excluding sample pretreatment. Human milk lysozyme can be quantified over the concentration range 0.09–1.50 g/L, with within- and between-run coefficients of variation <5%. Changes in the lysozyme concentration of human milk during lactation were determined in 636 samples. Lysozyme concentrations (mean ± SE) decreased from colostrum (0.36 ± 0.02 g/L) to transitional milk (0.30 ± 0.01 g/L) and mature milk during days 15–42 (0.30 ± 0.01 g/L), then increased in the mature milk during days 43–56 (0.35 ± 0.01 g/L) and especially during days 57–84 (0.83 ± 0.05 g/L). The proportion of lysozyme contributing to total protein was found to rise during lactation and was as follows: colostrum (1.7%), transitional milk (2.3%), and mature milk from days 15–28 (2.7%), days 29–42 (3.1%), days 43–56 (3.8%), and days 57–84 (7.3%). The assay developed for milk was also suitable for the determination of lysozyme in other human body fluids.
Assessment of current National Cholesterol Education Program guidelines for total cholesterol, triglyceride, HDL-cholesterol, and LDL-cholesterol measurements
Authors: Caudill Samuel P , Cooper Gerald R , Smith S Jay , Myers Gary L
Published: January 2020

We examine the effect of systematic bias and random error, quality control, and intraperson biological variation on the National Cholesterol Education Program (NCEP) clinical classifications for reported lipid measurements. We consider misclassification to occur if a true lipid homeostatic set point is within a desirable range but the reported lipid value is in a high-risk range, or if a true lipid homeostatic set point is in a high-risk range but the reported lipid value is in a desirable range. To evaluate the overall adequacy of the NCEP guidelines to ensure correct patient classification, we construct operating characteristic curves for total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. We demonstrate that if laboratories are meeting the NCEP guidelines for inherent bias and analytic precision and are using standard quality-control (QC) procedures incorporating at least two QC samples per analytical run from each of two QC pools (for a total of 4 QC samples), the current NCEP guidelines are adequate to ensure (probability >0.90) correct patient classifications regardless of the size of the systematic bias of the laboratory or increased random analytic error. Thus we suggest that at least two concentrations of QC material be included in the QC scheme to ensure that the measurement system is operating within desired specifications across the entire range of desirable and high-risk lipid concentrations and to ensure with high probability that patients are correctly classified.
Development of an enzyme-linked immunosorbent assay with monoclonal antibody for quantification of homovanillic in human urine samples
Authors: Shi Run Zhang , Ho Yee-Ping , Yeung John Hok Keung , Or Penelope Mei Yu , To Kenneth Kin Wah , Lau Mary Wai Man , Arumanayagam Mano
Published: January 2020

A monoclonal antibody to homovanillic acid (HVA) was prepared by synthesis of a HVA-protein conjugate (HVA-ovalbumin) as an immunogen, immunization of mice, and the subsequent hybridization technique. Monoclonal antibodies were screened on the basis of sensitivity, specificity, and accuracy. An indirect ELISA was developed for quantification of HVA in human urine. The assay was characterized and shown to have high specificity, with cross-reactivities to vanillylmandelic acid and normetanephrine at 0.18% and <0.1%, respectively. The assay coefficients of variation were <10% within the working range of 0.5–40 mg/L. Initial results from testing urine samples of patients with neuroblastoma and other diseases were validated by HPLC, suggesting that this ELISA method is a reliable and convenient system for quantification of HVA in urine and can be used in the mass screening of neuroblastoma in infants.
Medicare coding and reimbursement for clinical laboratory services
Authors: Root Charles B
Published: January 2020

Medicare will continue to increase its efforts to cut spending through aggressive review of claims and the use of new fraud and abuse regulations. Providers must be especially careful to provide correct procedure codes that define precisely what services have been provided and accurate diagnosis codes that link those procedures or tests to an appropriate diagnosis. Medicare reimbursement rules for clinical laboratory procedures are explained, including the proper use of procedure and diagnosis codes. Coding and payment for new automated test panels are discussed, as well as the economic consequences of using smaller panels. Medicare coverage requirements, including medical necessity, are described, as well as the proper use of advance beneficiary notices and the Medicare appeals process.
Cardiac troponin T isoforms expressed in renal diseased skeletal muscle will not cause false-positive results by the second generation cardiac troponin T assay by Boehringer Mannheim
Authors: Ricchiuti Vincent , Voss Ellen M , Ney Arthur , Odland Mark , Anderson Page A W , Apple Fred S
Published: January 2020

The purpose of this study was to determine whether the two monoclonal anti-cardiac troponin T (cTnT) antibodies (MAbs) used in the second generation cTnT assay by Boehringer Mannheim (BM, capture Ab, M11.7; detection Ab, M7) would detect cTnT isoforms expressed in human skeletal muscle in response to chronic renal disease (CRD). cTnT expression was examined in skeletal muscle biopsies obtained from 45 CRD patients, as well as nondiseased human heart (n = 3) and skeletal muscle (n = 3). cTnT proteins were resolved by modified 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with the following anti-cTnT MAbs: M11.7; M7; JS-2, Lakeland Biomedical; and 13–11, Duke University. All four antibodies detected the cTnT isoforms (Ta, Te) expressed in human myocardium. In 20 of 45 skeletal muscle biopsies, MAb M11.7 recognized its epitope in one to three proteins, molecular mass 34–36 kDa, designated Te, Td, and Tc; the strongest signal was that of Te. The same proteins were recognized by MAbs JS-2 and 13–11. The BM M7 antibody did not detect the cTnT isoforms in the molecular mass range of 34–36 kDa. However, MAb M7 did detect a cTnT isoform, molecular mass 39 kDa, in 2 of 45 biopsies. This isoform had an electrophoretic mobility similar to the predominant heart cTnT isoform, Ta. We conclude that cTnT isoforms are expressed in the skeletal muscle of CRD patients. However, given the epitopes recognized by the BM MAbs M7 and M11.7 and the variable presence of these cTnT isoforms in skeletal muscle, the second generation BM cTnT assay will not detect these isoforms if they are released from skeletal muscle into the circulation.
Fixed polarizer ellipsometry for simple and sensitive detection of thin films generated by specific molecular interactions: applications in immunoassays and DNA sequence detection
Authors: Ostroff Rachel M , Maul Diana , Bogart Gregory R , Yang Shao , Christian Jennifer , Hopkins Deborah , Clark David , Trotter Brian , Moddel Garret
Published: January 2020

Biological thin films may form on a surface by specific molecular interactions. The fixed polarizer ellipsometer (FPE) is a sensitive instrument that detects biological thin films either qualitatively or quantitatively. The design is simple and inexpensive. The assays are formatted on an optical surface, and the FPE detection is based on the phase shift of linearly polarized light after reflection through a thin film. We have constructed mathematical models of the FPE response to reflection through single-layer and two-layer films that agree closely with experimental data. Several biological assays have been measured with the FPE to demonstrate the application of this technology to clinical targets, including ultrasensitive immunoassays for hepatitis B surface antigen (0.1 ng/mL) and α-fetoprotein (0.01 ng/mL) and DNA hybridization (0.5 fmol/μL target probe). A clinical study for detection of group A streptococcus from patient throat swabs demonstrated the qualitative application of the FPE to infectious disease targets. The flexibility and sensitivity of the FPE makes this technology suitable for numerous target analytes and applications.
Mass-sensing, multianalyte microarray immunoassay with imaging detection
Authors: Silzel John W , Cercek Bibijana , Dodson Charles , Tsay Tsong , Obremski Robert J
Published: January 2020

Miniaturization of ligand binding assays may reduce costs by decreasing reagent consumption, but it is less apparent that miniaturized assays can simultaneously exceed the sensitivity of macroscopic techniques by analyte “harvesting” to exploit the total analyte mass available in a sample. Capture reagents (avidin or antibodies) immobilized in 200-μm diameter zones are shown to substantially deplete analyte from a liquid sample during a 1–3-h incubation, and the assays that result sense the total analyte mass in a sample rather than its concentration. Detection of as few as 105 molecules of analyte per zone is possible by fluorescence imaging in situ on the solid phase using a near-infrared dye label. Single and multianalyte mass-sensing sandwich array assays of the IgG subclasses show the sensitivity and specificity of ELISA methods but use less than 1/100 the capture antibody required by the 96-well plate format.
Sensitive RIA for the Specific Determination of Insulin Lispro
Authors: Bowsher Ronald R , Lynch Renee A , Brown-Augsburger Patricia , Santa Paula F , Legan William E , Woodworth James R , Chance Ronald E
Published: January 2020

Insulin lispro is an insulin analog in which the primary sequence has been altered by the inversion of amino acids B28 and B29. To date, it has not been possible to specifically measure insulin lispro in the presence of endogenous insulin because of the high degree of homology between these peptides. However, the specific determination of insulin lispro offers advantages over quantifying total concentrations of immunoreactive insulin. We therefore immunized guinea pigs and screened for antibodies with increased affinity and selectivity for insulin lispro. We prepared a monospecific antiserum by a novel immunoadsorption strategy using despentapeptide insulin. The antiserum was used to develop a competitive RIA for insulin lispro. The RIA has a low limit of quantification (17.2 pmol/L); has no interference from insulin, proinsulin, or C-peptide; and has interassay CVs of 2.6–13.4%. The new RIA is useful for measuring serum concentrations of insulin lispro.
Automated Detection of the Factor V Leiden Mutation Using the LCx Microparticle Enzyme Immunoassay
Authors: Hunault Mathilde , Marsh-Scott Camile , Jou Cynthia , Marshall Ron , Scheffel Christi , Fiore Louis D , Bauer Kenneth A , Mitchell Michael J
Published: January 2020

The factor V Leiden mutation, a G→A transition at position 1691 in exon 10 of the gene that codes for factor V, produces an Arg506Gln substitution and is the most common genetic risk factor for venous thrombosis. We have developed a rapid, sensitive, and specific method to detect the factor V Leiden mutation in genomic DNA from whole blood by PCR amplification and microparticle enzyme immunoassay detection using the Abbott LCx instrument. We compared this automated method with the standard procedure using restriction endonuclease digestion of PCR products followed by gel electrophoresis in blinded experiments. In 130 patients (from Veterans Affairs medical centers) with deep venous thromboses, including 24 heterozygotes with the factor V Leiden mutation, there was complete agreement between the two methods. The assay was also able to distinguish heterozygotes from homozygotes. This method, which carries a low potential for cross-contamination of samples, should be a useful routine test for the factor V Leiden mutation in clinical laboratories with sufficient demand for molecular diagnostic assays using the LCx instrument.
Evaluation of Nicked Human Chorionic Gonadotropin Content in Clinical Specimens by a Specific Immunometric Assay
Authors: Kovalevskaya Galina , Birken Steven , Kakuma Tatsu , Schlatterer John , O’Connor John F
Published: January 2020

We report the development and characterization of an IRMA for the direct measurement of nicked human chorionic gonadotropin (hCGn) in blood and urine. hCGn derived from a reference preparation of hCG used as an immunogen elicits monoclonal antibodies (mAbs) with enhanced recognition of human luteinizing hormone epitopes. The most specific assay for pregnancy hCGn is an IRMA composed of one mAb to choriocarcinoma-derived hCGn (C5) and a second mAb developed from immunization with normal-pregnancy hCGn. This assay was used to evaluate hCGn profiles in normal, in vitro fertilization, Down syndrome, and ectopic pregnancies. In all pregnancies, hCGn was usually present in much lower concentrations than the non-nicked hCG isoform. Our results suggest that some form of physical separation from the overwhelming quantities of non-nicked hCG present in clinical specimens will be required before accurate immunochemical estimations of hCGn can be made.
Newer Portable Glucose Meters—Analytical Improvement Compared with Previous Generation Devices?
Authors: Weitgasser Raimund , Gappmayer Brigitta , Pichler Maximilian
Published: January 2020

Background: Newer glucose meters are easier to use, but direct comparisons with older instruments are lacking. We wished to compare analytical performances of four new and four previous generation meters.

Methods: On average, 248 glucose measurements were performed with two of each brand of meter on capillary blood samples from diabetic patients attending our outpatient clinic. Two to three different lots of strips were used. All measurements were performed by one experienced technician, using blood from the same sample for the meters and the comparison method (Beckman Analyzer 2). Results were evaluated by analysis of clinical relevance using the percentage of values within a maximum deviation of 5% from the reference value, by the method of residuals, by error grid analysis, and by the CVs for measurements in series.

Results: Altogether, 1987 blood glucose values were obtained with meters compared with the reference values. By error grid analysis, the newer devices gave more accurate results without significant differences within the group (zone A, 98–98.5%). Except for the One Touch II (zone A, 98.5%), the other older devices were less exact (zone A, 87–92.5%), which was also true for all other evaluation procedures.

Conclusions: New generation blood glucose meters are not only smaller and more aesthetically appealing but are more accurate compared with previous generation devices except the One Touch II. The performance of the newer meters improved but did not meet the goals of the latest American Diabetes Association recommendations in the hands of an experienced operator.
Observations on Centrifugation: Application to Centrifuge Development
Authors: Roberts Thomas , Smith Mathew , Roberts Barry
Published: January 2020

This report outlines the background to the development of an automated, serial, discrete centrifuge, reporting on the criteria considered essential in such an instrument. We established the criteria by examining the detailed logistics of centrifuge operation in a hospital laboratory. The mean sample load per run, using six centrifuges, was 13.6 samples, and the user-selectable cycle time ranged from 00:01:10 to 00:12:33 (hours:minutes:seconds) with a fixed g value of 1050. During the laboratory working window, (0900–1700), only 50% of the centrifuge capacity was utilized and more than one-third of the sample workload was delayed for >5 min because the centrifuges were not emptied promptly. In addition, 35% of the sample workload was centrifuged for less than the time prescribed in the operational specifications. Based on these findings, we designed a new continuous, serial centrifuge to overcome some of the deficiencies noted in the logistics study. The centrifuge operates continuously, nominally treating 150 samples/h, with a cycle time of 5 min at 1000g. The cycle time and g value are variable between limits, and their selection governs the throughput rate. Each sample is centrifuged separately in individual rotors mounted in a sturdy carousel with a periphery that traverses a load/unload station. There is no sample delay because of operator absence, and the capacity is fully utilized. The centrifuge can operate in a stand-alone capacity or has the capability of being integrated into a sample preparation system or as a direct front end for high-throughput analyzers.
Biochemical and Molecular Genetic Characteristics of the Severe Form of Tyrosine Hydroxylase Deficiency
Authors: Bräutigam Christa , Steenbergen-Spanjers Gerry CH , Hoffmann Georg F , Dionisi-Vici Carlo , van den Heuvel Lambert PWJ , Smeitink Jan AM , Wevers Ron A
Published: January 2020

Background: Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of the catecholamines dopamine, norepinephrine, and epinephrine. Recently, mutations were identified in cases of autosomal recessive dopa-responsive dystonia and infantile parkinsonism. We describe a patient with severe symptoms and a new missense mutation in TH.

Methods: Relevant metabolites in urine and cerebrospinal fluid were measured by HPLC with fluorometric and electrochemical detection. All exons of the TH gene were amplified by PCR and subjected to single-strand conformation polymorphism analysis. Amplimers displaying aberrant migration patterns were analyzed by DNA sequence analysis.

Results: The patient presented with severe axial hypotonia, hypokinesia, reduced facial mimicry, ptosis, and oculogyric crises from infancy. The major metabolite of dopamine, homovanillic acid, was undetectable in the patient’s cerebrospinal fluid. A low dose of l-dopa produced substantial biochemical but limited clinical improvement. DNA sequencing revealed a homozygous 1076G→T missense mutation in exon 10 of the TH gene. The mutation was confirmed with restriction enzyme analysis. It was not present in 100 control alleles. Secondary structure prediction based on Chou-Fasman calculations showed an abnormal secondary structure of the mutant protein.

Conclusions: We describe a new missense mutation (1076G→T, C359F) in the TH gene. The transversion is present in all known splice variants of the enzyme. It produces more severe clinical and biochemical manifestations than previously described in TH-deficient cases. Our findings extend the clinical and the biochemical phenotype of genetically demonstrated TH deficiency.
Capillary Electrophoresis for Detection of Inherited Disorders of Purine and Pyrimidine Metabolism
Authors: Adam Tomáš , Friedecký David , Fairbanks Lynette D , Ševc̆ík Juraj , Barták Petr
Published: January 2020

Background: Measurement of purine and pyrimidine metabolites presents complex problems for separations currently performed by HPLC and thin-layer chromatography in clinical practice. We developed a novel capillary electrophoresis method for this purpose.

Methods: Separations were performed in 60 mmol/L borate-2-amino-2-methyl-1-propanol-80 mmol/L sodium dodecyl sulfate (pH 9.6) at 35 °C.

Results: The conditions reported allowed separation of all diagnostic metabolites from major urinary constituents in an analysis time of 3 min and with a separation efficiency of 220 000 theoretical plates/m. The clinically important metabolites were detectable at concentrations of 0.85–4.28 μmol/L. The method was linear over the range 5–500 μmol/L (r >0.99). The within-run and intra- and interday imprecision (CV) was <5%. Characteristic abnormalities were detected in the electropherograms of urine samples from patients with purine and pyrimidine enzyme deficiencies. We provide the electrophoretic and spectral characteristics of many intermediates in purine and pyrimidine metabolism and describe common artifacts from medication and ultraviolet-absorbing compounds.

Conclusion: Capillary electrophoresis is a valuable screening tool in the detection of inborn errors of purine and pyrimidine metabolism.
Recombinant Human Tissue Transglutaminase ELISA for the Diagnosis of Gluten-sensitive Enteropathy
Authors: Sárdy Miklós , Odenthal Uwe , Kárpáti Sarolta , Paulsson Mats , Smyth Neil
Published: January 2020

Background: Tissue transglutaminase (TGc) has recently been identified as the major, if not the sole, autoantigen of gluten-sensitive enteropathy (GSE). We developed and validated an ELISA based on the human recombinant antigen and compared it to existing serological tests for GSE [guinea pig TGc ELISA and endomysium antibody (EMA) test].

Methods: Human TGc was expressed in the human embryonic kidney cell line 293-EBNA as a C-terminal fusion protein with the eight-amino acid Strep-tag II allowing one-step purification via streptavidin affinity chromatography. We carried out ELISA assays for IgA antibodies against TGc using calcium-activated human and guinea pig TGc. The sera were also tested on monkey esophagus sections by indirect immunofluorescence for IgA EMA. We examined 71 serum samples from patients with GSE (38 with celiac disease, 33 with dermatitis herpetiformis), including 16 on therapy, and 53 controls.

Results: The human TGc could be expressed and purified as an active enzyme giving a single band on a Coomassie-stained gel. The mean intra- and interassay CVs for the human TGc ELISA were 3.2% and 9.2%, respectively. The area under the ROC curve was 0.999. The specificity and sensitivity were 98.1% (95% confidence interval, 95.7–100%) and 98.2% (95.9–100%), respectively.

Conclusions: The human TGc ELISA was somewhat superior to the guinea pig TGc ELISA, and was as specific and sensitive as the EMA test. The human TGc-based ELISA is the method of choice for easy and noninvasive screening and diagnosis of GSE.
Comparison of Serum and Plasma Methylmalonic Acid Measurements in 13 Laboratories: An International Study
Authors: Pfeiffer Christine M , Smith S Jay , Miller Dayton T , Gunter Elaine W
Published: January 2020

Background: Detection of cobalamin deficiency is increasingly important, and methylmalonic acid (MMA) appears to be a useful marker. Information on interlaboratory variation and on methodological differences for MMA in serum and plasma is limited.

Methods: Using gas chromatography/mass spectrometry, 13 laboratories participated in a 2-day analysis of 8 serum and 11 EDTA-plasma specimens. Results were analyzed for imprecision, recovery, and differences among laboratories and methods.

Results: The mean among-laboratory imprecision (CV) was 19% and 21% for serum and plasma samples, respectively, and 9.3% and 7.8% for serum and plasma samples with added MMA, respectively. The mean within-laboratory (among-run) CV was 13% for both serum and plasma samples and 5.2% and 4.9% for serum and plasma samples with added MMA. Within-method imprecision was the same or higher than among-method imprecision. The mean among-laboratory recovery of MMA was 105% and 95% in serum and plasma, respectively. Most laboratories showed a proportional bias relative to the consensus mean of up to 15%. Two laboratories reported results that on average were almost 30% higher than the consensus mean.

Conclusions: No method differences were found, but significant among-laboratory imprecision was found in the present study. Improvements are needed to reduce the analytical imprecision of most laboratories, and attention must be focused on calibration issues. Differences among laboratories can be improved by introducing high-quality reference materials and by instituting external quality assessment programs.
Thyroid Function during Pregnancy
Authors: Fantz Corinne R , Dagogo-Jack Samuel , Ladenson Jack H , Gronowski Ann M
Published: January 2020

Background: This Case Conference reviews the normal changes in thyroid activity that occur during pregnancy and the proper use of laboratory tests for the diagnosis of thyroid dysfunction in the pregnant patient.

Case: A woman in the 18th week of pregnancy presented with tachycardia, increased blood pressure, severe vomiting, increased total and free thyroid hormone concentrations, a thyroid-stimulating hormone (TSH) concentration within the reference interval, and an increased human chorionic gonadotropin (hCG) β-subunit concentration.

Issues: During pregnancy, normal thyroid activity undergoes significant changes, including a two- to threefold increase in thyroxine-binding globulin concentrations, a 30–100% increase in total triiodothyronine and thyroxine concentrations, increased serum thyroglobulin, and increased renal iodide clearance. Furthermore, hCG has mild thyroid stimulating activity. Pregnancy produces an overall increase in thyroid activity, which allows the healthy individual to remain in a net euthyroid state. However, both hyper- and hypothyroidism can occur in pregnant patients. In addition, two pregnancy-specific conditions, hyperemesis gravidarum and gestational trophoblastic disease, can lead to clinical hyperthyroidism. The normal changes in thyroid activity and the association of pregnancy with conditions that can cause hyperthyroidism necessitates careful interpretation of thyroid function tests during pregnancy.

Conclusion: Assessment of thyroid function during pregnancy should be done with a careful clinical evaluation of the patient’s symptoms as well as measurement of TSH and free, not total, thyroid hormones. Measurement of thyroid autoantibodies may also be useful in selected cases to detect maternal Graves disease or Hashimoto thyroiditis and to assess risk of fetal or neonatal consequences of maternal thyroid dysfunction.
Tetrahydropalmatine Poisoning: Diagnoses of Nine Adult Overdoses Based on Toxicology Screens by HPLC with Diode-Array Detection and Gas Chromatography–Mass Spectrometry
Authors: Lai Chi-Kong , Chan Albert Yan-Wo
Published: January 2020

Background: Tetrahydropalmatine (THP) is a neuroactive alkaloid with analgesic and hypnotic action. Its analysis is important because cases of human poisonings have emerged as a result of unregulated use of some proprietary biopharmaceuticals containing purified THP. Methods: We established analytical parameters for HPLC with diode-array detection (HPLC-DAD) and gas chromatography–mass spectrometry (GC-MS) for the detection of THP in serum and urine. Nine acutely THP-poisoned adults were thus screened over 16 months. Results: All patients recovered quickly after mild neurological disturbance. In general, THP was metabolized rapidly and excreted as polar metabolites in urine. Serum THP was measured in five cases and found to be <0.1–1.2 mg/L (<0.3–3.4 μmol/L). Paired analyses of urine with and without glucuronidase treatment clarified the disposition of THP. Our GC–MS method with trimethylsilane derivatization identified O-desmethyl metabolites. With a uniform solid-phase extraction, the HPLC-DAD procedure detected intact glucuronide metabolites. Conclusion: Intact glucuronide metabolites of THP are sensitive markers for THP exposures. Our methods and findings provide practical tools and information for surveillance of intoxication caused by excessive THP intake.
Study of Calcium Metabolism in Idiopathic Hypercalciuria by Strontium Oral Load Test
Authors: Vezzoli Giuseppe , Caumo Andrea , Baragetti Ivano , Zerbi Simona , Bellinzoni Piera , Centemero Antonella , Rubinacci Alessandro , Moro GianLuigi , Adamo Donatella , Bianchi Giuseppe , Soldati Laura
Published: January 2020

Calcium excretion and absorption were evaluated in hypercalciuric calcium stone formers by the study of Sr2+ excretion and absorption after an oral load. Ca2+ stone formers (n = 140) were studied, and the results were compared in the 83 of them who had idiopathic hypercalciuria and in the 57 who had Ca2+ excretion within reference values. Hypercalciuric patients showed increased renal Sr2+ clearance (CRE; 5.26 ± 0.358 vs 3.29 ± 0.277 mL/min; P <0.001), whereas Sr2+ absorption [assessed as the area under the serum concentration–time curve (AUC)] was increased at 30 and 60 min (1.53 ± 0.087 vs 1.21 ± 0.071 mmol · L−1 · min; P <0.05), but not at 240 min after the load. In hypercalciuric patients, the AUCs were positively correlated with urinary Sr2+ fractional excretion (P <0.001). Conversely, in normocalciuric patients plasma parathyroid hormone (PTH) was negatively correlated with the AUCs (P <0.01) and CRE (P <0.05), whereas 1,25-dihydroxyvitamin D plasma concentrations normalized to PTH were positively correlated with the AUCs (P <0.05). The results of Sr2+ load tests suggest that in the hypercalciuric population, Ca2+ absorption is altered predominantly in the duodenum and that the normal regulation exerted by calciotropic hormones on tubular and enteral Ca2+ handling is lost.
Quantitative Study of the Variability of Hepatic Iron Concentrations
Authors: Emond Mary J , Bronner Mary P , Carlson Timothy H , Lin Masami , Labbe Robert F , Kowdley Kris V
Published: January 2020

Background: The hepatic iron concentration (HIC) is widely used in clinical practice and in research; however, data on the variability of HIC among biopsy sites are limited. One aim of the present study was to determine the variability of HIC within both healthy and cirrhotic livers.

Methods: Using colorimetric methods, we determined HIC in multiple large (microtome) and small (biopsy-sized) paraffin-embedded samples in 11 resected livers with end-stage cirrhosis. HIC was also measured in multiple fresh samples taken within 5 mm of each other (“local” samples) and taken at sites 3–5 cm apart (“remote” samples) from six livers with end-stage cirrhosis and two healthy autopsy livers.

Results: The within-organ SD of HIC was 13–1553 μg/g (CV, 3.6–55%) for microtome samples and 60–2851 μg/g (CV, 15–73%) for biopsy-sized samples. High variability of HIC was associated with mild to moderate iron overload, because the HIC SD increased with increasing mean HIC (P <0.002). Livers with mean HIC >1000 μg/g exhibited significant biological variability in HIC between sites separated by 3–5 cm (remote sites; P <0.05). The SD was larger for biopsy-sized samples than for microtome samples (P = 0.02).

Conclusion: Ideally, multiple hepatic sites would be sampled to obtain a representative mean HIC.
Nitrogen Dioxide Formation during Inhaled Nitric Oxide Therapy
Authors: Sokol Gregory M , Van Meurs Krisa P , Wright Linda L , Rivera Oswaldo , Thorn William J , Chu Pamela M , Sams Robert L
Published: January 2020

Background: Nitrogen dioxide (NO2) is a toxic by-product of inhalation therapy with nitric oxide (NO). The rate of NO2 formation during NO therapy is controversial.

Methods: The formation of NO2 was studied under dynamic flows emulating a base case NO ventilator mixture containing 80 ppm NO in a 90% oxygen matrix. The difficulty in measuring NO2 concentrations below 2 ppm accurately was overcome by the use of tunable diode laser absorption spectroscopy.

Results: Using a second-order model, the rate constant, k, for NO2 formation was determined to be (1.19 ± 0.11) × 10−11 ppm−2s−1, which is in basic agreement with evaluated data from atmospheric literature.

Conclusions: Inhaled NO can be delivered safely in a well-designed, continuous flow neonatal ventilatory circuit, and NO2 formation can be calculated reliably using the rate constant and circuit dwell time.
Automated Homogeneous Immunoassay for Gentamicin on the Dimension Clinical Chemistry System
Authors: Wei Tie Q , Chu Victor P , Craig Alan R , Duffy James E , Obzansky David M , Kilgore Daniel , Masulli Ignazio S , Sanders Connie M , Thompson John C
Published: January 2020

Background: Monitoring of the concentration of gentamicin in serum and plasma during therapy is widely recommended and practiced in hospitals. Our aim was to develop a homogeneous immunoassay based on particle-enhanced turbidimetric inhibition immunoassay technology to quantify gentamicin on the Dimension® clinical chemistry system.

Methods: Assay performance was assessed on each of the Dimension models in a 15-instrument interlaboratory comparison study. A split-sample comparison (n = 1171) was also performed between the gentamicin methods on the Dimension system and the Abbott®TDx® analyzer, using multiple reagent and calibrator lots on multiple instruments.

Results: The Dimension method was linear to 25.1 μmol/L (12.0 μg/mL) with a detection limit of 0.63 μmol/L (0.3 μg/mL). Calibration was stable for 30 days. The within-run imprecision (CV) was <1.3%, and total imprecision ranged from 1.8% to 3.2% between 4.2 μmol/L (2.0 μg/mL) and 16.7 μmol/L (8.0 μg/mL) gentamicin. Linear regression analysis of the results on the Dimension method (DM) vs the Abbott TDx yielded the following equation: DM = 0.98TDx − 0.42; r = 0.987. Minimal interference was observed from structurally related compounds such as sagamicin, netilmicin, and sisomicin.

Conclusion: The monoclonal antibody used in this method has similar reactivities toward the individual gentamicin subspecies C1, C1a, and C2, thus providing analytical recovery not significantly dependent on relative subspecies concentrations.
Defect in Dimethylglycine Dehydrogenase, a New Inborn Error of Metabolism: NMR Spectroscopy Study
Authors: Moolenaar Sytske H , Poggi-Bach Jo , Engelke Udo FH , Corstiaensen Jacqueline MB , Heerschap Arend , de Jong Jan GN , Binzak Barbara A , Vockley Jerry , Wevers Ron A
Published: January 2020

Background: A38-year-old man presented with a history of fish odor (since age 5) and unusual muscle fatigue with increased serum creatine kinase. Our aim was to identify the metabolic error in this new condition.

Methods: We used 1H NMR spectroscopy to study serum and urine from the patient.

Results: The concentration of N,N-dimethylglycine (DMG) was increased ∼100-fold in the serum and ∼20-fold in the urine. The presence of DMG as a storage product was confirmed by use of 13C NMR spectroscopy and gas chromatography–mass spectrometry. The high concentration of DMG was caused by a deficiency of the enzyme dimethylglycine dehydrogenase (DMGDH). A homozygous missense mutation was found in the DMGDH gene of the patient.

Conclusions: DMGDH deficiency must be added to the differential diagnosis of patients complaining of a fish odor. This deficiency is the first inborn error of metabolism discovered by use of in vitro 1H NMR spectroscopy of body fluids.
Near-Patient Test for C-Reactive Protein in General Practice: Assessment of Clinical, Organizational, and Economic Outcomes
Authors: Dahler-Eriksen Bjarne Steen , Lauritzen Torsten , Lassen Jens Flensted , Lund Erik D , Brandslund Ivan
Published: January 2020

Background: The benefits of near-patient, point-of-care tests have not been fully examined. We have assessed the clinical, organizational, and economic outcomes of implementing a near-patient test for C-reactive protein (CRP) in general practice.

Methods: In a randomized crossover trial during intervention periods, general practitioners (GPs) were allowed to measure CRP within 3 min, using NycoCard® CRP. During control periods, they had to mail blood samples for CRP measurements to the hospital laboratory and received test results 24–48 h later. Twenty-nine general practice clinics participated (64 GPs), and 1853 patients were included in the study. Results were evaluated at both the level of participating GPs and the level of included patients.

Results: For participating GPs, the overall use of erythrocyte sedimentation rates (ESRs) decreased by 8% (95% confidence interval, 1–14%) during intervention periods, and the number of blood samples mailed to the hospital laboratory decreased by 6% (1–10%). No reduction in the prescription of antibiotics was seen. The proportion of study patients having a follow-up telephone consultation was reduced from 63% to 53% (P = 0.0001), and patients with CRP concentrations >50 mg/L had their antibiotic treatments started earlier when CRP was measured in general practices (P = 0.0161).

Conclusion: The implementation of the near-patient CRP test was cost-effective mainly on the basis of a reduction in the use of services from the hospital laboratory by GPs. If the implementation is followed by education and clinical guidelines, opportunities exist for additional reduction in the use of ESR and for a more appropriate use of antibiotics.
Comparison of Three Different Plasma Homocysteine Assays with Gas Chromatography–Mass Spectrometry
Authors: Ubbink Johan B , Delport Rhena , Riezler Reiner , Vermaak WJ Hayward
Published: January 2020

Background: Various methods are available to measure plasma total homocyst(e)ine (tHcy) concentrations, but whether plasma tHcy assays may be used interchangeably is not known.

Methods: Results from three different methods [HPLC with fluorescence detection, enzyme immunoassay (EIA), and fluorescence polarization immunoassay (FPIA)] to determine fasting (n = 163) and post-methionine load (n = 80) plasma tHcy concentrations were compared with those obtained by gas chromatography–mass spectrometry (GC-MS). Difference plots on non-transformed and log-transformed data were used to assess the agreement between HPLC and GC-MS, EIA and GC-MS, and FPIA and GC-MS.

Results: The closest agreement between methods was observed between GC-MS and FPIA for fasting tHcy concentrations, with 95% of the FPIA values between 19% above and 24% below the corresponding GC-MS results. Post-methionine load tHcy concentrations measured by EIA showed the least agreement with GC-MS, with 95% of values measured by EIA ranging between 52% above and 16% below the GC-MS values. With respect to GC-MS, the above-mentioned methods showed a negative bias for fasting tHcy concentrations, but a positive bias for both immunoassays for post-methionine load tHcy concentrations.

Conclusions: The agreement among methods is insufficient to allow them to be used interchangeably. The intermethod differences emphasize the need for standardization of plasma tHcy assays.
Promoting Safe and Effective Genetic Tests in the United States: Work of the Task Force on Genetic Testing
Authors: Holtzman Neil A
Published: January 2020

The Task Force on Genetic Testing was created to review genetic testing in the United States and, when necessary, to make recommendations to ensure the development of safe and effective genetic tests. A survey to explore the state of genetic testing was undertaken for the Task Force and completed in early 1995. The survey, as well as literature reports and other information collected for the Task Force, showed problems affecting safety and effectiveness, as defined by the Task Force: validity and utility of predictive tests, laboratory quality, and appropriate use by healthcare providers and consumers. On the basis of these findings, the Task Force made several recommendations to ensure safe and effective genetic testing. The Secretary of Health and Human Services followed up one recommendation by creating the Secretary’s Advisory Committee on Genetic Testing. One of its functions will be to implement other recommendations of the Task Force.