bluuebunny/crossref_metadata_2025 huggingface dataset:
Analytical and Clinical Evaluation of Two Homogeneous Assays for LDL-Cholesterol in Hyperlipidemic Patients
Authors: Esteban-Salán Margarita , Guimón-Bardesi Amada , de la Viuda-Unzueta Jesús María , Azcarate-Ania María Nerea , Pascual-Usandizaga Pilar , Amoroto-Del-Río Eduardo
Published: January 2020

Background: LDL-cholesterol (LDL-C) concentrations are the primary basis for treatment guidelines established for hyperlipidemic patients. LDL-C concentrations are commonly monitored by means of the Friedewald formula, which provides a relative estimation of LDL-C concentration when the triglyceride concentration is <2000 mg/L and there are no abnormal lipids. The Friedewald formula has several limitations and may not meet the current total error requirement of <12% in LDL-C measurements.

Methods: We evaluated the analytical and clinical performance of two direct methods (Roche and Wako) by analyzing 313 fresh serum samples obtained from dyslipidemic patients in a lipid clinic and comparing them with modified β-quantification.

Results: Both homogeneous assays displayed excellent precision (CV <2%). The Roche method showed a mean total error of 7.72%, and the Wako method showed a mean total error of 4.46% over a wide range of LDL-C concentrations. The Roche method correlated highly with the modified β-quantification assay (r = 0.929; y = 1.052x − 168 mg/L; n = 166) and showed a bias of −4.5% as a result of the assigned standard value. The Wako method also correlated highly with β-quantification (r = 0.966; y = 0.9125x + 104.8 mg/L; n = 145) without significant bias. The Roche method correctly classified 97% of patients with triglycerides <2000 mg/L, 75% of patients with type IIb hyperlipemia (HPL), and 84% of patients with type IV HPL based on the cutpoints of 1300 and 1600 mg/L, compared with 98%, 78.4%, and 89%, respectively, for the Wako method. In dysbetalipoproteinemic patients, both methods have a 30% mean positive bias compared with β-quantification.

Conclusions: Both direct methods can be a useful alternative when ultracentrifugation is not available for the diagnosis and control of lipid-lowering medication for patients with mixed HPL, but not for patients with type III hyperlipidemia.
Flow Cytometry: Principles and Clinical Applications in Hematology
Authors: Brown Michael , Wittwer Carl
Published: January 2020

The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. This is attributable in part to the development of smaller, user-friendly, less-expensive instruments and a continuous increase in the number of clinical applications. Flow cytometry measures multiple characteristics of individual particles flowing in single file in a stream of fluid. Light scattering at different angles can distinguish differences in size and internal complexity, whereas light emitted from fluorescently labeled antibodies can identify a wide array of cell surface and cytoplasmic antigens. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This report reviews the general principles in flow cytometry and selected applications of flow cytometry in the clinical hematology laboratory.
Reference Reagents for Prostate-specific Antigen (PSA): Establishment of the First International Standards for Free PSA and PSA (90:10)
Authors: Rafferty Brian , Rigsby Peter , Rose Matthew , Stamey Thomas , Gaines Das Rose
Published: January 2020

Background: Prostate-specific antigen (PSA) measurements in serum by immunoassay are widely used in the screening, diagnosis, and monitoring of patients with prostate cancer although the lack of common reference reagents has led in the past to wide differences in estimates. We report here the results of a WHO international collaborative study in which two preparations of PSA representative of the main immunoreactive components in serum, free PSA and PSA 90:10, and a preparation of recombinant DNA-derived PSA were assessed as potential standards for the calibration of diagnostic immunoassays for PSA.

Methods: Coded vials of the candidate materials and serum preparations containing PSA in the clinically important range were provided to the 10 laboratories in the study, and participants were asked to perform PSA assays currently in use in their laboratories. Data from 89 immunoassays by 26 different method-laboratory combinations were contributed to the study and analyzed centrally at the National Institute for Biological Standards and Control.

Results: Potency estimates of the preparations relative to the in-house calibrators were in good agreement with the target value of 1 μg of total PSA/vial, the preparation of free PSA giving 1.10 μg/vial (95% confidence interval, 0.99–1.21 μg/vial) and PSA 90:10, 1.11 μg/vial (95% confidence interval, 1.04–1.18 μg/vial). No immunoreactivity was detected in ampoules containing the recombinant material. Use of a common standard of PSA 90:10 significantly reduced the between-laboratory geometric coefficients of variation for serum samples included in the study and gave a much narrower range of potency estimates.

Conclusions: The preparation of free PSA was established by WHO as the First International Standard for PSA (free) with an assigned content of 1 μg of total PSA per vial. In addition, the preparation of bound PSA was established as the First International Standard for PSA (90:10) with an assigned content of 1 μg of total PSA per vial.
Determination of Acid α-Glucosidase Protein: Evaluation as a Screening Marker for Pompe Disease and Other Lysosomal Storage Disorders
Authors: Umapathysivam Kandiah , Whittle Alison M , Ranieri Enzo , Bindloss Colleen , Ravenscroft Elaine M , van Diggelen Otto P , Hopwood John J , Meikle Peter J
Published: January 2020

Background: In recent years, there have been significant advances in the development of enzyme replacement and other therapies for lysosomal storage disorders (LSDs). Early diagnosis, before the onset of irreversible pathology, has been demonstrated to be critical for maximum efficacy of current and proposed therapies. In the absence of a family history, the presymptomatic detection of these disorders ideally can be achieved through a newborn screening program. One approach to the development of such a program is the identification of suitable screening markers. In this study, the acid α-glucosidase protein was evaluated as a marker protein for Pompe disease and potentially for other LSDs.

Methods: Two sensitive immunoquantification assays for the measurement of total (precursor and mature) and mature forms of acid α-glucosidase protein were used to determine the concentrations in plasma and dried blood spots from control and LSD-affected individuals.

Results: In the majority of LSDs, no significant increases above control values were observed. However, individuals with Pompe disease showed a marked decrease in acid α-glucosidase protein in both plasma and whole blood compared with unaffected controls. For plasma samples, this assay gave a sensitivity of 95% with a specificity of 100%. For blood spot samples, the sensitivity was 82% with a specificity of 100%.

Conclusions: This study demonstrates that it is possible to screen for Pompe disease by screening the concentration of total acid α-glucosidase in plasma or dried blood spots.
Clinical and Analytical Evaluation of an Enzyme Immunoassay for Myelin Basic Protein in Cerebrospinal Fluid
Authors: Ohta Mitsuhiro , Ohta Kiyoe , Ma Jie , Takeuchi Juji , Saida Takahiko , Nishimura Masataka , Itoh Nobuyuki
Published: January 2020

Background: RIA of myelin basic protein (MBP) in cerebrospinal fluid (CSF) is commonly used as a biochemical marker of demyelination in patients with multiple sclerosis (MS). Our aim was to develop a sufficiently sensitive ELISA for MBP and evaluate it clinically in patients with MS.

Methods: The ELISA used anti-bovine MBP antibody coated on plates and biotinylated anti-MBP antibody. The bound antibody complex was quantified with streptavidin-horseradish peroxidase. MBP was determined in CSF from 84 MS patients and 55 patients with other neurological diseases.

Results: The respective within- and between-assay CVs were 4.7% and 7.2% at 200 ng/L, and 6.3% and 8.8% at 2000 ng/L. The detection limit was 30 ng/L. Most of the MS patients with acute exacerbations had markedly increased MBP in the CSF. Longitudinal studies of six MS patients with recurrent exacerbation confirmed this observation. MBP concentrations from 78 MS patients, as tested with our ELISA, correlated well with those obtained by RIA (r = 0.9; P <0.01), but the detection limit of the ELISA was much lower than that of the RIA.

Conclusions: This convenient ELISA with higher sensitivity than the existing assays is a suitable routine assay that provides a diagnostic indicator of myelin breakdown in the central nervous system; moreover, it is an excellent indicator of MS disease activity.
Lower Cardiac Troponin T and I Results in Heparin-Plasma Than in Serum
Authors: Stiegler Hugo , Fischer Yuriko , Vazquez-Jimenez Jaime F , Graf Jürgen , Filzmaier Karsten , Fausten Bernd , Janssens Uwe , Gressner Axel M , Kunz Dagmar
Published: January 2020

Background: The use of plasma rather than serum for determination of cardiac troponins can improve turnaround time and potentially avoid incomplete serum separation that may produce falsely increased results. We investigated the influence of incomplete serum separation and the effect of heparin-plasma on cardiac troponin concentrations.

Methods: Serum and heparin-plasma samples were drawn simultaneously from 100 patients (50 patients with acute coronary syndrome and 50 patients after open heart surgery) and measured on three different analytical systems, two for determination of cardiac troponin I (cTnI; Abbott AxSYM and Bayer ACS:Centaur) and one for cardiac troponin T (cTnT; Roche Elecsys cTnT STAT). Serum samples were reanalyzed after a second centrifugation to assess the influence of incomplete serum separation.

Results: Mean results (± 95% confidence interval) in heparin-plasma compared with serum were 101% ± 2% (AxSYM cTnI), 94% ± 3% (ACS:Centaur cTnI), and 99% ± 3% (Elecsys cTnT). Differences >20% were seen in 11% of results on the ACS:Centaur, 9% of results on Elecsys cTnT, and 2% of results on the AxSYM. For the Elecsys cTnT assay, the magnitude of the difference between serum and plasma was independent of the absolute concentration and confined to individual samples, and was reversed by treatment with heparinase. A second centrifugation had no effect on serum results by any of the assays.

Conclusion: The concentrations of troponins measured in heparin-plasma are markedly lower than in serum in some cases.
Evaluation of Two Different Homogeneous Assays for LDL-Cholesterol in Lipoprotein-X-positive Serum
Authors: Fei Hua , Maeda Satoshi , Kirii Hirokazu , Fujigaki Suwako , Maekawa Naoya , Fujii Hidehiko , Wada Hisayasu , Saito Kuniaki , Seishima Mitsuru
Published: January 2020

Background: The purpose of this study was to evaluate the performance of two homogeneous assays for LDL-cholesterol (LDL-C), a polyethylene/cyclodextrin (PC) assay and a detergent (D) assay, which are based on different principles, in cholestatic serum.

Methods: We compared serum LDL-C concentrations determined by the two assays for healthy normolipidemic subjects (n = 42) and cholestatic patients (n = 51). LDL-C concentrations obtained with the homogeneous assays were also compared with those obtained by HPLC for patients’ sera. In the interference study, conjugated bile acids were added to normal serum, and their effects on the two assays were examined. The effects of lipoprotein-X (LP-X), intermediate-density lipoprotein (IDL), and apolipoprotein (apo) E-rich HDL on the LDL-C assays were also investigated by adding these lipoproteins to normal serum.

Results: The LDL-C concentrations obtained with the D assay were higher than those obtained with the PC assay in the serum with high LP-X. The bias for LDL-C vs LP-X in cholestatic serum correlated with LP-X concentration (r = 0.582; P <0.0001; n = 51). In the interference study, no effect of bile acids on the LDL-C assays was observed. However, the D assay measured 51.0% of the cholesterol in LP-X, whereas no reactivity was observed for LP-X in the PC assay. In addition, the D assay and the PC assay measured IDL-cholesterol at 31.2% and 52.4%, respectively, and measured apo E-rich HDL-C at 7.6% and 17.8%, respectively.

Conclusions: Although both homogeneous LDL-C assays are suitable for most cases, the present study showed that each homogeneous assay has a different limitation for cholestatic serum with gross alterations in lipoproteins.
Discrimination of Prohibited Oral Use of Salbutamol from Authorized Inhaled Asthma Treatment
Authors: Bergés Rosa , Segura Jordi , Ventura Rosa , Fitch Kenneth D , Morton Alan R , Farré Magı́ , Mas Marta , de la Torre Xavier
Published: January 2020

Background: The administration of salbutamol is permitted only by inhalation by the International Olympic Committee (IOC) for the management of asthma and exercise-induced asthma in athletes. The establishment of criteria to distinguish between the IOC authorized use (inhaled) and the IOC prohibited use (oral) of salbutamol appeared possible using simultaneous evaluation of variables based on the concentration of nonconjugated enantiomers of salbutamol excreted in urine.

Methods: Urine was collected from asthmatic and nonasthmatic swimmers who had received various preexercise doses of oral (five doses of 4 mg) or inhaled (two doses of 100 μg) salbutamol. Urine was also obtained from subjects who had received the maximum dosage of inhaled salbutamol advisable for competing athletes to provide protection from exercise-induced asthma and treatment of asthma (1600 μg in 24 h, 800 μg being in the last 4 h). All samples were analyzed to determine the total amount of unchanged salbutamol excreted in urine and the ratio between the S and R enantiomers.

Results: The discriminant function D = −3.776 + 1.46 × 10−3 {[S(+)] + [R(−)]} + 1.012 {[S(+)]/[R(−)]} can be used to classify data into two groups, inhaled and oral. The confirmatory criterion suggested (cutoff at D = 1.06, 4 SD from the mean D value of the inhaled distribution) has been verified in different sets of samples showing suspicious concentrations by conventional screening procedures in doping control. An 11.8% false-negative (oral classified as inhaled) rate is assumed with the confirmatory criterion proposed, but virtually no false positives (inhaled classified as oral) are obtained (<1 in 33 000).

Conclusions: The overall procedure recommended is to screen all samples and to apply the confirmation criterion proposed to samples showing free racemic salbutamol concentrations >500 μg/L by gas chromatography-mass spectrometry or free + conjugated racemic salbutamol concentrations >1400 μg/L by ELISA.
Streptavidin-Polyvinylamine Conjugates Labeled with a Europium Chelate: Applications in Immunoassay, Immunohistochemistry, and Microarrays
Authors: Scorilas Andreas , Bjartell Anders , Lilja Hans , Moller Christina , P. Diamandis Eleftherios
Published: January 2020

Background: The favorable properties of lanthanide chelates compared with conventional fluorescent probes have attracted considerable interest. A Eu3+ chelator, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), has been synthesized previously.

Methods: We here describe immunoassay, immunohistochemistry, and microarray applications of a new streptavidin-based universal polyvinylamine (PVA) detection reagent that is multiply labeled with the europium chelate of BCPDA. Solid-phase time-resolved immunofluorometric assays for biotinylated mouse IgG and prostate-specific antigen (PSA) were developed using the new conjugate as a detection reagent. The new conjugate was also used for the immunohistochemical localization of PSA expression in paraffin-embedded prostatic tissues. A model microarray with spotted biotinylated antibody as target was also performed.

Results: Approximately 50–100 BCPDA moieties were covalently bound to PVA, which was then linked to streptavidin via biotin interaction. The macromolecular complex successfully recognized and bound biotinylated detection reagents, e.g., antibodies. The new reagent enabled measurement of solid phase-immobilized biotinylated mouse IgG with a detection limit of ∼1 pg/assay and demonstrated excellent linearity. In an ELISA-type sandwich PSA assay that included two PSA monoclonal antibodies using the new conjugate as detection reagent, we detected 0.001 μg/L PSA (∼100 fg or ∼3 amol/assay). Serum samples analyzed for PSA by this method and a commercial assay gave highly correlated results. The new reagent enabled excellent immunohistochemical localization of PSA expression in prostate tissues. Using the new reagent in a model microarray experiment with biotinylated mouse IgG as target, we demonstrated excellent spatial resolution of 5- to 10-nL microspots.

Conclusions: The new detection reagent may find important applications in biotechnology.
Patient-derived Quality Specifications for Instruments Used in Self-Monitoring of Blood Glucose
Authors: Skeie Svein , Thue Geir , Sandberg Sverre
Published: January 2020

Background: Instruments for self-monitoring of glucose (SMBG) are increasingly used by diabetic patients. Information is limited on how patients use and interpret SMBG results, and no quality specifications for such instruments are based on the opinions of patients.

Methods: Type 1 diabetic patients (n = 201) filled in a questionnaire eliciting daily limits for blood glucose (BG) and changes of BG considered significant at different glucose concentrations. From these responses, patient-derived quality specifications were calculated in different clinical situations with low, intermediate, and high BG concentrations.

Results: Mean age of the patients was 31.8 years, mean diabetes duration was 14.7 years, and mean SMBG duration was 10.0 years with a mean frequency of 11.2 measurements/week. The threshold for hypoglycemic symptoms was 3.0 mmol/L (54 mg/dL), and the mean daily BG target window was 4.3–10.4 mmol/L (77–187 mg/dL). The mean absolute BG changes producing actions from the patients ranged from 1.1 mmol/L (20 mg/dL) to 3.6 mmol/L (65 mg/dL). The analytical quality specifications for imprecision depended on the clinical situation. Excluding the hypoglycemic situation, the analytical CV needed to fulfill the expectations of 75% of the patients was 6.4–9.7%. The analytical quality specification for CV at hypoglycemic concentrations was 3.1%.

Conclusions: Instruments for self-measurements of glucose with an imprecision (CV) of ≤5% and bias ≤5% meet the expectations of >75% of patients in clinical situations other than hypoglycemia.
Fluorescence-based, Nonradioactive Method for Efficient Detection of the Pentanucleotide Repeat (TTTTA)n Polymorphism in the Apolipoprotein(a) Gene
Authors: Rubin Jill , Pearson Thomas A , Reed Roberta G , Berglund Lars
Published: January 2020

Background: The apolipoprotein(a) [apo(a)] gene is a major predictor of plasma lipoprotein(a) concentrations, an independent risk factor for cardiovascular disease. The apo(a) gene contains a pentanucleotide repeat (PNR) polymorphism, 1.4 kb upstream from the apo(a) gene reading frame. This polymorphism has been suggested to be important in control of apo(a) gene expression.

Methods: We developed a fluorescence-based, nonradioactive procedure to detect the PNR polymorphism. After amplification of the polymorphism by PCR, the respective PCR products were separated by denaturing polyacrylamide gel electrophoresis and detected using a 3′-end fluorescently labeled oligonucleotide as a probe. We used the method to characterize the PNR polymorphism pattern in 313 individuals, 195 Caucasians and 118 African Americans. The new method efficiently separated DNAs corresponding to the different PNR repeats.

Results: Among both ethnic groups, alleles containing eight PNRs were most common. Smaller PNRs were more common among African Americans, and larger PNRs were more common among Caucasians.

Conclusions: We developed a nonradioactive technique that separates the PNR polymorphism in the apo(a) gene and can be used in other studies involving closely sized polymorphisms.
Hepatic Carnitine Palmitoyltransferase I Deficiency: Acylcarnitine Profiles in Blood Spots Are Highly Specific
Authors: Fingerhut Ralph , Röschinger Wulf , Muntau Ania C , Dame Torsten , Kreischer Jens , Arnecke Ralf , Superti-Furga Andrea , Troxler Heinz , Liebl Bernhard , Olgemöller Bernhard , Roscher Adelbert A
Published: January 2020

Background: In carnitine palmitoyltransferase I (CPT-I) deficiency (MIM 255120), free carnitine can be increased with no pathologic acylcarnitine species detectable. As inclusion of CPT-I deficiency in high-risk and newborn screening could prevent potentially life-threatening complications, we tested whether CPT-I deficiency might be diagnosed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS).

Methods: A 3.2-mm spot of whole blood dried on filter paper was extracted with 150 μL of methanol. After derivatization of carnitine and acylcarnitines to their butyl esters, the samples were analyzed by ESI-MS/MS with 37.5 pmol of l-[2H3]carnitine and 7.5 pmol of l-[2H3]palmitoylcarnitine as internal standards.

Results: In all dried-blood specimens from each of three patients with CPT-I deficiency, we found an invariably increased ratio of free carnitine to the sum of palmitoylcarnitine and stearoylcarnitine [C0/(C16 + C18)]. The ratio in patients was between 175 and 2000, or 5- to 60-fold higher than the ratio for the 99.9th centile of the normal newborn population in Bavaria (n = 177 842). No overlap with the values of children that were known to be supplemented with carnitine was detected [C0/(C16 + C18), 34 ± 30; mean ± SD; n = 27].

Conclusions: ESI-MS/MS provides a highly specific acylcarnitine profile from dried-blood samples. The ratio of free carnitine to the sum of palmitoylcarnitine and stearoylcarnitine [C0/(C16 + C18)] is highly specific for CPT-I deficiency and may allow presymptomatic diagnosis.
High-Throughput Genomic and Proteomic Analysis Using Microarray Technology
Authors: Huang Joe Xi , Mehrens Dorothy , Wiese Rick , Lee Sandy , Tam Sun W , Daniel Steve , Gilmore James , Shi Michael , Lashkari Deval
Published: January 2020

Background: High-density microarrays are ideally suited for analyzing thousands of genes against a small number of samples. The next step in the discovery process is to take the resulting genes of interest and rapidly screen them against thousands of patient samples, tissues, or cell lines to further investigate their involvement in disease risk or the response to medication.

Methods: We used a microarray technology platform for both single-nucleotide polymorphisms (SNPs) and protein expression. Each microarray contains up to 250 elements that can be customized for each application. Slides contained either a 16- or 96-microarray format (4000–24 000 elements per slide), allowing the corresponding number of samples to be rapidly processed in parallel.

Results: Results for SNP genotyping and protein profiling agreed with results of restriction fragment length polymorphism (RFLP) analysis or ELISA, respectively. Genotyping analyses, using the microarray technology, on large sample sets over multiple polymorphisms in the NAT2 gene were in full agreement with traditional methodologies, such as sequencing and RFLP analysis. The multiplexed protein microarray had correlation coefficients of 0.82–0.99 (depending on analyte) compared with ELISAs.

Conclusions: The integrated microarray technology platform is adaptable and versatile, while offering the high-throughput capabilities needed for drug development and discovery applications.
Denaturing HPLC for Identification of Clonal T-Cell Receptor γ Rearrangements in Newly Diagnosed Acute Lymphoblastic Leukemia
Authors: zur Stadt Udo , Rischewski Johannes , Schneppenheim Reinhard , Kabisch Hartmut
Published: January 2020

Background: Denaturing HPLC (DHPLC) can be used to screen DNA for known and unknown mutations. We describe a novel, HPLC-based method for discrimination among polyclonal, oligoclonal, and/or clonal T-cell receptor γ (TCR-γ) rearrangements in samples from children with newly diagnosed acute lymphoblastic leukemia.

Methods: TCR rearrangements were PCR amplified from initial leukemic samples and, after heteroduplex-induction, the clonality status of each product was evaluated. To attain this, we used DHPLC on a high-resolution micropellicular matrix. Running conditions were established by melting-curve analysis of known clonal and polyclonal products and melting-point prediction software. Elution profiles were studied at 50 °C (native) and, to achieve optimal separation, at different column temperatures between 56 and 64 °C.

Results: For VγI-Jγ1.3/2.3 rearrangements, an analysis temperature of 60 °C with a linear triethylammoniumacetate—acetonitrile gradient separated clonal bands from the polyclonal background amplification. Less than 15% clonal PCR product was detectable in mixtures of initial leukemic cell DNA and polyclonal DNA. Biallelic rearrangements produced two sharp peaks. Clonality of PCR products from 100 initial leukemic samples was completely identified in all investigated cases.

Conclusions: Heteroduplex analysis with standardized DHPLC conditions simplifies the detection of unknown clonal or polyclonal TCR rearrangements in newly diagnosed leukemias. Clonal targets for detection of minimal residual disease are available after a short, automated analysis of PCR amplified rearrangements.
Celiac Disease
Authors: Aleanzi Mabel , Demonte Ana María , Esper Cecilia , Garcilazo Silvia , Waggener Marta
Published: January 2020

Background: Selective deamidation of glutamine residues by tissue transglutaminase (tTG) turns gliadin peptides into stronger activators of T cells from celiac disease (CD) patients. We examined the possibility that these modified peptides could be more specific epitopes for circulating antibodies than are native peptides.

Methods: Two native synthetic peptides and their respective modified sequences were used as antigens for ELISA assays: peptide-1, with residues 56–75 of α-type gliadin; and peptide-2, with residues 134–153 of γ-type gliadin. We examined 40 CD patients [31 not being treated with a gluten-free diet (GFD) and 9 being treated with a GFD] and 30 non-CD patients.

Results: An enhanced response against deamidated peptides was observed in 4 (IgA) and 22 (IgG) of 31 untreated CD patients for peptide-1 and in 25 (IgA) and 29 (IgG) patients for peptide-2. Higher anti-gliadin antibody and anti-tTG IgA concentrations correlated with increased IgA reactivity to modified peptides. Among the nine treated CD patients, eight also displayed an improved IgG signal for the deamidated sequence. Deamidation of peptides did not increase the reactivity of non-CD sera.

Conclusions: Selective deamidation specifically increases circulating antibody recognition of gliadin peptides in CD patients. This suggests that deamidated gliadin peptides are more specific CD B-cell epitopes than native peptides; this finding may be relevant for designing improved diagnostic tests.
Determination of l-Pipecolic Acid in Plasma Using Chiral Liquid Chromatography-Electrospray Tandem Mass Spectrometry
Authors: Rashed Mohamed S , Al-Ahaidib Lujane Y , Aboul-Enein Hassan Y , Al-Amoudi Mohamed , Jacob Minnie
Published: January 2020

Background: l-Pipecolic acid (L-PA), an important biochemical marker for the diagnosis of peroxisomal disorders, is usually determined as the racemate. We developed a chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of L-PA in plasma.

Methods: We used a narrow bore chiral macrocyclic glycopeptide teicoplanin column for the enantioseparation of d-pipecolic acid (D-PA) and L-PA and interfaced the column directly to the electrospray source of a tandem mass spectrometer. We used phenylalanine-d5 as internal standard added to 50 μL of plasma followed by deproteinization, evaporation, and injection. The analysis was performed in the selected-reaction monitoring mode using two transitions: m/z 130→m/z 84 for PA, and m/z 171→m/z 125 for phenylalanine-d5. L-PA eluted at 7 min, and D-PA eluted at 11.7 min, whereas phenylalanine-d5 eluted at 6 min. The turnaround time for the assay was 20 min.

Results: Linear calibration curves were obtained in the range of 0.5–80 μmol/L. At a plasma concentration of 1.0 μmol/L, the signal-to-noise ratio was 50:1. The intra- and interassay variations were 3.1–7.9% and 5.7–13%, respectively, at concentrations of 1–50 μmol/L. Mean recoveries of L-PA added to plasma were 95% (5 μmol/L) and 102% (50 μmol/L). The method clearly distinguished between healthy individuals and peroxisomal disease patients.

Conclusions: The novel LC-MS/MS method is simple, rapid, and stereoselective, and uses only 50 μL of plasma, no derivatizing reagents, and a commercially available internal standard. Sample preparation is not complex and is faster than for all other methods.
The Ratio of Trypsin-2-α1-Antitrypsin to Trypsinogen-1 Discriminates Biliary and Alcohol-induced Acute Pancreatitis
Authors: Andersén Jan M , Hedström Johan , Kemppainen Esko , Finne Patrik , Puolakkainen Pauli , Stenman Ulf-Håkan
Published: January 2020

Background: Rapid determination of the etiology of acute pancreatitis (AP) enables institution of appropriate treatment. We evaluated the ability of trypsinogen-1, trypsinogen-2, trypsin-1-α1-antitrypsin (AAT), and trypsin-2-AAT in serum to identify the etiology of AP.

Methods: The study consisted of 67 consecutive patients with AP admitted to Helsinki University Central Hospital. Forty-two had alcohol-induced AP, 16 had biliary AP, and 9 had unexplained etiology. Serum samples were drawn within 12 h after admission. Trypsinogen-1, trypsinogen-2, trypsin-1-AAT, and trypsin-2-AAT were determined by time-resolved immunofluorometric assays. Logistic regression was used to estimate the ability of the serum analytes to discriminate between alcohol-induced and biliary AP. The validity of the tests was evaluated by ROC curve analysis.

Results: Patients with alcohol-induced AP had higher median values of trypsin-1-AAT (P = 0.065), trypsinogen-2 (P = 0.034), and trypsin-2-AAT (P <0.001) than those with biliary AP, who had higher values of amylase (P = 0.002), lipase (P = 0.012), and alanine aminotransferase (P = 0.036). The ratios of trypsin-2-AAT to trypsinogen-1, lipase, or amylase efficiently discriminated between biliary and alcohol-induced AP (areas under ROC curves, 0.92–0.96).

Conclusions: Trypsinogen-2 and trypsin-2-AAT are markedly increased in AP of all etiologies, whereas trypsinogen-1 is increased preferentially in biliary AP. The trypsin-2-AAT/trypsinogen-1 ratio is a promising new marker for discrimination between biliary and alcohol-induced AP.
Utility of Commonly Used Commercial Human Chorionic Gonadotropin Immunoassays in the Diagnosis and Management of Trophoblastic Diseases
Authors: Cole Laurence A , Shahabi Shohreh , Butler Stephen A , Mitchell Hugh , Newlands Edward S , Behrman Harold R , Verrill Harland L
Published: January 2020

Background: Patients with trophoblastic diseases produce ordinary and irregular forms of human chorionic gonadotropin (hCG; e.g., nicked hCG, hCG missing the β-subunit C-terminal segment, hyperglycosylated hCG, and free β subunit) that are recognized to differing extents by automated immunometric hCG (or hCGβ) assays. This has led to low or false-negative results and misdiagnosis of persistent disease. False-positive hCG immunoreactivity has also been detected, leading to needless therapy for trophoblastic diseases. Here we compare seven commonly used hCG assays.

Methods: Standards for five irregular forms hCG produced in trophoblastic diseases, serum samples from 59 patients with confirmed trophoblastic diseases, and serum samples from 12 women with previous false-positive hCG results (primarily in the Abbott AxSYM assay) were blindly tested by commercial laboratories in the Beckman Access hCGβ, the Abbott AxSYM hCGβ, the Chiron ACS:180 hCGβ, the Baxter Stratus hCG test, the DPC Immulite hCG test, the Serono MAIAclone hCGβ tests, and in the hCGβ RIA.

Results: Only the RIA and the DPC appropriately detected the five irregular hCG standards. Only the Beckman, DPC, and Abbott assays gave results similar to the RIA in the patients with confirmed trophoblastic diseases (values within 25% of RIA in 49, 49, and 54 of 59 patients, respectively). For samples that were previously found to produce false-positive hCG results, no false-positive results were detected with the DPC and Chiron tests (5 samples, median <2 IU/L), but up to one-third of samples were false positive (>10 IU/L) in the Beckman (1 of 5), Serono (2 of 9), and Baxter assays (1 of 5), and the hCGβ RIA (3 of 9; median for all assays, <5 IU/L). These samples, which produced false-positive results earlier in the Abbott AxSYM assay, continued to produce high values upon reassessment (median, 81 IU/L).

Conclusions: Of six frequently used hCG immunometric assays, only the DPC detected the five irregular forms of βhCG, agreed with the RIA, and avoided false-positive results in the samples tested. This assay, and similarly designed assays not tested here, seem appropriate for hCG testing in the diagnosis and management of trophoblastic diseases.
Evaluation of Nine Automated High-Sensitivity C-Reactive Protein Methods: Implications for Clinical and Epidemiological Applications. Part 2
Authors: Roberts William L , Moulton Linda , Law Terence C , Farrow Genesis , Cooper-Anderson Margaret , Savory John , Rifai Nader
Published: January 2020

Background: C-Reactive protein (CRP) can provide prognostic information about risk of future coronary events in apparently healthy subjects. This application requires higher sensitivity assays than have traditionally been available in the clinical laboratory.

Methods: Nine high-sensitivity CRP (hs-CRP) methods from Dade Behring, Daiichi, Denka Seiken, Diagnostic Products Corporation, Iatron, Kamiya, Olympus, Roche, and Wako were evaluated for limit of detection, linearity, precision, prozone effect, and comparability with samples from 388 apparently healthy individuals.

Results: All methods had limits of detection that were lower than the manufacturers’ claimed limit of quantification except for the Kamiya, Roche, and Wako methods. All methods were linear at 0.3–10 mg/L. The Diagnostic Products Corporation, Kamiya, Olympus, and Wako methods had imprecision (CVs) >10% at 0.15 mg/L. The Iatron, Olympus, and Wako methods demonstrated prozone effects at hs-CRP concentrations of 12, 206, and 117 mg/L, respectively. hs-CRP concentrations demarcating each quartile in a healthy population were method-dependent. Ninety-two to 95% of subjects were classified into the same quartile of hs-CRP established by the Dade Behring method by the Denka Seiken, Diagnostic Products Corporation, Iatron, and Wako methods. In contrast, 68–77% of subjects were classified into the same quartile by the Daiichi, Kamiya, Olympus, and Roche methods. No subject varied by more than one quartile by any method.

Conclusions: Four of the nine examined hs-CRP methods classified apparently healthy subjects into quartiles of hs-CRP similar to the classifications assigned by the comparison method. Additional standardization efforts are required because an individual patient’s results will be interpreted using population-based cutpoints.
Characteristics of an Albumin Cobalt Binding Test for Assessment of Acute Coronary Syndrome Patients: A Multicenter Study
Authors: Christenson Robert H , Duh Show Hong , Sanhai Wendy R , Wu Alan H B , Holtman Verena , Painter Pennell , Branham Elizabeth , Apple Fred S , Murakami MaryAnn , Morris Deborah L
Published: January 2020

Background: The ability of the N-terminal region of human albumin to bind cobalt is diminished by myocardial ischemia. The characteristics of an assay based on albumin cobalt binding were assessed in suspected acute coronary syndrome patients and in a control reference population. The ability of the Albumin Cobalt Binding (ACBTM) Test measurement at presentation to predict troponin-positive or -negative results 6–24 h later was also examined.

Methods: We enrolled 256 acute coronary syndrome patients at four medical centers. Blood specimens were collected at presentation and then 6–24 h later. The dichotomous decision limit and performance characteristics of the ACB Test for predicting troponin-positive or -negative status 6 h-24 h later were determined using ROC curve analysis. Results for 32 patients could not be used because the time of onset of ischemia appeared to have been >3 h before presentation or was uncertain. The reference interval was determined by parametric analysis to estimate the upper 95th percentile of a reference population (n = 109) of ostensibly healthy individuals.

Results: Increased cTnI was found in 35 of 224 patients. The ROC curve area for the ACB Test was 0.78 [95% confidence interval (CI), 0.70–0.86]. At the optimum decision point of 75 units/mL, the sensitivity and specificity of the ACB Test were 83% (95% CI, 66–93%) and 69% (95% CI, 62–76%). The negative predictive value was 96% (95% CI, 91–98%), and the positive predictive value was 33% (95% CI, 24–44%). The within-run CV of the ACB Test was 7.3%. Results for the reference population were normally distributed; the one-sided parametric 95th percentile was 80.2 units/mL.

Conclusions: This exploratory study suggests that the ACB Test has high negative predictive value and sensitivity in the presentation sample for predicting troponin-negative or -positive results 6–24 h later.
Real-Time Quantitative Telomeric Repeat Amplification Protocol Assay for the Detection of Telomerase Activity2
Authors: Hou Mi , Xu Dawei , Björkholm Magnus , Gruber Astrid
Published: January 2020

Background: Telomerase is a ribonucleoprotein enzyme associated with immortalization and transformation of human cells. The telomeric repeat amplification protocol (TRAP) is widely used for the detection of telomerase activity. The TRAP method, although highly sensitive and specific because it includes PCR amplification, is laborious and does not provide precise quantitative information.

Methods: We developed a real-time quantitative TRAP (RTQ-TRAP) system by combining a real-time PCR technique with the conventional TRAP method. Telomerase activity in human tumor cell lines and in 13 lymphoma samples was measured using the RTQ-TRAP assay, and the results obtained from the samples using the RTQ-TRAP method were compared with the conventional TRAP method.

Results: The RTQ-TRAP method was both accurate and reproducible in measuring telomerase activity in a dilution series of protein extracts from HL60 cells. Telomerase activity in 13 lymphoma samples, as determined by the RTQ-TRAP method, was ninefold lower than that measured by the conventional TRAP method. The half-life of telomerase activity in human tumor cells, as determined using RTQ-TRAP, was much shorter than the half-life reported previously.

Conclusions: Our results suggest that the conventional TRAP assay frequently overestimates telomerase activity in tumor samples. The RTQ-TRAP method is thus a useful tool to rapidly and precisely quantify telomerase activity.
Homogeneous Enzyme Immunoassay for Triiodothyronine in Serum
Authors: Karapitta Christina D , Sotiroudis Theodore G , Papadimitriou Athanassios , Xenakis Aristotelis
Published: January 2020

Background: The concentration of triiodothyronine (T3) in human serum is extremely low and can be determined only by very sensitive methods. We developed a homogeneous enzyme immunoassay for T3 analysis in unextracted serum.

Methods: A T3 derivative was conjugated to the −SH groups of glycogen phosphorylase b (GPb) from rabbit muscle. Conjugation caused inhibition of enzyme activity, and the enzyme conjugate was reactivated upon binding of anti-T3 antibody. Activation was blocked by the presence of non-antibody-bound T3; this was the basis for the development of the homogeneous enzyme immunoassay for T3 by determining GPb activity fluorometrically.

Results: We used furosemide to block the interaction of T3 with serum proteins with T3-binding sites, avoiding any serum treatment step. T3 was measured in the range 0.3–8 μg/L. T3 values obtained by this assay correlated well with those obtained by a RIA (y = 0.97x − 0.07 μg/L; r = 0.96; n = 92). Within- and between-run imprecision (CV) was 5–9% for normal and high concentrations and 16–20% for low concentrations.

Conclusions: Chemical modification of GPb with a T3 derivative allows the development of a simple homogeneous enzyme immunoassay for T3 in unextracted serum.
C677T and A1298C Polymorphisms of the Methylenetetrahydrofolate Reductase Gene: Incidence and Effect of Combined Genotypes on Plasma Fasting and Post-Methionine Load Homocysteine in Vascular Disease
Authors: Hanson Naomi Q , Aras Ömer , Yang Feng , Tsai Michael Y
Published: January 2020

Background: Moderately increased plasma concentrations of total homocysteine (tHcy) have been shown to be an important risk factor for vascular diseases. Two common polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene, the thermolabile C677T and a more recently reported A1298C polymorphism, may contribute to hyperhomocysteinemia.

Methods: Using PCR and restriction fragment length polymorphism analysis, we studied the prevalence of the C677T and A1298C MTHFR genotypes and the combined effect of these polymorphisms on plasma tHcy concentrations, as measured by HPLC with fluorometric detection, both fasting and post-methionine load (PML), in 1238 individuals.

Results: The prevalences of the C677T and A1298C genotypes did not differ significantly in 772 individuals with documented coronary artery disease (CAD), 137 individuals with deep-vein thrombosis (DVT), and 329 individuals without documented vascular disease. Individuals homozygous for the 677T allele had significantly increased fasting tHcy, particularly in the presence of low folate, compared with individuals homozygous for the wild-type allele. Neither the 1298AC nor the 1298CC genotype was associated with significantly increased fasting or PML tHcy concentrations irrespective of serum folate. Of the nine combined MTHFR genotypes, six were present in >10% of the population. Of these, the difference in mean fasting tHcy reached statistical significance (P <0.005) only in individuals with the 677TT/1298AA genotype compared with individuals with the wild-type 677CC/1298AA genotype. Differences in mean fasting tHcy did not reach statistical significance in individuals heterozygous for both MTHFR variants. We detected two 677CT/1298CC and three 677TT/1298AC individuals; only one, an 677TT/1298AC individual, had increased tHcy (both fasting and PML). No individuals had the 677TT/1298CC genotype.

Conclusions: The prevalences of the C677T and A1298C polymorphisms did not differ among individuals with CAD, DVT, or those without documented vascular disease. In contrast to the C677T polymorphism, the A1298C polymorphism is not associated with increased fasting tHcy. Although the two polymorphisms usually exist in trans configuration, crossover may occur rarely to form recombinant chromosomes.
Determination of RhD Zygosity: Comparison of a Double Amplification Refractory Mutation System Approach and a Multiplex Real-Time Quantitative PCR Approach
Authors: Chiu Rossa W K , Murphy Michael F , Fidler Carrie , Zee Benny C Y , Wainscoat James S , Lo Y M Dennis
Published: January 2020

Background: Rh isoimmunization and hemolytic disease of the newborn still occur despite the availability of Rh immunoglobulin. For the prenatal investigation of sensitized RhD-negative pregnant women, determination of the zygosity of the RhD-positive father has important implications. The currently available molecular methods for RhD zygosity assessment, in general, are technically demanding and labor-intensive. Therefore, at present, rhesus genotype assessment is most commonly inferred from results of serological tests. The recent elucidation of the genetic structure of the prevalent RHD deletion in Caucasians, as well as the development of real-time PCR, allowed us to explore two new approaches for the molecular determination of RhD zygosity.

Methods: Two methods for RhD zygosity determination were developed. The first was based on the double Amplification Refractory Mutation System (double ARMS). The second was based on multiplex real-time quantitative PCR. For the double ARMS assay, allele-specific primers were designed to directly amplify the most prevalent RHD deletion found in RhD-negative individuals in the Caucasian population. The multiplex real-time quantitative PCR assay, on the other hand, involved coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin, in a single PCR reaction. A ratio, ΔCt, based on the threshold cycle, was then determined and reflects the RHD gene dosage.

Results: The allele-specific primers of the double ARMS assay reliably amplified the RHD-deleted allele and therefore accurately distinguished homozygous from heterozygous RhD-positive samples. The results were in complete concordance with serological testing. For the multiplex real-time quantitative PCR assay, the ΔCt values clearly segregated into two distinct populations according to the RHD gene dosage, with mean values of 1.70 (SD, 0.17) and 2.62 (SD, 0.29) for the homozygous and heterozygous samples, respectively (P <0.001, t-test). The results were in complete concordance with the results of serological testing as well as with the double ARMS assay.

Conclusion: Double ARMS and real-time quantitative PCR are alternative robust assays for the determination of RhD zygosity.