bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative Analysis of Aldolase A mRNA in Liver Discriminates between Hepatocellular Carcinoma and Cirrhosis
Authors: Castaldo Giuseppe , Calcagno Giuseppe , Sibillo Raffaella , Cuomo Rosario , Nardone Gerardo , Castellano Luigi , Del Vecchio Blanco Camillo , Budillon Gabriele , Salvatore Francesco 
Published: January 2020

Abstract
Background: Chronic liver diseases can progress to cirrhosis and to hepatocellular carcinoma. Timely and unequivocal recognition of the neoplastic evolution of cirrhosis is critical. To this aim, we used a noncompetitive reverse transcription-PCR procedure to analyze aldolase A mRNA in liver tissue from patients with chronic liver diseases at different stages.
Methods: We studied 12 patients with hepatocellular carcinoma, 19 patients affected by chronic hepatitis C or cirrhosis, and 7 healthy controls. Aldolase A mRNA was reverse-transcribed to cDNA, which was then amplified by PCR. The amplified segments were “read” with a novel dot-blot procedure. A calibrator with the same sequence, synthesized in vitro using a T7 phage promoter, was processed at scalar dilutions in parallel to the target samples to generate a calibration curve and so quantify the target mRNA (detection limit, 0.03 amol; linearity spanning five orders of magnitude).
Results: Aldolase A mRNA was ∼10-fold higher in liver biopsies from patients with hepatocellular carcinoma vs patients with chronic hepatitis C or cirrhosis, and healthy individuals. Furthermore, aldolase A mRNA concentrations were 1.2- to 21.3-fold higher in 12 liver biopsies compared with the paired surrounding cirrhotic tissue.
Conclusions: The quantitative analysis of liver tissue aldolase A mRNA differentiates between nonneoplastic chronic liver diseases and hepatocellular carcinoma, which suggests that it has diagnostic potential.
Quantitative Analysis of Tyrosinase Transcripts in Blood
Authors: Johansson Malin , Pisa Eva K , Törmänen Vuokko , Årstrand Kerstin , Kågedal Bertil 
Published: January 2020

Abstract
Background: Tyrosinase is an enzyme unique to pigment-forming cells. Methods using this transcript for detection of melanoma cells in blood have given divergent results. Quantitative analytical procedures are therefore needed to study the analytical performance of the methods.
Methods: Mononucleated cells were isolated by Percoll centrifugation. RNA was isolated by each of three methods: UltraspecTM-II RNA isolation system, FastRNATM GREEN Kit, and QIAamp RNA Blood Mini Kit. cDNA was synthesized using random hexamer primers. A tyrosinase-specific product of 207 bp was amplified by PCR. As an internal standard (and competitor) we used a 207-bp cDNA with a base sequence identical to the tyrosinase target except for a 20-bp probe-binding region. The PCR products were identified by 2,4-dinitrophenol (DNP)-labeled probes specific for tyrosinase (5′DNP-GGGGAGCCTTGGGGTTCTGG-3′) and internal standard (5′DNP-CGGAGCCCCGAAACCACATC-3′) and quantified by ELISA.
Results: The calibration curves were linear and had a broad dynamic measuring range. A detection limit (2 SD above zero) of 48 transcripts/mL of blood was obtained from a low control. The analytical imprecision was 50% and 48% at concentrations of 1775 and 17 929 transcripts/mL (n = 12 and 14, respectively). With the cell line SK-Mel 28 added to blood and RNA extracted with the Ultraspec, Fast RNA, and QIAamp RNA methods, we found (mean ± SD) 1716 ± 1341, 2670 ± 3174, and 24 320 ± 5332 transcripts/mL of blood. Corresponding values were 527 ± 497, 2497 ± 1033, 14 930 ± 1927 transcripts/mL of blood when the cell line JKM86-4 was added. One high-risk patient was followed by repeated analysis of tyrosinase transcripts in blood. The melanoma marker 5-S-cysteinyldopa in serum and urine was within reference values, but tyrosinase mRNA was slightly increased (120–168 transcripts/mL of blood). The tyrosinase mRNA increased to 1860 transcripts/mL concomitant with the increase in 5-S-cysteinyldopa; later a spleen metastasis was found.
Conclusions: The results obtained with different RNA extraction methods illustrate the importance of quantitative methods for validation of methods. The use of QIAamp RNA improved the extraction efficiency considerably. Data from a case study suggest the assay is suitable in the follow-up of patients with high risk of developing metastases.
Flow Cytometric Analysis of Reverse Transcription-PCR Products: Quantification of p21WAF1/CIP1 and Proliferating Cell Nuclear Antigen mRNA
Authors: Wedemeyer Niels , Göhde Wolfgang , Pötter Thomas 
Published: January 2020

Abstract
Background: Reverse transcription-PCR (RT-PCR) is a powerful tool in clinical diagnostics for analyzing even small amounts of RNA, but sensitive assays for quantifying the amplification products are time-consuming or expensive. Here we describe a novel flow cytometry-based assay for rapid and sensitive determination of relative amounts of RT-PCR products.
Methods: For flow cytometric quantification, PCR products were labeled with both digoxigenin and biotin during amplification. Subsequently, amplicons were simultaneously bound to anti-digoxigenin microparticles and fluorescently labeled with streptavidin-R-phycoerythrin. Fluorescence intensity per bead was determined by flow cytometry. To study this assay, we examined the expression of the p21WAF1/CIP1 gene and the proliferating cell nuclear antigen (PCNA) gene in ultraviolet irradiation-exposed human keratinocytes lacking functional p53.
Results: Fluorescence was linear with 60–10 000 pg of PCR product. As little as 0.4 fmol (40 pg of a 163-bp amplicon) of PCR product could be distinguished from background. The between-run CV of the fluorescent signal for 10 ng of p21 cDNA was 12% (n = 10). The fluorescence-template curve was sigmoidal. p21WAF1/CIP1 mRNA was decreased after ultraviolet irradiation of keratinocytes, whereas PCNA mRNA was markedly increased.
Conclusion: The flow cytometric assay permits rapid (25 min) and reproducible identification of changes in mRNA abundance.
An IgM λ Antibody to Escherichia coli Produces False-Positive Results in Multiple Immunometric Assays
Authors: Covinsky Michael , Laterza Omar , Pfeifer John D , Farkas-Szallasi Tunde , Scott Mitchell G 
Published: January 2020

AbstractBackground: Interferences in immunometric assays as a result of human anti-immunoglobulin antibodies frequently have been described in the literature. The etiology of these interfering antibodies is usually not known but has been associated with rheumatoid factors in some assays. It is known that microorganisms in experimental settings can induce anti-immunoglobulin antibodies.Methods: Following Escherichia coli septicemia, a 56-year-old male patient had increased immunoassay results for cardiac troponin I, thyrotropin, human chorionic gonadotropin, α-fetoprotein, and CA-125 that were consistent with myocardial infarction, hyperthyroidism, and pregnancy, and suggestive of an occult neoplasm such as hepatic or ovarian cancer. None of these diagnoses were consistent with the rest of his medical exam. In addition, the patient had a restricted IgM λ paraprotein by immunofixation. Plasma from the patient was incubated with Sepharose-conjugated protein A, irrelevant murine monoclonal antibodies, and formalin-killed E. coli organisms from his infection to determine whether these immunoassay values were falsely increased.Results: Incubation of the patient’s plasma with irrelevant murine monoclonal antibodies or the E. coli organism produced normal immunoassay values and removed the IgM λ paraprotein.Conclusions: The patient produced a very restricted IgM λ antibody response to the E. coli infection that had anti-immunoglobulin activity and caused falsely increased values in numerous immunometric assays. Microorganism-induced anti-immunoglobulin antibodies are discussed in the context of this patient.
The CBC at the Turn of the Millennium: An Overview
Authors: Ward Patrick C J 
Published: January 2020

AbstractThis review offers a birds-eye view of the state of automated hematology at the turn of the millennium. Despite its shortcomings (mainly flaggings and labor-intensive demands for confirmation), instrument-driven hematology provides much accurate and precise data to clinicians. Advances in technology over the next few decades will undoubtedly improve on the categorization of currently ambiguous mononuclear cells and even introduce channels for the detection and subclassification of poikilocytes. Until then, familiarity with the morphology of blood cell variants will be mandatory for technologists attending to the demands of flagging.
Cobalamin and Folate Evaluation: Measurement of Methylmalonic Acid and Homocysteine vs Vitamin B12 and Folate
Authors: Klee George G 
Published: January 2020

AbstractVitamin B12 and folate are two vitamins that have interdependent roles in nucleic acid synthesis. Deficiencies of either vitamin can cause megaloblastic anemia; however, inappropriate treatment of B12 deficiency with folate can cause irreversible nerve degeneration. Inadequate folate nutrition during early pregnancy can cause neural tube defects in the developing fetus. In addition, folate and vitamin B12 deficiency and the compensatory increase in homocysteine are a significant risk factor for cardiovascular disease. Laboratory support for the diagnosis and management of these multiple clinical entities is controversial and somewhat problematic. Automated ligand binding measurements of vitamin B12 and folate are easiest to perform and widely used. Unfortunately, these tests are not the most sensitive indicators of disease. Measurement of red cell folate is less dependent on dietary fluctuations, but these measurements may not be reliable. Homocysteine and methylmalonic acid are better metabolic indicators of deficiencies at the tissue level. There are no “gold standards” for the diagnosis of these disorders, and controversy exists regarding the best diagnostic approach. Healthcare strategies that consider the impact of laboratory tests on the overall costs and quality of care should consider the advantages of including methylmalonic acid and homocysteine in the early evaluation of patients with suspected deficiencies of vitamin B12 and folate.
Laboratory Investigation of Hemoglobinopathies and Thalassemias: Review and Update
Authors: Clarke Gwendolyn M , Higgins Trefor N 
Published: January 2020

AbstractStructural hemoglobin (Hb) variants typically are based on a point mutation in a globin gene that produce a single amino acid substitution in a globin chain. Although most are of limited clinical significance, a few important subtypes have been identified with some frequency. Homozygous Hb C and Hb S (sickle cell disease) produce significant clinical manifestations, whereas Hb E and Hb D homozygotes may be mildly symptomatic. Although heterozygotes for these variants are typically asymptomatic, diagnosis may be important for genetic counseling. Thalassemia, in contrast, results from quantitative reductions in globin chain synthesis. Those with diminished β-globin chains are termed β-thalassemias, whereas those with decreased α-chain production are called α-thalassemias. Severity of clinical manifestations in these disorders relates to the amount of globin chain produced and the stability of residual chains present in excess. The thalassemia minor syndromes are characterized clinically by mild anemia with persistent microcytosis. Thalassemia intermedia (i.e., Hb H disease) is typified by a moderate, variably compensated hemolytic anemia that may present with clinical symptoms during a period of physiologic stress such as infection, pregnancy, or surgery. The thalassemia major syndromes produce severe, life-threatening anemia. α-Thalassemia major usually is incompatible with extrauterine life; β-thalassemia major presents in infancy and requires life-long transfusion therapy and/or bone marrow transplantation for successful control of the disease. Double heterozygosity for certain structural variants and/or thalassemia syndromes may also lead to severe clinical disease. Several guidelines have been published that outline the required steps for hemoglobinopathy and thalassemia investigation. The availability of HPLC has streamlined many of these requirements, allowing an efficient stepwise diagnostic strategy for these complex disorders.
Immobilization of Antibodies on Ultraflat Polystyrene Surfaces
Authors: Qian Weiping , Yao Danfeng , Yu Fang , Xu Bin , Zhou Rong , Bao Xiang , Lu Zuhong 
Published: January 2020

Abstract
Background: Functional antibody surfaces were prepared on ultraflat polystyrene surfaces by physical adsorption, and the uniform distribution of monoclonal antibodies against hepatitis B surface antigen (anti-HBs) on such surfaces and the presence of dense hepatitis B surface antigen (HBsAg) particles captured by immobilized antibodies were identified.
Methods: A model polystyrene film was spin-coated directly onto a silicon wafer surface. Atomic force microscopy was used to directly monitor the immobilization of anti-HBs antibodies and their specific molecular interaction with HBsAg. Enzyme immunoassay was also used to characterize functional antibody surfaces.
Results: A mean roughness of 2 Å for areas of 25 μm2 was produced. We found a uniform distribution of anti-HBs antibodies on ultraflat polystyrene surfaces and the presence of dense HBsAg particles bound to such anti-HBs surfaces after incubation with HBsAg.
Conclusions: This study confirmed the potential of preparing dense, homogeneous, highly specific, and highly stable antibody surfaces by immobilizing antibodies on polystyrene surfaces with controlled roughness. It is expected that such biofunctional surfaces could be of interest for the development of new solid-phase immunoassay techniques and biosensor techniques.
Use of Free Radical Chemistry in an Immunometric Assay for 17β-Estradiol
Authors: Buscarlet Laure , Volland Hervé , Dupret-Carruel Jacqueline , Jolivet Michel , Grassi Jacques , Créminon Christophe , Taran Frédéric , Pradelles Philippe 
Published: January 2020

AbstractBackground: We wished to develop an enzyme immunometric assay for 17β-estradiol (E2) in human serum using solid-phase immobilized epitope immunoassay (SPIE-IA) technology and free radical chemistry.Methods: We used an anti-estradiol monoclonal antibody as capture antibody and Fenton-like reagents to cross-link it to E2. The same antibody, labeled with acetylcholinesterase, was used for detection. Serum was diluted 10-fold before assay.Results: After correction by the dilution factor, the detection limit was 5 ng/L for human serum and intra- and interassay CVs were &lt;7% and 15%, respectively, at concentrations of 169-2845 ng/L. No cross-reactivity was seen with other natural steroids. In comparison with a competitive commercial RIA performed on 88 undiluted human sera, the slope (SD) of the regression line was 1.05 (± 0.02) and the intercept was 47 (±27) ng/L (Sy|x = 186 ng/L) at concentrations of 20–5000 ng/L (r2 = 0.97).Conclusions: The use of Fenton-like chemistry in SPIE-IA technology allows a sensitive measurement of E2 in human serum and could be a new approach for the development of sensitive immunoassays.
Molecular Diagnosis of Intermediate and Severe α1-Antitrypsin Deficiency: MZ Individuals with Chronic Obstructive Pulmonary Disease May Have Lower Lung Function Than MM Individuals
Authors: Dahl Morten , Nordestgaard Børge G , Lange Peter , Vestbo Jørgen , Tybjærg-Hansen Anne 
Published: January 2020

AbstractBackground: We tested whether intermediate (MZ, SZ) and severe (ZZ) α1-antitrypsin deficiency affects lung function in the population at large.Methods: We performed spirometry [forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC)] and genotyping of 9187 individuals from the adult general population of Copenhagen, Denmark.Results: As expected, the frequencies of individuals with MM, MS, SS, MZ, SZ, and ZZ genotypes were 0.891, 0.054, 0.001, 0.052, 0.001, and 0.001, respectively. Genotype interacted with clinically established chronic obstructive pulmonary disease (COPD) on the percentage of the predicted FEV1 (P = 0.004): the percentage of the predicted FEV1 was reduced in MZ compared with MM individuals among those with clinically established COPD, but not among those without COPD. Furthermore, SZ compound heterozygotes had lower FEV1/FVC ratios than MM individuals (P &lt;0.05), and ZZ homozygotes had lower percentages of the predicted FEV1 and FEV1/FVC ratios than MM, MS, SS, and MZ individuals (all Ps &lt;0.01). Reduced lung function in SZ and ZZ vs MM individuals could be demonstrated in current and ex-smokers, but not in nonsmokers. Compared with MM individuals in the same groups, FEV1 was reduced 655 mL in MZ individuals with clinically established COPD, 364 mL in SZ current smokers, and 791 mL in ZZ current smokers.Conclusions: In the population at large, MZ was associated with reduced pulmonary function in individuals with clinically established COPD, whereas SZ and ZZ were associated with reduced pulmonary function in smokers. The presence of the α1-antitrypsin MZ genotype may in certain circumstances produce marked aggravation of airway obstruction in individuals prone to develop COPD.
Radioiodinated Tyrosyl-Ouabain and Measurement of a Circulating Ouabain-like Compound
Authors: Vakkuri Olli , Arnason Sighvatur S , Joensuu Päivi , Jalonen Jorma , Vuolteenaho Olli , Leppäluoto Juhani 
Published: January 2020

Abstract
Background: Assays for endogenous ouabain, a cardiac glycoside believed to be involved in blood pressure and volume regulation, are characterized by laboratory-specific plasma values that are measured by different assays. Because of this variability, our study focused on the development of a new 125I-labeled ouabain derivative for RIA of high sensitivity.
Methods: We generated rabbit antisera against a ouabain-thyroglobulin conjugate. A tyrosylated ouabain derivative for radioiodination was synthesized using periodate and sodium cyanoborohydride reagents.
Results: Mass spectrometric analyses showed that the main product of the tyrosylating reaction was tyrosyl-ouabain (molecular mass, 702 Da). This was radioiodinated with Chloramine-T and used as a tracer in a RIA, which gave an assay detection limit of 5 pmol/L (4 ng/L), 2–100 times lower than that in the corresponding 3H-RIAs and 2–20 times lower than ouabain ELISAs, making it possible to measure low plasma concentrations of immunoreactive ouabain. Different amounts of SepPak C18-extracted plasma samples displaced the 125I-labeled tyrosyl-ouabain tracer at the same rate at which authentic ouabain was displaced. Plasma immunoreactive ouabain coeluted with authentic ouabain in two different HPLC conditions. Using the new RIA, we found plasma ouabain concentrations, assayed as immunoreactive equivalents, of 10.0 ± 1.3 pmol/L in healthy women and 12.0 ± 0.9 pmol/L in healthy men (mean ± SE; n = 10), as well as 41.2 ± 9.6 pmol/L in rats. The concentrations were 2–90 times lower than those previously reported using different assay methods.
Conclusions: Our ouabain 125I-RIA enables reliable measurements of low endogenous concentrations of a ouabain-like compound for both physiological and clinical purposes.
Tau and Aβ42 in Cerebrospinal Fluid from Healthy Adults 21–93 Years of Age: Establishment of Reference Values
Authors: Sjögren Magnus , Vanderstichele Hugo , Ågren Hans , Zachrisson Olof , Edsbagge Mikael , Wikkelsø Carsten , Skoog Ingmar , Wallin Anders , Wahlund Lars-Olof , Marcusson Jan , Nägga Katarina , Andreasen Niels , Davidsson Pia , Vanmechelen Eugeen , Blennow Kaj 
Published: January 2020

Abstract
Background: Tau protein and the 42-amino acid form of β-amyloid (Aβ42) measured in cerebrospinal fluid (CSF) have been proposed as potential biochemical diagnostic markers for Alzheimer disease. For the introduction of these assays in clinical practice, adequate reference values are of importance.
Methods: CSF samples were obtained from 231 neurologically and psychiatrically healthy individuals, 21–93 years of age, all with a MiniMental State examination score of 28 or above. Standardized ELISAs were used to measure tau and Aβ42 in CSF. Following IFCC recommendations, we used a rank-based method; the 0.90 and 0.10 fractiles were estimated to establish reference values for CSF-tau and CSF-Aβ42, respectively. Putative confounding factors, such as the influence of the passage of proteins from peripheral blood to CSF, influence of dysfunction of the blood-brain barrier, and freezing and thawing of CSF, were investigated.
Results: A correlation with age was found for CSF-tau (r = 0.60; P &lt;0.001). Therefore, separate reference values for different age groups were established for CSF-tau: &lt;300 ng/L in the group 21–50 years of age, &lt;450 ng/L in the group 51–70 years of age, and &lt;500 ng/L in the group 71–93 years of age. CSF-Aβ42 did not correlate with age (r = −0.045), and the reference value was set to &gt;500 ng/L. No correlation was found between blood-brain barrier function and CSF-tau or CSF-Aβ42.
Conclusions: These reference values can be applied when using CSF-tau and CSF-Aβ42 in clinical practice.
Age-dependent Prevalence of Vascular Disease-associated Polymorphisms among 2689 Volunteer Blood Donors
Authors: Hessner Martin J , Dinauer David M , Kwiatkowski Robert , Neri Bruce , Raife Thomas J 
Published: January 2020

AbstractBackground: The development of vascular disease involves the interaction of genetic and environmental factors. Because vascular disease is a major contributor to mortality in Western societies, we hypothesized that deleterious polymorphisms associated with hemostasis decrease in frequency among a healthy population as a function of age.Methods: The frequencies of factor V G1691A Leiden (FVL), factor II (FII) G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, glycoprotein Ia (GPIa) C807T, glycoprotein IIIa (PlA1/PlA2) T1565C, and angiotensin-converting enzyme (ACE) intron 16 insertion/deletion (I/D) alleles were determined among 2689 healthy Caucasian whole-blood donors. For analysis, participants were divided into three age groups: 17–39 years (n = 979; 505 males and 474 females), 40–59 years (n = 900; 526 males and 374 females), and 60–85 years (n = 810; 530 males and 280 females).Results: The PlA2 allele frequency decreased from 17.5% to 15.7% and 14.1% in the 17–39 years, 40–59 years, and 60–85 years age groups, respectively (n = 5094 alleles; P = 0.025). Among ACE DD males, the PlA2 allele frequency decreased from 20.8% to 16.1% and 9.1% in the same groups, respectively (n = 810 alleles; P = 0.001). No statistically significant decrease in genotype or allele frequency was observed among carriers of FVL, FII 20210A, MTHFR 677T, GPIa 807T, or ACE D.Conclusions: These data suggest that PlA2 carriers, especially those who are ACE DD, are statistically less prevalent among older healthy blood donors compared with their younger counterparts. These observations suggest an important, deleterious, time-dependent impact of the PlA2 allele, as well as the ACE DD/PlA2 allelic combination, on overall health and longevity.