bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Analysis of Tyrosinase Transcripts in Blood
Authors: Johansson Malin , Pisa Eva K , Törmänen Vuokko , Årstrand Kerstin , Kågedal Bertil
Published: January 2020

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

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

Background: 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

This 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

Vitamin 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

Structural 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

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

Background: 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 <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

Background: 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 <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 <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

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

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 <0.001). Therefore, separate reference values for different age groups were established for CSF-tau: <300 ng/L in the group 21–50 years of age, <450 ng/L in the group 51–70 years of age, and <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 >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

Background: 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.
Silicon-based Biosensors for Rapid Detection of Protein or Nucleic Acid Targets
Authors: Jenison Robert , La Helen , Haeberli Ayla , Ostroff Rachel , Polisky Barry
Published: January 2020

Background: We developed a silicon-based biosensor that generates visual, qualitative results or quantitative results for the detection of protein or nucleic acid targets in a multiplex format.

Methods: Capture probes were immobilized either passively or covalently on the optically coated surface of the biosensor. Intermolecular interactions of the immobilized capture probe with specific target molecules were transduced into a molecular thin film. Thin films were generated by enzyme-catalyzed deposition in the vicinity of the surface-bound target. The increased thickness on the surface changed the apparent color of the biosensor by altering the interference pattern of reflected light.

Results: Cytokine detection was achieved in a 40-min multiplex assay. Detection limits were 4 ng/L for interleukin (IL)-6, 31 ng/L for IL1-β, and 437 ng/L for interferon-γ. In multianalyte experiments, cytokines were specifically detected with signal-to-noise ratios ranging from 15 to 80. With a modified optical surface, specificity was also demonstrated in a nucleic acid array with unambiguous discrimination of single-base changes in a 15-min assay. For homozygous wild-type and homozygous mutant samples, signal-to-noise ratios of ∼100 were observed. Heterozygous samples yielded approximately equivalent signals for wild-type and mutant capture probes.

Conclusions: The thin-film biosensor allows rapid, sensitive, and specific detection of protein or nucleic acid targets in an array format with results read visually or quantified with a charge-coupled device camera. This biosensor is suited for multianalyte detection in clinical diagnostic assays.
New Thiocholine Ester Substrates for the Assay of Human Serum Cholinesterase
Authors: Yamada Magohei , Marui Yoji , Hayashi Chozo , Miki Yasuyoshi , Takemura Shoji
Published: January 2020

Background: Several thiocholine alkanoyl esters were newly synthesized and explored as substrates for the assay of human serum cholinesterase after being subjected to the Ellman reaction (Arch Biochem Biophys 1958;74:443–50 and Arch Biochem Biophys 1959;82:70–7).

Methods: We synthesized thiocholine ester iodides by the method of Renshow et al. (J Am Chem Soc 1938;60:1765–70). We examined solubility in H2O, substrate specificity serum for cholinesterase, (spontaneous) self-hydrolysis, storage stability, and reaction conditions for measurement of the activity of the enzyme.

Results: Isobutyryl and cyclohexane-carboxyl esters showed the best efficiency for the specific and stable assay of human serum cholinesterase. Aqueous solubility of each was >10 mmol/L, and the reactivity with acetylcholinesterase was negligible. For isobutyryl and cyclohexane-carboxyl esters, respectively, spontaneous hydrolysis in the aqueous phase was ∼1/25 and ∼1/175 slower than the enzymatic hydrolysis, and assays with these substrates were linear to 1800 and 3000 U/L, respectively. The Km values of these acylthiocholines with human cholinesterase were almost equivalent (6.9 × 10−3 mmol/L). The substrates were stable in aqueous solution and in the solid state as the iodides for at least 5 years at 5 °C.

Conclusions: The isobutyrate and cyclohexane-carboxylate of thiocholine are suitable for the specific assay of human serum cholinesterase.
Concentration Ratios of Morphine to Codeine in Blood of Impaired Drivers as Evidence of Heroin Use and not Medication with Codeine
Authors: Ceder Gunnel , Jones Alan Wayne
Published: January 2020

Background: Both the illicit drug heroin and the prescription drug codeine are metabolized to morphine, which tends to complicate interpretation of opiate-positive samples. We report here the concentrations of morphine and codeine, the morphine/codeine ratios, and 6-acetylmorphine (6-AM) in blood specimens from individuals arrested for driving under the influence of drugs (DUID) in Sweden. The results were compared with positive findings of 6-AM in urine as evidence of heroin intake.

Methods: In 339 DUID suspects, both blood and urine specimens were available for toxicologic analysis. In another 882 cases, only blood was available. All specimens were initially analyzed by immunoassay, and the positive results were verified by isotope-dilution gas chromatography–mass spectrometry. In routine casework, the limits of quantification (LOQs) for unconjugated opiates were 5 ng/g for blood and 20 μg/L for urine.

Results: The median concentration of morphine in blood was 30 ng/g with 2.5 and 97.5 percentiles of 5 and 230 ng/g, respectively (n = 979). This compares with a median codeine concentration of 20 ng/g and 2.5 and 97.5 percentiles of 5 and 592 ng/g, respectively (n = 784). The specific metabolite of heroin, 6-AM, was identified in only 16 of 675 blood specimens (2.3%). This compares with positive findings of 6-AM in 212 of 339 urine samples (62%) from the same population of DUID suspects. When 6-AM was identified in urine, the morphine/codeine ratio in blood was always greater than unity (median, 6.0; range, 1–66). In 18 instances, 6-AM was present in urine, although morphine and codeine were below the LOQ in blood. The morphine/codeine ratio in blood was greater than unity in 85% of DUID cases when urine was not available (n = 506), and the median morphine and codeine concentrations were 70 ng/g and 10 ng/g, respectively. When morphine/codeine ratios in blood were less than unity (n = 76), the median morphine and codeine concentrations were 10 ng/g and 180 ng/g, respectively.

Conclusions: Only 2.3% of opiate-positive DUID suspects were verified as heroin users on the basis of positive findings of 6-AM in blood. A much higher proportion (62%) were verified heroin users from 6-AM identified in urine. When urine was not available for analysis, finding a morphine/codeine concentration ratio in blood above unity suggests heroin use and not medication with codeine. This biomarker indicated that 85% of opiate-positive DUID blood samples were from heroin users.
Identification of α1-Antitrypsin Variants in Plasma with the Use of Proteomic Technology
Authors: Mills Kevin , Mills Philippa B , Clayton Peter T , Johnson Andrew W , Whitehouse David B , Winchester Bryan G
Published: January 2020

Background: Proteomic technology permits the investigation of genetic metabolic diseases at the level of protein expression. Changes in the expression, polypeptide structure, and posttranslational modification of individual proteins can be detected in complex mixtures of proteins.

Methods: We used high-resolution two-dimensional polyacrylamide gel electrophoresis to separate isoforms of plasma proteins and detect abnormalities of mass and/or charge. We confirmed the identity of the separated proteins by in-gel digestion with proteases and N-glycanases and then analyzed the released peptides and glycans by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry.

Results: Complete characterization of the polypeptide sequences and glycosylation of α1-antitrypsin isoforms was achieved in plasma from controls and from patients with three different known α1-antitrypsin deficiencies and congenital disorder of glycosylation type Ia.

Conclusions: This study shows that proteomic techniques are a powerful and sensitive means of detecting changes in the amino acid sequence and abnormal posttranslational modifications of specific proteins in a complex biologic matrix.
Hurler-like Phenotype
Authors: Chamoles Néstor A , Blanco Mariana B , Gaggioli Daniela , Casentini Carina
Published: January 2020

Background: Clinical differentiation among mucopolysaccharidosis, oligosaccharidosis, and mucolipidosis II and III is difficult. We describe methods for the assay of 8 lysosomal enzymes in dried blood spots on filter paper that allow screening for 12 lysosomal storage diseases that present with a Hurler-like phenotype.

Methods: To test tubes containing 3-mm blood spots, we added elution liquid and fluorescent or radioactive substrate solution. After incubation at 37 °C, the reaction was terminated by the addition of a stop buffer. The amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. Sample stability was studied during storage for 21 days and during shipment of samples. We measured enzyme activities in 85 healthy controls (35 newborn, 50 adult), 57 patients suffering from 11 lysosomal storage diseases, and 46 obligate carriers.

Results: Intra- and interassay CVs were <9% and <15%, respectively. Mean activity losses during transportation or storage for up to 21 days at 4 °C were ≤27%. Enzyme activities in all patients were outside the ranges of values seen for carriers and controls.

Conclusions: The described methodology distinguishes between patients and controls with samples that are sufficiently stable to be mailed to the testing laboratory.
Early Blood Chemistry in Britain and France
Authors: Coley Noel G
Published: January 2020

I review here key research in the early years of the field of blood chemistry. The review includes successes and limitations of animal chemistry in the critical period of the eighteenth and nineteenth centuries. Eighteenth century medical theories emphasized the primacy of body solids. Body fluids were governed by the tenets of humoral pathology. After Boerhaave sparked interest in the chemistry of the body fluids, a new humoralism developed. With the rise of animal chemistry in the eighteenth century, two complementary ideas came into play. The concept of vital force was introduced in 1774, and the chemical composition of animal matters, including the blood, began to be investigated. In the early nineteenth century, the development of new methods of analysis encouraged such chemical studies. Prominent chemists led the field, and physicians also became involved. Physiologists were often opposed to the chemical tradition, but François Magendie recognized the importance of chemistry in physiology. Liebig linked the formation and functions of the blood to general metabolism and so extended the scope of animal chemistry from 1842. About the same time, microscopic studies led to discoveries of the globular structure of the blood, and Magendie’s famous pupil, Claude Bernard, began the animal chemistry studies that led him to new discoveries in hematology. This review addresses discoveries, controversies, and errors that relate to the foundations of clinical chemistry and hematology and describes contributions of instrumental investigators.
K-ras Mutation Detection by Hybridization to a Polypyrrole DNA Chip
Authors: Lopez-Crapez Evelyne , Livache Thierry , Marchand Joseph , Grenier Jean
Published: January 2020

Background: Detection of mutations in cancer-related genes is of major importance for both basic knowledge and clinical practice. Several strategies have been developed to diagnose these alterations. We describe a method based on polypyrrole DNA chip technology to detect K-ras gene mutations in tumors.

Methods: An oligodeoxynucleotide array was constructed on a silicon device by copolymerization of 5′-pyrrole-labeled oligodeoxynucleotides and pyrrole. The samples to be analyzed were then amplified by PCR, and the single-stranded biotin-labeled amplified DNA was specifically hybridized to the addressed probes. Perfectly matched duplexes were detected by fluorescence microscopy using R-phycoerythrin as the detection label. The developed methodology was applied to genotype assignment of K-ras in human samples. The genotypes of 75 DNA genomic samples from colorectal cancer patients were analyzed side by side using direct DNA sequencing and a polypyrrole DNA chip.

Results: The chip method unequivocally defined all of the genotypes. Mutations present at <10% of the wild-type DNA concentration could be distinguished.

Conclusions: This probe array assay is a rapid and reliable procedure that may be used to detect mutations.
Quality Specifications for Glucose Meters: Assessment by Simulation Modeling of Errors in Insulin Dose
Authors: Boyd James C , Bruns David E
Published: January 2020

Background: Proposed quality specifications for glucose meters allow results to be in error by 5–10% or more of the “true” concentration. Because meters are used as aids in the adjustment of insulin doses, we aimed to characterize the quantitative effect of meter error on the ability to identify the insulin dose appropriate for the true glucose concentration.

Methods: Using Monte Carlo simulation, we generated random “true” glucose values within defined intervals. These values were converted to “measured” glucose values using mathematical models of glucose meters having defined imprecision (CV) and bias. For each combination of bias and imprecision, 10 000–20 000 true and measured glucose concentrations were matched with the corresponding insulin doses specified by selected insulin-dosing regimens. Discrepancies in prescribed doses were counted and their frequencies plotted in relation to bias and imprecision.

Results: For meters with a total analytical error of 5%, dosage errors occurred in ∼8–23% of insulin doses. At 10% total error, 16–45% of doses were in error. Large errors of insulin dose (two-step or greater) occurred >5% of the time when the CV and/or bias exceeded 10–15%. Total dosage error rates were affected only slightly by choices of sliding scale among insulin dosage rules or by the range of blood glucose. To provide the intended insulin dosage 95% of the time required that both the bias and the CV of the glucose meter be <1% or <2%, depending on mean glucose concentrations and the rules for insulin dosing.

Conclusions: Glucose meters that meet current quality specifications allow a large fraction of administered insulin doses to differ from the intended doses. The effects of such dosage errors on blood glucose and on patient outcomes require study.
Determination of Carbohydrate-deficient Transferrin Using Capillary Zone Electrophoresis
Authors: Wuyts Birgitte , Delanghe Joris R , Kasvosve Ishmael , Wauters Annick , Neels Hugo , Janssens Jacques
Published: January 2020

Background: Current methods for carbohydrate-deficient transferrin (CDT) often suffer from low precision, complexity, or risk of false positives attributable to genetic variants. In this study, a new capillary zone electrophoresis (CZE) method for CDT was developed.

Methods: CZE was performed on a P/ACE 5000 using fused-silica capillaries [50 μm (i.d.) × 47 cm] and the CEOFIX CDT buffer system with addition of 50 μL of anti-C3c and 10 μL of anti-hemoglobin. Native sera were loaded by high-pressure injection for 3 s, separated at 28 kV over 12 min, and monitored at 214 nm.

Results: CDT was completely resolved by differences in migration times (di-trisialotransferrin, 9.86 ± 0.05 min; monosialotransferrin, 9.72 ± 0.05 min; asialotransferrin, 9.52 ± 0.04 min), with a CV of 0.15%. The number of theoretical plates was 312 000 ± 21 000 for the mono- and 199 000 ± 6500 for the di-trisialylated transferrin. Genetic CB and CD variants showed prominent peaks with migration times of 10.12 ± 0.06 and 9.89 ± 0.03 min, respectively, and the carbohydrate-deficient glycoprotein syndrome could be detected, excluding false-positive results. CZE results (as a percentage; y) correlated with the Axis %CDT TIATM (x) values by Deming regression analysis: y = 1.92x − 7.29; r = 0.89. CDT values in 130 healthy nonalcoholics were determined. The 2.5th and 97.5th percentiles were 1.84% and 6.79%.

Conclusions: CZE without sample pretreatment can determine CDT with good precision, allows detection of variants, and correlates with ion-exchange chromatography.
HPLC Analysis of Reduced and Oxidized Coenzyme Q10 in Human Plasma
Authors: Tang Peter H , Miles Michael V , DeGrauw Antonius , Hershey Andrew , Pesce Amadeo
Published: January 2020

Background: The percentage of reduced coenzyme Q10 (CoQ10H2) in total coenzyme Q10 (TQ10) is decreased in plasma of patients with prematurity, hyperlipidemia, and liver disease. CoQ10H2 is, however, easily oxidized and difficult to measure, and therefore reliable quantification of plasma CoQ10H2 is of clinical importance.

Methods: Venous blood was collected into evacuated tubes containing heparin, which were immediately placed on ice and promptly centrifuged at 4 °C. The plasma was harvested and stored in screw-top polypropylene tubes at −80 °C until analysis. After extraction with 1-propanol and centrifugation, the supernatant was injected directly into an HPLC system with coulometric detection.

Results: The in-line reduction procedure permitted transformation of CoQ10 into CoQ10H2 and avoided artifactual oxidation of CoQ10H2. The electrochemical reduction yielded 99% CoQ10H2. Only 100 μL of plasma was required to simultaneously measure CoQ10H2 and CoQ10 over an analytical range of 10 μg/L to 4 mg/L. Intra- and interassay CVs for CoQ10 in human plasma were 1.2–4.9% across this range. Analytical recoveries were 95.8–101.0%. The percentage of CoQ10H2 in TQ10 was ∼96% in apparently healthy individuals. The method allowed analysis of up to 40 samples within an 8-h period.

Conclusions: This optimized method for CoQ10H2 analysis provides rapid and precise results with the potential for high throughput. This method is specific and sufficiently sensitive for use in both clinical and research laboratories.
Year-Long Validation Study and Reference Values for Urinary Amino Acids Using a Reversed-Phase HPLC Method
Authors: Venta Rafael
Published: January 2020

Background: Reversed-phase HPLC (RP-HPLC) has become an alternative to ion-exchange chromatography for amino acid analysis in biological fluids. However, validation studies for its urine application are limited, and the corresponding reference values have not been reported extensively. We studied the long-term performance of a commercial HPLC method for urine amino acid analysis and established specific age-related reference values for urine amino acid excretion.

Methods: Method performance was continuously assessed by recovery and precision studies with urine samples and controls, respectively. Healthy individuals were prospectively analyzed throughout a 5-year period. Excretion of individual amino acids, expressed as mmol/mol of creatinine, was included in six age-related groups for random urine samples (0–1 month, 1–12 months, 1–3 years, 3–8 years, 8–16 years, and >16 years) and in two groups for 24-h urine collections (8–16 years and >16 years).

Results: Over a 1-year period, CVs for retention times were <0.5% and 3.3% for within- and between-run imprecision, respectively. For amino acid concentrations, within-run CVs were 2.9–17% and between-run CVs were 7.1–46% for the same period. Amino acid recoveries were 78–122%. Reference intervals for 35 amino acids were calculated and compared with the concentrations observed in patients diagnosed with specific pathologies. A few statistically significant differences were found between the reference intervals derived using random and 24-h urine collections.

Conclusions: Long-term reliability of the RP-HPLC method for urine amino acid analysis has been demonstrated. Representative age-related reference intervals for the RP-HPLC method in both random urine and 24-h urine collections have been established, and their feasibility for diagnosis of aminoaciduria has been shown. These intervals could serve as a guide for laboratories changing to HPLC methods.
ζ-, ε-, and γ-Globin mRNA in Blood Samples and CD71+ Cell Fractions from Fetuses and from Pregnant and Nonpregnant Women, with Special Attention to Identification of Fetal Erythroblasts
Authors: Høgh Anne Mette , Hviid Thomas Vauvert F , Christensen Britta , Sørensen Steen , Larsen Rasmus D , Smidt-Jensen Steen , Bang Jens , Philip John
Published: January 2020

Background: Information about the appearance of γ-, ε-, and ζ-globin mRNAs in fetal erythroblasts during gestation and about the presence and amounts of these mRNAs in pregnant and nonpregnant women is important from the perspective of using these molecules as a marker of fetal erythroblasts. A specific marker is necessary for isolation and identification of fetal nucleated red blood cells from maternal blood samples for use in antenatal diagnosis of fetal genetic or chromosomal abnormalities.

Methods: We used a very sensitive reverse transcription-PCR (RT-PCR) method, coamplification analysis of γ- and ε-globin cDNA, and quantitative analysis of γ-globin mRNA based on competitive RT-PCR to investigate these aspects.

Results: All adult whole-blood samples were negative for ε- and ζ-globin mRNA. Analyses of CD71+ cell fractions showed that specimens from 19 of 20 nonpregnant and 10 of 14 pregnant women (at 9–13 weeks of gestation) were positive for γ-globin mRNA (Fisher’s exact test, P = 0.13), and those from 3 of 20 nonpregnant and 5 of 14 pregnant women were positive for ζ-globin mRNA (Fisher’s exact test, P = 0.23). No ε-globin mRNA was detected in CD71+ cell fractions from 1-mL blood samples from adults. CD71+ cell fractions from eight fetal blood samples (at 17–20 weeks of gestation) were positive for all three globin mRNAs. We found no statistically significant difference between the amounts of γ-globin mRNA in pregnant and nonpregnant women.

Conclusions: This study indicates that ε-globin mRNA might function as a marker for fetal CD71+ cells early in pregnancy. Although γ-globin mRNA can be detected in CD71+ cell fractions from most adults, these transcripts also may be of use because of a marked difference between adult and fetal values.
Polymorphic Short Tandem Repeats for Diagnosis of the Charcot-Marie-Tooth 1A Duplication
Authors: Latour Philippe , Boutrand Laetitia , Levy Nicolas , Bernard Rafaëlle , Boyer Amandine , Claustrat Francine , Chazot Guy , Boucherat Michel , Vandenberghe Antoon
Published: January 2020

Background: A 1.5-Mb microduplication containing the gene for peripheral myelin protein 22 (PMP22) on chromosome 17p11.2-12 is responsible for 75% of cases of the demyelinating form of Charcot-Marie-Tooth disease (CMT1A). Methods for molecular diagnosis of CMT1A use Southern blot and/or amplification by PCR of polymorphic poly(AC) repeats (microsatellites) located within the duplicated region, or the detection of junction fragments specific for the duplication. Difficulties with both strategies have led us to develop a new diagnostic strategy with highly polymorphic short tandem repeats (STRs) located inside the CMT1A duplicated region.

Methods: We tested 10 STRs located within the duplication for polymorphic behavior. Three STRs were selected and used to test a set of 130 unrelated CMT1A patients and were compared with nonduplicated controls. The study was then extended to a larger population of patients. Alleles of interest were sequenced. A manual protocol using polyacrylamide electrophoresis and silver staining and an automated capillary electrophoresis protocol to separate fluorescently labeled alleles were validated.

Results: We identified three new STRs covering 0.55 Mb in the center of the CMT1A duplication. One marker, 4A, is located inside the PMP22 gene. The two others, 9A and 9B, more telomerically positioned, have the highest observed heterozygosity reported to date for CMT1A markers: 0.80 for 9A, and 0.79 for 9B. Tetra- and pentanucleotide repeats offered clear amplification, accurate sizing, and easy quantification of intensities.

Conclusions: Combined use of the three STRs allows robust diagnosis with almost complete informativeness. In our routine diagnosis for CMT1A, they have replaced the use of other polymorphic markers, either in a manual adaptation or combined with fluorescence labeling and allele sizing on a DNA sequencer.
Evaluation of the Performance and Clinical Impact of a Rapid Intraoperative Parathyroid Hormone Assay in Conjunction with Preoperative Imaging and Concise Parathyroidectomy
Authors: Johnson Lawrence R , Doherty Gerard , Lairmore Terry , Moley Jeffrey F , Brunt L Michael , Koenig John , Scott Mitchell G
Published: January 2020

Background: 99mTc-sestamibi scans and rapid, intraoperative intact parathyroid hormone (PTH) assays allow preoperative identification of diseased glands and intraoperative confirmation of diseased gland removal, respectively. Use of these two new technologies may facilitate simpler, more concise surgery, shorter hospital stays, and decreased costs for frozen-section analysis. One major drawback to this new strategy has been the high cost of rapid point-of-care PTH assays.

Methods: We performed rapid PTH assays with the DPC Turbo PTH assay on the DPC IMMULITE automated analyzer. The number of intraoperative frozen sections, type of anesthesia, surgical approach, length of hospital stay, and pre- and postoperative calcium values were compared between a group of 49 patients undergoing parathyroidectomy where the intraoperative PTH assay was used in conjunction with preoperative imaging, and a historical control group of 55 patients before the use of these two technologies in our institution.

Results: Comparison of the Turbo PTH assay to the standard IMMULITE PTH assay gave the following: y = 1.08x − 4.36 (r = 0.97; n = 48). For the 49 patients, the median turnaround time for each intraoperative PTH determination was 19 min (range, 14–40 min). The median decrease in PTH values from baseline was 88% (range, 33–99%). Thirty-seven patients required two PTH determinations, 7 required three, 4 had four, and 1 required five determinations. The average laboratory cost for the rapid intraoperative PTH assays was <$100 per patient (range, $55 to $113). Compared with the control group, the experimental group had significantly fewer frozen sections (1.4 vs 2.5; P <0.0001), shorter hospital stays (17 discharged on the day of surgery vs none discharged on the day of surgery; P <0.0001), greater use of local anesthesia (33% vs 0%; P <0.001), and more unilateral, rather than bilateral neck explorations (65% vs 0%; P <0.001).

Conclusions: The combination of intraoperative Turbo PTH assay and preoperative 99mTc-sestamibi scans can lead to significant decreases in laboratory and surgical pathology costs, hospital stays, and exposure to general anesthesia by facilitating concise parathyroidectomy surgery.