bluuebunny/crossref_metadata_2025 huggingface dataset:
Ribonuclease-resistant RNA Controls (Armored RNA) for Reverse Transcription-PCR, Branched DNA, and Genotyping Assays for Hepatitis C Virus
Authors: WalkerPeach Cindy R , Winkler Matthew , DuBois Dwight B , Pasloske Brittan L
Published: January 2020

Background: Comparison and evaluation of molecular diagnostic assays for the detection and quantification of hepatitis C virus (HCV) RNA have been limited by the lack of RNA controls and calibrators. Armored RNA® technology is a means for producing RNA that is completely protected from plasma ribonucleases. This method produces recombinant pseudoviral particles that are noninfectious and contain predefined RNA sequences.

Methods: A consensus 412-base sequence from the 5′NCR/Core region of HCV subtype 2b was derived from 34 individually sequenced HCV genotype 2b variants. A DNA fragment encoding the consensus HCV-2b sequence was synthesized de novo, cloned, and expressed as an Armored RNA control. The resulting HCV-2b Armored RNA (AR-HCV-2b) contained the complete HCV-2b consensus RNA sequence encapsidated within a protective protein coat.

Results: AR-HCV-2b was fully recoverable from human plasma incubated at 4 °C for >300 days. The particles were tested in three clinical assay formats: AmplicorTM HCV Monitor 1.0, QuantiplexTM HCV RNA 2.0, and INNO-LiPATM HCV II. When added into seronegative, nonviremic plasma, AR-HCV-2b showed reproducible signals and linear dilutions in both the Amplicor and Quantiplex assays. AR-HCV-2b was correctly identified as subtype 2b in the INNO-LiPA line probe assay.

Conclusion: The HCV-2b Armored RNA control is a versatile, durable, ribonuclease-resistant viral RNA control that is compatible in three different clinical assay formats.
Application of a Thermodynamic Nearest-Neighbor Model to Estimate Nucleic Acid Stability and Optimize Probe Design: Prediction of Melting Points of Multiple Mutations of Apolipoprotein B-3500 and Factor V with a Hybridization Probe Genotyping Assay on the LightCycler
Authors: von Ahsen Nicolas , Oellerich Michael , Armstrong Victor William , Schütz Ekkehard
Published: January 2020

Background: PCR-based mutation detection is prone to methodological errors, e.g., in restriction length fragment polymorphism (RFLP) and allele-specific amplification (ASA), false PCR results may occur because of technical faults or atypical new mutations.

Methods: We investigated the ability of a genotyping assay based on hybridization of labeled oligonucleotides to detect and discriminate known and as yet unknown mutations in the factor V and apolipoprotein B-100 genes. Expected melting points were calculated using a nearest-neighbor model for nucleic acid duplex stability and compared with experimental findings derived from LightCycler melting curves. A method for genotyping the apolipoprotein B-100 G10699A and C10698T mutations is presented.

Results: All mismatches tested for in the probed sequence could be detected with a single probe. The measured melting points were in good agreement with their values predicted using the nearest-neighbor model (r = 0.96; y = 0.98x + 1.18; Sy|x= 0.96; n = 24).

Conclusions: This procedure not only allows the identification of the mutation of interest but also enables the discrimination from other potential mutations in the vicinity of the former. The nearest-neighbor model is valid for hybridization probe assays on the LightCycler and should be of general value in setting up such assays. We have shown for two clinically relevant genotyping examples that the LightCycler mutation detection system has superior discriminatory performance compared with conventional RFLP or ASA PCR-based methods for molecular diagnostic purposes. With this method, in every hybridization probe assay, all mutations under a properly designed probe should be detectable, but they will not necessarily be discriminated from each other in all cases.
Hyperglycosylated Human Chorionic Gonadotropin (Invasive Trophoblast Antigen) Immunoassay: A New Basis for Gestational Down Syndrome Screening
Authors: Cole Laurence A , Shahabi Shohreh , Oz Utku A , Bahado-Singh Ray O , Mahoney Maurice J
Published: January 2020

Background: Serum human chorionic gonadotropin (hCG) and hCG free β-subunit tests are used in combination with unconjugated estriol and α-fetoprotein in the triple screen test, and with the addition of inhibin-A in the quadruple marker test for detecting Down syndrome in the second trimester of pregnancy. These tests have a limited detection rate for Down syndrome: ∼40% for hCG or free β-subunit alone, ∼60% for the triple screen test, and ∼70% for the quadruple marker test, all at 5%, or a relatively high, false-positive rate. New tests are needed with higher detection and lower false rates. Hyperglycosylated hCG (also known as invasive trophoblast antigen or ITA) is a new test. It specifically detects a unique oligosaccharide variant of hCG associated with Down syndrome pregnancies. We evaluated this new Down syndrome-directed test in prenatal diagnosis.

Methods: Hyperglycosylated hCG was measured in urine samples from women undergoing amniocentesis for advanced maternal age concerns at 14–22 weeks of gestation, 1448 with normal karyotype and 39 with Down syndrome fetuses.

Results: The median hyperglycosylated hCG value was 9.5-fold higher in Down syndrome cases (9.5 multiples of the normal karyotype median). The single test detected 80% of Down syndrome cases at a 5% false-positive rate. Urine hyperglycosylated hCG was combined with urine β-core fragment (urine breakdown product of serum hCG free β-subunit), serum α-fetoprotein, and maternal age-related risk. This urine-serum combination detected 96% of Down syndrome cases at a 5% false-positive rate, 94% of cases at a 3% false-positive rate, and 71% of cases at a 1% false-positive rate. These detection rates exceed those of any previously reported combination of biochemical markers.

Conclusions: Hyperglycosylated hCG is a new base marker for Down syndrome screening in the second trimester of pregnancy. The measurement of hyperglycosylated hCG can fundamentally improve the performance of Down syndrome screening protocols.
Analysis of Nicotine, 3-Hydroxycotinine, Cotinine, and Caffeine in Urine of Passive Smokers by HPLC-Tandem Mass Spectrometry
Authors: Tuomi Tapani , Johnsson Tom , Reijula Kari
Published: January 2020

Background: A method is described for the simultaneous analysis of nicotine and two of its major metabolites, cotinine and 3-hydroxycotinine, as well as for caffeine from urine samples. The method was developed to assess exposure of restaurant and hotel workers to environmental tobacco smoke.

Methods: The method includes sample pretreatment and reversed-phase HPLC separation with tandem mass spectrometric identification and quantification using electrospray ionization on a quadrupole ion trap mass analyzer. Sample pretreatment followed standard protocols, including addition of base before liquid-liquid partitioning against dichloromethane on a solid matrix, evaporation of the organic solvent using gaseous nitrogen, and transferring to HPLC vials using HPLC buffer. HPLC separation was run on-line with the electrospray ionization-tandem mass spectrometric detection.

Results: The detection limits of the procedure were in the 1 μg/L range, except for nicotine (10 μg/L of urine). Still lower detection limits can be achieved with larger sample volumes. Recoveries of the sample treatment varied from 99% (cotinine) to 78% (3-hydroxycotinine).

Conclusions: The method described is straightforward and not labor-intensive and, therefore, permits a high throughput of samples with excellent prospects for automation. The applicability of the method was demonstrated in a small-scale study on restaurant employees.
Increased Serum Transferrin Saturation Is Associated with Lower Serum Transferrin Receptor Concentration
Authors: Looker Anne C , Loyevsky Mark , Gordeuk Victor R
Published: January 2020

Background: Serum transferrin receptor (sTfR) concentrations are increased in iron deficiency. We wished to examine whether they are decreased in the presence of potential iron-loading conditions, as reflected by increased transferrin saturation (TS) on a single occasion.

Methods: We compared sTfR concentrations between 570 controls with normal iron status and 189 cases with increased serum TS on a single occasion; these latter individuals may be potential cases of iron overload. Cases and controls were selected from adults who had been examined in the third National Health and Nutrition Examination Survey (1988–1994) and for whom excess sera were available to perform sTfR measurements after the survey’s completion. Increased TS was defined as >60% for men and >55% for women; normal iron status was defined as having no evidence of iron deficiency, iron overload, or inflammation indicated by serum ferritin, TS, erythrocyte protoporphyrin, and C-reactive protein.

Results: Mean sTfR and mean log sTfR:ferritin were ∼10% and 24% lower, respectively, in cases than in controls (P <0.002). Cases were significantly more likely to have an sTfR value <2.9 mg/L, the lower limit of the reference interval, than were controls (odds ratio = 1.8; 95% confidence interval, 1.04–2.37).

Conclusion: Our results support previous studies that suggested that sTfR may be useful for assessing high iron status in populations.
Comparison of Pituitary and Recombinant Human Thyroid-stimulating Hormone (rhTSH) in a Multicenter Collaborative Study: Establishment of the First World Health Organization Reference Reagent for rhTSH
Authors: Rafferty Brian , Gaines Das Rose
Published: January 2020

Background: The increasing use of recombinant-DNA-derived materials in therapy and diagnosis poses a new challenge for biological standardization, that of developing reference preparations appropriate for both the native and recombinant products. Here we report the results of an international collaborative study that was carried out under the auspices of WHO to assess the suitability of a preparation of recombinant thyroid-stimulating hormone (rTSH; 94/674) to serve as a potential standard for the calibration of diagnostic immunoassays compared with the International Reference Preparation (IRP) for human TSH (80/558).

Methods: Coded samples were provided to the 33 laboratories in the study, and participants were asked to perform TSH assays currently in use in their laboratories. Twenty-eight laboratories contributed 93 immunoassays in 41 different method-laboratory combinations, and an additional 5 laboratories contributed bioassay data. All data were analyzed centrally at the National Institute for Biological Standards and Control.

Results: The results obtained in different laboratories and with different assay systems revealed significant variability between estimates of rTSH relative to the IRP. These ranged from 5.51 mIU (95% limits, 3.95–7.67 mIU) per ampoule by RIA to 7.15 mIU (95% limits, 6.7–7.63 mIU) per ampoule by immunofluorometric assay. However, the results showed that the assignment of a value of 6.70 mIU per ampoule of 94/674 would give reasonable continuity with the IRP in many assay systems.

Conclusions: The preparation was established as the First WHO Reference Reagent for TSH, human, recombinant, to provide a means of validating assay performance and to maintain continuity with the IRP without compromising clinical data.
Quantitative Abnormalities of Fetal DNA in Maternal Serum in Preeclampsia
Authors: Lo YM Dennis , Leung Tse N , Tein Mark SC , Sargent Ian L , Zhang Jun , Lau Tze K , Haines Christopher J , Redman Christopher WG
Published: January 2020

Background: There is much recent interest in the biologic and diagnostic implication of cell-free non-host DNA in the plasma and serum of human subjects. To determine if quantitative abnormalities of circulating non-host DNA may be associated with certain pathologic processes, we used circulating fetal DNA in preeclampsia as a model system. Methods: We studied 20 preeclamptic women and 20 control subjects of comparable gestational age (means, 32 and 33 weeks, respectively). Male fetal DNA in maternal serum was measured using real-time quantitative PCR for the SRY gene on the Y chromosome. Results: The imprecision (CV) of the assay was 2.7%. The median circulating fetal DNA was increased fivefold in 20 preeclamptic women compared with 20 control pregnant women (381 vs 76 genome-equivalents/mL, P <0.001). Conclusions: These observations suggest that preeclampsia is associated with disturbances in the liberation and/or clearance mechanisms of circulating DNA. These results also raise the possibility that measurement of circulating DNA may prove useful as a marker for the diagnosis and/or monitoring of preeclampsia.
Variability of Glutathione S-Transferase α in Human Liver and Plasma
Authors: Mulder Theo PJ , Court Daniel A , Peters Wilbert HM
Published: January 2020

Background: Glutathione S-transferases are a family of enzymes involved in the binding, transport, and detoxification of a wide variety of endogenous and exogenous compounds. Little information is available about the variability of class α glutathione S-transferases in human liver, where they are highly expressed, or in serum.

Methods: Both total class α glutathione S-transferase (GST-α, composed of GSTA1-1, GSTA1-2, and GSTA2-2) as well as GSTA1-1 concentrations were measured by specific and sensitive ELISA in liver cytosols of 35 organ donors and in plasma samples of 350 healthy controls.

Results: The mean total GST-α and GSTA1-1 in liver cytosols were 25.1 ± 9.4 and 10.7 ± 5.3 μg/mg protein, respectively, and did not correlate with activities of aspartate aminotransferase or alanine aminotransferase. The mean total GST-α in liver was significantly higher in females compared with males (28.8 ± 10.0 vs 22.0 ± 7.8 μg/mg protein; P <0.05). In contrast, the median total GST-α in plasma was lower in females compared with males (2.0 and 2.8 μg/L, respectively; P <0.0001). The median ratios for GSTA1-1/total GST-α in liver and plasma were 0.42 and 0.58, respectively.

Conclusions: GSTA1-1 constitutes approximately one-half of the total amount of α class GSTs in human plasma and liver. Total GST-α values are higher in female liver but lower in plasma compared with the respective values in males.
Performance and Specificity of Monoclonal Immunoassays for Cyclosporine Monitoring: How Specific Is Specific?
Authors: Steimer Werner
Published: January 2020

Background: Immunoassays designed for the selective measurement of cyclosporin A (CsA) inadvertently show cross-reactivity for CsA metabolites. The extent and clinical significance of the resulting overestimation is controversial. A comprehensive assessment of old and new methods in clinical specimens is needed.

Methods: In a comprehensive evaluation, CsA was analyzed in 145 samples with the new CEDIA® assay and compared with the Emit® assay with the old and new pretreatments, the TDx® monoclonal and polyclonal assays, the AxSYM®, and HPLC. All samples were from patients with liver and/or kidney transplants.

Results: The CEDIA offered the easiest handling, followed by the AxSYM, which showed the longest calibration stability. The TDx monoclonal assay provided the lowest detection limit and the lowest CVs. The mean differences compared with HPLC were as follows: Emit, 9–12%; CEDIA, 18%; AxSYM, 29%; and TDx monoclonal, 57%. The CycloTrac® RIA paralleled the Emit results. In contrast to the mean differences, substantial (>200%) and variable overestimations of the CsA concentration were observed in individual patient samples. Metabolic ratios, estimates of the overall concentrations of several cross-reacting metabolites (nonspecific TDx polyclonal/specific reference method), correlated with the apparent biases of the various monoclonal assays. Metabolic ratios varied up to 10-fold, which translated into biases for individual samples between −7% and +174%. The higher the cross-reactivity of an assay was, the higher was the range of biases observed. The interindividual differences markedly exceeded other factors of influence (organ transplanted, hepatic function).

Conclusion: Because assay bias cannot be predicted in individual samples, substantially erratic CsA dosing can result. The specificity of CsA assays for parent CsA remains a major concern.
TE671 Cell-based ELISA for Anti-Acetylcholine Receptor Antibody Determination in Myasthenia Gravis
Authors: Franciotta Diego , Martino Gianvito , Brambilla Elena , Zardini Elisabetta , Locatelli Vera , Bergami Alessandra , Tinelli Carmine , Desina Gaetano , Cosi Vittorio
Published: January 2020

Background: Acetylcholine receptor (AChR) from human muscles is the antigen used currently in radioimmunoprecipitation assays (RIPAs) for the determination of anti-AChR antibodies in the diagnosis of myasthenia gravis (MG). Our aim was to develop and validate an ELISA using TE671 cells as the source of AChR.

Methods: After TE671 cell homogenization, the crude AChR extract was used for plate coating. Anti-AChR antibodies were determined in 207 MG patients and in 77 controls.

Results: The mean intra- and interassay CVs (for two samples with different anti-AChR antibody concentrations) were 9.7% and 15.7%, respectively. Test sensitivity and specificity, for generalized MG, were 79.5% (95% confidence interval, 72.8–85.0%) and 96.1% (89.0–99.1%). The detection limit was 2 nmol/L. Anti-AChR antibody concentrations from 53 MG patients, as tested with our ELISA, showed good agreement with an RIPA with a mean difference (SD) of 1.0 (5.6) nmol/L.

Conclusion: Our ELISA is a simple screening test for the diagnosis of MG and enables rapid and inexpensive patient follow-up.
Major Interference from Leukocytes in Reverse Transcription-PCR Identified as Neurotoxin Ribonuclease from Eosinophils: Detection of Residual Chronic Myelogenous Leukemia from Cell Lysates by Use of an Eosinophil-depleted Cell Preparation
Authors: Hämäläinen Mauri M , Eskola Jarkko U , Hellman Jukka , Pulkki Kari
Published: January 2020

Background: The extraction of RNA from leukocytes for reverse transcription-PCR (RT-PCR) is time-consuming and contributes to variation in analysis of the Philadelphia (Ph1) chromosome of chronic myelogenous leukemia (CML) by RT-PCR. To detect residual CML after bone marrow transplantation, mRNA from at least 105 leukocytes should be analyzed, but the RNase activity of the cells precludes simple leukocytes lysis as an alternative to RNA extraction. We sought to identify the main source of RNase activity of leukocytes.

Methods: We used a three-step chromatographic process and amino acid sequence analysis. We selected eosinophil-free granulocytes by using a biotinylated CD16 antibody and selected mononuclear cells by fractionating the leukocytes with a Ficoll-Paque® density gradient.

Results: Chromatography and amino acid sequencing identified eosinophil-derived neurotoxin (EDN) as the main source of leukocyte RNase. Depletion of eosinophils reduced the EDN content of cell lysates by ∼90%, allowing a signal from a lysate of 50 K562 Ph1-positive cells mixed with 105 CD16+ granulocytes that was equivalent to 77% of the signal in the absence of leukocytes. A similar lysate with mononuclear cells gave a signal equivalent to 53% of that without mononuclear cells. RNA extraction gave a signal equivalent to only 24% of the leukocyte-free control.

Conclusion: The depletion of eosinophils during the preparation of leukocyte samples for RT-PCR efficiently reduces the risk of mRNA degradation by ribonucleases, enabling RT-PCR analysis directly from cell lysates with a better signal than can be obtained by RNA extraction.
Epitope Analysis of a Prostate-specific Antigen (PSA) C-Terminal-specific Monoclonal Antibody and New Aspects for the Discrepancy between Equimolar and Skewed PSA Assays
Authors: Nagasaki Hiroshi , Watanabe Motoyuki , Komatsu Naoki , Kaneko Takashi , Dubé Jean Y , Kajita Tadahiro , Saitoh Yoshihiro , Ohta Yohsuke
Published: January 2020

Background: Immunoassays to measure prostate-specific antigen (PSA) often give different values for the same patient samples, and the calibrators among commercial immunoassays are not interchangeable. We developed three novel assays to quantify the free and complexed forms of PSA in serum.

Methods: We synthesized 46 peptides, which encompassed the entire PSA molecule, and determined the interactions between selected monoclonal antibodies (MAbs) and those peptides or the intact PSA molecule.

Results: MAb PA313 did not cross-react with human glandular kallikrein (hK2), which has 78% amino acid homology to PSA. This MAb bound with KD = 40 nmol/L to the C-terminal peptide of PSA and distinguished between a synthetic peptide derived from PSA (PSA46A: NH2-C-R226KWIKDTIVANP237-COOH) that differed from one derived from hK2 (PSA46B: NH2-C-R226KWIKDTAANP237-COOH) by a single amino acid. Only the MAb combination of PA313/PA121 showed equimolar reactivity with PSA and with PSA complexed with α1-antichymotrypsin (PSA-ACT). The free form of PSA (F-PSA) was determined by MAbs PA313/FPA503, and the amount of complexed PSA (C-PSA) in PSA-ACT was determined by αACT/PA313. The total PSA (T-PSA) measured by either of the equimolar assays (PA313/PA121 or Tandem-R) was consistent with the sum of F-PSA and C-PSA. In contrast, T-PSA by a skewed assay (IMx) was higher than F-PSA + C-PSA when the ratio of F-PSA to T-PSA (F/T) was >0.15. T-PSA measured by IMx was nearly equal to F-PSA/0.55 + C-PSA. The coefficient 0.55 reflected different reactivities of the IMx assay with PSA-ACT and PSA.

Conclusion: The discrepancy between the values measured by equimolar and skewed assays depends on the ratio of free to total PSA in the sample.
Apolipoprotein E in Cerebrospinal Fluid: Relation to Phenotype and Plasma Apolipoprotein E Concentrations
Authors: Yamauchi Kazuyoshi , Tozuka Minoru , Nakabayashi Tetsuo , Sugano Mitsutoshi , Hidaka Hiroya , Kondo Yoshiyuki , Katsuyama Tsutomu
Published: January 2020

Background: Apolipoprotein (apo) E may be related to the development of Alzheimer disease, but data on apoE in cerebrospinal fluid (CSF) are limited. The aim of the present study was to measure apoE in CSF and relate its concentrations to apoE phenotype and CSF lipids.

Methods: We adapted an assay for CSF apoE sensitivity using an ELISA. It allowed us to measure CSF apoE with sufficient reproducibility and precision.

Results: The within- and between-run CVs were <7%, and the detection limit was 0.025 mg/L. No cross-reaction was found for other apolipoproteins. No significant differences related to sex or apoE phenotype were observed in the CSF apoE concentration. The mean CSF apoE concentration was significantly higher in the 0–5 year group (n = 6; 18.47 ± 1.14 mg/L, mean ± SD) than in the >5 year group (n = 34; 8.82 ± 3.31 mg/L). The mean concentrations of total cholesterol (TC) and phospholipid (PL) in CSF were 2.68 ± 2.16 and 6.50 ± 2.84 mg/L (n = 52), respectively. Although no significant differences in TC or PL in the CSF were found with respect to sex or age, the concentrations in subjects with the apoE phenotype E4/E3 were significantly lower than in those with E3/E3 and E3/E2. The concentrations of apoE, TC, and PL in CSF did not correlate with those in plasma. The time-related fluctuations in CSF apoE were independent of those in total protein and IgG. CSF apoE was significantly correlated with TC and PL concentrations in the CSF, but not with the number of cells in the CSF.

Conclusions: These findings support the idea that apoE and lipids are unable to cross the blood-brain barrier and that their concentrations in CSF may reflect production in central nervous tissue.
A Practical Approach to Determine Cutoff Concentrations for Opiate Testing with Simultaneous Detection of Codeine, Morphine, and 6-Acetylmorphine in Urine
Authors: Paul Buddha D , Shimomura Eric T , Smith Michael L
Published: January 2020

Background: Both the Department of Defense (DoD) and the Department of Health and Human Services (DHHS) currently require two confirmation tests to verify use of heroin, one test for total morphine and a separate test for 6-acetylmorphine (6-AM). Our aim was to determine appropriate free-codeine, free-morphine, and 6-AM cutoff concentrations that could be substituted for total-morphine, total-codeine, and 6-AM cutoff concentrations and to develop a less labor-intensive method for measuring codeine, morphine, and 6-AM.

Methods: Urine samples containing opiates were extracted, derivatized, and analyzed using gas chromatography–mass spectrometry with selective ion monitoring.

Results: The limits of detection for codeine, morphine, and 6-AM were 6, 5, and 0.5 μg/L, respectively. Recoveries were >90%. Quantification was linear over the concentration range of 6–1000 μg/L for codeine, 5–5000 μg/L for morphine, and 0.5–800 μg/L for 6-AM. Cutoff concentrations for confirmation of opiates were 100, 100, and 10 μg/L for free codeine, free morphine, and 6-AM.

Conclusion: The proposed cutoff concentrations for free morphine and 6-AM provide better detection windows for morphine and heroin use than the cutoff concentrations for total morphine and 6-AM used at present. Detection of free codeine, instead of total codeine, simplifies interpretation of codeine use. The single-extraction method enables simultaneous, less labor-intensive analysis of morphine, codeine, and 6-AM.
Library of Sequence-specific Radioimmunoassays for Human Chromogranin A
Authors: Børglum Jensen Tine , Hilsted Linda , Rehfeld Jens F
Published: January 2020

Background: Human chromogranin A (CgA) is an acidic protein widely expressed in neuroendocrine tissue and tumors. The extensive tissue- and tumor-specific cleavages of CgA at basic cleavage sites produce multiple peptides.

Methods: We have developed a library of RIAs specific for different epitopes, including the NH2 and COOH termini and three sequences adjacent to dibasic sites in the remaining part of CgA.

Results: The antisera raised against CgA(210–222) and CgA(340–348) required a free NH2 terminus for binding. All antisera displayed high titers, high indexes of heterogeneity (∼1.0), and high binding affinities (Keff0 ∼ 0.1 × 1012 to 1.0 × 1012 L/mol), implying that the RIAs were monospecific and sensitive. The concentration of CgA in different tissues varied with the assay used. Hence, in a carcinoid tumor the concentration varied from 0.5 to 34.0 nmol/g tissue depending on the specificity of the CgA assay. The lowest concentration in all tumors was measured with the assay specific for the NH2 terminus of CgA. This is consistent with the relatively low concentrations measured in plasma from carcinoid tumor patients by the N-terminal assay, whereas the assays using antisera raised against CgA(210–222) and CgA(340–348) measured increased concentrations.

Conclusion: Only some CgA assays appear useful for diagnosis of neuroendocrine tumors, but the entire library is valuable for studies of the expression and processing of human CgA.
Affinity Chromatography: A Review of Clinical Applications
Authors: Hage David S
Published: January 2020

Affinity chromatography is a type of liquid chromatography that makes use of biological-like interactions for the separation and specific analysis of sample components. This review describes the basic principles of affinity chromatography and examines its use in the testing of clinical samples, with an emphasis on HPLC-based methods. Some traditional applications of this approach include the use of boronate, lectin, protein A or protein G, and immunoaffinity supports for the direct quantification of solutes. Newer techniques that use antibody-based columns for on- or off-line sample extraction are examined in detail, as are methods that use affinity chromatography in combination with other analytical methods, such as reversed-phase liquid chromatography, gas chromatography, and capillary electrophoresis. Indirect analyte detection methods are also described in which immunoaffinity chromatography is used to perform flow-based immunoassays. Other applications that are reviewed include affinity-based chiral separations and the use of affinity chromatography for the study of drug or hormone interactions with binding proteins. Some areas of possible future developments are then considered, such as tandem affinity methods and the use of synthetic dyes, immobilized metal ions, molecular imprints, or aptamers as affinity ligands for clinical analytes.
When Is a Heterophile Antibody Not a Heterophile Antibody? When It Is an Antibody against a Specific Immunogen
Authors: Kaplan Irina V , Levinson Stanley S
Published: January 2020

Heterophile antibodies are antibodies produced against poorly defined antigens. These are generally weak antibodies with multispecific activities. Human anti-animal antibodies that develop as a result of treatments with animal immunoglobulins are antibodies with strong avidities, produced against well-defined antigens. Although heterophile antibodies and human anti-animal antibodies interfere with immunological assays by similar mechanisms, modes for identifying the sources of the antibodies and for circumventing or retarding the interference may differ. Unfortunately, there has not been a well-organized attempt to encourage correct definition of these antibodies. This problem of inexact definition is highlighted by recent articles in this Journal. In the present discussion, we examine the history leading to this problem and discuss the origins and the reasons that the nature of the antibody is important for rectifying the problem. We propose a simple nomenclature for general usage that should appropriately characterize these antibodies in most cases.
Single-Nucleotide Polymorphisms in Intron 2 of CYP21P: Evidence for a Higher Rate of Mutation at CpG Dinucleotides in the Functional Steroid 21-Hydroxylase Gene and Application to Segregation Analysis in Congenital Adrenal Hyperplasia
Authors: Jiddou Renée R , Wei Wan-Li , Sane Kumud S , Killeen Anthony A
Published: January 2020

Background: Intron 2 of CYP21, the functional steroid 21-hydroxylase gene contains several single-nucleotide polymorphisms (SNPs). We tested the hypothesis that intron 2 of the pseudogene, CYP21P, might also be polymorphic and provide markers for segregation analysis of this region of the genome, including observable markers for segregation analysis of CYP21 gene deletions. A comparison of SNPs in both genes might provide insights into the rates of mutation in these duplicated genes.

Methods: After amplification with PCR, we examined restriction site polymorphisms in intron 2 of CYP21P in 24 members of the parental generation of the Centre d’Étude du Polymorphisme Humain families and selected offspring.

Results: Intron 2 of CYP21P contains frequent SNPs around nucleotide 398 and nucleotide 509, which can be typed by PCR/restriction enzyme digestion with HaeIII. Of the 48 CYP21P alleles examined, 44 could be characterized unambiguously. Of these 44 alleles, 4 were deleted, and the frequencies of restriction at the polymorphic HaeIII sites were 20 of 40 at nucleotide 398 and 30 of 40 at nucleotide 509. Both polymorphisms result from C→T transitions that occur at CpG dinucleotides. The frequencies of C at these nucleotides in CYP21P are significantly higher than at the corresponding nucleotides in CYP21 of the same individuals (P <0.01).

Conclusion: These data suggest that these CpG dinucleotides are more frequently mutated in CYP21 than in CYP21P, and that several mutations at CpG dinucleotides in the coding regions of CYP21 might result from CpG instability rather than the more usually proposed mechanism of gene conversion. These frequent SNPs provide useful markers for studying both allelic segregation of CYP21, particularly for chromosomes with known CYP21 deletions, and for investigating the origin of these polymorphisms.
Anti-Free Prostate-specific Antigen Monoclonal Antibody Epitopes Defined by Mimotopes and Molecular Modeling
Authors: Michel Sandrine , Deléage Gilbert , Charrier Jean-Philippe , Passagot Jacques , Battail-Poirot Nicole , Sibai Geneviève , Jolivet Michel , Jolivet-Reynaud Colette
Published: January 2020

Background: Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer, and the free PSA/total PSA ratio has been shown to be efficient for distinguishing prostate cancer from benign prostatic hyperplasia. We report here the characterization of seven mouse monoclonal antibodies (mAbs) and the partial localization of two conformational epitopes identified by anti-free PSA mAbs.

Methods: The mAbs were studied by competition and sandwich assays, and the epitope localization of the two anti-free PSA mAbs (6C8D8 and 5D3D11) was performed using phage displayed peptide libraries and molecular modeling.

Results: The seven mAbs were classified into three groups according to their recognition specificities and their ability to inhibit the enzymatic activity of PSA and the formation of PSA-α1-antichymotrypsin (ACT) complex. Among the anti-free PSA mAb group, 6C8D8 recognized the phage displayed peptide RKLRPHWLHFHPVAV, two parts of which presented similarities with two regions distant on the PSA sequence but joined in the tridimensional structure. mAb 5D3D11 recognized the peptide DTPYPWGWLLDEGYD, which is similar to a PSA region located on the board of the groove containing the PSA enzymatic site. Both epitopes were located in the theoretical ACT binding site described previously. Moreover, these mAbs were able to inhibit the enzymatic activity of PSA.

Conclusions: These epitope localizations are in agreement with the ability of both mAbs to inhibit enzymatic activity and ACT fixation. The results presented here could bring information for the generation of clinically relevant PSA assays.
Combined Inhibin and CA125 Assays in the Detection of Ovarian Cancer
Authors: Robertson David M , Cahir Nicholas , Burger Henry G , Mamers Pamela , McCloud Philip I , Pettersson Kim , McGuckin Michael
Published: January 2020

Background: The reproductive hormone inhibin has been used as a diagnostic marker of ovarian mucinous and granulosa cell cancers. The aims of this study were to develop a new inhibin immunofluorometric assay (αC IFMA) to replace an inhibin RIA as a diagnostic marker of these ovarian cancers and to assess whether the αC IFMA in combination with CA125, which detects serous cancers, leads to an improved biochemical diagnosis of all ovarian cancers.

Methods: Serum inhibin concentrations were determined in healthy postmenopausal women (n = 165) and women with ovarian cancers (n = 154), using an inhibin RIA and an αC IFMA, which detects inhibin forms containing the αC subunit as well as the free αC subunit.

Results: The αC IFMA gave a similar or better discrimination of mucinous (90% vs 71%) and granulosa cell (100% vs 100%) cancers compared with the inhibin RIA. Combination of CA125 and αC IFMA values by canonical variate analysis or by multiROC analysis showed that the percentage of all ovarian cancers detected was significantly increased compared with either CA125 or αC IFMA alone.

Conclusions: The αC IFMA shows a similar or better specificity compared with the RIA, but with increased sensitivity. In combination with CA125, the αC IFMA provides an effective dual test for the detection of the majority (90%) of ovarian cancers.
Detection of Human Serum Tumor Necrosis Factor-α in Healthy Donors, Using a Highly Sensitive Immuno-PCR Assay
Authors: Saito Kaori , Kobayashi Daisuke , Sasaki Masateru , Araake Hiroshi , Kida Takashi , Yagihashi Atsuhito , Yajima Tomomi , Kameshima Hidekazu , Watanabe Naoki
Published: January 2020

Background: Tumor necrosis factor-α (TNFα) is an important mediator of inflammatory and autoimmune diseases. Analysis of its pathophysiologic roles has been difficult because low concentrations of TNFα, including those in healthy controls, cannot be measured by existing methods.

Methods: We developed a sensitive immuno-PCR assay for the detection of TNFα in human serum. The DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to the biotinylated third antibody. TNFα sandwiched by antibodies was detected by amplification of the DNA label using PCR.

Results: The limit of detection of the assay was 0.001 ng/L, an ∼5 × 104-fold improvement compared with a conventional ELISA. The mean serum TNFα concentration (± SD) in healthy donors was 0.021 ± 0.044 ng/L in men (n = 29) and 0.033 ± 0.065 ng/L in women (n = 25).

Conclusion: This method may be useful for analyzing the significance of TNFα concentration in various diseases.
Serum Galactosyl Hydroxylysine as a Biochemical Marker of Bone Resorption
Authors: Al-Dehaimi Abdul Wahed , Blumsohn Aubrey , Eastell Richard
Published: January 2020

Background: Serum-based biochemical markers of bone resorption may provide better clinical information than urinary markers because direct comparison with serum markers of bone formation is possible and because the within-subject variability of serum markers may be lower. We describe a method for the measurement of free β-1-galactosyl-O-hydroxylysine (Gal-Hyl) in serum.

Methods: The assay used preliminary ultrafiltration of serum, dansylation, and separation by reversed-phase HPLC with fluorescence detection. Healthy subjects were recruited from population-based studies of bone turnover.

Results: The within-run (n = 15) and between-run (n = 15) CVs were 7% and 14%, respectively, at a mean value of 48 nmol/L. In women and pubertal girls, serum free Gal-Hyl correlated with urine free Gal-Hyl (r = 0.84; P <0.001). Serum Gal-Hyl was higher during puberty and increased after menopause. The fractional renal clearance of free Gal-Hyl relative to that of creatinine was 0.90 (95% confidence interval, 0.82–0.98). Serum free Gal-Hyl decreased by 36% (SE = 4%) in 14 patients with mild Paget disease treated with an oral bisphosphonate, and this decrease was significantly (P <0.001) greater than that seen for either serum tartrate-resistant acid phosphatase (9%; SE = 4%) or serum C-terminal telopeptide of collagen I (19%; SE = 8%).

Conclusion: Serum free Gal-Hyl may be useful as a serum marker of bone resorption.
The Role of Food and Drug Administration Regulation of In Vitro Diagnostic Devices—Applications to Genetics Testing
Authors: Gutman Steven
Published: January 2020

The Food and Drug Administration (FDA) has been involved in the regulation of in vitro diagnostic devices (IVDs or laboratory tests) since the introduction of the Medical Device Amendments of 1976. IVDs developed as kits or systems intended for use in multiple laboratories require review by the FDA before being marketed to ensure appropriate performance and labeling. IVDs developed as in-house, or so-called “home-brew”, tests or laboratory test services are considered medical devices, but historically have not been subject to premarket review as a matter of enforcement discretion. FDA recently established a new regulatory paradigm for in-house tests based on classification of the active ingredients or building blocks of these tests as analyte-specific reagents (ASRs). ASRs are exempt from premarket review but subject to both manufacturing and labeling controls. Currently, genetic tests are received and reviewed by the FDA in the same manner as other in vitro diagnostic tests. The FDA currently is in the process of chartering a new genetics advisory panel to provide the agency with outside expertise to deal with genetic testing issues. We are also continuing to work with other agencies within the Department of Health and Human Services to determine how we can cooperatively help foster this important new area of testing.
Measurement of the Complex between Prostate-specific Antigen and α1-Protease Inhibitor in Serum
Authors: Zhang Wan-Ming , Finne Patrik , Leinonen Jari , Vesalainen Satu , Nordling Stig , Stenman Ulf-HÅkan
Published: January 2020

Background: Prostate-specific antigen (PSA) occurs in serum both free and in complex with protease inhibitors. The complex with α1-antichymotrypsin (ACT) is the major form in serum, and the proportion of PSA-ACT is higher in prostate cancer (PCa) than in benign prostatic hyperplasia (BPH). PSA also forms a complex with α1-protease inhibitor (API) in vitro, and the PSA-ACT complex has been detected in serum from patients with prostate cancer. The aim of the present study was to develop a quantitative method for the determination of PSA-API and to determine the serum concentrations in patients with PCa and BPH.

Methods: The assay for PSA-API utilizes a monoclonal antibody to PSA as capture and a polyclonal antibody to API labeled with a Eu-chelate as a tracer. For calibrators, PSA-API formed in vitro was used. Serum samples were obtained before treatment from 82 patients with PCa, from 66 patients with BPH, and from 22 healthy females.

Results: The concentrations of PSA-API are proportional to the concentrations of total PSA. PSA-API comprises 1.0–7.9% (median, 2.4%) of total immunoreactive PSA in PCa and 1.3–12.2% (median, 3.6%) in BPH patients with serum PSA concentrations >4 μg/L. In patients with 4–20 μg/L total PSA, the proportion of PSA-API serum is significantly higher in BPH (median, 4.1%) than in PCa (median, 3.2%; P = 0.02).

Conclusions: The proportion of PSA-API in serum is lower in patients with PCa than in those with BPH. These results suggest that PSA-API is a potential adjunct to total and free PSA in the diagnosis of prostate cancer.
Impact of Antibody Specificity and Calibration Material on the Measure of Agreement between Methods for Cardiac Troponin I
Authors: Newman David J , Olabiran Yemi , Bedzyk William D , Chance Suzette , Gorman Eileen G , Price Christopher P
Published: January 2020

Background: Available assays for cardiac troponin I (cTnI) yield numerically different results. The aim of this study was to compare patient values obtained from four cTnI immunoassays.

Methods: We studied the Stratus® II assay, the Opus® II assay, the Access® assay, and a research-only cTnI heterogeneous immunoassay that uses the Dade Behring aca® plus immunoassay system equipped with two new noncommercial monoclonal antibodies. Because the aca plus cTnI assay is for research only, we first evaluated and analytically validated it for serum and citrated plasma. Initially, each method was calibrated using the method-specific calibrator supplied by each manufacturer; however, the aca plus cTnI assay was calibrated using patient serum pools containing cTnI and selected on the basis of increased creatine kinase MB isoenzyme and with values assigned by use of the Stratus cTnI assay. For method comparisons, individual patient sample cTnI values were determined and compared with the Stratus II assay.

Results: Passing and Bablock regression analysis yielded slopes of 1.44 (r = 0.96; n = 72) for the Opus II vs Stratus II assays; 0.07 (r = 0.91; n = 72) for the Access vs Stratus II assays; and 0.90 (r = 0.91, n = 72) for the aca plus vs Stratus II assays. The recalibration of each method with a Stratus II-assigned serum pool improved, but did not entirely eliminate, the slope differences between the different assays (range, 1.00–1.16). The observed scatter in the correlation curves remained.

Conclusion: There is a need to further explore the specificities of these assays with respect to the different circulating forms of cTnI.
Improved Immunoradiometric Assay for Plasma Renin
Authors: Deinum Jaap , Derkx Frans HM , Schalekamp Maarten ADH
Published: January 2020

Background: Our renin IRMA overestimated renin in plasmas with high prorenin-to-renin ratios. We suspected that the overestimation of renin was caused less by cross-reactivity of the renin-specific antibody with prorenin than by a conformational change of prorenin into an enzymatically active form during the assay.

Methods: Because the inactive form of prorenin converts slowly into an active form at low temperature, we raised the assay temperature from 22 °C to 37 °C, simultaneously shortening the incubation time from 24 to 6 h. The former IRMA was performed in <1 working day with these modifications.

Results: The comeasurement of prorenin as renin was eliminated. Reagents were stable at 37 °C, and the new and old IRMAs were comparable in terms of precision and accuracy. The functional lower limit of the assay (4 mU/L) was below the lower reference limit (9 mU/L). The modified IRMA agreed closely with the activities measured with an enzyme-kinetic assay. Results were not influenced by the plasma concentration of angiotensinogen. At normal angiotensinogen concentrations, the IRMA closely correlated with the classical enzyme-kinetic assay of plasma renin activity.

Conclusion: The modified IRMA, performed at 37 °C, avoids interference by prorenin while retaining the desirable analytical characteristics of the older IRMA and requiring less time.
Quantification of Riboflavin, Flavin Mononucleotide, and Flavin Adenine Dinucleotide in Human Plasma by Capillary Electrophoresis and Laser-induced Fluorescence Detection
Authors: Hustad Steinar , Ueland Per Magne , Schneede Jørn
Published: January 2020

Background: Riboflavin is the precursor of flavin mononucleotide (FMN) and FAD, which serve as cofactors for several redox enzymes. We have developed a capillary electrophoresis method for the determination of riboflavin and its two coenzyme forms in human plasma.

Methods: Trichloroacetic acid-treated plasma was subjected to solid-phase extraction on reversed-phase columns. The analytes were separated by micellar electrokinetic capillary chromatography in uncoated fused- silica capillaries filled with borate buffer containing 50 mmol/L sodium dodecyl sulfate, methanol, and N-methylformamide. Native fluorescence was monitored at 530 nm, using an argon laser operating at 488 nm as excitation source.

Results: The assay was linear over a concentration range of two orders of magnitude, and the limit of detection was far below physiological concentrations for all vitamers. The within-day and between-day coefficients of variation were 4–9% and 6–12%, respectively. The reference values (median, 5–95 percentiles) obtained by analyzing plasma from 63 healthy subjects were 8.6 nmol/L (2.7–42.5 nmol/L) for riboflavin, 7.0 nmol/L (3.5–13.3 nmol/L) for FMN, and 57.9 nmol/L (44.5–78.1 nmol/L) for FAD.

Conclusions: Capillary electrophoresis with laser-induced fluorescence detection allows determination of all riboflavin vitamers far below physiological concentrations. The method may become a useful tool for the assessment of riboflavin status in humans.
Degradation of Cardiac Troponin I in Serum Complicates Comparisons of Cardiac Troponin I Assays
Authors: Shi Qinwei , Ling Mingfu , Zhang Xiaochen , Zhang Minyuan , Kadijevic Lilly , Liu Shigui , Laurino Joseph P
Published: January 2020

Background: Up to a 20-fold variation in serum cardiac troponin I (cTnI) concentration may be observed for a given patient sample with different analytical methods. Because more limited variation is seen for control materials and for purified cTnI, we explored the possibility that cTnI was present in altered forms in serum.

Methods: We used four recombinantly engineered cTnI fragments to study the regions of cTnI recognized by the Stratus®, Opus®, and ACCESS® immunoassays. The stability of these regions in serum was analyzed with Western blot.

Results: The measurement of several control materials and different forms of purified cTnI using selected commercial assays demonstrated five- to ninefold variation. Both the Stratus and Opus assays recognized the N-terminal portion (NTP) of cTnI, whereas the ACCESS assay recognized the C-terminal portion (CTP) of cTnI. Incubation of recombinant cTnI in normal human serum produced a marked decrease in cTnI concentration as determined with the ACCESS, but not the Stratus, immunoassay. Western blot analysis of the same samples using cTnI NTP- and CTP-specific antibodies demonstrated preferential degradation of the CTP of cTnI.

Conclusions: The availability of serum cTnI epitopes is markedly affected by the extent of ligand degradation. The N-terminal half of the cTnI molecule was found to be the most stable region in human serum. Differential degradation of cTnI is a key factor in assay-to-assay variation.