bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Filter Paper Blood Lead Methods: Results of a Pilot Proficiency Testing Program
Authors: Stanton Noel V , Maney Jane M , Jones Robert
Published: January 2020

Background: Lead testing on dried filter paper (FP) blood spots is used routinely by some laboratories for lead poisoning screening. Proficiency testing (PT) as required under CLIA ‘88 laboratory regulations has not been available for these methods.

Methods: We describe a suitable PT scheme and evaluate FP laboratory performance based on program results. Monthly testing events consisting of five FP specimens were provided to six participating laboratories. Results were evaluated against target values determined by referee laboratories.

Results: Preliminary FP laboratory results showed poor agreement with specimen target values, exhibiting a mean absolute bias of 0.29 μmol/L (5.9 μg/dL). Five of six participating laboratories demonstrated significant improvement in later testing events, with bias decreasing to 0.12 μmol/L (2.5 μg/dL). Performance varied widely between the participating laboratories and appeared to be method dependent. When evaluated using CLIA blood lead acceptability criteria, the proportion of acceptable individual specimen results (n = 35) ranged from 54% to 100%. On a testing event basis (n = 7), the proportion of acceptable events ranged from 29% to 100%.

Conclusions: A suitable FP PT program now exists to capably assist and monitor FP laboratories. Based on overt PT results, properly utilized FP testing methods can accurately measure blood lead concentration.
Efficiency of Two Different Nine-Loci Short Tandem Repeat Systems for DNA Typing Purposes
Authors: Sacchetti Lucia , Calcagno Giuseppe , Coto Iolanda , Tinto Nadia , Vuttariello Emilia , Salvatore Francesco
Published: January 2020

Background: Genotyping based on short tandem repeat (STR) regions is widely used in human identification and parentage testing, in gene mapping studies, and as an approach to studies on the etiopathogenesis and diagnosis of hereditary diseases. We wished to study a new analytical approach that uses capillary electrophoresis and multicolor fluorescence in place of slab gel electrophoresis. Methods: We evaluated the efficiency for parentage and forensic purposes of the AmpFLSTR Profiler PlusTM typing kit that is used with the ABI Prism 310 Genetic Analyzer (System-2 STR), and that of a widely used panel of nine STRs analyzed with conventional slab-gel electrophoresis followed by radioactive detection (System-1 STR). System-2 STR, based on automated capillary electrophoresis and automated sizing of the alleles by Genotyper 2.0 software, was used to determine the allele frequency of the nine loci in 157 Caucasian subjects from southern Italy. On the basis of the data obtained, we submitted 40 trios to parentage testing. Results: A higher median probability of paternity attribution and power of exclusion were obtained with System-2 STR vs System-1 STR: respectively, 99.99% and 99.95% (P <0.05) for attribution; and five and four excluding loci (P <0.05) for exclusion. The most informative and highly discriminating loci were D18S51, D21S11, and FGA. The combined probability of matching-by-chance for all nine STRs was 1.36 × 10−12 for System-2 compared with 1.11 × 10−7 obtained with the other system. The internal standard and allelic ladder of the System-2 STR facilitated accurate and precise genotyping; furthermore, System-2 STR and was faster than the conventional System-1 STR. Conclusions: The System-2 STR allows rapid testing with higher probabilities of attribution and a higher power of exclusion than with the comparison method with slab-gel electrophoresis.
Cobalamin Deficiency in General Practice. Assessment of the Diagnostic Utility and Cost-Benefit Analysis of Methylmalonic Acid Determination in Relation to Current Diagnostic Strategies
Authors: Hølleland Geir , Schneede Jørn , Ueland Per Magne , Lund Per Kristian , Refsum Helga , Sandberg Sverre
Published: January 2020

Diagnosing cobalamin deficiency is often difficult. We investigated the diagnostic strategies that 224 general practitioners used to assess cobalamin status and the criteria on which they based their decisions to supplement patients. From all serum cobalamin analyses carried out at a single laboratory during 1993, individuals with serum cobalamin concentrations <300 pmol/L were identified, and one patient per general practitioner was included. When serum methylmalonic acid (s-MMA) values >0.376 μmol/L were used as the “reference standard” for cobalamin deficiency, the serum cobalamin assay had a diagnostic sensitivity of 0.40 and a specificity of 0.98. With the same reference standard, the diagnostic accuracy of the physicians’ decision to supplement patients had the same specificity but a higher sensitivity (0.51). Cost-benefit analysis indicated that measurement of s-MMA can be recommended in patients with serum cobalamin >60–90 pmol/L and <200–220 pmol/L, depending on its diagnostic accuracy.
Simultaneous Rapid Measurement of Whole Blood Myoglobin, Creatine Kinase MB, and Cardiac Troponin I by the Triage Cardiac Panel for Detection of Myocardial Infarction
Authors: Apple Fred S , Christenson Robert H , Valdes Roland , Andriak Alexander J , Berg Amy , Duh Show-Hong , Feng Yue-Jin , Jortani Saeed A , Johnson Nancy A , Koplen Brenda , Mascotti Kristin , Wu Alan HB
Published: January 2020

This multicenter study evaluated the Biosite Triage® Cardiac Panel as a quantitative, multimarker, whole blood system for the detection of acute myocardial infarction (MI). Optimum cutoffs for the discrimination of acute MI (n = 192 patients, 59 with MI) as determined by ROC curve analyses were as follows: 0.4 μg/L for cardiac troponin I (cTnI); 4.3 μg/L for the creatine kinase MB isoenzyme (CK-MB); and 107 μg/L for myoglobin. The Triage Panel showed the following concordances for detection or rule-out of MI compared with established devices: cTnI >89%; CK-MB >81%; myoglobin >69%. No significant differences were present between methods for the same marker. Diagnostic efficiencies demonstrated comparable sensitivities and specificities for the diagnosis of MI in patients presenting with symptoms compared with the Dade, Beckman, and Behring CK-MB, cTnI, and myoglobin assays; the ratio of sensitivity to specificity for each marker was as follows: cTnI, 98%:100%; CK-MB, 95%:91%; myoglobin, 81%:92%. The areas under the ROC curves for the Biosite myoglobin, CK-MB, and cTnI were 0.818, 0.905, and 0.970, respectively; the areas were significantly different, P <0.05. In patients with skeletal muscle injury and renal disease, the Triage cTnI showed 94% and 100% specificity, respectively. The Triage panel offers clinicians a whole blood, point-of-care analysis of multiple cardiac markers that provides excellent clinical sensitivity and specificity for the detection of acute MI.
Determination of Cardiac Troponin I Forms in the Blood of Patients with Acute Myocardial Infarction and Patients Receiving Crystalloid or Cold Blood Cardioplegia
Authors: Giuliani Isabelle , Bertinchant Jean-Pierre , Granier Claude , Laprade Michel , Chocron Sidney , Toubin Gérard , Etievent Joseph-Philippe , Larue Catherine , Trinquier Sylvie
Published: January 2020

To determine the forms of cardiac troponin I (cTnI) circulating in the bloodstream of patients with acute myocardial infarction (AMI) and patients receiving a cardioplegia during heart surgery, we developed three immunoenzymatic sandwich assays. The first assay involves the combination of two monoclonal antibodies (mAbs) specific for human cTnI. The second assay involves the combination of a mAb specific for troponin C (TnC) and an anti-cTnI mAb. The third assay was a combination of a mAb specific for human cardiac troponin T (cTnT) and an anti-cTnI mAb. Fifteen serum samples from patients with AMI, 10 serum samples from patients receiving crystalloid cardioplegia during heart surgery, and 10 serum samples from patients receiving cold blood cardioplegia during heart surgery were assayed by the three two-site immunoassays. We confirmed that cTnI circulates not only in free form but also complexed with the other troponin components (TnC and cTnT). We showed that the predominant form in blood is the cTnI-TnC binary complex (IC). Free cTnI, the cTnI-cTnT binary complex, and the cTnT-cTnI-TnC ternary complex were seldom present, and when present, were in small quantities compared with the binary complex IC. Similar results were obtained in both patient populations studied. These observations are essential for the development of new immunoassays with improved clinical sensitivity and for the selection of an appropriate cTnI primary calibrator.
One-Step Direct Assay for Mature-type Adrenomedullin with Monoclonal Antibodies
Authors: Ohta Hideki , Tsuji Tetsuo , Asai Shigeru , Sasakura Kazuyuki , Teraoka Hiroshi , Kitamura Kazuo , Kangawa Kenji
Published: January 2020

Adrenomedullin (AM) is a potent hypotensive peptide. Plasma contains mature-type AM (m-AM), which is amidated at the carboxy terminus, and an intermediate, AM-Gly. We developed a one-step two-site IRMA specific for determining human m-AM with monoclonal antibodies. The detection limit was 0.5 pmol/L, and the working range (CV <15%) was 1–300 pmol/L. Dilution of plasma samples showed good linearity. The recovery of added AM was 91–118%. The intra- and interassay imprecision values (CVs) were 4.4–8.2% and 5.5–8.3%, respectively. The assay had no cross-reactivity with AM-Gly or other peptides similar to AM. The mean (± SD) plasma human m-AM concentration of 61 healthy subjects was 1.18 ± 0.65 pmol/L. In conclusion, our IRMA makes it possible to specifically measure m-AM, using a small amount of plasma sample (0.2 mL) by a one-step overnight assay without prior extraction. Our simplified method would be suitable for clinical studies on AM, especially when large numbers of samples must be processed.
System to Monitor a Portion of the Total Testing Process in Medical Clinics and Laboratories: Evaluation of a Split-Specimen Design
Authors: Shahangian Shahram , Cohn Richard D , Gaunt Edward E , Krolak John M
Published: January 2020

To evaluate a split-specimen design to identify problems in the testing process in hospital and physician office laboratories, we examined the testing for serum total cholesterol (n = 646) and potassium (n = 732) at 11 medical clinics evaluating 30–199 patients (mean, 125). Clinic personnel collected three tubes of blood from each patient. One specimen was processed routinely, the second was sent to a referral laboratory (RL), and the third specimen was sent to a holding facility for storage. The corresponding stored sample was retrieved and divided into three audit samples randomly and when result difference for the first two specimens exceeded critical values; one audit sample was sent to the original participant, the second to the RL, and the third to a referee laboratory. When three criteria were used, the result discrepancy rates were 2.5–8.7% for potassium and 1.5–4.6% for cholesterol. The split-specimen design could be implemented and evaluated as a monitoring system for a portion of the testing process.
Urinary Cotinine and Exposure to Parental Smoking in a Population of Children with Asthma
Authors: Oddoze Christiane , Dubus Jean Christophe , Badier Monique , Thirion Xavier , Pauli Anne Marie , Pastor Jean , Bruguerolle Bernard
Published: January 2020

Background: Studies of the effects of tobacco smoke often rely on reported exposure to cigarette smoke, a measure that is subject to bias. We describe here the relationship between parental smoking exposure as assessed by urinary cotinine excretion and lung function in children with asthma.

Methods: We studied 90 children 4–14 years of age, who reported a confirmed diagnosis or symptoms of asthma. In each child, we assessed baseline pulmonary function (spirometry) and bronchial responsiveness to carbachol stimulation. Urinary cotinine was measured by HPLC with ultraviolet detection.

Results: Urinary cotinine concentrations in the children were significantly correlated (P <0.001) with the number of cigarettes the parents, especially the mothers, smoked. Bronchial responsiveness to carbachol (but not spirometry test results) was correlated (P <0.03) with urinary cotinine in the children.

Conclusion: Passive smoke exposure increases the bronchial responsiveness to carbachol in asthmatic children.
Immunoradiometric Assay for Intact Human Osteocalcin(1–49) without Cross-Reactivity to Breakdown Products
Authors: Colford John W , Lueddecke Bruce A , Salvati Michael , Hanna Dave , Sailer Dawn , Khosla Sundeep , Riggs B Lawrence , Langman Craig B
Published: January 2020

Background: Osteocalcin (Oc), a serum marker of bone turnover, circulates in several forms. We developed an assay for intact human Oc and investigated its clinical features.

Methods: We generated goat antibodies and N- and C-terminal Oc. The former was used on solid phase (polystyrene beads), and the latter was used as the tracer in an IRMA.

Results: The assay was linear with no cross-reactivity to Oc(1–43), total imprecision (CV) of <10%, and recovery of 100% ± 10%. Assay values for intact Oc in EDTA plasma samples were unchanged at 18–25 °C for 6 h. Values for intact Oc in serum, EDTA plasma, and heparin plasma samples did not change after storage on ice for 8 h. Serum samples from patients with various conditions were stored at −70 or −135 °C for up to 5 years and yielded z-scores comparable to an Oc(1-43) IRMA for all conditions except for renal failure. In renal failure, the Oc(1–43) assay values were increased, whereas the intact assay values were in the reference interval.

Conclusion: Decreases in Oc assay values are inhibited by calcium chelation, and slowed by reduced temperatures. The described assay for intact Oc allows improved specificity for bone compared with an assay for Oc(1–43).
Quantitative Reverse Transcription-PCR Measurement of Thyroglobulin mRNA in Peripheral Blood of Healthy Subjects
Authors: Wingo Susan T , Ringel Matthew D , Anderson Jeffrey S , Patel Aneeta D , Lukes Yvonne D , Djuh Yin-Ying , Solomon Barbara , Nicholson Diamaud , Balducci-Silano Pina L , Levine Michael A , Francis Gary L , Tuttle R Michael
Published: January 2020

Background: Thyroglobulin mRNA can be detected qualitatively in the peripheral blood of patients with metastatic thyroid cancer, thyroid cancer patients with residual thyroid bed uptake, and individuals with no known thyroid disease with intact thyroid glands by use of a lengthy, highly sensitive extraction technique. To improve and broaden the clinical usefulness of this assay, we developed a quantitative reverse transcription (RT)-PCR assay for thyroglobulin mRNA, using RNA recovered from whole blood with a simplified extraction technique.

Methods: Whole blood was drawn from 32 healthy subjects in standard EDTA blood collection tubes. Total RNA was extracted from whole blood, using the PUREscript RNA Isolation Kit. RT-PCR using intron-spanning primers was used to quantitatively amplify thyroglobulin mRNA, using the ABI PRISM 7700 Sequence Detection System with a fluorescent-labeled, thyroglobulin-specific oligonucleotide probe. Thyroid RNA calibration curves were created using total RNA recovered from a single nondiseased thyroid gland.

Results: Qualitative RT-PCR demonstrated the presence of thyroglobulin mRNA in the whole blood sample of each healthy subject. The mean concentration of thyroglobulin mRNA detected in these subjects was 433 ± 69 ng of total thyroid RNA per liter of whole blood (range, 26–1502 ng/L). Overall assay imprecision (CV) was 24% for five samples analyzed 10 times each in separate analytical runs on different days.

Conclusions: Thyroglobulin mRNA can be accurately detected and quantified in peripheral blood from healthy subjects. This new quantitative technique may improve the clinical utility of circulating thyroglobulin mRNA detection in patients with thyroid disease.
Falsely Increased Immunoassay Measurements of Total and Unbound Phenytoin in Critically Ill Uremic Patients Receiving Fosphenytoin
Authors: Roberts William L , De Barun K , Coleman John P , Annesley Thomas M
Published: January 2020

Background: Fosphenytoin, a phosphate ester prodrug of phenytoin, is metabolized to phenytoin in vivo. Phenytoin metabolites accumulate in renal insufficiency and cross-react in some phenytoin immunoassays. Our aim was to determine the accuracy of phenytoin immunoassays in renal patients treated with fosphenytoin.

Methods: We measured phenytoin with HPLC and with the aca, ACS:180, TDx phenytoin II, Vitros, and AxSYM methods. Specimens were collected 2–120 h after fosphenytoin administration from 17 patients with renal insufficiency.

Results: The AxSYM, TDx phenytoin II, ACS:180, and Vitros assays displayed falsely increased phenytoin results up to 20 times higher than the HPLC results. The aca Star results for these specimens were comparable to the HPLC results. Although fosphenytoin can cross-react with phenytoin immunoassays, no fosphenytoin was detected by a sensitive HPLC method in any sample that was tested for its presence.

Conclusion: These results are consistent with the formation of one or more novel metabolites or adducts of fosphenytoin that accumulate in some critically ill patients with renal insufficiency and that display significant cross-reactivity with some, but not all, phenytoin immunoassay methods.
Quantitative Bedside Assay for Cardiac Troponin T: A Complementary Method to Centralized Laboratory Testing
Authors: Müller-Bardorff Margit , Rauscher Tilmann , Kampmann Markus , Schoolmann Sven , Laufenberg Franz , Mangold Dieter , Zerback Rainer , Remppis Andrew , Katus Hugo A
Published: January 2020

Background: In the evaluation of chest pain patients, whole blood bedside assays of highly specific cardiac molecules may be an attractive alternative to centralized clinical chemistry testing. We now report on an optimized test strip device for cardiac troponin T (cTnT) that can be analyzed by a cardiac reader for quantitative assessment of the test result.

Methods and Results: The cTnT test strip reader measures, via a CCD camera, the reflectance of the signal line. For quantitative analysis, a calibration curve was constructed from 1030 samples of 252 consecutive patients with acute coronary syndromes. In a method comparison of 140 samples, the quantitative results of the cTnT test strip reader correlated closely with the results of the cTnT ELISA (r = 0.98; y = 0.85x + 0.002). Within-run and day-to-day (n = 10) mean CVs were between 11% and 16%, respectively. The cross-reactivity with skeletal troponin T was <0.02%. In patients with myocardial infarction, 45% and 91% of all samples were positive on admission and at 4–8 h after the onset of symptoms, respectively. ROC curve analysis demonstrated a comparable efficiency of the cTnT test strip reader and the laboratory-based cTnT ELISA in patients with suspected myocardial infarction.

Conclusions: It is now possible to quantitatively determine cTnT at the patient’s bedside with a rapid and convenient test device. This will facilitate the diagnostic work up of patients with suspected myocardial cell necrosis.
The Apparent Inhibition of Inosine Monophosphate Dehydrogenase by Mycophenolic Acid Glucuronide Is Attributable to the Presence of Trace Quantities of Mycophenolic Acid
Authors: Korecka Magdalena , Nikolic Dejan , van Breemen Richard B , Shaw Leslie M
Published: January 2020

Background: Mycophenolic acid glucuronide, the primary metabolite of the immunosuppressive agent mycophenolic acid, affords weak inhibition of proliferating and resting lymphocytes and recombinant human inosine monophosphate dehydrogenase in comparison to the active drug. We evaluated the hypothesis that mycophenolic acid is a trace contaminant of the glucuronide metabolite preparation and that this accounts for the observed effects of mycophenolic acid glucuronide on human inosine monophosphate dehydrogenase catalytic activity both in lymphocytes and the pure enzyme.

Methods: We used negative ion electrospray HPLC-mass spectrometry (HPLC-MS) and HPLC-tandem MS (HPLC-MS-MS) to identify mycophenolic acid as a contaminant of mycophenolic acid glucuronide. Quantification of the mycophenolic acid contaminant was achieved using a negative ion electrospray HPLC-MS method in the selected-ion monitoring mode.

Results: Trace amounts of mycophenolic acid were detected and definitively identified in the mycophenolic acid glucuronide preparation by the HPLC-MS-MS analysis. In addition to having identical HPLC retention times, pure mycophenolic acid and the contaminant produced the following major fragments upon HPLC-MS-MS analysis: deprotonated molecular ion, m/z 319; and fragment ions, m/z 275, 243, 205, and 191 (the most abundant fragment ion). Using the negative ion electrospray HPLC-MS procedure in the selected-ion monitoring mode, the quantity of the contaminant mycophenolic acid was determined to be 0.312% ± 0.0184% on a molar basis.

Conclusion: These data provide strong support for the proposal that the apparent inhibition of the target enzyme inosine monophosphate dehydrogenase by mycophenolic acid glucuronide is attributable to the presence of trace amounts of contaminant mycophenolic acid.
Plasma Porphyrins in the Porphyrias
Authors: Hindmarsh J Thomas , Oliveras Linda , Greenway Donald C
Published: January 2020

Background: As an aid in the diagnosis and management of porphyria we have developed a method to fractionate and quantify plasma porphyrins and have evaluated its use in various porphyrias.

Methods: We used HPLC with fluorometric detection to measure plasma concentrations of uroporphyrin I and III, heptacarboxyl III, hexacarboxyl III, pentacarboxyl III, and coproporphyrin I and III. We studied 245 healthy subjects, 32 patients with classical porphyria cutanea tarda (PCT), 12 patients with PCT of renal failure, 13 patients with renal failure, 8 patients with pseudoporphyria of renal failure, 3 patients with acute intermittent porphyria, 5 patients with variegate porphyria, 5 patients with hereditary coproporphyria, and 4 patients with erythropoietic protoporphyria.

Results: Between-run CVs were 5.4–13%. The recoveries of porphyrins added to plasma were 71–114% except for protoporphyrin, which could not be reliably measured with this technique. Plasma porphyrin patterns clearly identified PCT, and its clinical sensitivity equaled that of urine porphyrin fractionation. The patterns also allowed differentiation of PCT of renal failure from pseudoporphyria of renal failure.

Conclusions: The assay of plasma porphyrins identifies patients with PCT and appears particularly useful for differentiating PCT of renal failure from pseudoporphyria of renal failure.
Evaluation of Cation-Exchange HPLC Compared with Isoelectric Focusing for Neonatal Hemoglobinopathy Screening
Authors: Campbell Michelle , Henthorn Joan S , Davies Sally C
Published: January 2020

Background: Central Middlesex Hospital, in northwest London, has screened neonates for hemoglobinopathies, using the established manual technique of isoelectric focusing (IEF) since 1989. Recently, this laboratory has faced a large increase in the number of samples tested per year. This study compared the detection of hemoglobin abnormalities between the existing manual IEF method and that of automated cation-exchange HPLC to determine the reliability of HPLC and whether an automated system would save time in the laboratory.

Methods: Over a 15-month period, 25 750 blood samples, collected by heel prick onto filter paper, were tested using HPLC, and the results were compared with those obtained with IEF.

Results: HPLC and IEF each identified 568 patients with FAS, 151 with FAC, 49 with FAD-Punjab, 23 with FS, 3 with FC, 6 with FSC, 5 with FE, and 1 with FD. IEF detected 62 patients with FAE, whereas HPLC detected 63. This additional FAE was observed on repeat IEF. One additional heterozygote detected by HPLC was initially not observed by IEF, but was detected on repeat IEF. HPLC detected all but six cases of Hb Barts observed by IEF. One double heterozygote and four heterozygotes were detected by IEF, but not by HPLC. The detection of hemoglobin variants expressed at low concentrations was comparable for the two methods, and carryover was not observed in routine analysis on HPLC.

Conclusions: HPLC is a sensitive, efficient, and time-saving alternative to IEF for the neonatal screening of common hemoglobinopathies.
TaqMan PCR-based Gene Dosage Assay for Predictive Testing in Individuals from a Cancer Family with INK4 Locus Haploinsufficiency
Authors: Laurendeau Ingrid , Bahuau Michel , Vodovar Nicolas , Larramendy Claire , Olivi Martine , Bieche Ivan , Vidaud Michel , Vidaud Dominique
Published: January 2020

Background: A genetic syndrome of cutaneous malignant melanoma and nervous system tumors recently has been characterized and shown to be linked to the INK4 locus in the 9p21 region. Hemizygosity at adjacent physically mapped microsatellite markers indicated deletion of p16, p19, and p15 clustered tumor suppressors. Because individuals from this family could benefit from predictive testing in terms of cancer prevention, we developed a direct test without need to analyze parental DNAs to comply with the rules of individual consent and secrecy.

Methods: We developed an assay using TaqManTM real-time quantitative PCR, with p15 as the test sequence and albumin (ALB) as the reference gene. The normalized ratio of p15/ALB is expected to yield a value of ∼1 in individuals without the deletion, whereas a ratio of ∼0.5, indicating p15 haploinsufficiency, is expected in predisposed individuals.

Results: All patients harboring the previously defined at-risk haplotype were correctly identified using this approach. In six individuals with deletions, the p15/ALB ratios were 0.472–0.556 (SD, 0.013–0.078). In the five individuals without deletions, the ratios were 0.919–1.019 (SD, 0.006–0.075).

Conclusions: This is the first report of a high-throughput, automatable gene dosage assay successfully applied to the identification of a germ-line deletion. This approach, not limited by marker informativeness or the need for harvesting live cells, can be applied to any condition with haploinsufficiency and extended to the characterization of most abnormalities of the ploidy.
Optimization of Allopurinol Challenge: Sample Purification, Protein Intake Control, and the Use of Orotidine Response as a Discriminative Variable Improve Performance of the Test for Diagnosing Ornithine Carbamoyltransferase Deficiency
Authors: Arranz José A , Riudor Encarnació , Rodés Margarita , Roig Manuel , Climent Consuelo , Rubio Vicente , Sentís Margarita , Burlina Alberto
Published: January 2020

Background: The diagnosis of heterozygosity for X-linked ornithine carbamoyltransferase (OCT) deficiency has usually been based on measurement of the increase of orotate and orotidine excretion after an allopurinol load. We examined the choices of analyte, cutoff, and test conditions to obtain maximal test accuracy.

Methods: Urine orotate/orotidine responses to allopurinol load in 37 children (13 OCT-deficient and 24 non-OCT-deficient) and 24 women (7 at risk for carrier status and 17 not related to OCT-deficient children) were analyzed by liquid chromatography after sample purification by anion-exchange chromatography. Diagnostic accuracy was evaluated by nonparametric ROC curves.

Results: Sample purification was necessary to prevent interferences. Orotate and orotidine excretion increased with increased protein intake during the test. At a cutoff of 8 mmol orotidine/mol creatinine, sensitivity was 1.0 and specificity was 0.92 in mild forms of OCT deficiency. Results in monoplex carrier women may differ greatly from those expected because of the genetics of this deficiency.

Conclusions: Standardization of protein intake is required in the allopurinol loading test. A negative response in the face of clinical suspicion should be followed with a repeat test during a protein intake not <2.5 g · kg−1 · day−1. Measurements of orotidine provide better clinical sensitivity than measurements of orotate.
Rapid, Homogeneous Genotyping of the 4G/5G Polymorphism in the Promoter Region of the PAI1 Gene by Fluorescence Resonance Energy Transfer and Probe Melting Curves
Authors: Nauck Markus , Wieland Heinrich , März Winfried
Published: January 2020

Background: Many studies have convincingly shown that survivors of myocardial infarction have impaired fibrinolytic activity because of increased concentrations of plasma plasminogen activator inhibitor-1 (PAI-1). A single guanosine insertion/deletion polymorphism in the promoter region of the PAI1 gene, commonly called 4G/5G, has been shown to be associated with plasma PAI-1 activity. Our aim was to develop and validate a homogeneous assay for rapid genotyping of the 4G/5G polymorphism.

Methods: In this report we present a single-tube method for genotyping of the 4G/5G polymorphism that combines both rapid-cycle PCR with real-time monitoring of the amplification process and generation of allele-specific fluorescent probe melting profiles on the LightCyclerTM. Two fluorescently labeled hybridization probes recognizing adjacent sequences in the amplicon were present in the reaction mixture. The shorter detection probe spanned the polymorphic site, perfectly matching the 5G allele. After annealing, the fluorophores were in resonance energy transfer, providing real-time monitoring of the amplification process. At the completion of the PCR, fluorescence was monitored as the temperature increased through the Tm of the probe/product duplex, and a characteristic melting profile for each genotype was obtained.

Results: With this method, 32 samples were genotyped within 30 min without the need of any post-PCR sample manipulation. The genotypes of 100 DNA samples determined with the LightCycler were identical to those obtained with conventional PCR and restriction fragment length analysis.

Conclusion: The genotyping of the 4G/5G polymorphism with the LightCycler is a rapid, reliable method that is suitable for typing both small and large numbers of samples.
Isocitrate as Calcium Ion Activity Buffer in Coagulation Assays
Authors: RÅnby Mats , Gojceta Tony , Gustafsson Kerstin , Hansson Kenny M , Lindahl Tomas L
Published: January 2020

Background: Ca2+ activity close to the physiological concentration of 1.3 mmol/L is essential in blood coagulation. Is this also true for the performance of global diagnostic coagulation assays? We searched for compounds that would buffer Ca2+ activity at ∼1.3 mmol/L without disturbing coagulation reactions and investigated whether such Ca2+ buffering improves diagnostic efficacy in global diagnostic coagulation tests.

Methods: Buffering was investigated by mixing CaCl2 and 11 candidate compounds and determining Ca2+ activity. The best candidates were added to mixtures of plasma and thromboplastin to detect interference with coagulation reactions. The best of these candidates, isocitrate, was used to modify an activated partial thromboplastin time (APTT), buffering final Ca2+ activity to ∼1.3 mmol/L. Plasma samples from 22 healthy individuals and 120 patients were analyzed with original and modified APTT to determine whether diagnostic efficacy was improved.

Results: Two suitable Ca2+ buffers, citrate and isocitrate, were found. Isocitrate was preferred as being less coagulation inhibitory, a better Ca2+ buffer, and possibly a better anticoagulant. The isocitrate-modified APTT showed a final Ca2+ activity of 1.60 ± 0.07 mmol/L, compared with 2.73 ± 0.20 mmol/L for the original APTT. The means and SDs for the healthy individuals were determined for both procedures, and the values were used to express patient deviation from normality (difference from mean divided by SD). The deviation was greater for the modified APTT; 4.3 ± 5.7, compared with 3.6 ± 5.0 (P <0.005) for the original APTT.

Conclusions: Isocitrate can be used to buffer Ca2+ activity at physiological concentrations and can serve as an anticoagulant. APTT with isocitrate-buffered Ca2+ activity shows signs of improved diagnostic efficacy.
Raging Hormones: Closing the Loop from Bench to Bedside. The Twenty-Second Arnold O. Beckman Conference in Clinical Chemistry
Authors: Ashwood Edward R
Published: January 2020

The Twenty-second Annual 1999 Arnold O. Beckman Conference was held on February 21 and 22, 1999, in San Antonio, Texas. In keeping with the tradition of the conference, the goal of this year’s meeting was to strengthen attendees’ knowledge of clinical endocrinology and the use of the clinical laboratory. The conference was divided into four sessions: basic science, diabetes, bone, and diagnostic endocrinology.
Quantitative Reverse Transcription-PCR Assay with an Internal Standard for the Detection of Prostate-specific Antigen mRNA
Authors: Ylikoski Alice , Sjöroos Minna , Lundwall Åke , Karp Matti , Lövgren Timo , Lilja Hans , Iitiä Antti
Published: January 2020

Background: Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA.

Methods: The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates.

Results: The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 106 copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 ± 200 to 44 100 ± 4900 (mean ± SD) in the samples, corresponding to ∼1–100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 ± 100 and 2000 ± 900 (mean ± SD) PSA mRNA copies in 5 mL of blood for the 2 males].

Conclusions: The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.
Analytical Validation of the PRO-Trac II ELISA for the Determination of Tacrolimus (FK506) in Whole Blood
Authors: MacFarlane Gordon D , Scheller Daniel G , Ersfeld Diana L , Shaw Leslie M , Venkatarmanan Raman , Sarkozi Laszlo , Mullins Richard , Fox Bonnie R
Published: January 2020

Background: The analytical validation of multiple lots of the PRO-TracTM II ELISA (DiaSorin) for the determination of tacrolimus in whole blood is described.

Methods: The analytical parameters assessed included analytical sensitivity, dilution linearity, functional sensitivity, values in samples containing no tacrolimus, intra- and interassay precision, supplementation and recovery, metabolite cross-reactivity, interference studies, and method comparisons HPLC-tandem mass spectrometry (HPLC/MS/MS) and the IMx® Tacrolimus II multiparticle enzyme immunoassay. Where appropriate, assessments were performed according to NCCLS guidelines.

Results: The mean analytical detection limit was <0.25 μg/L for all lots, whereas the functional sensitivity was 1.0 μg/L. Excellent linear correlation (r = 0.985) was observed for dilution linearity. The intraassay imprecision was <7%, and the total imprecision by ANOVA was <10%. Recovery was 109% ± 11%. Metabolite cross-reactivity was consistent with previous reports for this antibody. No interference was observed for 35 tested drugs. Method comparison with HPLC/MS/MS showed no statistically significant differences. Samples exhibited stability through four freeze/thaw cycles and for 1 week at room temperature.

Conclusion: These data demonstrate that the PRO-Trac II ELISA is a robust, accurate, and precise tool for the assessment and management of tacrolimus blood concentrations in transplant patients.
Simple Method for Determination of Thiocyanate in Urine
Authors: Haque M Rezaul , Bradbury J Howard
Published: January 2020

Background: It would be useful to develop a simple kit method for determination of thiocyanate in urine, which could be used to monitor cyanide overload in cassava-consuming populations.

Methods: The method was based on the quantitative oxidation of thiocyanate in acid permanganate at room temperature in a closed vial with liberation of HCN, which reacted with a picrate paper. For semiquantitative analysis in the field, the colored picrate paper was matched with a color chart prepared using known amounts of KSCN. In the laboratory, a more accurate result was obtained by elution of the colored complex in water and measurement of the absorbance at 510 nm. Over the range 0–100 mg/L, there was a linear relationship given by the equation: thiocyanate content (mg/L) = 78 × absorbance.

Results: The picrate thiocyanate method gave no interference with urine samples containing protein at less than 7 g/L, 21 amino acids, histamine, glucose, NaCl, urea, blood, and linamarin. For 53 urine samples analyzed by an accurate column method and the thiocyanate picrate method, a regression line gave very good agreement (r2 = 1.000). Quantitative recoveries of thiocyanate added to urine samples were obtained with the picrate method.

Conclusions: A simple picrate kit for determination of thiocyanate in urine was developed and is available free of charge for workers in developing countries.
Codeine Concentration in Hair after Oral Administration Is Dependent on Melanin Content
Authors: Kronstrand Robert , Förstberg-Peterson Sophie , KÅgedal Bertil , Ahlner Johan , Larson Göran
Published: January 2020

Background: Analysis of drugs in hair has been used on a qualitative basis to estimate earlier exposure to drugs. Clinical applications are rare because of the lack of dose–response relationships in the studies performed to date, and questions remain regarding the mechanisms of drug incorporation into hair. Several human studies have shown differences in drug accumulation between pigmented and nonpigmented hair. However, the melanin concentration in hair was not determined and correlated to the amount of drug incorporated.

Methods: Nine human subjects were given codeine as a single oral dose, and plasma codeine concentrations were determined for 24 h, using gas chromatography–mass spectrometry. Hair samples were obtained weekly for a month. Total melanin, eumelanin, and codeine were measured quantitatively in hair samples by spectrophotometry, HPLC, and gas chromatography–mass spectrometry, respectively.

Results: There was an exponential relationship between codeine and melanin concentrations in hair, (r2 = 0.95 with total melanin and r2 = 0.83 with eumelanin). After normalizing the results by the area under the curve for codeine in plasma, we obtained r2 = 0.86 for codeine vs total melanin and r2 = 0.90 vs eumelanin.

Conclusions: Our results stress the importance of melanin determination when measuring drugs in hair. We postulate that analysis of drug concentration in hair may be worthwhile in the monitoring of drug compliance if the results are normalized for melanin content.
A Collection Method and High-Sensitivity Enzyme Immunoassay for Sweat Pyridinoline and Deoxypyridinoline Cross-Links
Authors: Sarno Mark , Powell Helen , Tjersland Gayle , Schoendorfer Donald , Harris Holden , Adams Kimberly , Ogata Peggy , Warnick G Russell
Published: January 2020

Background: Collagen cross-link molecules such as pyridinoline (PYD), deoxypyridinoline (DPD), and N-terminal cross-linked peptides (NTX) have been measured in urine as indices of bone resorption. However, very little is known regarding the excretion of pyridinolines into other biological fluids. We report a collection device, normalizing analyte, and high-sensitivity immunoassay for quantitative analysis of free pyridinoline cross-links in sweat.

Methods: Flame atomic emission and ion-selective electrode techniques were used to measure potassium as a sweat volume marker. The Pyrilinks immunoassay for urine free pyridinolines was optimized to increase sensitivity for measurements in sweat. The precision, accuracy, and detection limit of this assay were characterized. To assess values and variability of sweat pyridinolines in human subjects, a nonocclusive skin patch was used to collect sweat samples from a reference group and from a mixed group experiencing accelerated bone resorption, postmenopausal women and men receiving gonadotropin-releasing hormone for prostate cancer.

Results: The immunoassay intra- and interassay variations were ≤10% and <16%, respectively, with a detection limit of 309 pmol/L. Linearity upon dilution and analytical recovery ranged from 93% to 109% and 85% to 122%, respectively. Sweat PYD values normalized to potassium output yielded a weekly intraindividual biological variability of 14.7%. The mean increase in the population experiencing increased bone resorption vs the reference group was 36% (P <0.05) for sweat PYD/K vs 23–40% (P <0.05) for urinary PYD/Cr, DPD/Cr, and NTX/Cr.

Conclusion: We conclude that this new platform sweat collection technology and PYD immunoassay show potential as an indicator of bone resorption.
Determination of Glucose in Dried Serum Samples by Fourier-Transform Infrared Spectroscopy
Authors: Petibois Cyril , Rigalleau Vincent , Melin Anne-Marie , Perromat Annie , Cazorla Georges , Gin Henri , Déléris Gérard
Published: January 2020

Background: Practical improvements are needed to allow measurement of glucose concentrations by Fourier- transform infrared (FT-IR) spectroscopy. We developed a new method that allows determination of the glucose concentration in dried sera.

Methods: We studied 32 serum samples after fourfold dilution and desiccation before FT-IR analyses on a spectrometer operated at a resolution of 2.0 cm−1. We integrated all spectral windows at the surface of the spectrum in the C—O region. For comparison, glucose was measured in the sera by a glucose oxidase method.

Results: One peak within the spectrum was most specific for glucose (997–1062 cm−1). Its surface integration showed a strong relationship with reference data (r = 0.998; P <0.001). FT-IR analyses of five glucose solutions were performed to determine its specific absorption at the same peak. In this way, glucose concentrations in serum spectra could be measured. For the first time while using FT-IR spectroscopy, no manipulation of spectra nor use of internal standard was necessary to obtain results in high accordance with glucose concentration measured by a conventional (glucose-oxidase) method (Sy|x = 0.25 mmol/L; r = 0.998).

Conclusions: FT-IR spectroscopy appears to be an easy and accurate method to determine glucose concentration and could be widely used to simultaneously identify and quantify several metabolites in biological fluids or tissues.
External Quality Assessment of Methylmalonic Acid and Total Homocysteine
Authors: Møller Jan , Rasmussen Karsten , Christensen Lene
Published: January 2020

Background: The use of analyses for methylmalonic acid (MMA) and total homocysteine (HCY) in plasma has become widespread. Realizing the need for external quality assessment for these measurements, we started a program in 1997. The results for 1998 are reviewed in this report.

Methods: Fourteen laboratories participated with 15 sets of results for MMA, and 28 laboratories participated with 34 sets of results for HCY. Results for four identical samples, made up from the same unmodified serum (MMA) or EDTA-plasma (HCY) pool, sent out under different identifications, were used for assessing the imprecision. Samples made up from the same pools supplemented with MMA or l-HCY to three concentrations were used for assessing the recovery. By using literature data for the biological variation, quality goals for both analytes were calculated.

Results: The overall within-laboratory CV was 12% for MMA and 7.5% for HCY. Gas chromatography–mass spectrometric HCY results had lower imprecision than the HPLC or immunoassay results. For MMA, no significant between-laboratory component of variance was found. Only results for HCY obtained with HPLC methods showed significant between-laboratory variance.

Conclusions: Eight of the 15 participants achieved the minimum imprecision goal for MMA vs 9 of the 34 participants for HCY. The minimum quality goals for bias as approximated by the recovery were achieved by 13 participants for MMA and 26 for HCY.
Preanalytical Factors (Biological Variation) and the Measurement of Serum Soluble Intercellular Adhesion Molecule-1 in Humans: Influence of the Time of Day, Food Intake, and Physical and Psychological Stress
Authors: Dugué Benoit , Leppänen Esa , Gräsbeck Ralph
Published: January 2020

Background: Adhesion proteins such as soluble intercellular adhesion molecule-1 (sICAM-1) may be important markers for early atherosclerosis and some other diseases. To devise optimum specimen collection procedures, we investigated the effects on serum sICAM-1 of several preanalytical factors (factors that occur before a specimen is analyzed) such as the time of the day, food ingestion, and physical and psychological stress.

Methods: Three sets of experiments were conducted: (a) 30 subjects were investigated during the morning, after an overnight fast, and then after the usual breakfast and at 1200; (b) 20 subjects were studied before and after exposure to thermal stress (sauna + swimming in ice-cold water); and (c) 15 volunteers were investigated after their driving license examination and during a (stress-free) control session. Conventional methods and kits were used to determine the blood picture and serum sICAM-1.

Results: All of these preanalytical factors induced a significant increase (∼10%) in the concentration of sICAM-1.

Conclusion: It is advisable to consider timing, food intake, and stress when collecting specimens and analyzing data on the concentration of sICAM-1 in serum.