bluuebunny/crossref_metadata_2025 huggingface dataset:
Errors, mistakes, blunders, outliers, or unacceptable results: how many?
Authors: Witte David L , VanNess Sue Ann , Angstadt Debbie S , Pennell Beverly J
Published: January 2020

We have studied 219 353 individual clinical chemistry results obtained in methods comparison studies. Each result was prospectively compared with its replicate, comparative, or repeat value to identify differences from expected values. Unacceptable results were defined as differing from the expected values by ≤7 SDs or CVs. We believe these differences represent special-cause variation and should be expressed as unacceptable rates per million results (ppm). We observed 447 ppm unacceptables: 196 ppm in control samples and 251 ppm in patients’ samples. Results judged likely to alter patient care occurred at a rate of 41 ppm. To better understand the magnitude of these rates, we compared these results with reports of error rates in HIV testing and the airline industry. The measurements reported were made for the purpose of quality improvement, not judgment or discovery. The significance of these findings for laboratorians, manufacturers, and regulators is discussed.
Immunofluorometric assay of pepsinogen C and preliminary clinical applications
Authors: Diamandis Eleftherios P , Nadkarni Sheila , Bhaumik Banani , Abdelrahman Aly , Melegos Dimitrios N , Borchert Gudrun , Black Margot H , Alonso Marta , Salas Ana , de los Toyos Juan R , Sampedro Andres , López-Otín Carlos
Published: January 2020

We developed mouse monoclonal antibodies (Abs) against pepsinogen C with highly purified antigen isolated from gastric mucosa. The Abs were used to construct a two-site sandwich-type assay for pepsinogen C with time-resolved fluorometry as a detection technique. The assay has a detection limit of 0.1 μg/L and is precise (within-run and day-to-day CVs <11%). We used this assay to measure pepsinogen C in seminal plasma, breast cyst fluid, amniotic fluid, male and female serum, serum from patients with prostate cancer, urine, breast tumor cytosolic extracts, breast milk, and cerebrospinal fluid. Highest pepsinogen C concentrations were in seminal plasma, followed by breast cyst fluid and amniotic fluid. We found no correlation between prostate-specific antigen concentrations and concentrations of pepsinogen C in serum of prostate cancer patients, and concluded that this marker is not useful for either diagnosing or monitoring prostatic carcinoma. The availability of a highly sensitive, reliable, and convenient method for quantifying pepsinogen C will allow investigations into the possible diagnostic value of this analyte in various clinical conditions, including benign breast diseases, breast cancer, fertility, and pregnancy.
Use of filter paper for sample collection and transport in steroid pharmacology
Authors: Howe Christopher J , Handelsman David J
Published: January 2020

Field studies of androgen pharmacology are complicated by the necessity to collect, process, and store blood samples in a central facility. We have assessed the feasibility of using capillary blood spots collected by fingerprick and dried on filter paper for pharmacokinetics and pharmacodynamic measurements with nandrolone and testosterone RIAs modified for extracts from capillary blood spots. Assays on punched spots of 7.9-mm diameter (14.9 μL of dried blood) permitted accurate quantification of testosterone down to 0.4 nmol/L from a single spot and nandrolone down to 0.9 nmol/L from two spots. Stability of the steroids in dried blood spots to adverse environmental conditions, notably increased temperatures, was investigated both in the laboratory and in field studies of dried spots sent through the postal system. Storage or postal transport under moderate conditions appeared to have no deleterious effects on apparent androgen concentrations. However, under extreme conditions of storage at 50 °C for a week or more, or transport to a very hot tropical location, a rise in the final concentration of nandrolone, and, to a lesser extent, testosterone when corrected for tracer recovery, was noticed. These effects were largely due to apparent susceptibility of tritiated tracer, but not unlabeled androgens, to thermal degradation. In a pilot pharmacological study involving intramuscular injection of 100 mg of nandrolone decanoate in 1 mL of arachis oil, nandrolone concentrations in concurrently collected plasma as well as venous and capillary blood spots showed good agreement. Testosterone concentrations in contemporaneously collected plasma and venous blood spots also showed very good agreement. We propose that these methods may allow patients and experimental subjects to self-collect samples at remote or field locations for convenient mailing to a central laboratory for androgen assay. Applications of this methodology are now under way.
Isolation and characterization of a digoxin-like immunoreactive substance from human urine by affinity chromatography
Authors: De Angelis Claudio , Riscazzi Massimiliano , Salvini Riccardo , Piccoli Alfonso , Ferri Claudio , Santucci Anna
Published: January 2020

A series of observations has suggested that one or more digoxin-like immunoreactive substances (DLIS) in biological fluids is able to cross-react with the antidigoxin antibody. Whether this substance is the endogenous inhibitor of Na+/K+ ATPase has not been well established. The aim of this study was to identify and characterize DLIS from human urine. Treated urine from healthy men was run on an affinity chromatography column at a flow rate of 1 mL/min in which the ligand was an antibody (antiserum) to digoxin. Eluates from affinity chromatography were applied onto analytical reversed-phase HPLC. The active material was eluted with a linear gradient of acetonitrile (from 350 to 650 mL/L) and water. A second step in HPLC was carried out isocratically with 280 mL/L acetonitrile in water. We found a single peak showing cross-reactivity with antidigoxin antibody as measured by RIA. It showed the same retention time as that of a digoxin calibrator. This highly purified substance is able to displace [3H]ouabain from dog kidney-derived Na+/K+ ATPase, to inhibit Na+/K+ ATPase activity as measured by the86Rb+ uptake in red blood cells and by coupled enzyme assay. Our results are consistent with the hypothesis that DLIS, as isolated by this particular digoxin antibody, is a single substance and an inhibitor of Na+/K+ ATPase.
Amino acid determination in biological fluids by automated ion-exchange chromatography: performance of Hitachi L-8500A
Authors: Le Boucher Jacques , Charret Christelle , Coudray-Lucas Colette , Giboudeau Jacqueline , Cynober Luc
Published: January 2020

The Hitachi L-8500A is a newly available apparatus for amino acid (AA)analysis that allows automatic on-line mixing of the ninhydrin reagent. The within-run precision (human plasma pools at three different concentrations) showed CVs <3.8% except for the lowest concentration of citrulline (4.4%), Tyr (4.5%), and α-aminobutyric acid (7.6%), and for the intermediate concentration of Asp (8.7%). Between-run precision (CV) was <3.1% for 17 AAs and <8.0% for 24 of 25 AAs (CV Asp = 12.0%). For retention times, within-run precision was <0.4% and between-run precision <1.8%. Excellent relations were found between the results from the Hitachi L-8500A and the widely used Beckman 6300 analyzer (0.929≤ r ≤0.999). The detection was still linear at 5 μmol/L except for Pro and hydroxyproline (20 μmol/L). The upper limit was at least 2500 μmol/L for 13 AAs and at least 1000 μmol/L for 27 of 29 AAs (anserine = 500, Val = 600 μmol/L). Values from 100 human plasma samples agreed with previously published data. We conclude that the results obtained with the Hitachi L-8500A are satisfactory when compared with those of other AA analyzers utilizing the same method. Furthermore, the Hitachi L-8500A displays several advantages including programming flexibility, microsample capacity, low noise plotting, ammonia filtering, and manual repacking of the analytical column.
Suitability of chemical in vitro models to investigate LDL oxidation: study with different initiating conditions in native and α-tocopherol-supplemented LDL
Authors: Seccia Milfred , Albano Emanuele , Bellomo Giorgio
Published: January 2020

Isolated human LDL, used in the native form or supplemented with α-tocopherol (αT), were oxidized with Cu2+, 2,2′-azobis-(2-amidino propane) hydrochloride (AAPH), and H2O2 plus horseradish peroxidase (HRP). The oxidation kinetics were measured spectrophotometrically at 234 nm to follow the formation of conjugated dienes and evaluated as resistance to oxidation (lag phase, LP) and maximal oxidation rate (propagation rate, PR). The duration of LP in nonsupplemented LDL was different with the three prooxidant stimuli (LP, in min: 96 ± 19 for Cu2+, 28.7 ± 6.7 for HRP, and 67.1 ± 11.2 for AAPH). No correlation was found between the values obtained with Cu2+ and AAPH or HRP, but a significant correlation was found with AAPH and HRP (r = 0.798, P <0.002). In vitro αT supplementation prolonged the LP and decreased the PR with all the stimuli. The extent of increase in LP was highly correlated (r = 0.872, P <0.001 for Cu2+ and HRP; r = 0.603, P <0.03 for Cu2+ and AAPH; r = 0.749, P <0.005 for AAPH and HRP). Although the evaluation of ex vivo LDL oxidation is dependent on the prooxidant stimulus, the three prooxidant conditions used detect equally well the efficiency of αT supplementation in preventing LDL oxidation.
Hepatitis B: diagnosis, prevention, and treatment
Authors: Gitlin Norman
Published: January 2020

Hepatitis B virus (HBV) infection occurs worldwide and is an important cause of acute and chronic viral hepatitis in the US. In this review, I describe the virus, risk factors for infection, clinical features of infection, results of laboratory tests during infection, and standard and emerging treatment for chronic infection. Although 95% of adult patients recover completely from HBV infection, 90% of children ≤4 years of age develop chronic infection. Active vaccination is highly efficacious.
Comparison of analytical performance and biological variability of three bone resorption assays
Authors: Ju Hsin-Shan J , Leung Sunny , Brown Bradley , Stringer Matthew A , Leigh Scott , Scherrer Christy , Shepard Karen , Jenkins Dean , Knudsen Jane , Cannon Robert
Published: January 2020

We have compared the analytical performance and biological variability of three commercially available bone resorption assays: Pyrilinks®-D, Osteomark®, and CrossLaps™, for the measurement of urinary free deoxypyridinoline (Dpd), cross-linked N-telopeptides of type I collagen (NTx), and linear C-telopeptides of type I collagen (CTx), respectively. The intraassay and interassay CVs for precision of the Dpd and NTx assays were <10% for analyte concentrations greater than the second calibrator (i.e., 3 nmol/L Dpd or 30 nmol bone collagen equivalents/L NTx). The CTx assay demonstrated poor precision for analyte concentration lower than the third calibrator (i.e., 200 μg/L). The NTx assay exhibited nonlinear recovery for sample dilutions prepared in buffer; however, this nonlinear recovery could be corrected for sample dilutions made in urine at a low analyte concentration. Supplement recoveries of each of the three assays were within 100% ± 10% on average. All three analytes showed stability through five freeze–thaw cycles. The mean day-to-day variations were 16% for Dpd, and 23% for both NTx and CTx. Similar diurnal rhythm was observed for all three assays on average, with the peak in the early morning and the nadir in the afternoon. Mean amplitude of the diurnal variation was 37% for Dpd and NTx, and 57% for CTx. Variations within the reference intervals for a healthy premenopausal population were 28% for Dpd, 57% for NTx, and 56% for CTx. Pyrilinks-D has demonstrated analytical precision and accuracy equal or superior to Osteomark and CrossLaps in all areas. Dpd exhibits the least biological variability day-to-day, within individuals across the diurnal cycle, and within a healthy premenopausal population.
Analytical performance of Bayer Immuno 1™ estradiol and progesterone assays
Authors: Lévesque Ann , Letellier Marc , Dillon Paul W , Grant Andrew
Published: January 2020

We evaluated the analytical performance of new estradiol and progesterone assays performed on the Bayer Immuno 1™system. Within-run and between-day CVs for estradiol at concentrations of 116.8–6645.8 pmol/L were ≤6.4% and for 5.54–103.95 nmol/L progesterone were ≤7.7%, thus meeting published analytical goals. The detection limits (2 SDs from mean of zero calibrator) were 27.1 pmol/L for estradiol (n = 72 over 20 days) and 0.51 nmol/L for progesterone (n = 47 over 20 days). The assays were linear to 9766 pmol/L and 113.0 nmol/L, respectively. Estradiol results agreed well with the Diagnostic Products Corporation (DPC) assays, except for serum samples from patients receiving estrogen replacement therapy; results for these samples agreed closely with the DPC estradiol-6 assay. The progesterone assay agreed closely with the DPC assay, except for samples from uremic patients. Reference values were estimated by the study of 29 women throughout the menstrual cycle with 15 samples per subject. We concluded that both assays demonstrate suitable precision, linearity, and intermethod agreement to allow their use in the clinical laboratory.
Multicenter study of Abbott AxSYM® Digoxin II assay and comparison with 6 methods for susceptibility to digoxin-like immunoreactive factors
Authors: Azzazy Hassan M E , Duh Show-Hong , Maturen Andrew , Schaller Elisabeth , Shaw Leslie , Grimaldi Robert , Shock Gloria , Christenson Robert H
Published: January 2020

Performance characteristics of the Abbott nonpretreatment AxSYM® Digoxin II assay were evaluated for quantification of digoxin at four independent sites. Correlation of digoxin measurements with the Abbott pretreatment AxSYM®, Baxter Stratus® II, Abbott TDx/TDxFLx II®, Abbott IMx®, Emit® 2000, and Beckman Synchron CX® digoxin assays showed acceptable agreement, as indicated by: slope values >0.84, r >0.90, y-intercepts for all comparisons at or below the assay detection limit, and Sy|x ranging between 7.5% and 15.4% of the average digoxin value. Susceptibility to interference from digoxin-like immunoreactive factors (DLIFs) was examined in 233 samples from renal patients, liver disease patients, cord blood, and third-trimester pregnancies; the AxSYM Digoxin II assay demonstrated the least DLIFs interference. DLIF susceptibility for four of the methods was significantly greater (P <0.05) than in the AxSYM Digoxin II assay; susceptibilities of the Stratus II and Emit 2000 methods were similar to the AxSYM Digoxin II assay.
Evaluation of new immunoenzymometric assay for measuring soluble transferrin receptor to detect iron deficiency in anemic patients
Authors: Suominen Pauli , Punnonen Kari , Rajamäki Allan , Irjala Kerttu
Published: January 2020

During the last few years the measurement of serum-soluble transferrin receptor (sTfR) has been introduced as a tool to detect iron deficiency and as an analyte to differentiate between anemia caused by iron deficiency (IDA) and that caused by chronic disease (ACD). Commercially available methods have emerged to make diagnostics by sTfR more readily accessible. We documented the analytical performance of a newly introduced IDeATM sTfR immunoenzymometric assay (IEMA) by Orion Diagnostica. We also evaluated its clinical performance in 98 consecutive anemic patients, with information derived from bone marrow aspirate samples as the reference for iron status. The clinical usefulness of two other commercially available sTfR assays was assessed for comparison. The analytical performance and clinical applicability of the IDeA were sufficient to support reliable clinical work. We conclude that IDA and iron deficiency in the presence of inflammatory states can be differentiated efficiently from ACD with this new commercial test to measure sTfR.
Detection of hepatitis B surface antigen in whole blood by coupled particle light scattering (Copalis™)
Authors: Benecky Michael J , Post Diane R , Schmitt Susan M , Kochar Manish S
Published: January 2020

Coupled particlelight scattering (Copalis™) is a homogeneous immunoassay technology that permits simultaneous determination of multiple analytes in serum, plasma, or whole blood. Copalis differentiates monomeric latex microparticles from latex aggregates and cells on the basis of their unique light scatter properties. Copalis readily discriminates small (∼0.1 μm) differences in latex microparticle size. Therefore, multiple simultaneous assays are configured by the use of mixtures of different-size latex microparticles. The Copalis research immunoassay for hepatitis B surface antigen (HBsAg) is configured in a sandwich format where the extent of light scatter histogram broadening due to HBsAg-mediated binding of colloidal gold to latex provides the basis for antigen quantification. Simultaneous Copalis forward- and wide-angle light scatter measurements allow discrimination of latex microparticles from the cell components of whole blood. Consequently, direct detection of HBsAg in unprocessed whole-blood samples by Copalis is feasible.
Ethylene glycol poisoning: toxicokinetic and analytical factors affecting laboratory diagnosis
Authors: Eder Anne F , McGrath Cindy M , Dowdy Yvonne G , Tomaszewski John E , Rosenberg Frances M , Wilson Robert B , Wolf Bryan A , Shaw Leslie M
Published: January 2020

Ethylene glycol poisoning is an important toxicological problem in medical practice because early diagnosis and treatment can prevent considerable morbidity and mortality. When ingested in the form of antifreeze or other automotive products, ethylene glycol results in central nervous system depression, cardiopulmonary compromise, and renal insufficiency. Metabolism of ethylene glycol to organic acids is required for metabolic derangement and organ damage. Laboratory features of ethylene glycol poisoning include increased anion gap metabolic acidosis, increased osmolal gap, calcium oxalate crystalluria, and detectable ethylene glycol in serum. This Case Conference integrates discussion of the toxicokinetic and analytical variables that affect the laboratory diagnosis of ethylene glycol intoxication.
Clinical utility of the soluble transferrin receptor and comparison with serum ferritin in several populations
Authors: Mast Alan E , Blinder Morey A , Gronowski Ann M , Chumley Cara , Scott Mitchell G
Published: January 2020

Soluble transferrin receptor (sTfR) and ferritin concentrations were measured in a variety of clinical settings to compare the ability of these two tests to identify iron deficiency. Among 62 anemic patients who either had a bone marrow aspirate performed or had a documented response to iron therapy, the diagnostic sensitivity and specificity of sTfR (at a diagnostic cutoff of >2.8 mg/L) were 92% and 84%, respectively, with a positive predictive value of 42% in this population. Ferritin (≤12 μg/L) had a sensitivity of 25% and a specificity of 98%. However, the sensitivity and specificity of ferritin could be improved to 92% and 98%, respectively, by using a diagnostic cutoff value of ≤30 μg/L, resulting in a positive predictive value of 92%. Ferritin and sTfR were also measured in 267 outpatient samples and 112 medical students. In the outpatient group, the two tests agreed in 73% of the samples; however, 25% of the samples had ferritin values >12 μg/L and increased sTfR. Among the medical students, there was 91% agreement between the two tests, but 7% of the samples had ferritin ≤12 μg/L and normal sTfR. Together, these data suggest that measurement of sTfR does not provide sufficient additional information to ferritin to warrant routine use. However, sTfR may be useful as an adjunct in the evaluation of anemic patients, whose ferritin values may be increased as the result of an acute-phase reaction.
X-Chromosome inactivation in healthy females: incidence of excessive lyonization with age and comparison of assays involving DNA methylation and transcript polymorphisms
Authors: El Kassar Nahed , Hetet Gilles , Brière Jean , Grandchamp Bernard
Published: January 2020

Skewed lyonization in healthy females represents the major disadvantage of X-chromosome-based clonality assays. Because most techniques are based on the difference in DNA methylation between active and inactive X-chromosomes, incomplete DNA digestion may occur, giving an unreliable clonality result. Here, we compare two different techniques carried out in healthy females belonging to three age groups and in a group of patients with essential thrombocythemia. The first technique involved the human androgen receptor gene, the second the transcript analysis of the iduronate-2-sulfatase, P55, and glucose-6-phospate dehydrogenase genes. Results between both techniques were concordant in most cases except in neonates, and the same pattern was observed in all fractions in healthy females. We conclude that: (a) clonality assays involving DNA and RNA polymorphisms are usually concordant except in neonates; (b) appropriate control tissue embryologically related to the sample must be chosen to eliminate excessive lyonization; (c) acquired skewing increases with age, whereas nonrandom lyonization is a rare phenomenon.
Comprehensive analytical strategy for mutation screening in 21-hydroxylase deficiency
Authors: Krone Nils , Roscher Adelbert Anton , Schwarz Hans Peter , Braun Andreas
Published: January 2020

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease with a wide range of clinical manifestations. It is most often caused by deficiency of steroid 21-hydroxylase, reflecting any of a wide range of mutations in the 21-hydroxylase (CYP21) gene. A major challenge in molecular diagnostics of CAH is the high homology between the CYP21 gene and the CYP21P pseudogene and the phenomenon of apparent gene conversion, which inactivates the functional gene. In this study we devised an improved stepwise diagnostic procedure involving nonradioactive Southern blotting and direct DNA sequencing. This strategy led to a successful elucidation of the molecular cause of the disease in 181 out of 182 unrelated alleles in a total of 91 clinically and biochemically characterized patients. We were able to identify all classical known disease-causing mutations of the 21-hydroxylase gene and a novel nonsense mutation (bp 670, A→C, Y97X). Our method also allows the reliable, secure diagnosis of the heterozygous configuration and may therefore be used for pre-, peri-, and postnatal diagnosis of CAH, even when informative data of the index patient are lacking. Furthermore, it can be used to confirm the diagnosis of CAH in newborns detected in 17-hydroxyprogesterone screening programs.
The precursor form of the human kallikrein 2, a kallikrein homologous to prostate-specific antigen, is present in human sera and is increased in prostate cancer and benign prostatic hyperplasia
Authors: Saedi Mohammad S , Hill Timothy M , Kuus-Reichel Kristine , Kumar Abhay , Payne Janice , Mikolajczyk Stephen D , Wolfert Robert L , Rittenhouse Harry G
Published: January 2020

Prostate-specific antigen (PSA, hK3) is a diagnostic marker for prostatic cancer but lacks the specificity to sufficiently distinguish between prostatic cancer and benign prostatic hyperplasia (BPH). Human glandular kallikrein 2 (hK2) has been proposed as a potential diagnostic marker for prostate cancer that could complement the current PSA test. Recently we demonstrated that proPSA is present in prostate cancer sera. This study examines the expression of prohK2 in prostate cells and its presence in human sera. Western blot analysis was used to assess prohK2 expression in the human carcinoma cell line, LNCaP. A highly specific and sensitive dual monoclonal immunoassay for prohK2 was developed and used to assess the presence of prohK2 in human sera. prohK2 was detected in the spent media of LNCaP cells. Furthermore, prohK2 was present at immunodetectable concentrations in human sera, and its concentration was increased in prostatic cancer and BPH. These results indicate for the first time that prohK2 is secreted by human prostate cells and is a major component of uncomplexed (free) hK2 in human sera. In addition, prohK2 in human sera is associated with prostate disease and thus may be a useful marker for prostatic cancer and BPH.
Urinary free deoxypyridinoline by chemiluminescence immunoassay: analytical and clinical evaluation
Authors: Rosano Thomas G , Peaston Robert T , Bone Henry G , Woitge Henning W , Francis Roger M , Seibel Markus J
Published: January 2020

We evaluated an automated chemiluminescence immunoassay (CLIA) developed for the measurement of urinary free deoxypyridinoline (DPD). The new DPD method by CLIA is based on the competition of DPD with particle-bound pyridinoline for a limited amount of monoclonal mouse anti-DPD antibody. Total imprecision (CV) was 3.2–9.0% at 30–270 nmol/L. Regression analysis of urinary DPD concentration (second morning-void) measured by CLIA (y) and enzyme immunoassay (EIA) for adult volunteers (n = 449) with and without bone disease revealed a best fit equation of: y = 1.08 ± 0.03x − 1.15 ± 0.98 nmol/L (r = 0.964, Sy‖x = 14 nmol/L). CLIA and EIA methods were correlated with HPLC measurement of urinary free DPD (r = 0.846 and 0.871, respectively). For healthy adults, the creatinine-normalized excretion of DPD (mean ± SD) measured by CLIA for 61 men (4.1 ± 1.2 μmol DPD/mol creatinine) and 76 premenopausal women (5.3 ± 1.8 μmol DPD/mol creatinine) did not differ significantly (P >0.05) from DPD excretion measured by EIA, and both immunoassays showed a significant gender difference (P <0.001) in reference intervals. In a clinical trial, DPD excretion (μmol DPD/mol creatinine) measured by CLIA differed substantially from the reference population for 54 untreated pagetic (12.7 ± 8.0 SD), 255 untreated osteoporotic (7.5 ± 4.1), 21 osteomalacic (12.4 ± 8.5), 17 primary hyperparathyroid (9.4 ± 4.4), and 14 secondary hyperparathyroid (9.2 ± 5.1) patients. Clinical sensitivities of the CLIA and EIA methods range from 38% to 80% in bone disorders and limit the use of the DPD measurement in disease detection. DPD excretion after pamidronate treatment in a subgroup of the pagetic patients fell dramatically as assessed by CLIA or EIA. We conclude that the automated CLIA method for DPD is a convenient and reliable method that may aid in the evaluation and management of bone disease and is applicable to high volume testing in the routine clinical laboratory.
Hemoglobin Rambam (β69[E13]Gly→Asp), a pitfall in the assessment of diabetic control: characterization by electrospray mass spectrometry and HPLC
Authors: Bissé E , Zorn N , Eigel A , Lizama M , Huaman-Guillen P , März W , Van Dorsselaer A , Wieland H
Published: January 2020

Hemoglobin (Hb) Rambam, or β69[E13]Gly→Asp, has been identified in a German woman also suffering from non-insulin-dependent diabetes mellitus and chronic obstructive pulmonary disease. This is the first observation of this Hb variant in a German family thus far. The detailed evaluation of its structure using electrospray mass spectrometry revealed new minor glycohemoglobin components and showed that the attachment of glucose to the β NH2 terminus occurred at an almost identical rate in both wild-type and mutant β-chains. However, the introduction of a carboxyl group at β69 seems to increase the glycation of ε-amino groups of lysine residues. The glycemic state in the propositus was well reflected by the total glycohemoglobin concentrations but not by the Hb A1C values, which did not reflect hemoglobin glycation in this patient. This case demonstrates that Hb A1C cannot be used reliably in the management of diabetic patients carrying Hb variants such as Hb Rambam. Functional studies of the whole blood of the heterozygous carrier demonstrated extremely low oxygen affinity, which may have been caused by increased 2,3-diphosphoglycerate related to chronic obstructive pulmonary disease and hyperthyroidism. None of the clinical symptoms could be directly associated to Hb Rambam.
Clinical evaluation of the Serum CrossLaps One Step ELISA, a new assay measuring the serum concentration of bone-derived degradation products of type I collagen C-telopeptides
Authors: Christgau Stephan , Rosenquist Christian , Alexandersen Peter , Bjarnason Nina Hannover , Ravn Pernille , Fledelius Christian , Herling Christian , Qvist Per , Christiansen Claus
Published: January 2020

The Serum CrossLapsTM One Step ELISA is a sandwich assay using two monoclonal antibodies specific for a β-aspartate form of the epitope EKAHDGGR derived from the carboxy-terminal telopeptide region of type I collagen α1-chain. Our objective was to assess the clinical value of the Serum CrossLaps assay for monitoring antiresorptive therapy in osteoporosis treatment. Samples obtained from postmenopausal women treated with different doses of cyclic or continuous hormone replacement therapy (HRT) with an estrogen analog (tibolone) or with a bisphosphonate (ibandronate) were measured in the Serum CrossLaps One Step ELISA at baseline and at various time points during therapy. The corresponding urine samples were measured in the urine CrossLapsTM ELISA and corrected for creatinine excretion. The serum CrossLaps measurements and corresponding urinary CrossLaps measurements were highly correlated (r >0.8 for all studies). The serum and urine CrossLaps measurements showed a significant decrease among the women treated with clinically relevant doses of either of the antiresorptive agents. Furthermore, the annual percentage change in bone mineral density (BMD) correlated with the measured changes in CrossLaps concentration. The serum CrossLaps assay showed a specificity of 83–100% and a sensitivity of 59–83% for assessing BMD changes. The corresponding values for the creatinine-corrected urinary measurements were 83–92% specificity and 68–79% sensitivity. We conclude that performance of the convenient Serum CrossLaps One Step ELISA is at least equivalent to that of the urine text for follow up of antiresorptive treatment in osteoporosis. Further studies are needed to optimize its use in this and other clinical applications.
Lipoprotein(a) as a risk factor for ischemic heart disease: metaanalysis of prospective studies
Authors: Craig Wendy Y , Neveux Louis M , Palomaki Glenn E , Cleveland Meghan M , Haddow James E
Published: January 2020

Although in vitro studies support a pathophysiologic role for lipoprotein(a) [Lp(a)] in the development of atherosclerosis, and retrospective studies consistently report that there is a relationship between Lp(a) and ischemic heart disease (IHD), the conclusions drawn from prospective studies about this relationship have been inconsistent. To address this issue, we have performed a metaanalysis of data available from prospective studies. Lp(a) concentrations expressed as mass units vary markedly between studies, reflecting the need for assay standardization. In 12 of 14 prospective studies, Lp(a) concentrations are higher in subjects who later develop IHD (cases) than in those who do not (controls), although there is variation in the size of the effect. Sample storage temperature may contribute to this variability. When the studies are analyzed collectively, Lp(a) concentrations are significantly higher in cases than in controls, and the extent of the effect is similar in men and women. These findings provide evidence in support of a causal role for Lp(a) in the development of atherosclerosis. Measurement of Lp(a) may be useful to guide management of individuals with a family history of IHD or with existing disease. The separation in values between cases and controls is not, however, sufficient to allow the use of Lp(a) as a screening test in the general population.
Use of phenylalanine-to-tyrosine ratio determined by tandem mass spectrometry to improve newborn screening for phenylketonuria of early discharge specimens collected in the first 24 hours
Authors: Chace Donald H , Sherwin John E , Hillman Steven L , Lorey Fred , Cunningham George C
Published: January 2020

We compared the screening interpretation of fluorometric analytical results for phenylketonuria (PKU) with tandem mass spectrometry (MS/MS) in filter paper blood spots collected from newborns <24 h of age. In MS/MS, both Phe and Tyr are quantified. Two hundred and eight blood spots collected from infants <24 h of age were retrieved from storage from the California newborn screening program. These samples had been categorized on the basis of fluorometric analysis as initial negative, initial positive for hyperphenylalaninemia with negative determination on recall, or initial positive for hyperphenylalaninemia and confirmed on follow up as PKU or variant hyperphenylalaninemia. The retrieved samples were analyzed in a blinded fashion using MS/MS. Correlation analysis of fluorometry vs MS/MS for Phe concentration was high, with a Pearson correlation coefficient of 0.817. When 180 μmol/L was used as the cutoff Phe concentration for MS/MS and 258 μmol/L was used as the cutoff for fluorometry, all infants with confirmed classical PKU and variant hyperphenylalaninemia were detected. MS/MS analysis reduced the number of false-positive results from 91 to 3. Simultaneous quantification of Phe and Tyr by MS/MS with the use of a cutoff Phe/Tyr molar ratio of 2.5 further reduced the number of false positives to 1. Samples from affected infants showed a discernible trend of increasing Phe concentration and Phe/Tyr molar ratio with age of collection. These results demonstrate the utility of MS/MS in the routine PKU screening of early-discharge newborns. MS/MS reduces the false-positive rate of fluorometric screening almost 100-fold because of the improved accuracy and precision of Phe measurement and simultaneous confirmation with the Phe/Tyr molar ratio. In addition to the detection of PKU, MS/MS can also detect other aminoacidopathies and disorders of fatty acid and organic acid metabolism with lower false-positive rates than other methods currently used in newborn screening programs.
Characterization of frequent polymorphisms in intron 2 of CYP21: application to analysis of segregation of CYP21 alleles
Authors: Killeen Anthony A , Jiddou Renée R , Sane Kumud S
Published: January 2020

The gene encoding adrenal steroid 21-hydroxylase, CYP21, is located in the MHC class III region. Most cases of congenital adrenal hyperplasia (CAH) are caused by mutations in this gene, and most mutations appear to arise from gene conversion-like events involving the transfer of deleterious sequences from the pseudogene, CYP21P, which is located within 30 kb of CYP21. Approximately 20–30% of mutations are caused by deletions of CYP21. The second intron of CYP21 is polymorphic, and several base substitutions that include nt395, nt453, and nt601 have been reported; however, the frequencies of these polymorphisms are unknown. Using a combination of cleavase fragment length polymorphism analysis and direct sequencing, we examined the sequence of intron 2 in seven wild-type CYP21 genes and determined the frequency of polymorphisms at nt395, nt453, and nt601 in 48 chromosomes from the parental generation of Centre d’Étude du Polymorphisme Humain families. The observed frequencies of bases at these positions were as follows: 395C, 0.17; 395T, 0.83; 453C, 0.71; 453T, 0.29; 601A, 0.1; and 601C, 0.9. Using a PCR/restriction digestion approach to examine these intragenic markers, we could follow the segregation of alleles in informative families with 21-hydroxylase deficiency and identify deletions of CYP21. We emphasize that this method should be used in conjunction with other molecular genetic techniques for diagnosis of CAH. In addition to their potential use in families with CAH, these markers may be of use in genetic studies of the MHC in humans.
Quantitative reverse transcription-PCR assay of the RNA component of human telomerase using the TaqMan fluorogenic detection system
Authors: Yajima Tomomi , Yagihashi Atsuhito , Kameshima Hidekazu , Kobayashi Daisuke , Furuya Daisuke , Hirata Koichi , Watanabe Naoki
Published: January 2020

We established the validity of a quantitative reverse transcription (RT)-PCR assay for the RNA component of human telomerase (hTR), using the TaqMan fluorogenic detection system. Using this assay, we quantified hTR expression in two human pancreatic cancer cell lines, ASPC-1 and MIAPaCa-2. Our results indicated that hTR expression in MIAPaCa-2 was 1.99-fold higher than that in ASPC-1 cells. This TaqMan RT-PCR assay appears to be useful in determining the amount of hTR in clinical specimens.
Routine analysis of plasma busulfan by gas chromatography–mass fragmentography
Authors: Lai Wai-Kai , Pang Chi-Pui , Law Lap-Kay , Wong Raymond , Li Chi-Kong , Yuen Patrick Man-Pan
Published: January 2020

Busulfan (BU) is a widely used alkylating agent for antineoplastic therapy and marrow ablation in preparation for bone marrow transplantation (BMT). High-dose BU often leads to successful preparation and low relapse but is associated with veno-occlusive disease of liver. We established a protocol to determine postdosage plasma BU concentrations by gas chromatography–mass fragmentography in an attempt to relate clinical outcome to plasma BU concentrations. We used nonisotopic pusulfan as the internal standard. After extraction into ethyl acetate, BU and pusulfan were iodinated into 1,4-diiodobutane and 1,5-diiodopentane, respectively. Gas chromatography–mass spectrometry (GC–MS) analysis was carried out on an Hewlett–Packard (HP) 5890II gas chromatograph with a 30-m 100% methyl silicon narrow bore, fused-silica capillary column interfaced with an HP 5970A mass spectrometer. Helium was the carrier gas. The sample molecules were identified by total ion monitoring and quantified by selective ion monitoring of m/z 183 and 197. The calibration curve was linear to 4 mg/L. The limit of quantification was 0.04 mg/L, and the analytical recovery was ∼97%. The within-day and between-day imprecision (CV) was <6% and 9%, respectively. In a preliminary study of 12 children, the BU areas under the BU-time curve were 616–949 μmol · min/L after the first dose and 793-1143 μmol · min/L after the fifth dose. We conclude that the GC–MS procedure is suitable for routine analysis of plasma BU.
Increased concentrations of prostate-specific antigen in maternal serum from pregnancies affected by fetal Down syndrome
Authors: Lambert-Messerlian Geralyn M , Canick Jacob A , Melegos Dimitrios N , Diamandis Eleftherios P
Published: January 2020

Down syndrome is one of the most common causes of mental retardation in the industrialized world. Prenatal serum screening to identify mothers at risk of carrying a fetus affected with Down syndrome is presently part of routine obstetrical care. Prostate-specific antigen (PSA) concentration was measured in stored second-trimester maternal serum samples from 19 pregnancies affected with fetal Down syndrome and in 95 samples from unaffected pregnancies, with each case matched to five controls for gestational age and duration of frozen sample storage. Concentrations of PSA in Down syndrome pregnancy were significantly higher (case median = 2.28 multiples of the median; P = 0.02) than in unaffected pregnancy. PSA concentrations were not significantly correlated with the current serum screening analytes, alpha-fetoprotein, unconjugated estriol, or human chorionic gonadotropin in either cases or controls. The increased maternal serum PSA concentrations in Down syndrome pregnancy and their relative independence from other markers suggest the possible utility of PSA as a prenatal screening marker for fetal Down syndrome.
Development of FlexSure® HP—an immunochromatographic method to detect antibodies against Helicobacter pylori
Authors: Schrier Wayne H , Schoengold Ronald J , Baker Josefina T , Norell Joyce L , Jaseph Corey L , Okin Yelena , Doe Jeam Y , Chandler Howard
Published: January 2020

We describe a solid-phase immunochromatographic serologic test, FlexSure® HP, to detect IgG antibodies against Helicobacter pylori. H. pylori colonize the stomach and proximal duodenum, cause ulcer disease and mucosa-associated lymphoid tissue lymphoma, and have a role in the development of other disorders, including gastric adenocarcinoma. FlexSure HP consists of a test strip, conjugate pad, and absorbent pad, in a novel reverse-flow chromatography format. In these studies, FlexSure HP was demonstrated to be specific for IgG antibodies against H. pylori. The reactive cutoff of the test was consistent with [13C]urea breath test and commercially available ELISAs. FlexSure HP had 94% sensitivity, 88% specificity, and 91% accuracy relative to [13C]urea breath test; and 95% sensitivity, 94% specificity, and 95% overall agreement relative to high-molecular-mass cell-associated protein enzyme immunoassay (HM-CAP EIA). FlexSure HP is a simple-to-perform, visually read test requiring no specialized training, equipment, or instrumentation, and yields rapid, accurate, qualitative results.
Multivariate discrimination for phenylketonuria (PKU) and non-PKU hyperphenylalaninemia after analysis of newborns’ dried blood-spot specimens for six amino acids by ion-exchange chromatography
Authors: Reilly Andrew A , Bellisario Ronald , Pass Kenneth A
Published: January 2020

Ion-exchange HPLC was developed for testing dried blood-spot specimens from newborns. The method is suitable for quantitative confirmatory testing of abnormal specimens detected in the New York State Newborn Screening Program. Positive specimens were initially identified among all New York State newborns with semiquantitative bacterial inhibition assays (BIA) for aminoacidopathies, including phenylketonuria (PKU) and non-PKU hyperphenylalaninemia (HP), maple syrup urine disease, and homocystinuria. A selection of 1346 specimens from routine BIA screening, including 131 newborns with PKU or persistent HP, were tested by HPLC. Of 179 BIA results that were falsely positive, 98 (55%) were also falsely positive by HPLC in which the Phe/Tyr ratio was the discriminator and the threshold was set to attain 100% sensitivity. Investigation of three multivariate discriminatory methods revealed that linear discriminant analysis excluded all but 35 (20%) of the BIA false-positives.
Plasma lipoprotein profiles change significantly during cardiac catheterization
Authors: Miida Takashi , Otsuka Hideaki , Tsuchiya Atsushi , Okada Masahiko
Published: January 2020

Most patients in acute myocardial infarction (AMI) undergo emergent coronary angiography (CAG). However, when to analyze lipoprotein profiles in AMI is not clear. To determine whether lipoprotein profiles change during catheterization, we measured serum lipid and apolipoprotein concentrations in 65 patients (51 men and 14 women) before and after catheterization. Heparin was injected at 50 units/kg for CAG and 200 units/kg for percutaneous transluminal coronary angioplasty (PTCA). We found that cholesterol and triglyceride decreased by 9.4% (P <0.001) and 53.1% (P <0.001), respectively, after catheterization. Apolipoproteins also decreased significantly. Variables decreased two to five times more after PTCA than after CAG. Lipoprotein lipase mass was higher after PTCA (267.8 ± 135.3 μg/L) than after CAG (93.3 ± 48.4 μg/L; P <0.05). In conclusion, lipoprotein profiles change during catheterization. We recommend avoiding analysis of lipoprotein profiles after emergent CAG in AMI.
Homogeneous assay for measuring low-density lipoprotein cholesterol in serum with triblock copolymer and α-cyclodextrin sulfate
Authors: Sugiuchi Hiroyuki , Irie Tetsumi , Uji Yoshinori , Ueno Tomohiro , Chaen Toshiko , Uekama Kaneto , Okabe Hiroaki
Published: January 2020

We have developed a fully automated method for measuring LDL-cholesterol (LDL-C) in human serum without the need for prior separation, using a nonionic surfactant, polyoxyethylene–polyoxypropylene block copolyether (POE-POP), and a sodium salt of sulfated cyclic maltohexaose, α-cyclodextrin sulfate. Of the surfactants tested, POE-POP with a higher molecular mass of the POP block and a greater hydrophobicity reduced the reactivity of cholesterol in lipoprotein fractions; the reactivity in descending order was LDL ≫ VLDL > chylomicron ≈ HDL. Gel filtration chromatographic studies revealed that POE-POP removed lipids selectively from the LDL fraction and allowed them to participate in the cholesterol esterase–cholesterol oxidase coupling reaction system. By contrast, α-cyclodextrin sulfate reduced the reactivity of cholesterol, especially in chylomicrons and VLDL. A combination of POE-POP with α-cyclodextrin sulfate provided the required selectivity for the determination of LDL-C in serum in the presence of magnesium ions and a small amount of dextran sulfate without precipitating lipoprotein aggregates. There was a good correlation between the results of LDL-C assayed by the proposed method and the beta-quantification reference method involving 161 sera with triglyceride concentrations ranging from 0.3 to 22.6 mmol/L.
Simultaneous determination of 6-thioguanine and methyl 6-mercaptopurine nucleotides of azathioprine in red blood cells by HPLC
Authors: Dervieux Thierry , Boulieu Roselyne
Published: January 2020

6-thioguanine (6-TGN) and methyl 6-mercaptopurine nucleotides (Me6-MPNs) are the two major metabolites found in erythrocytes after administration of azathioprine. In an attempt to understand the role of these metabolites in the pharmacologic and toxic activity of thiopurines, we have developed a HPLC method for the simultaneous determination of 6-TGNs and Me6-MPNs in erythrocytes. A simple and rapid treatment procedure based on deproteinization by perchloric acid with dithiothreitol is described. The nucleotides were hydrolyzed to their own bases by heating the sample for 45 min at 100 °C. During acid hydrolysis Me6-MP was converted into a compound analyzed on a Purospher RP18-e column with 0.02 mol/L dihydrogenophosphate buffer–methanol as eluents. With this procedure, mean recoveries of 73.1% and 84.0% for 6-TGN and Me6-MPN derivatives, respectively, were found
A robust approach to reference interval estimation and evaluation
Authors: Horn Paul S , Pesce Amadeo J , Copeland Bradley E
Published: January 2020

We propose a new methodology for the estimation of reference intervals for data sets with small numbers of observations or for those with substantial numbers of outliers. We propose a prediction interval that uses robust estimates of location and scale. The SAS software can be readily modified to do these calculations. We compared four reference interval procedures (nonparametric, transformed, robust with a nonparametric lower limit, and transformed robust) for sample sizes of 20, 40, 60, 80, 100, and 120 from χ2 distributions of 1, 4, 7, and 10 df. χ2 distributions were chosen because they simulate the skewness of distributions often found in clinical chemistry populations. We used the root mean square error as the measure of performance and used computer simulation to calculate this measure. The robust estimator showed the best performance for small sample sizes. As the sample size increased, the performance values converged. The robust method for calculating upper reference interval values yields reasonable results. In two examples using real data for haptoglobin and glucose, the robust estimator provides slightly smaller upper reference limits than the other procedures. Lastly, the robust estimator was compared with the other procedures in a population where 5% of the values were multiplied by a factor of 5. The reference intervals were calculated with and without outlier detection. In this case, the robust approach consistently yielded upper reference interval values that were closer to those of the true underlying distributions. We propose that robust statistical analysis can be of great use for determinations of reference intervals from limited or possibly unreliable data.