bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrarapid drug metabolism: PCR-based detection of CYP2D6 gene duplication
Authors: Steijns Linda S W , Van Der Weide Jan
Published: January 2020

The enzyme debrisoquine 4-hydroxylase (CYP2D6), which metabolizes many widely used drugs, is highly polymorphic. The activity of the enzyme ranges between subjects from ultrafast to a complete absence. Therefore, metabolic capacity varies, producing intersubject differences in therapeutic efficacy and side effects at standard recommended doses. Up to 7% of Caucasians may demonstrate ultrarapid drug metabolism (UM) because of inherited alleles with multiplicate functional CYP2D6 genes, causing an increased amount of enzyme to be expressed. Identification of UM subjects is of potential clinical importance for adjustment of doses in drug therapy, as well as to avoid misidentification of noncompliance. In our study, we tested recently designed PCR assays for the detection of the UM genotype. We found a 3.5% prevalence of UMs carrying duplicate active CYP2D6 genes in a population consisting of 202 psychiatric patients.
Biological variations and genetic reference values for apolipoprotein E serum concentrations: results from the STANISLAS cohort study
Authors: Vincent-Viry Monique , Schiele Françoise , Gueguen René , Bohnet Karin , Visvikis Sophie , Siest Gérard
Published: January 2020

Serum apolipoprotein (apo) E concentrations were determined by immunoturbidimetry in 4284 subjects from 4 to 71 years of age and belonging to 1003 nuclear families recruited for the STANISLAS cohort study between January 1994 and August 1995. Values for apo E ranged from 16 to 169 mg/L, with a geometric mean ± SD values of 46.6 ± 13.8 mg/L in the overall sample. The interindividual variability varied from 24.6% to 32.0% among family members. Females exhibited higher apo E values than males until the age of 17–26 years. Conversely, after the age of 26 years, serum apo E concentrations were higher in men than in women. Biological factors affecting serum apo E concentrations were described in fathers, mothers, sons, and daughters and explained up to 32.0% of the apo E variability in daughters and 19.0% in fathers. The main biological factors affecting apo E concentrations were the following: apo E polymorphism, waist-to-hip ratio, oral contraceptive intake, puberty, body mass index, age, and gender. Given the importance of apo E polymorphism in the regulation of apo E concentrations, we recommend the use of genetic-based reference values for the clinical interpretation of serum apo E concentrations.
Sodium dodecyl sulfate-agarose gel electrophoresis of urinary proteins: application to multiple myeloma
Authors: Le Bricon Thierry , Erlich Danielle , Bengoufa Djaouida , Dussaucy Michelle , Garnier Jean-Pierre , Bousquet Bernard
Published: January 2020

We evaluated a new sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) for urinary protein analysis in patients with multiple myeloma (MM; n = 47; ages, 62 ± 2 years, mean ± SE). Abnormal proteinuria (mean = 1872 ± 360 mg/24 h) was present in 95% of the samples; 75% of the patients had some sign of renal dysfunction (glomerular and/or tubular) according to their SDS-AGE pattern. A band suggesting Bence Jones proteinuria (BJP) was detected in 40 vs 33 specimens by routine AGE. Immunofixation identified BJP in 38 patients; the calculated sensitivity of SDS-AGE for BJP was 97%. Excellent correlation (P <0.0001) was obtained with routine AGE (r = 0.994) and immunonephelometry (r = 0.963) for light chain quantification. SDS-AGE allows easy evaluation of renal dysfunction and shows high sensitivity for BJP detection. In a specialized laboratory, it is useful for following the progress of MM patients through the semiquantification of BJP.
Low percentage of aluminoxamine and ferrioxamine in uremic serum after desferrioxamine administration
Authors: Menéndez-Fraga Pilar , Fernández-Martín Jose L , Blanco-González Elisa , Cannata-Andía Jorge B
Published: January 2020

HPLC was used to study the effectiveness of two different desferrioxamine (DFO) administration strategies (15 mg/kg DFO, 1 h or 44 h before dialysis) on generation of aluminoxamine and ferrioxamine in five hemodialysis patients. The percentage of ultrafilterable aluminum and iron in these patients was also investigated by electrothermal atomic absorption spectrometry. The administration of DFO in both schemes increased the ultrafilterable serum aluminum concentrations from a mean of 17.1 ± 1.6% to a mean of 75.7 ± 14.1%. However, 1 h after DFO infusion, only 38.8 ± 7.7% of the total serum aluminum was bound to DFO; 44 h after DFO infusion, only 15.8 ± 8.0% was bound. Similar results were obtained for ferrioxamine. These results suggest that the ultrafilterable serum fraction contains aluminum and iron chelated by DFO and by DFO metabolites, which retain similar metal-chelating abilities.
Predictive value of determinations of zinc protoporphyrin for increased blood lead concentrations
Authors: Froom Paul , Kristal-Boneh Estella , Benbassat Jochanan , Ashkanazi Ruth , Ribak Joseph
Published: January 2020

Blood lead (PbB) and red cell zinc protoporphyrin (ZPP) concentrations are widely used biomarkers for lead toxicity. It is uncertain, however, whether either or both are needed for monitoring lead exposure and how discordant PbB and ZPP values should be interpreted. We reviewed the results of PbB and ZPP determinations in 94 workers in a lead-battery plant over a 13-year period and retrieved all 807 sets of tests in which both PbB and ZPP were available, with a follow-up PbB value 6 months later. PbB exceeded 1.93 μmol/L (40 μg/dL) in 414 (51%), and 2.90 μmol/L (60 μg/dL) in 105 (14%) of the blood samples. We derived the test properties of various ZPP concentrations for concurrent “toxic” PbB concentrations, defined as ≥1.93 and 2.90 μmol/L (40 and 60 μg/dL). The results indicated that, given a population of lead-exposed workers with a 10% prevalence of PbB of ≥2.90 μmol/L (60 μg/dL), a policy of testing PbB only in those with ZPP >0.71 μmol/L (40 μg/dL) would obviate 42% of the PbB tests, but would miss about three cases with toxic PbB concentrations in every 200 workers at risk. A finding of increased ZPP concentrations with a concurrent “nontoxic” PbB was associated with an increased risk of a toxic PbB concentration 6 months later. We conclude that (a) screening by testing only ZPP does not safeguard exposed persons against lead toxicity, and (b) the frequency of PbB monitoring should be guided by estimates of the risk of future lead toxicity in individual workers.
Effect of serum-clot contact time on clinical chemistry laboratory results
Authors: Zhang Dongbo J , Elswick R K , Miller W Greg , Bailey Jimmy L
Published: January 2020

The effect of serum-clot contact time on laboratory results was studied by dividing each blood specimen into four blood collection tubes. The control sera were separated from the clot within 30 min of the collection. The other tubes were incubated at 32 °C, and the sera were separated at 3, 6, and 24 h. The sera were stored at 4 °C and analyzed at the same time. The stability of the tests was determined by comparing the results of the 3-, 6-, and 24-h samples with the values from the 30-min samples. The acceptable limits around the 30-min values were derived from the analytical and intraindividual biological variation of the tests. A total of 63 analytes were studied. Potassium, phosphorous, and glucose were the least stable, and the serum should be separated from the clot within 3 h for these analytes. Albumin, bicarbonate, chloride, C-peptide, HDL-cholesterol, iron, LDL-cholesterol, and total protein should be separated within 6 h. The other analytes were stable for 24 h of serum-clot contact.
Correlation of plasma concentrations of cystatin C and creatinine to inulin clearance in a pediatric population
Authors: Stickle Douglas , Cole Barbara , Hock Karl , Hruska Keith A , Scott Mitchell G
Published: January 2020

Measurement of blood concentrations of cystatin C (cysC), a cysteine protease inhibitor present in human plasma, has been suggested for use as an indicator of glomerular filtration rate (GFR) in a manner analogous to the use of plasma creatinine (SCR). In this study, cysC and SCR were measured in plasma from pediatric patients (4–19 years) with renal disease for whom a “gold standard” measurement of GFR via inulin clearance (CIN) was available. The data analyses were divided into two age groups: group A (4–12 years, n = 26) and group B (12–19 years, n = 34). For both age groups, the linear correlation coefficient of [cysC]−1 vs CIN (mL/min/1.73 m2) (r = 0.765 for group A and r = 0.869 for group B) was less than that of the linear correlation coefficient of [SCR]−1 vs CIN (r = 0.841 for group A and r = 0.892 for group B). As a single measurement for detection of abnormal GFR, however, the optimum receiver-operator characteristic point for cysC measurement (for group A at cysC >1.2 mg/L, sensitivity = 80%, specificity = 91%; and for group B at cysC >1.4 mg/L, sensitivity = 87%, specificity = 100%) was numerically superior to that for SCR measurement (for group A at SCR >8.0 mg/L, sensitivity = 67%, specificity = 100%; and for group B at SCR >9.0 mg/L, sensitivity = 91%, specificity = 91%), using a reference value for normal GFR of CIN > 90 mL/min/1.73 m2. However, these differences were not statistically significant. CysC measurement appears to be broadly equivalent to SCR measurement for estimation of GFR in pediatric patients.
Cardiac troponins I and T in patients with suspected acute coronary syndrome: a comparative study in a routine setting
Authors: Hetland Øyvind , Dickstein Kenneth
Published: January 2020

We compared cardiac troponin I (cTnI), using Access®, Sanofi Pasteur, and cardiac troponin T (cTnT), using Elecsys®, Boehringer Mannheim, in the first two routine blood samplings in a routine panel of cardiac markers for the biochemical diagnostic evaluation of patients with symptoms of acute myocardial infarction (AMI). No significant differences in the overall clinical performances of cTnI and cTnT were observed for the diagnosis of AMI (n = 68), but cTnI demonstrated lower initial sensitivity and higher specificity compared with cTnT. cTnT was increased to higher relative values than cTnI (P = 0.023). Discordances were found between cTnI and cTnT in sample I but not in sample II; positive cTnT/negative cTnI was more common than the opposite discordance (P = 0.027). cTnT was more frequently increased in patients with unstable angina pectoris (UAP) than cTnI (P = 0.038), with no significant differences between sample I and sample II; discordant results with respect to cTnI and cTnT appeared in 6 (33%) of these patients, all of which were positive for cTnT and negative for cTnI. Four patients with UAP (22%) developed AMI within 4 months; three were associated with increased cTnI and cTnT at the time of initial testing, and one was discordant (positive cTnT). In patients classified with no acute coronary syndrome (n = 84), five concordant positives for cTnI and cTnT were observed, indicating the existence of a myocardial injury of recent origin in these patients. AMI evolved in one of these patients 5 months later. We conclude that cTnT and cTnI detect acute myocardial injury with equal clinical performance in AMI patients classified by WHO criteria. cTnT was more frequently increased in patients with UAP than cTnI, but the clinical significance of this discordance could not be determined from this study.
Response of several markers of bone collagen degradation to calcium supplementation in postmenopausal women with low calcium intake
Authors: Kamel Saïd , Fardellone Patrice , Meddah Boumedienne , Lorget-Gondelmann Florence , Sebert Jean Luc , Brazier Michel
Published: January 2020

We investigated the response of bone-specific resorption markers in fasting urine samples from postmenopausal women with low daily dietary calcium (Ca) intake (<800 mg/day) who received either Ca supplementation (1200 mg/day, n = 18) or placebo (n = 14) for 2 months. We measured urinary hydroxyproline, total pyridinoline, and deoxypyridinoline by HPLC, and free deoxypyridinoline (i-F-Dpd) and N- and C-telopeptide fragments of type I collagen (NTX and CTX) by immunoassays. Before supplementation, the urine concentrations of bone resorption markers in the 32 subjects were not statistically different from those measured in 21 subjects with daily dietary Ca intake >800 mg/day. In contrast to the placebo group, Ca supplementation decreased all collagen-related degradation markers except i-F-Dpd as early as the first month. The magnitude of response after 2 months of Ca supplementation, expressed as mean percentage of decrease from baseline values or as individual Z scores, was greatest for the telopeptide assays. Furthermore, the percentage of change assessed at 2 months was greater than the within-person biological variability (CV) assessed in the placebo-treated women for NTX and CTX, whereas for the other markers the percentage of change was very close of the within-person CVs. We conclude that cross-linked telopeptide fragments of type I collagen most sensitively reflect the change in bone resorption after Ca supplementation.
Cross-reactivity of fosphenytoin in two human plasma phenytoin immunoassays
Authors: Kugler Alan R , Annesley Thomas M , Nordblom Gerald D , Koup Jeffrey R , Olson Stephen C
Published: January 2020

The cross-reactivity of fosphenytoin, a phosphate ester prodrug of phenytoin, was investigated in the Abbott phenytoin TDx®/TDxFLxTM fluorescence polarization immunoassay (TDx) and the Behring Diagnostics phenytoin Emit® 2000 enzyme-multiplied immunoassay (Emit). The first part of our study investigating cross-reactivity utilized in vitro correlation of the two immunoassays with a validated and specific phenytoin HPLC method used to assay plasma samples prepared in several phenytoin and fosphenytoin concentration combinations. Fosphenytoin cross-reacted with both immunoassays, but to a greater extent with TDx. In the second part of the study, empirically-derived models that best explained the in vitro data were used to predict “immunoassay-derived” phenytoin concentrations in plasma samples collected from actual patients after intravenous (IV) or intramuscular (IM) fosphenytoin dosing. The greatest degree of phenytoin concentration overestimation occurred at times when fosphenytoin concentrations were highest: within 1 to 2 h after IV infusion or during the first 2 to 4 h after IM injection. It is recommended that phenytoin concentrations not be monitored using these or other potentially nonspecific immunoanalytical methods for at least 2 h after IV fosphenytoin infusion or 4 h after IM fosphenytoin injection.
Biological variation of cystatin C: implications for the assessment of glomerular filtration rate
Authors: Keevil Brian G , Kilpatrick Eric S , Nichols Simon P , Maylor Paul W
Published: January 2020

To assess the inherent potential for detecting mild to moderate reductions in glomerular filtration rate, this study determined the biological variability of serum cystatin C and creatinine in 12 healthy subjects. After accounting for analytical variation, interindividual variance accounted for 93% and intraindividual variance accounted for 7% of serum creatinine biological variation. As such, to lie outside the assay reference interval, some subjects must exceed 13 SD from their usual mean value, whereas in others, a change of only 2 SD would be sufficient. For cystatin C, interindividual variation explained 25% and intraindividual variance explained 75% of biological variability. Therefore, the upper limit of the population reference interval for cystatin C is seldom more than 3–4 SD from the mean value of any healthy individual. The critical difference for sequential values significant at P ≤0.05 was calculated as 37% for serum cystatin C and 14% for serum creatinine. We conclude that cystatin C is potentially a better marker for detecting impaired renal function than serum creatinine, but serum creatinine is probably still the better marker for detecting temporal changes of renal function in individuals with established renal disease.
Evaluating stat testing options in an academic health center: therapeutic turnaround time and staff satisfaction
Authors: Kilgore Meredith L , Steindel Steven J , Smith John A
Published: January 2020

We compared centralized vs distributed methods for delivering “stat” test results for blood gas, glucose, and electrolyte assays. The parameters for comparison were as follows: (a) laboratory turnaround time (TAT), (b) therapeutic TAT, and (c) staff satisfaction. Therapeutic TAT, defined as the time from the initiating order to the receipt of the result and the implementation of any indicated change in treatment, was obtained by direct observation of testing procedures at the bedside and timing each step in the process. Observing therapeutic TAT yields information on the impact of laboratory testing methods in the context of clinical decision making. Therapeutic TAT was 1–2 min shorter for bedside testing compared with a satellite laboratory and 9–14 min shorter in the satellite laboratory compared with centralized testing. Satellite laboratories received the highest staff satisfaction scores, followed by bedside testing, with the central laboratory receiving the lowest scores.
Correlation of sperm motility with mitochondrial enzymatic activities
Authors: Ruiz-Pesini Eduardo , Diez Carmen , Lapeña Ana Cristina , Pérez-Martos Acisclo , Montoya Julio , Alvarez Enríque , Arenas Joaquín , López-Pérez Manuel J
Published: January 2020

Until now, little attention has been paid to the contribution of mitochondrial dysfunction to germinal tissue disorders. The target of this study was to investigate the relationship between sperm motility and mitochondrial respiratory chain enzyme activities. The results obtained showed that semen samples of control individuals (n = 33) have substantially higher activities of complexes I, II, and IV compared with those of asthenozoospermic subjects (n = 86). Moreover, a direct and positive correlation was found in the whole population studied between spermatozoa motility and all the mitochondrial respiratory complex activities assayed (I, II, I+III, II+III, and IV). The ratio of these enzymes to citrate synthase (a reliable enzymatic marker of mitochondrial volume) activities did not correlate with sperm motility. This suggests that motility depends largely on the mitochondrial volume within the sperm midpiece. These observations could be of physiopathological relevance because they suggest that factors affecting the mitochondrial energy production could be then responsible for particular cases of idiopathic asthenozoospermia.
Medicare’s demonstration of competitive bidding for clinical laboratory services: what it means for clinical laboratories
Authors: Hoerger Thomas J , Lindrooth Richard C , Eggleston Jennifer L
Published: January 2020

The purpose of Medicare’s Demonstration of Competitive Bidding for Clinical Laboratory Services is to determine whether competitive bidding can be used to provide quality laboratory services at prices below current Medicare reimbursement rates. Here, we present key features of the preliminary design for the demonstration. The following areas are covered: scope of the demonstration, bidding process, selection of winners, reimbursement, quality, and administration and monitoring. The role of the Health Care Financing Administration’s Laboratory Technical Advisory Committee is also described, and the future of competitive bidding in a Medicare managed care environment is evaluated. We close with some brief comments on how to succeed in competitive bidding for Medicare services.
Role of nitric oxide in cardiovascular disease: focus on the endothelium
Authors: Cannon Richard O
Published: January 2020

Nitric oxide is a soluble gas continuously synthesized by the endothelium. This substance has a wide range of biological properties that maintain vascular homeostasis, including modulation of vascular dilator tone, regulation of local cell growth, and protection of the vessel from injurious consequences of platelets and cells circulating in blood. A growing list of conditions, including those commonly associated as risk factors for atherosclerosis such as hypertension and hypercholesterolemia, are associated with diminished release of nitric oxide into the arterial wall either because of impaired synthesis or excessive oxidative degradation. Diminished nitric oxide bioactivity may cause constriction of coronary arteries during exercise or during mental stress and contribute to provocation of myocardial ischemia in patients with coronary artery disease. Additionally, diminished nitric oxide bioactivity may facilitate vascular inflammation that could lead to oxidation of lipoproteins and foam cell formation, the precursor of the atherosclerotic plaque. Numerous therapies have been investigated to assess the possibility of reversing endothelial dysfunction by enhancing the release of nitric oxide from the endothelium, either through stimulation of nitric oxide synthesis or protection of nitric oxide from oxidative inactivation and conversion to toxic molecules such as peroxynitrite. Accordingly, causal relationships between improved endothelial function and reduction in myocardial ischemia and acute coronary events can now be investigated.
Erythrocyte folate analysis: a cause for concern?
Authors: Wright Anthony J A , Finglas Paul M , Southon Susan
Published: January 2020

Neural tube defects can be prevented by adequate intake of periconceptional folate, and inverse associations between folate status and cardiovascular disease and various cancers have been noted. Thus, there is renewed interest in the analysis of red cell folate (RCF) as an indicator of folate deficiency risk. Assessment of the assumptions that underpin RCF assays indicates that many are false. Published literature suggests that increased deoxy-hemoglobin (which can bind RCF electrostatically) yields more assayable folate, and increased oxy-hemoglobin (which cannot bind RCF) yields less assayable folate. It is argued that as deoxy-hemoglobin picks up oxygen and switches quaternary structure, any bound folate must, on purely theoretical grounds, become physically “trapped”. Venous blood taken for analysis is 65% to 75% saturated with oxygen, and pro-rata “trapping” will lead to serious underestimation of RCF. Hence, doubt is cast over the validity of all previous RCF values. Some strategies for accurately assessing RCF are suggested.
Simple method for the routine determination of betaine and N,N-dimethylglycine in blood and urine
Authors: Laryea Maurice D , Steinhagen Folkert , Pawliczek Sandra , Wendel Udo
Published: January 2020

A simple and convenient method using commercially available derivatization reagents is described for the measurement of betaine and N,N-dimethylglycine (DMG) in blood and urine. Precolumn derivatization of plasma or urine is performed directly in acetonitrile without extraction with p-bromophenacyl bromide and crown ether as catalyst. The p-bromophenacyl ester derivatives are then separated by high-performance liquid chromatography, using an isocratic system of acetonitrile and water containing choline. Effluent was monitored at 254 nm. The limit of detection was 5 μmol/L for betaine and 2 μmol/L for DMG. Analytical recovery was >97% for both analytes. Total and within-day CVs were 2.0–4.4% and 0.9–2.2% for DMG. For betaine, the total and within-day CVs were 1.3–5.3% and 0.4–3.8%, respectively. The method is precise and cost-effective and has been used successfully to determine the concentrations of DMG and betaine in human plasma and urine.
Ligand assays: from electrophoresis to miniaturized microarrays
Authors: Ekins Roger P
Published: January 2020

The main developments in the “ligand assay” field in which I have been involved are traced. These include the original development of “first generation” competitive assays relying on radiolabeled analyte markers; the development of the first “second generation”, noncompetitive (ultrasensitive) methods, which rely on the use of labeled (monoclonal) antibodies and high specific activity nonisotopic labels (leading to the transformation of the immunodiagnostic field in the 1980s); and the development of the first “third generation” miniaturized, chip-based, microarray methods, which permit the simultaneous ultrasensitive measurement of many analytes in the same small sample. The latter—applicable both to immunoassay and to DNA/RNA analysis—are likely to revolutionize the diagnostic and pharmaceutical fields in the next decade.
Automated Flow Cytometry Compared with an Automated Dipstick Reader for Urinalysis
Authors: Langlois Michel R , Delanghe Joris R , Steyaert Sophia R , Everaert Karel C , De Buyzere Marc L
Published: January 2020

Recently, the Sysmex UF-100 flow cytometer was developed to automate urinalysis. We compared UF-100 test results with those of an automated dipstick reader. A cross-check of UF-100, dipstick, and microscopic sediment data was performed in 1001 urine samples. Good agreements (P <0.001) were obtained between UF-100 and dipstick data for erythrocytes (r = 0.636) and leukocytes (r = 0.785). Even in urine with low conductivity, the UF-100 could detect lysed erythrocytes. The UF-100 bacterial count was higher among nitrite-positive urine samples (P <0.0001) and was positively correlated with the UF-100 leukocyte count (r = 0.745; P <0.001). In stored urine (24 h), bacterial counts increased, whereas the forward light scatter of leukocytes decreased (P <0.01). Casts and yeast cells reported by the UF-100 should be confirmed by microscopic review because false positives occurred. We suggest that a computer-assisted cross-check of UF-100 and dipstick data allows a clinically acceptable sieving system to reduce the workload of microscopic sediment urinalysis.
Interference of Plant Peroxidases with Guaiac-based Fecal Occult Blood Tests Is Avoidable
Authors: Sinatra Marc A , St. John D James B , Young Graeme P
Published: January 2020

Peroxidase-rich fruits and vegetables are reputed to interfere with guaiac-based fecal occult blood tests. We added horseradish peroxidase to fecal samples and tested them with Hemoccult®, Hemoccult SENSA®, and hydrated Hemoccult. Positivity rates with Hemoccult and Hemoccult SENSA decreased rapidly as the time between smearing (preparation) and development increased, whereas they remained high with hydrated Hemoccult. For samples with added blood, positivity rates did not decrease with time. When 61 volunteers were tested on a standard restriction and on a challenge diet high in plant peroxidase, no positive results occurred during standard restriction. During the challenge diet, one volunteer was positive with Hemoccult and Hemoccult SENSA when development was delayed 24 h, and no volunteers were positive when it was delayed 48 h and 72 h. However, with hydrated Hemoccult, positives occurred in 13 of 61 volunteers at 24 h, 8 of 61 at 48 h, and 5 of 61 at 72 h. Thus, peroxidase-rich plant foods do not need to be excluded from the diet with Hemoccult and Hemoccult SENSA if development is delayed for at least 48 h after smearing. A delay of this duration will not solve the problem of plant peroxidase interference with hydrated Hemoccult.
Immunoreactivity, Ultrastructural Localization, and Transcript Expression of Prostate-specific Antigen in Human Neuroblastoma Cell Lines
Authors: Mannello Ferdinando , Malatesta Manuela , Luchetti Francesca , Papa Stefano , Battistelli Serafina , Gazzanelli Giancarlo
Published: January 2020

Prostate-specific antigen (PSA) is considered a highly specific biochemical marker of the human prostate gland, and it currently is used for prostate cancer diagnosis and monitoring. Recently, PSA production and secretion were found in nondiseased and diseased cells, tissues, and fluids from women. In this study, we characterized the presence of PSA in two human neuroblastoma cell lines with biochemical, ultrastructural, and molecular approaches. Using reverse transcription-PCR, we identified PSA mRNA, and Western blotting revealed a substantial amount of complexed form of PSA protein, which is localized mainly in free ribosomes. Although the role of PSA in human neuroblastoma cell lines is still unknown, our study supports the hypothesis that this serine protease may be involved in controlling the growth of human brain tumor cells, adding more support to the notion that PSA is a widespread kallikrein-like protease with biological functions much more complex than recently thought.
Presence of Donor- and Recipient-derived DNA in Cell-free Urine Samples of Renal Transplantation Recipients: Urinary DNA Chimerism
Authors: Zhang Jun , Tong Kwok-Lung , Li Philip KT , Chan Albert YW , Yeung Chung-Kwong , Pang Calvin CP , Wong Teresa YH , Lee Kam-Cheong , Lo YM Dennis
Published: January 2020

Background: Previous studies have indicated that microchimerism is present in body tissues, peripheral blood, and plasma of recipients after organ transplantation. We hypothesize that donor-derived DNA may also be present in cell-free urine of renal transplant recipients and that the concentrations of urine DNA may be correlated with graft rejection.

Methods: Thirty-one female patients who had renal transplantation were enrolled in the study. In women with male organ donors, the SRY gene on the Y chromosome was used as a marker for donor-derived DNA. Real-time quantitative PCR for the SRY and β-globin genes was carried out on cell-free urinary DNA from these patients. Serial urine samples from a female renal transplant recipient undergoing an acute rejection episode were also collected and analyzed with the β-globin quantitative PCR system.

Results: SRY sequences were detected in the urine of 14 of 17 female patients with male organ donors. None of the 14 patients with female organ donors had detectable SRY sequences in urinary DNA. The median fractional concentration of donor-derived DNA was 8.7% (interquartile range, 1.9–26.4%). During the acute rejection episode, urinary concentrations of the β-globin gene were markedly increased, with the concentrations returning rapidly to normal following antirejection treatment.

Conclusions: Our results demonstrate that urinary DNA chimerism is present following renal transplantation. The measurement of urinary DNA using quantitative PCR may be useful for the diagnosis and monitoring of graft rejection.
Evaluation of a Rapid Whole Blood ELISA for Quantification of Troponin I in Patients with Acute Chest Pain
Authors: Heeschen Christopher , Goldmann Britta U , Langenbrink Lukas , Matschuck Guido , Hamm Christian W
Published: January 2020

Background: Troponin I (cTnI) provides important prognostic information in patients with chest pain. We wished to evaluate a rapid, whole-blood analyzer for quantitative point-of-care testing.

Methods: A quantitative point-of-care test system (Stratus CS®; Dade-Behring) for cTnI with an incorporated centrifuge was evaluated in 412 patients with chest pain less than 12 h.

Results: Results were available within 15 min. CVs were 4.5% at 0.1 μg/L, 4.2% at 0.25 μg/L, and 6.5% at 0.82 μg/L. The detection limit was 0.01 μg/L. The 97.5% percentile in a healthy population was 0.08 μg/L. Based on ROC curve analysis, a threshold of 0.15 μg/L was calculated for the detection of acute myocardial infarction (AMI). With it, sensitivity for the detection of patients with AMI (n = 62) was 63% at arrival and 98% after 4 h (Stratus II®, 48% and 85%, respectively; P <0.01). In 42% of patients with unstable angina (n = 121), cTnI was ≥0.08 μg/L (Stratus II, 28%; P <0.01). During 30 days, death or AMI occurred in 25.5% of these cTnI-positive vs 2.9% of cTnI-negative patients (Stratus II, 29.4% vs 5.8%).

Conclusion: The Stratus CS provided better analytical performance and comparable or better prognostic information than the Stratus II.
Aminopeptidase Activities in Breast Cancer Tissue
Authors: Martínez José M , Prieto Isabel , Ramírez María J , Cueva Cristobal , Alba Francisco , Ramírez Manuel
Published: January 2020

Background: Endopeptidases such as cathepsins help determine the prognosis of breast cancer (BC). However, little information is available about the role in BC of aminopeptidases (APs), which have been implicated in the metabolism of several local hormonal factors.

Methods: Using aminoacyl-β-naphthylamides as substrates, we measured fluorometrically alanyl-AP, arginyl-AP, cystinyl-AP, glutamyl-AP, aspartyl-AP, and pyroglutamyl-AP activities in their soluble and membrane-bound forms in surgically removed BC tissue from which we separated samples of neoplastic, adjacent tumoral, and unaffected surrounding tissue.

Results: Compared with unaffected tissue, neoplastic tissue had significantly higher activities of soluble alanyl-AP (553.9 ± 82.8 vs 1615.2 ± 183.0 pmol/mg protein; P <0.001), arginyl-AP (372.4 ± 56.6 vs 1027.2 ± 143.5 pmol/mg protein; P <0.001), and cystinyl-AP (74.8 ± 10.0 vs 282.9 ± 37.2 pmol/mg protein; P <0.001), and of membrane-bound arginyl-AP (457.7 ± 97.9 vs 886.6 ± 140.0 pmol/mg protein; P <0.01). However, membrane-bound aspartyl-AP activity was significantly lower in neoplastic tissue (17.3 ± 1.4 vs 9.2 ± 1.2 pmol/mg protein; P <0.05) and pyroglutamyl-AP activity was significantly lower in neoplastic and adjacent tissues (12.8 ± 0.9 vs 7.0 ± 1.2 and 8.0 ± 1.3 pmol/mg protein; P <0.001 for both comparisons).

Conclusions: The present results document changes in AP activities in BC tissue. These changes may reflect the functional status of the AP substrates, which can be selectively activated or inhibited locally in the affected tissue as a result of specific conditions brought about by the tumor.
Serum Sialic Acid in a Random Sample of the General Population
Authors: Pönniö Maritta , Alho Hannu , Nikkari Seppo T , Olsson Ulf , Rydberg Ulf , Sillanaukee Pekka
Published: January 2020

Background: The serum sialic acid (SA) concentration has been reported to be a potentially useful but nonspecific disease marker. We wanted to study which factors influence SA concentration in a well-characterized healthy population.

Methods: SA was determined in 97 women and 96 men with a colorimetric Warren method.

Results: The mean ± SD concentrations of SA were 634 ± 109 (95% confidence interval, 612–656) and 630 ± 106 (95% confidence interval, 608–651) mg/L for women and men, respectively. The serum SA showed a significant positive association with body mass index and with systolic and diastolic blood pressure among both women and men. SA also correlated significantly with the use of contraceptive pills and age among women and with smoking among men.

Conclusions: Our study suggests that SA does not increase with age in men but appears to increase with female menopause. The strong positive association with blood pressure may explain why SA predicts cardiovascular mortality.
Molecular Diagnostics on Microfabricated Electrophoretic Devices: From Slab Gel- to Capillary- to Microchip-based Assays for T- and B-Cell Lymphoproliferative Disorders
Authors: Munro Nicole J , Snow Karen , Kant Jeffrey A , Landers James P
Published: January 2020

Background: Current methods for molecular-based diagnosis of disease rely heavily on modern molecular biology techniques for interrogating the genome for aberrant DNA sequences. These techniques typically include amplification of the target DNA sequences followed by separation of the amplified fragments by slab gel electrophoresis. As a result of the labor-intensive, time-consuming nature of slab gel electrophoresis, alternative electrophoretic formats have been developed in the form of capillary electrophoresis and, more recently, multichannel microchip electrophoresis.

Methods: Capillary electrophoresis was explored as an alternative to slab gel electrophoresis for the analysis of PCR-amplified products indicative of T- and B-cell malignancies as a means of defining the elements for silica microchip-based diagnosis. Capillary-based separations were replicated on electrophoretic microchips.

Results: The microchip-based electrophoretic separation effectively resolved PCR-amplified fragments from the variable region of the T-cell receptor-γ gene (150–250 bp range) and the immunoglobulin heavy chain gene (80–140 bp range), yielding diagnostically relevant information regarding the presence of clonal DNA populations. Although hydroxyethylcellulose provided adequate separation power, the need for a coated microchannel for effective resolution necessitated additional preparative steps. In addition, preliminary data are shown indicating that polyvinylpyrrolidone may provide an adequate matrix without the need for microchannel coating.

Conclusions: Separation of B- and T-cell gene rearrangement PCR products on microchips provides diagnostic information in dramatically reduced time (160 s vs 2.5 h) with no loss of diagnostic capacity when compared with current methodologies. As illustrated, this technology and methodology holds great potential for extrapolation to the abundance of similar molecular biology-based techniques.
Activated Protein C Resistance: Automated Detection of the Factor V Leiden Mutation by Mismatch Hybridization
Authors: Klingler Karl R , Junold Thomas , Wielckens Klaus
Published: January 2020

Background: A single point mutation in the factor V gene has been demonstrated to be the cause of factor Va resistance to proteolytic cleavage by activated protein C. Knowledge of the patient’s genetic disposition is of great importance in situations such as pregnancy, surgery, use of oral contraceptives, and immobilization.

Methods: We have developed a rapid, automated test for the detection of the factor V mutation that makes use of differences in thermal stability between perfect-match and non-perfect-match hybrids. A DNA fragment spanning the mutation is amplified with a biotin-labeled primer. Ruthenium-labeled oligonucleotides, perfectly matching either the biotinylated wild-type strand or the biotinylated mutation strand, are added. Heating to 95 °C and subsequent cooling lead to the formation of double-stranded DNA. Under the conditions chosen, ruthenium-labeled oligonucleotides form stable, double-stranded DNA with the biotinylated strand only if both strands perfectly match each other. The ruthenium signal is measured on a modified Elecsys 1010 system (Roche Diagnostics).

Results: The ratio between the signals obtained with perfectly matching and non-perfectly matching oligonucleotides reflects the genetic status. Analyzed samples can be divided into three nonoverlapping groups based on these ratios. We confirmed the reliability of the method by analyzing several samples of known genetic status; the results were identical in every single instance.

Conclusions: The test discriminates unambiguously between the heterozygous and the homozygous states. Because of its low costs and easy handling, the assay is suitable for use in routine laboratories of clinical chemistry.
New Electrochemiluminescent Immunoassay for the Determination of CYFRA 21-1: Analytical Evaluation and Clinical Diagnostic Performance in Urine Samples of Patients with Bladder Cancer
Authors: Sánchez-Carbayo Marta , Espasa Antonia , Chinchilla Virtudes , Herrero Enrique , Megías Julián , Mira Antonio , Soria Federico
Published: January 2020

Background: A new electrochemiluminescent immunoassay (ECLIA) has been developed for the determination of cytokeratin 19 (CYFRA 21-1) in the Elecsys 2010 immunoassay system. Urinary CYFRA 21-1 might have a role in the diagnosis of bladder cancer.

Methods: We performed an analytical evaluation of the CYFRA 21-1 ECLIA for serum and urine samples. The clinical value of urinary CYFRA 21-1 for the detection of bladder cancer was evaluated through its measurement in 226 urine samples from symptomatic and asymptomatic controls.

Results: At concentrations of 2–30 μg/L, within-assay imprecision (CV) was below 2.1% for sera and 3.3% for urines, with interassay CVs below 3.3% for sera and 4.9% for urines. The day-to-day CV was <20% at concentrations >0.2 μg/L (functional sensitivity). Measurement of diluted samples showed that the assay estimated CYFRA 21-1 between 98% and 103% for sera and 98% and 105% for urines. Recovery of added CYFRA 21-1 was 99–105% for sera and 96–115% for urines. We separately compared serum and urine CYFRA 21-1 ECLIA results with those obtained with an IRMA (CIS bio international). Regression analysis for sera was: CYFRA 21-1 (ECLIA) = 0.520 + 1.018 CYFRA 21-1 (IRMA); [95% confidence interval (CI) (y-intercept), −0.260 to 1.309]; 95% CI (slope), 0.978–1.060; n = 100; Sy|x = 3.242; r2 = 0.987. For urine samples it was: CYFRA 21-1 (ECLIA) = 0.716 + 0.966 CYFRA 21-1 (IRMA); 95% CI (y-intercept), 0.009–1.422; 95% CI (slope), 0.956–0.976; n = 100; Sy|x = 4.136; r2 = 0.986. In urine samples voided by patients with and without bladder cancer, the best ROC analysis discrimination provided 81.0% (95% CI, 72.7–87.7%) sensitivity and 97.2% (95% CI, 90.2–99.6%) specificity at a threshold value of 5.7 μg/L.

Conclusions: Our initial evaluation showed reliable analytical performance for urinary CYFRA 21-1, which might assist urologists in the detection of bladder cancer as a noninvasive adjunct to cystoscopy.
The Combination of Human Glandular Kallikrein and Free Prostate-specific Antigen (PSA) Enhances Discrimination Between Prostate Cancer and Benign Prostatic Hyperplasia in Patients with Moderately Increased Total PSA
Authors: Magklara Angeliki , Scorilas Andreas , Catalona William J , Diamandis Eleftherios P
Published: January 2020

Background: Prostate-specific antigen (PSA) is the most reliable tumor marker available and is widely used for the diagnosis and management of prostate cancer. Unfortunately, PSA cannot distinguish efficiently between benign and malignant disease of the prostate, especially within the range of 4–10 μg/L. Among the refinements developed to enhance PSA specificity is the free/total PSA ratio, which is useful in discriminating between the two diseases within the diagnostic “gray zone”. Recent data indicate that human glandular kallikrein (hK2), a protein with high homology to PSA, may be an additional serum marker for the diagnosis and monitoring of prostate cancer.

Methods: We analyzed 206 serum samples (all before treatment was initiated) from men with histologically confirmed benign prostatic hyperplasia (n = 100) or prostatic carcinoma (n = 106) with total PSA in the range of 2.5–10 μg/L. Total and free PSA and hK2 were measured with noncompetitive immunological procedures. Statistical analysis was performed to investigate the potential utility of the various markers or their combinations in discriminating between benign prostatic hyperplasia and prostatic carcinoma.

Results: hK2 concentrations were not statistically different between the two groups of patients. There was a strong positive correlation between hK2 and free PSA in the whole patient population. hK2/free PSA ratio (area under the curve = 0.69) was stronger predictor of prostate cancer than the free/total PSA ratio (area under the curve = 0.64). At 95% specificity, the hK2/free PSA ratio identified 30% of patients with total PSA between 2.5–10 μg/L who had cancer. At 95% specificity, the hK2/free PSA ratio identified 25% of patients with total PSA between 2.5 and 4.5 μg/L who had cancer.

Conclusions: Our data suggest that hK2 in combination with free and total PSA can enhance the biochemical detection of prostate cancer in patients with moderately increased total PSA concentrations. More specifically, the hK2/free PSA ratio appears to be valuable in identifying a subset of patients with total PSA between 2.5 and 4.5 μg/L who have high probability of cancer and who should be considered for biopsy.
RNA Expression of Cardiac Troponin T Isoforms in Diseased Human Skeletal Muscle
Authors: Ricchiuti Vincent , Apple Fred S
Published: January 2020

Background: The expression of multiple cardiac troponin T (cTnT) isoforms has been demonstrated in diseased human skeletal muscle. However, cardiac troponin I (cTnI) expression has been described only in heart muscle. The goal of this study was to determine whether mRNA for cTnT, slow skeletal troponin T (sTnT), or cTnI was expressed in skeletal muscle biopsies obtained from patients with end-stage renal disease (ESRD) and Duchenne muscular dystrophy (DMD).

Methods: Total mRNA was extracted from healthy human heart (n = 4), healthy human skeletal muscle (n = 5), and skeletal muscle from patients with ESRD (n = 7) and DMD (n = 5). Total RNA (1 μg) was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase. The reverse-transcribed cDNAs were amplified by PCR using oligonucleotide primers specific for cTnT, sTnT, and cTnI sequences (GenBank accession numbers X74819, m19308, and X54163, respectively).

Results: In all heart specimens, a 150-bp cTnT amplicon was detected. Skeletal muscle from four of seven patients with ESRD and two of five patients with DMD showed expression of a 150-bp amplicon. Using DNA sequencing and a comparison program, the 150-bp amplicons found in heart and diseased skeletal muscle specimens were 100% identical and specific to the cTnT mRNA sequence. No cTnT mRNA expression was found in healthy skeletal muscle. No evidence of sTnT mRNA was found in heart muscle. A 200-bp sTnT amplicon specific to a human sTnT sequence was detected in all skeletal muscle specimens. A 250-bp cTnI amplicon specific to the cTnI sequence was detected in all heart specimens. However, no cTnI mRNA expression was found in healthy or diseased skeletal muscle specimens. cTnT mRNA expression in both heart and diseased skeletal muscles corresponded with cTnT isoform expression, respectively, as determined by Western blot analysis.

Conclusion: Our findings demonstrate cTnT mRNA expression, but no cTnI mRNA expression, by reverse transcription-PCR in diseased human skeletal muscle that expresses cTnT isoforms.
New Method for Determining Cystine in Leukocytes and Fibroblasts
Authors: de Graaf-Hess Adriana , Trijbels Frans , Blom Henk
Published: January 2020

Background: Cystinosis is a rare inborn error of cystine transport, leading to accumulation of cystine in the lysosomes. To diagnose cystinosis and monitor treatment with cysteamine, adequate measurements of cystine concentrations in leukocytes and cultured fibroblasts are required.

Methods: Cells were sonicated in the presence of excess N-ethylmaleimide to prevent oxidation of cysteine to cystine and disulfide exchange reactions of cystine with available sulfhydryl moieties. Cystine was measured as cysteine after reduction with sodium borohydride and derivatization with monobromobimane, followed by separation with automated HPLC and fluorescence detection.

Results: The assay was linear to 200 μmol/L cysteine. Within-run and day-to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 μmol/L. Added cysteine, up to 200 μmol/L, was completely removed, and recovery of added cystine was 69–86%. Cystine was stable for at least 2 months in leukocytes frozen in liquid nitrogen and stored at −80 °C

Conclusions: Oxidation of cysteine to cystine and disulfide exchange reactions of cystine with sulfhydryl moieties are prevented by N-ethylmaleimide. The detection limit for the determination of cystine is adequate to measure cystine in leukocytes and cultured fibroblasts for diagnosis of cystinosis and monitoring treatment with cysteamine.