bluuebunny/crossref_metadata_2025 huggingface dataset:
Age- and gender-specific reference intervals for total homocysteine and methylmalonic acid in plasma before and after vitamin supplementation
Authors: Rasmussen K , Møller J , Lyngbak M , Pedersen A M , Dybkjaer L
Published: January 2020

We present reference intervals for total homocysteine and methylmalonic acid in plasma based on samples from 126 women (ages 20-85 years, median 49 years) and 109 men (ages 20-84 years, median 50 years). The central 0.95 interval for methylmalonic acid was 0.08-0.28 micromol/L. Supplementation with cyanocobalamin caused a nonsignificant decrease in methylmalonic acid. Supplementation with folic acid caused a decrease in homocysteine concentrations, with data analysis identifying two significantly different clusters: 182 subjects with the lowest initial concentrations (7.76 +/- 1.54 micromol/L, mean +/- SD) and the smallest decrease (1.26 +/- 0.96 micromol/L), and 53 subjects with the highest initial concentrations (12.33 +/- 2.04 micromol/L) and greatest decrease (4.14 +/- 1.32 micromol/L). We argue in favor of the age- and gender-specific central 0.95 intervals obtained for the 182 subjects before being supplemented with folic acid: 4.6-8.1 micromol/L for subjects at <30 years; 4.5-7.9 micromol/L for women, ages 30-59 years; 6.3-11.2 micromol/L for men, ages 30-59 years; and 5.8-11.9 micromol/L for subjects at >60 years.
Urine trypsinogen-2 as marker of acute pancreatitis
Authors: Hedström J , Sainio V , Kemppainen E , Puolakkainen P , Haapiainen R , Kivilaakso E , Schauman K O , Stenman U H
Published: January 2020

We examined the clinical utility of urine trypsinogen-2 as a marker of acute pancreatitis (AP). Fifty-nine patients with AP, 42 with acute abdominal diseases of extrapancreatic origin, and 63 without evidence of acute abdominal disease were studied. Urine trypsinogen-2 was determined by a time-resolved immunofluorometric assay. As reference methods we used serum trypsinogen-2, urine amylase, and serum amylase. The diagnostic accuracy of the markers was evaluated by receiver-operating characteristic (ROC) analysis. At admission, urine trypsinogen-2 differentiated patients with AP from controls with high accuracy. The area under the ROC curve (AUC) was 0.978, which was equal to that of serum trypsinogen-2 (0.998) and serum amylase (0.969) and significantly larger than that of urine amylase. For differentiation between severe and mild AP, urine trypsinogen-2 (0.730) was equal to serum trypsinogen-2 (0.721), and clearly better than amylase in serum and urine. These results suggest that determination of urine trypsinogen-2 is a useful test to detect AP and to evaluate disease severity.
Impact of point-of-care testing on patients' length of stay in a large emergency department
Authors: Parvin C A , Lo S F , Deuser S M , Weaver L G , Lewis L M , Scott M G
Published: January 2020

We prospectively investigated whether routine use of a point-of-care testing (POCT) device by nonlaboratory operators in the emergency department (ED) for all patients requiring the available tests could shorten patient length of stay (LOS) in the ED. ED patient LOS, defined as the length of time between triage (initial patient interview) and discharge (released to home or admitted to hospital), was examined during a 5-week experimental period in which ED personnel used a hand-held POCT device to perform Na, K, Cl, glucose (Gluc), and blood urea nitrogen (BUN) testing. Preliminary data demonstrated acceptable accuracy of the hand-held device. Patient LOS distribution during the experimental period was compared with the LOS distribution during a 5-week control period before institution of the POCT device and with a 3-week control period after its use. Among nearly 15 000 ED patient visits during the study period, 4985 patients (2067 during the experimental period and 2918 during the two control periods) had at least one Na, K, Cl, BUN, or Gluc test ordered from the ED. However, no decrease in ED LOS was observed in the tested patients during the experimental period. Median LOS during the experimental period was 209 min vs 201 min for the combined control periods. Stratifying patients by presenting condition (chest pain, trauma, etc.), discharge/admit status, or presence/absence of other central laboratory tests did not reveal a decrease in patient LOS for any patient subgroup during the experimental period. From these observations, we consider it unlikely that routine use of a hand-held POCT device in a large ED such as ours is sufficient by itself to impact ED patient LOS.
Three routine methods for measuring high-density lipoprotein cholesterol compared with the Reference Method
Authors: Harris N , Galpchian V , Rifai N
Published: January 2020

We compared the performance of three methods for quantifying high-density lipoprotein cholesterol (HDL-C) with the Reference Method for HDL-C, using samples with a wide range of triglyceride (TG) concentrations (290-18000 mg/L). All three comparison assays-- utilizing a magnetic dextran sulfate precipitating reagent, a direct method, and a standard MgCl2-dextran sulfate reagent--were precise, with a run-to-run CV of less than or equal to 4.1%. However, the systematic error of these assays exceeded the National Cholesterol Education Program (NCEP) performance goal of less than or equal to 10% in half of the concentration ranges tested. Nevertheless, the total error of the assays generally meets the current 22% limit set by the NCEP. Although both the magnetic dextran sulfate precipitation reagent and the direct assay can be performed more rapidly than the MgCl2-dextran sulfate assay, the direct assay involves no sample preparation and requires only 4 microL of sample excluding the dead space. Although precipitation is frequently inadequate with the MgCl2-dextran sulfate reagent at TG concentrations >6000 mg/L, both the magnetic and the direct reagent show no interference from high TG concentrations as great as 18 000 mg/L.
A critical review of personnel standards
Authors: Peddecord K M
Published: January 2020

Qualified personnel are necessary to ensure quality results, but there is no evidence on what minimal educational requirements are necessary. In the future, cost-effectiveness analysis may be useful for policy questions such as the utility of personnel regulations. CLIA '88 regulations require explicit assessment of personnel competency. Survey evidence suggests that previously regulated laboratories tend to have highly formalized competency assessment programs, while physician office laboratory programs are minimal. Responding laboratory supervisors had no uniform definition of competency, but technical abilities, productivity, and professionalism were regarded as essential. Suggestions for contemporary strategies by laboratory professionals and their organizations in the current increasingly competitive managed care environment are described. Faced with limited empirical evidence on personnel, laboratory directors and supervisors should focus on the total testing process, the needs of clinicians, and the uses of testing information to guide personnel assignments.
The case for cardiac troponin T: marker for effective risk stratification of patients with acute cardiac ischemia
Authors: Alonsozana G L , Christenson R H
Published: January 2020

Availability of markers such as cardiac troponin T (cTnT) has brought new insights into ischemic heart disease (IHD). cTnT is a distinct protein that differs from other markers in biological function, molecular mass, and cytosolic pool. cTnT has been utilized for diagnosis of acute myocardial infarction (AMI) and risk stratification of patients with IHD. For AMI diagnosis, cTnT showed high sensitivity (94-100%) but generally lower specificity (46-99%), possibly because of increases in non-AMI patients with minor myocardial damage. Outcome studies have demonstrated that IHD patients with increased cTnT are at significantly greater risk for cardiac events; revascularization in patients with increased cTnT may improve outcome. Estimated costs for batched ES 300 cTnT results and for a cTnT rapid assay run "on demand" were $17.48 and $21.65, respectively. cTnT currently has no specific common procedure test code; expected reimbursement is $18.32 for the ES 300 and is not established for the rapid assay.
Why is the laboratory an afterthought for managed care organizations?
Authors: Forsman R W
Published: January 2020

Market forces have dramatically influenced the environment in which healthcare is delivered, but these changes do not need to be interpreted negatively by community laboratorians. Only total vertical integration of laboratory medicine can control episode-of-care cost. Opportunities also exist for horizontal integration with community partners to provide geographical coverage and to compete favorably for managed care contracts. Lowering cost through "economies of scale" may apply to the procurement of supplies and equipment, but the delivery of services must be considered in the context of their overall effect on episode-of-care cost. Laboratory services may make up 5% of a hospital's budget but leverage 60-70% of all critical decision-making such as admittance, discharge, and medication. Laboratory outreach can help the medical center's financial stability by: (a) providing tests and service that can reduce or avoid a hospital stay; (b) using the additional volume of testing to distribute existing fixed costs and lower unit cost; and (c) adding revenue as a direct contribution to margin. To successfully compete for contracted managed care services, the laboratory must network with other providers to demonstrate comprehensive access and capacity. Community hospital laboratories perform 50% of all laboratory tests in this country and have adequate excess capacity to fulfill the remaining community needs.
How is telepathology being used to improve patient care?
Authors: Weinberg D S
Published: January 2020

Telepathology, as a subspecialty of telemedicine, involves the use of telecommunications technologies to transmit images to distant sites for the purpose of communicating diagnostic information or for teaching. Recent advances in technology have greatly increased the feasibility of performing diagnosis by telepathology, but there are still significant obstacles to implementation. In this review, I will discuss the technologies and organizations involved in telepathology, with examples of current practice in the US and abroad.
Biological and clinical importance of the p53 tumor suppressor gene
Authors: Velculescu V E , El-Deiry W S
Published: January 2020

The p53 tumor suppressor gene controls cellular growth after DNA damage through mechanisms involving growth arrest and apoptosis. Mutations that inactivate p53 occur commonly in virtually all human malignancies and can be detected by sequencing of the p53 gene, immunohistochemical staining of tumor tissue with anti-p53 antibodies, single-strand conformation polymorphisms, or other biological assays. Identification of p53 mutation in the germ line is diagnostic of the cancer-prone Li-Fraumeni syndrome. Alterations of the p53 gene result in defective cellular responses after DNA damage and predispose cells to dysregulated growth, tumor formation and progression, and potential resistance (of tumor cells) to certain chemotherapeutic agents or ionizing radiation. A variety of tumors involving mutant p53 have a worse prognosis than tumors of the same type containing no p53 mutations. New diagnostic and therapeutic strategies are evolving as the p53 pathways of cell-cycle arrest and apoptosis become elucidated.
Interlaboratory variability of bilirubin measurements
Authors: Vreman H J , Verter J , Oh W , Fanaroff A A , Wright L L , Lemons J A , Shankaran S , Tyson J E , Korones S B , Bauer C R , Stoll B J , Papile L A , Donovan E F , Ehrenkranz R A , Stevenson D K
Published: January 2020

During an 8-month study, 14 laboratories used automated analytical systems to measure total bilirubin concentrations in lyophilized bovine specimens containing 38, 169, and 253 micromol/L bilirubin (2.2, 9.9, and 14.8 mg/dL, respectively). The measured mean +/- SD (n, range) were: 39 +/- 7 micromol/L (n = 90, 31-53) [2.3 +/- 0.4 mg/dL (1.8-3.1)]; 176 +/- 29 micromol/L (n = 89, 146-222) [10.3 +/- 1.7 mg/dL (8.5-13.0)]; and 260 +/- 43 micromol/L (n = 103, 208-316) [15.2 +/- 2.5 mg/dL (12.1-18.5)]. In comparison with target values, measurements were consistently lower at 4, higher at 6, and within +/- 4% at 4 laboratories for each of the three concentrations. The measured values for each concentration remained fairly constant during the study at each laboratory. We conclude that bilirubin measurements differed significantly from the established target values at most of the participating laboratories.
Greater frequency of K-ras Val-12 mutation in colorectal cancer as detected with sensitive methods
Authors: Carpenter K M , Durrant L G , Morgan K , Bennett D , Hardcastle J D , Kalsheker N A
Published: January 2020

Assays for tumor mutations need to be sufficiently sensitive that a mutation can be readily detected in a high background of wild-type sequence. We have evaluated competitive allele-specific oligonucleotide (cASO) hybridization, the amplification refractory mutation system (ARMS), and a combination of cASO with mutant-enriched polymerase chain reaction (ME-PCR) for their respective sensitivities in detecting the K-ras Val-12 mutation. cASO hybridization detected 1 mutant allele in 100 wild-type alleles and identified 6 of 74 (8%) colorectal tumors as having the Val-12 mutation. ARMS detected 1 mutant in 1000 and 12 of 74 (16%) tumor samples, and the ME-PCR with cASO hybridization detected 1 in 5000 and 20 of 74 (27%) tumor samples. The mutation was not detected in normal bowel mucosa from selected Val-12 tumor-positive patients. ME-PCR combined with cASO resulted in a threefold higher detection rate than has previously been reported and suggests that other studies may have underestimated the frequency of K-ras mutations.
Methylmalonic acid quantification by stable isotope dilution gas chromatography-mass spectrometry from filter paper urine samples
Authors: McCann M T , Thompson M M , Gueron I C , Lemieux B , Giguère R , Tuchman M
Published: January 2020

A specific method for quantification of methylmalonic acid (MMA) from urine samples dried onto filter paper is described. The method involves stable isotope dilution gas chromatography-mass spectrometry with [methyl-2H3]-MMA as the internal standard. MMA is stable in dry paper samples stored at room temperature for at least 2 weeks. The extraction efficiency of MMA from paper was 56-58%. The concentration of urinary MMA in dried filter paper specimens from 190 normal controls was 1.21 +/- 1.34 (mean +/- SD) mmol/mol of urinary creatinine. Age-related reference values are also reported. The concentrations, normalized to the urinary creatinine concentration, decrease with age. The applicability of this method to rapid screening for cobalamin (vitamin B12)-related disorders and methylmalonic aciduria is demonstrated.
Sequential microenzymatic assay of cholesterol, triglycerides, and phospholipids in a single aliquot
Authors: Nanjee M N , Miller N E
Published: January 2020

The assay of multiple analytes in a single aliquot can be advantageous from both measurement and economic standpoints. The objective of this study was to develop a simple and sensitive microenzymatic method for the determination of three biologically important lipids. Triglycerides (as glycerol), phospholipids (as choline), and total cholesterol (as unesterified cholesterol) were assayed, in that order, by sequential addition of sample, reagents, and microbial enzymes directly into a single microtiter plate well, accompanied by continuous monitoring of a common reporter reaction in which hydrogen peroxide is quantified either by colorimetry with 4-aminoantipyrine and 3-hydroxy-2,4,6-triiodobenzoic acid or by ultraviolet fluorometry with p-hydroxyphenylacetic acid. The detection limit of the method is in the subnanomole mass range for all three lipids. Results obtained with either fluorescence or colored endpoints were in excellent agreement with alternative individual chemical and enzymatic procedures.
Apolipoprotein B 3' hypervariable repeat genotype: association with plasma lipid concentration, coronary artery disease, and other restriction fragment polymorphisms
Authors: Wu J H , Chern M S , Lo S K , Wen M S , Kao J T
Published: January 2020

Apolipoprotein B gene 3' variable number tandem repeat (VNTR) and related regions were amplified by PCR and analyzed by agarose gel electrophoresis. Eighteen VNTR alleles (VNTR25, 26, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 58, 60) and 45 genotypes were observed in 477 Taiwanese subjects. The VNTR35 allele and genotype VNTR35/35 were observed most frequently in this population. The polymorphism information content was 0.62. Some minor alleles, such as VNTR25 and 60, were found only in coronary artery disease (CAD) and stroke patients in our sampling, and no statistically significant difference was observed in VNTR allelic frequency between control and CAD or stroke patients. Significant differences in allelic distribution of some VNTR alleles were observed between our normal Taiwanese population and a Caucasian group studied by others. VNTR43-47 and AluI+ (coding Ala591) restriction fragment length polymorphism (RFLP) as well as VNTR49-60 and EcoRI- (coding Lys4154) RFLP were found to be highly coinherited. No apparent association between the VNTR genotype and plasma lipid concentration was observed; however, for the same genotype, the CAD and stroke patients frequently showed higher lipoprotein(a) and lower HDL cholesterol concentrations than the control group.
Daidzein and genistein concentrations in human milk after soy consumption
Authors: Franke A A , Custer L J
Published: January 2020

Soy isoflavones were quantified from human milk by a fast, precise, and selective HPLC method after enzymatic hydrolysis of conjugated isoflavones and extraction with ethyl acetate. Isoflavone aglycones and their mammalian metabolites equol and O-desmethylangolensin were separated selectively and identified by absorbance patterns, fluorometric and electrochemical detection, gas chromatography-mass spectrometric analysis after trimethylsilylation, and with internal and external authentic standards. HPLC injections of 20 microL of human milk showed detection limits of 1-3 pmol for all analytes by using diode-array detection. The detection limit could be improved by as much as 1000-fold by extended concentration through partitioning with ethyl acetate, by using electrochemical detection, by increasing the injection volumes, or by combining these techniques. We used the proposed method to monitor isoflavone concentrations in human milk and in human urine after challenge with 5, 10, and 20 g of roasted soybeans in the diet. Implications of the results for the potential of isoflavones to prevent cancer in newborn infants exposed to these agents are discussed.
Ultrafiltration discrepancies in recovery of myoglobin from urine
Authors: Loun B , Copeland K R , Sedor F A
Published: January 2020

We investigated the efficiency, accuracy, and reliability of the ultrafiltration/dipstick methodology commonly used to diagnose myoglobinuria. Twenty-five myoglobin-containing urine specimens were filtered by centrifugation for 15 min at 1500g through a Centricon-30 membrane filter. Both the original specimen and filtrate were assayed for myoglobin. The amount of myoglobin recovered subsequent to filtration varied from <1-38%. This poor and variable recovery was independent of sample matrix or precentrifugation of the specimens. This was most critical for urine specimens with myoglobin concentrations <60 000 microg/L. Fourteen of 18 such filtrates had concentrations <350 microg/L, a concentration below which a negative result would be obtained by using conventional dipstick methods. Thus, the use of this procedure has the potential to misdiagnose patients with myoglobin concentrations associated with increased risk of subsequent renal dysfunction, in particular when urine myoglobin concentrations are <60 000 microg/L.
Evolution of serum lipids in two male bodybuilders using anabolic steroids
Authors: Lajarin F , Zaragoza R , Tovar I , Martinez-Hernandez P
Published: January 2020

We followed weekly the evolution of serum lipid concentrations in two bodybuilders undergoing a cycle of treatment with anabolic steroids. These drugs caused maximum depression of high-density lipoprotein cholesterol concentrations by 69.1% in the fifth week after the beginning of the cycle for subject 1, and by 72.4% in the fourth week for subject 2. Maximum increases in low-density lipoprotein cholesterol concentrations were 144% and 156%, respectively. Total cholesterol and apolipoprotein (apo) B were highly increased with anabolic steroid use. We also saw depression of apo A-I by 84% and 91%, and lipoprotein(a) decreased to undetectable amounts in both cases. These effects were reversed 10 weeks after the end of the steroid cycle in subject 1, but subject 2 still presented abnormal concentrations of serum lipids 13 weeks after drug cessation. The periods until reversibility of anabolic steroid effects on lipids were longer than those reported in previous studies.
Molecular diagnosis of Charcot-Marie-Tooth 1A disease and hereditary neuropathy with liability to pressure palsies by quantifying CMT1A-REP sequences: consequences of recombinations at variant sites on chromosomes 17p11.2-12
Authors: Vandenberghe A , Latour P , Chauplannaz G , Chapon F , Pouget J , Dumas R , Laguenay A , Ollagnon E , Bost M , Duthel S , Chazot G , Boucherat M
Published: January 2020

The most frequent form of Charcot-Marie-Tooth disease (CMT1A; OMIM118.220) is the result of a duplication on chromosome 17 in pll.2-p12. This region contains PMP22, a gene expressed in peripheral myelin. The mutation results from an unequal crossing-over involving repeated sequences, CMT1A-REP, located on both sides of the duplicated region. The reciprocal product of this recombination is a deletion of the same region, which is associated with hereditary neuropathy with liability to pressure palsies (HNPP; OMIM162.500). Proximal and distal CMT1A-REP sequences can be distinguished by the presence of a variant EcoRI site. We quantified the number of these repeat sequences in 36 CMT1A and 40 HNPP patients. CMT1A-REP sequences are involved in almost all of the mutations. The majority of recombination breakpoints occur distally from the variant EcoRI site. However, a few have a breakpoint proximal to this site, which creates the risk of misinterpretation with respect to a duplicated/deleted status.
Analytical performance and clinical validity of two free prostate-specific antigen assays compared
Authors: Jung K , Stephan C , Lein M , Henke W , Schnorr D , Brux B , Schürenkämper P , Loening S A
Published: January 2020

We compared two recently introduced commercial assays (CanAg and Immulite) for measuring free prostate-specific antigen (f-PSA), total PSA (t-PSA), and the ratio of t-PSA/f-PSA (f-PSA%) in control materials and sera of 54 healthy men, 50 patients with benign prostatic hyperplasia (BPH), and 45 patients with prostate cancer (PCa). The lower detection limits for f-PSA were 0.038 microgram/L and 0.004 microgram/L for the CanAg and Immulite assays, respectively. The within-run and between-day precisions of the Immulite assay were < 5%; the CanAg assay showed a poorer precision. Whereas f-PSA values differed between controls and patients but not between BPH and PCa patients, the f-PSA% values were lower in PCa patients than in BPH patients and controls. The receiver-operating characteristic (ROC) curve showed an improved diagnostic power of f-PSA% compared with t-PSA to discriminate between BPH and PCa. Discrimination limits of 16% (CanAg assay), and 15% (Immulite assay) are recommended for f-PSA%.
Long-term stability of blood glutathione and cysteine in humans
Authors: Richie J P , Abraham P , Leutzinger Y
Published: January 2020

Glutathione (GSH) availability is an important factor in the maintenance of health. Accordingly, blood GSH has been proposed as an indicator of health status. To validate the use of blood GSH in population studies, we investigated the long-term intraindividual variation of blood GSH and cyst(e)ine (Cys and cystine) concentrations in healthy adults. In a longitudinal study of 10 subjects, GSH and cyst(e)ine were measured in blood samples collected weekly over 8 months. The average within-person CV for GSH was only 9.1% compared with an observed interindividual CV of 20%. Blood cyst(e)ine was more variable within individuals (mean CV 14.7%) than GSH, whereas the interindividual CV for cyst(e)ine was lower (8.6%). The results demonstrate the stability of blood GSH in free-living subjects and the utility of GSH measurements as an indicator of long-term GSH status.
High sensitivity of the single-strand conformation polymorphism method for detecting sequence variations in the low-density lipoprotein receptor gene validated by DNA sequencing
Authors: Jensen H K , Jensen L G , Hansen P S , Faergeman O , Gregersen N
Published: January 2020

We designed oligonucleotide primer pairs to amplify the promoter region, the translated exon sequences, and the flanking intron sequences of all 18 exons of the LDL receptor gene to compare the ability of the PCR single-strand conformation polymorphism (PCR-SSCP) method with semiautomated solid-phase genomic DNA sequencing to detect sequence variations. In 20 apparently unrelated Danish patients with a clinical diagnosis of heterozygous familial hypercholesterolemia (FH), we identified 13 different mutations in the LDL receptor gene: two silent (C331C, N494 N); five missense (W66G, E119K, T383P, W556S, T7051); one nonsense (W23X); three splice-site (313 + 1G-->A, 1061-8T-->C, 1846-1G-->A); and two frameshift (335del10, 1650delG) mutations. Four of these mutations, N494 N, T383P, 1061-8T-->C, and W556S, have not been reported earlier. The pathogenicity of the T383P, 1061-8T-->C, and W556S mutations remains to be established by in vitro mutagenesis and transfection studies. One patient had three mutations (335del10, 1061-8T-->C, and T705I) on the same allele. Further, nine well-known polymorphisms were detectable with this methodological setup. Direct DNA sequencing of the PCR products used for the SSCP analysis did not reveal any sequence variations not detected by the PCR-SSCP method. In two patients we did not detect any mutation by either method. We conclude that the PCR-SSCP analysis, performed as described here, is as sensitive and efficient as DNA sequencing in the ability to identify the sequence variations in the LDL receptor gene of the patients with heterozygous FH of this study.
Noncompetitive ELISA for human serum insulin-like growth factor-I
Authors: Khosravi M J , Diamandi A , Mistry J , Lee P D
Published: January 2020

Measurement of serum insulin-like growth factor I (IGF-I) is important in assessing the growth hormone/IGF axis. Competitive immunoassay methods for IGF-I are complicated by substantial interference from IGF-binding proteins (IGFBPs) due to the relatively low ratio of specific antibody to IGFBPs in the sample, even after standard acid-ethanol sample extraction. We report of development of a noncompetitive ELISA for human IGF-I that avoids this problem by utilizing excess amounts of capture and detection antibodies. Serum samples were prepared by using an abbreviated acid-ethanol extraction method. Neutralized supernatant was added to microwells coated with IGF-I capture antibody; horseradish peroxidase-labeled detection antibody was then added, incubated for 2 h, and then developed. Compared with the "gold standard" method of acid-column chromatography, the simplified acid-ethanol extraction yields a mean +/- SD recovery of 103% +/- 5.5% despite the presence of residual IGFBPs in the extracted sample. Comparisons with a centrifugal filtration sample extraction method are also shown. The ELISA is specific for IGF-I with an absolute sensitivity of 0.03 microgram/L and inter- and intraassay CVs of 3.9-8.8% and 2.6-6.7%, respectively. The availability of a rapid IGF-I ELISA combined with a simple and reliable sample preparation procedure should facilitate clinical and research studies of this important growth factor.
Effects of androgen suppression by gonadotropin-releasing hormone agonist and flutamide on lipid metabolism in men with prostate cancer: focus on lipoprotein(a)
Authors: Denti L , Pasolini G , Cortellini P , Ferretti S , Sanfelici L , Ablondi F , Valenti G
Published: January 2020

No clear relation between lipoprotein(a) [Lp(a)] and endogenous gonadal hormones has been demonstrated. In this study, we compared the effects on Lp(a) of pharmacological castration in 50 patients with prostate cancer who were undergoing therapy with a gonadotropin-releasing hormone agonist (goserelin), with effects on 58 age-matched controls. We also studied 16 untreated patients under baseline conditions and after 3 months of therapy with goserelin alone or combined with an antiandrogen (flutamide). Neither cross-sectional nor prospective studies showed any significant effects of therapy on Lp(a). However, cluster analysis identified a subgroup of patients showing slight but significant increases in Lp(a) concentrations, as well as greater declines of testosterone and estradiol, suggesting that androgen, like estrogen, can exert some slight, though not easily detectable, influence on Lp(a).
Distribution of blood viscosity values and biochemical correlates in healthy adults
Authors: Rosenson R S , McCormick A , Uretz E F
Published: January 2020

Increases in the viscosity of blood and plasma predict clinical manifestations of atherothrombotic vascular disease. The clinical utility of viscosity measurements in cardiovascular risk factor analysis requires reference values established from a healthy disease-free population. A cohort of 126 (71 men, 55 women) healthy nonsmoking adults had fasting blood analysis after a 12-14-h fast. Viscosity measurements were made on samples of whole blood, plasma, and serum at 37 degrees C with a coaxial cylinder microviscometer. The mean blood viscosity at shear rates of 100, 50, and 1 s-1 were 3.26 +/- 0.43, 4.37 +/- 0.60, and 5.46 +/- 0.84 mPa.s, respectively. Men had significantly higher blood viscosity values than women at each shear rate. The differences in blood viscosity did not remain significant after blood viscosity values were normalized to a hematocrit of 45%, except at 100 s-1. For the entire group, normalized blood viscosity values at each measured rate correlated inversely with HDL cholesterol and positively with fibrinogen. The mean plasma viscosity was 1.39 +/- 0.08 mPa.s and the mean serum viscosity was 1.27 +/- 0.06 mPa.s. Plasma viscosity correlated with fibrinogen (r = 0.51, P < 0.0001), total serum protein (r = 0.33, P < 0.0001), and triglyceride concentrations (r = 0.33, P < 0.0015). Serum viscosity correlated with total serum protein (r = 0.50, P < 0.0001) and LDL cholesterol (r = 0.24, P = 0.0065). This study provides reference values for the viscosity of blood, plasma, and serum that may assist in evaluating hemorheological profiles.
Evaluation of a two-site immunoradiometric assay for measuring noncomplexed (free) prostate-specific antigen
Authors: Cuny C , Pham L , Kramp W , Sharp T , Soriano T F
Published: January 2020

Serum prostate-specific antigen (PSA) in men is present as two different molecular forms separable by gel-filtration chromatography (GFC). We have evaluated a two-site IRMA that measures only the noncomplexed (free) form of PSA (F-PSA). Verification that the F-PSA assay measures solely F-PSA was obtained by assaying GFC-fractionated serum samples with both the F-PSA IRMA and a commercial PSA assay that measures total PSA (T-PSA: F-PSA plus alpha 1-antichymotrypsin-complexed PSA). The F-PSA assay detected only the 30-kDa peak corresponding to the free form of PSA, whereas the T-PSA assay detected two peaks: complexed PSA at approximately 90 kDa and F-PSA at approximately 30 kDa. The F-PSA assay had an analytical detection limit of 0.03 microgram/L and a measuring range up to 50 micrograms/L. The intraassay CV was 1.7-10% in the concentration range of 0.2-30 micrograms/L. The interassay CV was 3.4-12.5% in the same concentration range. Dilution and recovery studies showed no significant deviation from linearity across the assay range. The assay was insensitive to interference from hemoglobin, bilirubin, and total lipids up to concentrations of 5, 0.2, and 10 g/L, respectively. No significant loss of immunological activity (analyte stability) was seen day-to-day ( < or = 5) or after repeated freeze/thaw ( < or = 5) cycles. We conclude that the F-PSA IRMA is an accurate, precise, and reliable tool for measuring F-PSA in human serum.
Calpastatin autoantibodies: detection, epitope mapping, and development of a specific peptide ELISA
Authors: Schlosser U , Lackner K J , Scheckenhofer C , Schmitz G
Published: January 2020

Autoantibodies against calpastatin (CAST) were detected in a 53-year-old female patient with a history of arthritis and thrombosis. The specificity of the autoantibodies was determined by screening expression cDNA libraries, sequence analysis of positive clones, and subsequent Western blotting against recombinant antigen. Because the Western blot lacked satisfactory reproducibility, an ELISA for anti-CAST antibodies was established. The major epitope recognized by 24 Western blot-positive sera was located within the C-terminal 27 amino acids. The ELISA was therefore based on a synthetic peptide representing these amino acids. The assay was calibrated with serial dilutions of a positive reference serum. Intraassay precision is high with a CV of approximately 4% for low- and high-titer samples. Interassay precision (CV) was 5.6-8.2% for sera with low and intermediate titers [10-60 arbitrary units (AU), where 1:100 dilution of the positive reference serum = 100 AU, 1:1000 dilution = 10 AU, etc.], which increases with higher titers ( > 60 AU). Among 205 healthy blood donors, the mean + 3 SD (after logarithmic transformation) was 30 AU; higher values were seen in 2.9% of 138 hospitalized patients. The newly developed ELISA will be a useful tool for further clinical studies on the association of anti-CAST antibodies to disease, because it permits rapid and reproducible analysis of patient sera.
Measurement of ascorbic acid in human plasma and serum: stability, intralaboratory repeatability, and interlaboratory reproducibility
Authors: Margolis S A , Duewer D L
Published: January 2020

We demonstrate that total ascorbic acid (TAA, the sum of ascorbic acid and dehydroascorbic acid) in properly prepared human plasma is stable at -70 degrees C for at least 6 years when preserved with dithiothreitol. TAA in human plasma or serum preserved with metaphosphoric acid degrades slowly, at the rate of no more than 1% per year. As assessed from our stability data and from data obtained from 23 laboratories over a period of > 2 years, the intralaboratory repeatability of TAA measurement is approximately 2 mumol/L, irrespective of TAA concentration. Nonchromatographic analytical methods involving dinitrophenylhydrazine and 0-phenylenediamine yield biased results relative to chromatographic methods. Within groups of laboratories that use roughly similar analytical methods, the interlaboratory measurement reproducibility CV for TAA is 15%.
Differential diagnosis between hepatocellular carcinoma and cirrhosis through a discriminant function based on results for serum analytes
Authors: Castaldo G , Oriani G , Lofrano M M , Cimino L , Topa M , Budillon G , Salvatore F , Sacchetti L
Published: January 2020

We applied a multivariate analysis to a large series of serum biochemical tests in an attempt to identify a function that could efficiently discriminate cirrhosis from hepatocellular carcinoma (HC). We analyzed two successive temporal cohorts (1987-90; 1991-94) of HC and cirrhotic patients, all histologically classified (first cohort: 69 cirrhosis and 39 HC; second cohort: 66 cirrhosis and 38 HC). Using data from the first temporal cohort of patients, we obtained a discriminant function based on seven serum analytes: alpha-fetoprotein, the hepatic isoenzyme of alkaline phosphatase, lactate dehydrogenase isoenzyme 5, total gamma-glutamyltransferase (GGT), GGT isoforms complexed with low-density lipoprotein, aspartate aminotransferase, and copper. The same panel of analytes emerged when the second cohort was tested and also when both cohorts were tested together. In the two successive cohorts (total, 212 patients) with a prevalence of cirrhosis vs HC of approximately 2:1, the discriminant function correctly classified 93% of cases, the highest percentage of correct classification of the two diseases obtained so far by laboratory approaches. Validation with the jackknife reallocation statistical algorithm confirmed these results. In addition, of six patients with liver cirrhosis for whom we had the opportunity of following up and observing the evolution to HC, five were classified as HC at diagnosis by the multivariate discriminant analysis; i.e., discriminant analysis provided a diagnostic lead time of 6-12 months over histology. This discriminant function, based on easy-to-perform serum biochemical tests, may help solve a fundamental problem of differential diagnosis in the evolution of chronic liver diseases from cirrhosis to HC.
Quantification of vanadium in serum by electrothermal atomic absorption spectrometry
Authors: Heinemann G , Vogt W
Published: January 2020

We describe an ultrasensitive and reliable method for determining vanadium in human serum by electrothermal atomic absorption spectrometry. After lyophilization, the serum is digested in acid at high pressure, and the digests are evaporated to a small volume. Vanadium in the digests is complexed with cupferron, extracted, and dried. The residue is redissolved in formic acid, where it is 15-fold more concentrated than in the original serum sample. To enhance the furnace sensitivity, we injected six 40-microL aliquots (total, 240 microL) of the concentrated extract. The median concentration of vanadium in 108 persons was 50 ng/L, in good agreement with previously reported results by neutron activation analysis. The characteristic mass obtained (the mass required to give a signal of 0.0044A. s) was 28 pg, the limit of detection 11 ng/L, the limit of quantification 17 ng/L, and the total imprecision (CV) 5.5% at 1.54 micrograms/L. In two assays of Standard Reference Material 1577a (certified vanadium content 99 +/- 8 ng/g), we obtained values of 94.1 and 97 ng/g.
Epidemiology of illicit and abused drugs in the general population, emergency department drug-related episodes, and arrestees
Authors: Rouse B A
Published: January 2020

National trends in substance abuse are presented: the civilian noninstitutionalized general population; drug-related emergency department episodes; and booked arrestees. Major metropolitan differences are also noted. This study was based on the primary national data systems for these groups: The Substance Abuse and Mental Health Services Administration (SAMHSA) National Household Survey on Drug Abuse, SAMHSA's Drug Abuse Warning Network (DAWN), and the National Institute of Justice Drug Use Forecasting (DUF) system. While the most prevalent drug differed in the three data sources, all three showed recent increases in marijuana. Despite the general decline in drug use seen in the general population, both the number of drug-related cases in the DAWN system and the drug use detected in the DUF arrestees showed recent increases.
Advances in poison management
Authors: Bayer M J , McKay C
Published: January 2020

This article advances the most current concepts in the management of poisoned patients including the use of ipecac, lavage, activated charcoal, whole-bowel irrigation, and specific antidotes. The benefits vs the risks of each of these procedures are reviewed.
Preimplantation diagnosis by whole-genome amplification, PCR amplification, and solid-phase minisequencing of blastomere DNA
Authors: Paunio T , Reima I , Syvänen A C
Published: January 2020

We have developed a new method for preimplantation diagnosis of inherited diseases. Our procedure for the identification of point mutations in single cells combines whole-genome amplification using 15-mer random primers (primer extension preamplification, PEP) with a single locus-specific PCR amplification, followed by detection of the mutation by solid-phase minisequencing. The procedure was evaluated by detecting three disease-causing mutations and seven polymorphic nucleotides located on different human chromosomes from single granuloma and blastomere cells. The correct genotype of the cell was identified at 96% of the nucleotide positions analyzed, showing that a representative part of the genome is amplified during PEP. We estimate that PEP yielded at least 1000 copies of the genome. The quantitative nature of the solid-phase minisequencing method allowed us to notice that preferential amplification of one allele occurs at heterozygous loci during PEP, which is a potential problem in preimplantation diagnosis.
Analysis of low-dose benzodiazepines by HPLC with automated solid-phase extraction
Authors: Akerman K K , Jolkkonen J , Parviainen M , Penttilä I
Published: January 2020

We describe a simple, specific, and sensitive reversed-phase HPLC method with automated solid-phase extraction (SPE) for analyzing alprazolam, clonazepam, and nitrazepam concentrations in human serum or plasma. We prepare samples and calibrators with an automated sample preparer, using 100-mg Bond-Elut C18 SPE columns. The HPLC method uses isocratic elution with acetonitrile:methanol:dipotassium hydrogen phosphate, 10 nmol/L, pH 3.7 (30:2:100 by vol), at a flow rate of 1.5 mL/min to separate the drugs. We detect the benzodiazepines with diode-array detector at 240 nm; analyses of peak purity are performed at 210-365 nm. The recoveries were between 94% and 100%. The intraassay and interassay CVs were between 1.0% and 4.1%. The detection limit is 5 nmol/L. The antiepileptic and antidepressant drugs tested did not interfere with the assay. We developed the method for use in a clinical laboratory for therapeutic drug monitoring.
Sensitive and specific quantification of sirolimus (rapamycin) and its metabolites in blood of kidney graft recipients by HPLC/electrospray-mass spectrometry
Authors: Streit F , Christians U , Schiebel H M , Napoli K L , Ernst L , Linck A , Kahan B D , Sewing K F
Published: January 2020

Sirolimus (rapamycin) has a macrolide structure and is under clinical investigation as an immunosuppressant after organ transplantation. An HPLC/mass spectrometry assay to quantify sirolimus in blood was developed. 28-O-Acetyl sirolimus was used as internal standard. Blood samples were extracted with C18 columns. The extracts were injected into an HPLC system and isocratically eluted with methanol/1% formic acid (90/10 by vol) from a 150 X 4 mm C18 analytical column. The HPLC system was connected to a triple-stage quadrupole mass spectrometer with an electrospray interface and positive ions were detected. The limit of quantification in 1 mL of blood was 0.25 microgram/L and the calibration curve in blood was linear up to 250 microgram/L. The recovery from blood was 88 +/- 26% and interassay variation at 1 microgram/L was 19% and at 15 microgram/L 9.3%. Hydroxy, dihydroxy, demethyl, and didemethyl sirolimus as well as sirolimus were detected in blood of kidney graft patients.
Tacrolimus (FK506) metabolite patterns in blood from liver and kidney transplant patients
Authors: Gonschior A K , Christians U , Winkler M , Linck A , Baumann J , Sewing K F
Published: January 2020

The metabolite patterns of tacrolimus in blood were evaluated in 41 kidney and liver graft recipients. Trough concentrations of tacrolimus and its metabolites were measured by HPLC-mass spectrometry and microparticle enzyme immunoassay in parallel. A statistically significant correlation between results of both assays was observed for kidney and liver transplant patients (r = 0.77, P <0.001 and r = 0.71, P <0.001, respectively). The main metabolites in blood were demethyl, demethylhydroxy, didemethyl, didemethylhydroxy, and hydroxy tacrolimus. These metabolites added up to 42% (range 0-145%) of the tacrolimus concentration in liver transplant patients and to 44.8% (range 16-152%) in kidney transplant patients. During episodes of impaired liver function, concentrations of tacrolimus and its metabolites were increased compared with normal liver function, indicating accumulation of metabolites, in particular second-generation metabolites such as didemethyl and didemethylhydroxy tacrolimus. Stepwise regression analysis including tacrolimus, its metabolites, and liver function parameters suggested a model including serum activities of gamma-glutamyltransferase, alkaline phosphatase, and alanine aminotransferase as predictors for increased concentrations of demethyl tacrolimus, didemethyl tacrolimus, and the parent drug.
Clinical evaluation of the Triage analytic device for drugs-of-abuse testing
Authors: de la Torre R , Domingo-Salvany A , Badia R , Gonzàlez G , McFarlane D , San L , Torrens M
Published: January 2020

In the present clinical evaluation of Triage immunoassay for detection of drugs of abuse, we examined a possible connection between the laboratory skills of the personnel using it and the quality of analytical results; we also evaluated the degree of concordance between the Triage results and those obtained by an instrumented fluorescence polarization immunoassay (FPIA). Three groups of evaluators with different laboratory skills and two sets of urine samples from subjects with different rates of drug consumption prevalence were included in the study. Urines were analyzed by the Triage analytical device at the collection site. A second analysis was performed in a toxicology laboratory, and the results were compared with those obtained by FPIA. Nonconcordant results were confirmed by gas chromatography-mass spectrometry and HPLC. Results were independent of the laboratory skills of the evaluators. The agreement for each drug calculated for two populations with differences in prevalence of drug consumption was almost the same, except for benzodiazepines. No differences between Triage and FPIA results were observed for samples clearly over or under cutoff concentrations. Globally, the Triage device, in a clinical situation, demonstrated performance comparable with that of an instrument-based immunoassay and, in some cases (e.g., for benzodiazepines), the Triage performance was even better.
Monoclonal antibody-based dual-label time-resolved fluorometric assays in a simplified one-step format
Authors: Meriö L , Pettersson K , Lövgren T
Published: January 2020

We have performed a one-step all-in-one dual-label dry reagent time-resolved fluorometric assay measuring free and total prostate-specific antigen (PSA). All assay-specific components were predried in an ordinary microtitration well, including the Eu and Tb chelate-labeled monoclonal antibodies for the recognition of free and total PSA, respectively. Only the sample and assay buffer needed to be added. Optimally, equilibrium was reached in <15 min. New intrinsically fluorescent Eu and Tb chelates were used, which made it possible to read the fluorescence directly from the solid phase. The developed dual-label assay with the simple protocol is ideal for easy automation, as the reaction wells can be of alternative design. The performance characteristics are equivalent to those of the present state-of-the-art single-label assays. The detection limits for free and total PSA were 0.03 and 0.02 microg/L, respectively, and the assays were linear over the range tested (up to 300 microg/L).