bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of a Transformation Method for Estimation of Normal Range
Authors: Reed Allen H , Wu Gaw Tzu
Published: January 2020

We have evaluated a recently proposed method for estimating normal limits by prior transformation of data to gaussian-like form. Computer simulation techniques are used to generate random data from frequency distributions having various shapes. For each such distribution, the transformation method is compared with the method of nonparametric percentile estimation. We conclude that the nonparametric method should continue to be the method of choice unless data have a nearly symmetrical frequency distribution. The present state of methods for estimating normal ranges is discussed.
Increased Uropepsinogen Excretion in Diabetes Mellitus
Authors: Belfiore Francesco , Lo Vecchio Luigi , Napoli Elena , Borzi Vito , Rabuazzo Agata M
Published: January 2020

The average value for 24-h excretion of uropepsinogen increased by 64% (P < 0.05) in 44 uncomplicated diabetics as compared to 33 normal subjects. Uropepsinogen excretion was correlated with the daily output of urine (r = 0.43, P < 0.05) in the normals, but not in the diabetics (r = 0.24, P > 0.10). This shows that the enhanced excretion in diabetics is not merely a result of increased urine output, and suggests that diabetes is an interfering factor that affects uropepsinogen excretion. No correlation was found with age (in normal persons or diabetics), duration of disease, fasting glycemic level, or daily insulin requirement. Because gastric secretion is depressed in persons with diabetes mellitus, the increased uropepsinogen excretion is tentatively attributed to alterations of gastric mucosa, known to occur in this disease, which might result in a change of the "exocrine-endocrine partition" of pepsinogen in favor of the "endocrine" fraction, i.e., the fraction that enters the blood.
Quality-Control System for Blood pH and Gas Measurements, with Use of a Tonometered Bicarbonate—Chloride Solution and Duplicate Samples of Whole Blood
Authors: Noonan Daniel C , Burnett Robert W
Published: January 2020

We describe a procedure for the simultaneous quality control of pH, pco2,and po2 measurement. A solution of NaCl (150 mmol/liter) and NaHCO3 (50 mmol/liter) is equilibrated at room temperature (24 ± 2 °C) with a gas having the composition 21% O2, 12% CO2, 67% N2. When this solution is analyzed at 37 °C in a conventional blood-gas analyzer, a pH of 7.20, a pco2 of 110 mmHg (14.6 kPa) and a po2 of 170 mmHg (22.6 kPa) are obtained. We show that data from such an analysis, performed daily, can be simply used to meet many of the unique demands of quality control in blood pH and gas determinations. This same approach may also become the basis for devising an ideal aqueous system for blood gas calibration.
Evaluation of Alternative Counting Methods for Radioimmunoassay of Hepatitis-Associated Antigen (HB Ag)
Authors: Jordan William C , Spiehler Vina , Haendiges Robert , Helman Edith Zak
Published: January 2020

Alternative counting methods for the Abbott "Aus-RIA" hepatitis-associated antigen test have been devised and evaluated. These methods involve solubilization of the tube-bound radiolabeled complex with glacial acetic acid, transfer of the eluate to a counting vial of choice, and measurement by either a gamma scintillation or a liquid scintillation system. These methods, when compared to the direct counting of the AusRIA tube for sera from more than 250 patients and controls, gave a correlation coefficient of 0.94 to 0.99. Iodine-125 counting efficiencies averaged 65% both for direct counting of the AusRIA tube and for gamma counting of the eluate. By liquid scintillation counting, iodine-125 counting efficiency averaged 50%. This method avoids high background, chemiluminescence, and quench, and is easy to perform. By using glacial acetic acid to solubilize the labeled material, the eluate can be counted by various types of gamma counters as well as by liquid scintillation systems.
Fluorometric Analysis for N'-Formylkynurenine in Plasma and Urine
Authors: Buxton T , Guilbault G G
Published: January 2020

We describe a fluorometric method of analysis for N'-formylkynurenine. The method depends on the rate of enol formation when a solution of the compound is made basic. The enol is more fluorescent than the neutral compound. The increase in fluorescence is measured, with use of 430-nm emission and 319-nm excitation wavelengths. This method is applied to the determination of N'-formylkynurenine in protein-free filtrates of plasma or urine. To our knowledge, this is the first quantitative method for determination of N'-formylkynurenine.
Determination of Unbound Bilirubin in the Serum of Newborns
Authors: Jacobsen Jorgen , Wennberg Richard P
Published: January 2020

An enzymatic assay is described for non-albuminbound bilirubin in the serum of newborn infants. Unbound bilirubin is oxidized to colorless compounds by ethyl hydroperoxide in the presence of horseradish peroxidase (EC 1.11.1.7), while albumin-bound bilirubin is protected from oxidation. Because the equilibrium between albumin and bilirubin occurs rapidly, the oxidation step is rate limiting, and the initial oxidation velocity of total bilirubin is proportional to the unbound bilirubin concentration. By titrating serum with bilirubin in vitro, the association constant and binding capacity of high-affinity sites for albumin binding can be determined. Normal human serum albumin tightly binds 1 mole of bilirubin per mole of albumin (binding constant, 2-4 x 108 liter/mol). Although weaker secondary binding occurs, the unbound bilirubin fraction increases rapidly after the high-affinity binding sites are saturated. Compromised newborns may have a decreased apparent binding capacity and (or) binding affinity. The method can be used to assess the risk of a jaundiced infant for bilirubin encephalopathy.
Chemical and Clinical Evaluation of the Continuous-flow Analyzer "SMAC"
Authors: Schwartz Morton K , Bethune Victor G , Fleisher Martin , Pennacchia Gina , Menendez-Botet Celia J , Lehman Donald
Published: January 2020

"SMAC" (Sequential Multiple Analyzer plus Computer) is a high-speed computer-controlled multitest analyzer. A 20-channel prototype SMAC (glucose, urea nitrogen, creatinine, carbon dioxide content, total bilirubin, calcium, phosphorus, cholesterol, iron, uric acid, chloride, sodium, potassium, total protein, albumin, creatine kinase, alkaline phosphatase, lactate dehydrogenase, and aspartate and alanine aminotransferases) has been evaluated for: (a) method precision during within-day runs and on a day-to-day basis over a period of time; (b) method linearity over a range established on a chemical basis and related to clinical requirements, with use of both aqueous standards and protein matrix reference material; and (c) correlation of SMAC values with those obtained by the methods routinely in use in our department.
Simultaneous Determination of Methemoglobin and Total Hemoglobin by a Continuous-flow Method
Authors: Groff William A , Kaminskis Andris , Cucinell Samuel A
Published: January 2020

We describe a sensitive, accurate automated method for the simultaneous measurement of methemoglobin and total hemoglobin in blood. The method can be used for either continuous in vivo monitoring or intermittent sampling at a rate of 20/h. Standardization procedures are described, in which human blood treated with sodium nitrite is used. Repeatability of methemoglobin and total hemoglobin measurements, expressed as coefficient of variation in percent, are within ±4.1 and ±2.6%, respectively. Normal methemoglobin concentration’s in humans and dogs did not exceed 0.55 g/dl (3.3% of the total hemoglobin).
Optimized Determination of γ-Glutamyltransferase by Reaction-Rate Analysis
Authors: Rosalki Sidney B , Tarlow David
Published: January 2020

We describe a method for measuring γ-glutamyltransferase (EC 2.3.2.2) activity in serum, which can be used with automated enzyme analyzers (such as the LKB 8600 Reaction Rate Analyzer) that require enzyme reactions to be initiated with substrate. The method also permits optimal determination conditions to be obtained at 37 °C. The enzymatic reaction is commenced by adding γ-glutamyl-p-nitroanilide dissolved in dilute hydrochloric acid to samples pre-incubated with tris(hydroxymethyl)aminomethane—glycylglycine buffer. The p-nitroaniline liberated is continously monitored at 37 °C at 405 nm. The pH of the pre-incubation buffer is such that the optimal pH for the enzyme reaction results from addition of the acid substrate solution.
New Method for Direct Determination of "True" Creatinine
Authors: Yatzidis Hippocrates
Published: January 2020

Determination of serum creatinine by the Jaffé reaction may give erroneous results because of interfering noncreatinine chromogens. Studies of the Jaffé reaction in alkaline picrate media of various pH demonstrated that creatinine, proteins, and certain chromogens have characteristic properties as regards the appearance and intensity of the color reaction. Thus a method for determining "true" creatinine has been developed in which simple spectrophotometric measurements are made at 500 nm in two alkaline picrate reagents, buffered at pH 9.65 and 11.50. Results agree well with those obtained by the use of an ion-exchange method. The proposed method is easy and rapid and offers the advantages of increased specificity and direct analysis of serum and urine.
Radioimmunoassay for Serum Triiodothyronine: Evaluation of Simple Techniques to Control Interference from Binding Proteins
Authors: Fang Victor S , Refetoff Samuel
Published: January 2020

Simple techniques for controlling interference from binding proteins in serum, such as thyroxine-binding globulin, in radioimmunoassay for triiodothyronine (T3) have been evaluated for their efficacy, and their effect on assay sensitivity and on recovery of added T3. Ethanol precipitation of serum proteins decreased the assay sensitivity, nonspecific binding was increased, and recoveries of added T3 were inconsistent. Heat-inactivation of thyroxine-binding globulin or use of 8-anilino-1naphthalene sulfonic acid (ANS) to displace T3 from thyroxine-binding globulin produced comparable recovery rates. The heat-inactivation method slightly decreased the sensitivity of the assay and prolonged the procedure, whereas use of ANS is simple, and the assay sensitivity is maintained. When sera contain a high concentration of thyroxine-binding globulin, a fixed concentration of ANS (175 µg/100 µl of serum) might be too low to displace T3 from all its binding sites, but a concentration of ANS greater than 200 µg/100 µl of serum interferes with T3 quantitation by competitively binding to the antibodies. The cross-reactivity of thyroxine to T3-antibodies varies with the antiserum. Thyroxine-binding globulin appears to be the only protein in serum that competes with the antibody for T3 binding.
Increased β-N-Acetyl-Glucosaminidase Activity in Diabetes Mellitus
Authors: Belfiore Francesco , Lo Vecchio Luigi , Napoli Elena , Borzi Vito
Published: January 2020

In 45 diabetics the 24-h urinary excretion of β-N-acetylglucosaminidase (E.C. 3.2.1.30) was increased by 40% (P < 0.05) compared to 35 control subjects. The enzyme excretion was correlated with glycemia (r = 0.58, P < 0.001), being little changed in diabetics with blood glucose concentrations of less than 200 mg/dl, and markedly elevated (+ 123%, P < 0.001) in those whose blood glucose was greater than 200 mg/dl. The rate of diuresis seemed to have no effect. These data indicate that the enhanced activity previously described in sera of diabetics for β-N-acetyl-glucosaminidase (as well as for other lysosomal enzymes) cannot be attributed to impairment of renal excretion, and support the hypothesis that in diabetes there is an "activation" of lysosomal enzymes in tissues that causes an increase in their activity in serum and, consequently, in urine.
"U" Protein and Migration Ratios in Paper Electrophoresis
Authors: Stern Joel R , Mais Roland F , Boggs Joseph D
Published: January 2020

Paper electrophoresis of serum from children with liver and kidney disease has led to recognition of a protein which migrates between the 2 and globulins. This protein has been observed by other workers, but because of confusion in nomenclature, a new designation, "U" protein, has been employed. The use of migration ratios in locating peaks of the 2 "U" protein, and globulin when these components resolve poorly has been discussed.

Characterization of "U" protein by employing the periodic acid-reduced fuchsin test for carbohydrate-protein complexes and Sudan black B for lipoprotein showed: (1)"U" protein varies in its content of protein-carbohydrate complex, and (2)"U" protein may be lipo-protein. Evidence based on benzidine tests and intentional hemolysis indicated that "U" protein was not an artifact resulting from accidental hemolysis, but a protein which is found in serum under certain physiological conditions.
Serum Copper and Iron on a Single Sample
Authors: Zak B , Ressler N
Published: January 2020

A procedure has been described for the quantitative determination of copper and iron in one serum sample. The sera are wet ashed to destroy organic material, leaving inorganic salts available in the residue for the subsequent spectrophotometric determination of iron as the ferrous-1,10-phenanthroline complex in the aqueous phase, and copper as the cuprous-2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline in an organic extract phase.
The Complexometric Titration of Micro and Ultramicro Quantities of Calcium in Blood Serum, Urine, and Inorganic Salt Solutions
Authors: Bachra Bernard N , Dauer Alan , Sobel Albert E
Published: January 2020

A method for the microdetermination and ultramicrodetermination of calcium in blood serum, urine, and inorganic salt solutions has been developed. Calcium was titrated with disodium ethylenediaminetetraacetate, and Cal-red as the indicator, using a Gilmont ultramicro-buret of 0.1-ml. capacity.

Amounts of 5 to 30 µg. of calcium per sample, of 0.05- to 1-ml. volumes, can be determined with a reproducibility of 1 to 2 per cent.

The difference between the values found for serum calcium with the Cal-red and the oxalate method had a standard deviation of ±3.32 per cent.

Serum magnesium can be determined as the difference of two simple titrations.

Phosphate levels up to 30 mg. per 100 ml. P did not interfere with the determination in serum, even when the calcium level was raised to about 20 mg. per 100 ml.

Up to products of 80(Ca X P, expressed in mg. per 100 ml.) there was no interference, in inorganic salt solutions. At higher products drift occurred, but the addition of some citrate to the solutions enabled the attainment of sharp end points at Ca X P products up to approximately 1000.

Urinary calcium was determined satisfactorily, without the occurrence of drift, if citrate was added to the solution.

Magnesium did not interfere appreciably, giving only 1 per cent higher readings at levels of 20 mg. per 100 ml. The interference of copper and iron was removed by the addition of sodium cyanide.
The Ferrohemoglobin Solubility Test
Authors: Goldberg C A J
Published: January 2020

1. Accuracy and precision of the ferrohemoglobin solubility test, a modification of the Itano procedure, have been discussed.

2. Excellent reproducibility was achieved using 10 buffers and 10 samples with a wide range of solubilities.

3. Ranges for normal adult hemoglobin, hemoglobins A and S, S in the presence of a small amount of fetal hemoglobin, S and C, and A and C are given, together with single values for hemoglobins A and D, A and I, A and L, and A and J.
Determination of Mercury in Urine
Authors: Nobel Sidney , Nobel Deana
Published: January 2020

A simple quantitative method for determination of mercury in urine has been described. The method is based on the reduction of mercury in urine to metallic mercury by a copper-hydrazine system. The metallic mercury is oxidized to mercuric ion by acid permanganate at room temperature. No special equipment or heat digestion is required. The mercury is estimated by a titrimetric dithizone technic. Industrial screening experience for mercurialism has been described.
Urinary Calculi
Authors: Nicholas Henry O , Leifeste H F
Published: January 2020

During the course of this investigation we have encountered two "fakes," both of which were represented by the patients to their respective physicians as having been "passed." They showed less than 2 per cent loss on ignition and proved to be calcium-iron-aluminum silicate rocks. We have also had about a dozen prostatic calculi which were analysed by the "ashing" method. This type of calculus consists of either a mixture of magnesium ammonium phosphate and calcium phosphate, or a mixture of calcium carbonate and calcium phosphate. The above-mentioned stones are, of course, not included in this picture. We hope that this survey and the simple method of analysis presented in these two articles will stimulate further work along this line in various parts of the country. The best similar survey we have seen is that of Leonard and Butt (1) on the types of calculi found in the Pensacola, Florida, area.
Residual Serum Thrombin Activity
Authors: Weiner Murray
Published: January 2020

A simple test for residual thrombin activity of serum is described. This test is believed to reflect the over-all antithrombin activity of the specimen.

These results are not altered by silicone vs. glass handling. The test is influenced by the presence or absence of contact with a fresh clot; by deficiencies in prothrombin or fibrinogen; by aging of plasma specimens and possibly by streptokinase administration in vivo, and by pancreatic disease.

The full significance of alteration in this test remains to be determined.
A Rapid Colorimetric Method for the Quantitative Determination of Copper Oxidase Activity (Ceruloplasmin)
Authors: Houchin O Boyd
Published: January 2020

A rapid procedure for determining quantitatively copper oxidase activity of plasma or serum is described. It is calibrated against determined amounts of ceruloplasmin and can be adapted to usual clinical laboratory equipment for the quantitative determination of ceruloplasmin.
DNA Microarray to Monitor the Expression of MAGE-A Genes
Authors: Zammatteo Nathalie , Lockman Laurence , Brasseur Francis , De Plaen Etienne , Lurquin Christophe , Lobert Pierre-Emmanuel , Hamels Sandrine , Boon Thierry , Remacle José
Published: January 2020

Background: The MAGE-A genes encode antigens that are of particular interest for antitumor immunotherapy because they are strictly tumor specific and are shared by many tumors. We developed a rapid method to identify the MAGE-A genes expressed in tumors.

Methods: A low-density DNA microarray was designed to discriminate between the 12 MAGE-A cDNAs amplified by PCR with only one pair of consensus primers. The assay involved reverse transcription of total RNA with oligo(dT) primer, followed by PCR amplification and hybridization on a microarray. Amplification in the presence of Biotin-16-dUTP allowed subsequent detection of the amplicons on the microarray carrying 12 capture probes, each being specific for a MAGE-A gene. Probe–amplicon hybrids were detected by a streptavidin-based method.

Results: PCR conditions were optimized for low detection limits and comparable amplification efficiencies among all MAGE-A nucleotide sequences. The microarray assay was validated with a panel of 32 samples, by comparison with well-established reverse transcription-PCR assays relying on amplification with primers specific for each gene. Virtually identical results were obtained with both methods, except for MAGE-A3 and MAGE-A5. Detection of MAGE-A5 was more sensitive with the microarray assay. Detection of MAGE-A3 was hampered by the presence of MAGE-A6, which is 98% identical: the MAGE-A3 capture probe cross-hybridized with MAGE-A6 amplicons because these sequences differed by only a single base.

Conclusions: This post-PCR microarray assay could be useful to evaluate MAGE expression in tumors before therapeutic vaccinations with MAGE-A gene products.
Use of Denaturing HPLC to Provide Efficient Detection of Mutations Causing Familial Hypercholesterolemia
Authors: Bodamer Olaf A , Bercovich Dan , Schlabach Michael , Ballantyne Christie , Zoch Danièle , Beaudet Arthur L
Published: January 2020

Background: Autosomal dominant familial hypercholesterolemia (FH) attributable to mutations in the LDL receptor (LDLR) gene is one of the most common genetic disorders associated with significant morbidity and mortality. Definitive diagnosis would help to initiate appropriate treatment to prevent premature cardiovascular disease. Currently, clinical diagnosis of FH is imprecise, and molecular diagnosis is labor-intensive and expensive because of the size of the LDLR gene and number of coding exons.

Methods: We used PCR to amplify all exons, including exon/intron boundaries, and the promoter of the LDLR gene. Nine individuals from five families with typical findings for a clinical diagnosis of heterozygous FH, 2 heterozygous FH cell lines, and 50 control individuals were screened for mutations by denaturing HPLC (DHPLC) followed by direct sequencing of aberrantly migrating fragments.

Results: Mutations that were previously reported to be disease causing were identified in eight of nine individuals with FH and both cell lines (V502M, C146X, E207X, C660X, C646Y, and delG197), but none were found in controls. The one individual with FH in whom no mutation was found had a previously unreported change in the 5′-untranslated region of unknown significance. In addition, we identified several previously reported polymorphism both in controls and individuals with FH.

Conclusions: DHPLC can be used to detect mutations causing FH. On the basis of our current experience with DHPLC, this method combined with confirmatory DNA sequencing is likely to be sensitive and efficient.
In-Cell PCR Method for Specific Genotyping of Genomic DNA from One Individual in a Mixture of Cells from Two Individuals: A Model Study with Specific Relevance to Prenatal Diagnosis Based on Fetal Cells in Maternal Blood
Authors: Hviid T Vauvert
Published: January 2020

Background: During recent years, much attention has been paid to the possibility of using fetal cells circulating in the pregnant woman’s blood for prenatal diagnosis of genetic or chromosomal abnormalities. Although successes have been achieved in enrichment procedures for fetal cells from maternal blood samples, the use of such an approach for genotyping by molecular biology techniques in a more routine setting has been hampered by the large contamination of maternal nucleated blood cells in the cell isolates. Therefore, a new method based on in-cell PCR is described, which may overcome this problem.

Methods and Results: Mixtures of cells from two different individuals were fixed and permeabilized in suspension. After coamplification of a DNA sequence specific for one of the individuals and the DNA sequence to be genotyped, the two PCR products were linked together in the fixed cells positive for both DNA sequences by complementary primer tails and further amplification steps. In a model system of mixtures of male and female CD71-positive cells from umbilical cord blood attached to immunomagnetic particles, a Y-chromosome-specific sequence (TSPY) was linked to a polymorphic HLA-DPB1 sequence only in the male cells, leading to the correct HLA-DPB1 genotyping of the male by DNA sequencing of a nested, linked TSPY-HLA-DPB1 PCR product.

Conclusion: This approach might be usable on mixed cell populations of fetal and maternal cells obtained after conventional cell-sorting techniques on maternal peripheral vein blood.
Structural Diversity of Cancer-related and Non-Cancer-related Prostate-specific Antigen
Authors: Isono Takahiro , Tanaka Tsutomu , Kageyama Susumu , Yoshiki Tatsuhiro
Published: January 2020

Background: Heterogeneity among the various molecular forms of prostate-specific antigen (PSA) has not been well characterized, despite the critical importance of PSA in the detection of prostate cancer. The purpose of this study was to examine PSA heterogeneity in cancerous and noncancerous materials by extensive and systematic protein analysis.

Methods: A catalog of molecular forms of PSA was established with the PSA purified from seminal fluid. This catalog was used to analyze PSA heterogeneity in cancerous and noncancerous materials by immunoblotting with polyclonal antibodies.

Results: PSA from noncancerous materials showed a wider range of molecular mass, from 6000 to 28 000 Da. PSA from cancerous materials did not contain lower molecular mass forms.

Conclusions: The PSA protein catalog may be useful for the analysis of differences among PSA forms in men with and without prostate cancer and for analysis of antibodies used to detect PSA.
Apolipoprotein E in Apolipoprotein B (apo B)- and Non-apo B-containing Lipoproteins in 3523 Participants in the Stanislas Cohort: Biological Variation and Genotype-specific Reference Limits
Authors: Schiele Françoise , Vincent-Viry Monique , Starck Marjorie , Beaud Brigitte , Hennache Geneviève , Siest Gérard , Visvikis Sophie , Herbeth Bernard
Published: January 2020

Background: Apolipoprotein (apo) E is a component of two major classes of plasma lipoproteins, apo B- (apo E-LpB) and non-apo B-containing (apo E-Lp-non-B) lipoproteins. The factors that affect total apo E in particles [lipoprotein E (LpE), apo E-Lp-non-B, and apo E-LpB], are incompletely characterized.

Methods: We studied the determinants of these lipoparticles in a sample population of presumably healthy individuals: 1784 children (age range, 8–18 years) and 1739 adults (age range, 19–50 years). Serum concentrations of LpE and apo E-Lp-non-B were measured by electroimmunoassays, and the concentration of apo E-LpB was calculated by a difference method.

Results: Serum LpE and apo E-Lp-non-B were higher in females than in males. Their concentrations decreased with age until 20–25 years and then increased in men but not in women. apo E-LpB concentrations increased up to 20–25 years and were similar in both sexes. Thereafter, adult men had higher values than women. Individuals carrying the ε2 allele had higher mean apo E-Lp-non-B concentrations and lower apo E-LpB concentrations than did individuals carrying the ε3 allele. Individuals with the ε4 allele showed an inverse profile compared with those with the ε2 allele. Age, gender, the common apo E polymorphism, puberty, serum lipid concentrations, and alcohol consumption were significantly associated with total LpE, apo E-Lp-non-B, and apo E-LpB concentrations. Reference limits were established according to age, gender, and the common apo E polymorphism.

Conclusions: Because measurements of LpE, apo E-Lp-non-B, and apo E-LpB concentrations may improve cardiovascular risk assessment, the proposed reference limits will aid interpretation of the results in clinical or therapeutic trials.
Stability of 11-Nor-Δ9-carboxy-tetrahydrocannabinol Glucuronide in Plasma and Urine Assessed by Liquid Chromatography-Tandem Mass Spectrometry
Authors: Skopp Gisela , Pötsch Lucia
Published: January 2020

Background: Unconjugated 11-nor-Δ9-carboxy-tetrahydrocannabinol (THCCOOH) in blood and urine has been proposed as a valuable marker, but the glucuronide (THCCOOglu) is present in considerably higher concentrations than the parent drug. Acyl glucuronides have been shown to be potentially reactive conjugates, which may affect the in vitro metabolite pattern.

Methods: Extraction procedures and a liquid chromatography-tandem mass spectrometry assay were developed and validated to investigate the stability of THCCOOglu in urine and plasma. Plasma and urine samples with added THCCOOglu were stored at −20, 4, 20, and 40 °C up to 10 days.

Results: The glucuronide was stable at −20 °C in both matrices, whereas THCCOOglu concentrations decreased at all other storage conditions. For a given storage time and temperature, the decrease in plasma was higher than that in urine. At 20 °C, a marked change in concentration could be observed within 2 days of storage. Degradation of THCCOOglu followed an apparent first-order process and led to the formation of THCCOOH. The sum of the molar concentrations of both analytes corresponded only to the initial THCCOOglu in plasma and urine samples stored at 4 °C.

Conclusions: The in vitro degradation of THCCOOglu prevents clinical conclusions based on the metabolite pattern or the concentration of unconjugated THCCOOH in samples stored at ≥4 °C for prolonged periods.
Evaluation of an Automated Preanalytical Robotic Workstation at Two Academic Health Centers
Authors: Holman J William , Mifflin Theodore E , Felder Robin A , Demers Laurence M
Published: January 2020

Background: Purchase of automated systems in today’s clinical laboratory needs justification based on demonstrable improvements in efficiency and a sound payback model. Few studies provide information on laboratory automation that focuses on the preanalytical portion of specimen processing.

Methods: We recently evaluated an automated preanalytical processing unit (GENESIS FE500) at two academic health centers. This preanalytical unit processes blood specimens through automated specimen sorting, centrifugation, decapping, labeling, aliquoting, and placement of the processed specimen in the analytical rack. We quantified the output of the FE500 by processing >3000 barcode-labeled specimens according to a protocol designed to test all of the features of this automated specimen-processing unit.

Results: Depending on the batch size, aliquot number requested, and percentage of tubes that required centrifugation, the mean system output performance varied between 93 and 502 total tubes/h. Throughput increased when the batch size expanded from 40 or 100 samples (mean = 211 total tubes processed/h) to batch sizes of 200 and 300 tubes (mean = 474 total tube processed/h). The GENESIS FE500 processed specimen tubes differing in size from 13 × 65 mm (width × height) to 16 × 100. At one site, the FE500 was operated by one person, compared with the three individuals required to perform the same tasks manually. Finally, the specimen-processing error rate determined at one of the institutions was significantly reduced.

Conclusions: We conclude that the GENESIS FE500 effectively reduces the labor associated with specimen processing; decreases the number of laboratory errors that occur with specimen sorting, labeling, and aliquoting; and improves the integrity of specimen handling throughout the steps of specimen processing.
Cystatin C: An Improved Estimator of Glomerular Filtration Rate?
Authors: Laterza Omar F , Price Christopher P , Scott Mitchell G
Published: January 2020

Background: Glomerular filtration rate (GFR) is routinely assessed by measuring the concentrations of endogenous serum markers such as blood urea nitrogen and serum creatinine (SCr). Although widely used, these endogenous markers are not ideal and do not perform optimally in certain clinical settings. The purpose of this review is to critically review the potential utility of cystatin C (CysC), especially in patient populations in which CysC may have an advantage over routinely used endogenous markers of GFR.

Approach: In a narrative approach, we extensively review publications, primarily from the last 5 years, that address the development of methods to measure CysC, reference intervals, and the diagnostic accuracy of CysC to assess GFR. Between June 2000 and September 2001 Medline was searched using “cystatin c” as a textword, and articles that examined >75 individuals (except for renal transplant studies) and/or used accepted “gold standards” for assessing GFR were selected for inclusion. A total of 17 studies are reviewed that provide reference interval data for several populations. A total of 24 studies make conclusions about the utility of CysC vs SCr and/or creatinine clearance, with 20 providing data on the sensitivity and specificity of CysC for detecting impaired GFR. These publications are organized into subgroups that deal with specific patient populations or clinical situations.

Content: This review focuses on two areas: (a) the evolution of immunoassays used to determine the concentration of CysC in serum, their analytic sensitivity, and reference intervals; and (b) the diagnostic performance of CysC against other renal markers in the general population and in specific subpopulations of patients.

Summary: Studies of reference intervals for CysC overwhelmingly demonstrated that CysC values in blood are independent of age and sex. Of the 24 studies that examined clinical utility, 15 concluded that CysC is superior to SCr, whereas 9 concluded that CysC is equivalent but provides no advantage. Summary ROC plot analysis of 20 studies that provide sensitivity and specificity data strongly suggests that CysC will be superior to SCr for detecting impaired GFR. Taken together, it is clear that CysC performs at least as well as SCr in the population at large and that it is likely to be superior to SCr in specific patient populations.
Diagnostic Value of Plasma Cystatin C as a Glomerular Filtration Marker in Decompensated Liver Cirrhosis
Authors: Orlando Rocco , Mussap Michele , Plebani Mario , Piccoli Pierpaolo , De Martin Sara , Floreani Maura , Padrini Roberto , Palatini Pietro
Published: January 2020

Background: Plasma creatinine concentration and calculated creatinine clearance are of limited value as glomerular filtration rate (GFR) markers in patients with decompensated liver cirrhosis. We assessed plasma cystatin C as an indicator of GFR in such patients.

Methods: We studied 36 patients with decompensated liver cirrhosis and 56 noncirrhotic controls, both groups including individuals with normal and impaired renal function. GFR was measured in all individuals by inulin clearance, with values <72 mL · min−1 · 1.73 m−2 considered decreased. We measured cystatin C and creatinine in plasma and calculated (from plasma concentrations) and measured creatinine clearances, using for them decision points of 1.25 mg/L, 115 μmol/L, and 72 and 72 mL · min−1 · 1.73 m−2, respectively.

Results: Plasma cystatin C concentrations were similar in controls and cirrhotics and, at the usual cutpoint, could detect decreased GFR with similar sensitivities in the two groups (73% and 88%, respectively). Serum creatinine was markedly lower in cirrhotics and remained mostly within the reference interval at all GFR values; the diagnostic sensitivity of creatinine was much lower in cirrhotics than in controls (23% vs 64%). Lower diagnostic sensitivity was also observed for calculated creatinine clearance (53% vs 100% in controls), whereas similar sensitivities were found for measured creatinine clearance (86% and 81%) in controls and cirrhotics, respectively. ROC analysis showed that all four variables had similar diagnostic accuracies in cirrhotic patients. However, it also revealed that good diagnostic accuracies for plasma creatinine and calculated creatinine clearance can be obtained only if reference intervals different from those used for the general population are adopted.

Conclusions: Plasma cystatin C concentration is an accurate GFR marker in cirrhotic patients. Plasma creatinine concentration and calculated creatinine clearance are of no practical value, as their reference values vary with the severity of the liver disease.
Single-Step Assays to Analyze CYP2D6 Gene Polymorphisms in Asians: Allele Frequencies and a Novel *14B Allele in Mainland Chinese
Authors: Ji Ling , Pan Shixiu , Marti-Jaun Jacqueline , Hänseler Edgar , Rentsch Katharina , Hersberger Martin
Published: January 2020

Background: Cytochrome P450-dependent monooxygenase 2D6 (CYP2D6) activity can be estimated by investigating the metabolism of model drugs or by genotyping the most common CYP2D6 alleles. For Caucasians, the CYP2D6 allele frequencies are well investigated, and single-step assays are available for genotyping, whereas allele analysis in mainland Chinese is limited.

Methods: Two tetra-primer assays and one allele-specific amplification assay were developed to easily genotype the CYP2D6 alleles *8, *10, and *14 previously detected in Asians. Applying these assays in combination with established single-tube assays, we analyzed 223 DNA samples from Chinese volunteers for the CYP2D6 alleles *3, *4, *5, *6, *8, *10, and *14 and for duplication of CYP2D6.

Results: Six different alleles were detected in mainland Chinese. The most frequent mutant allele was the intermediate metabolizer allele, CYP2D6*10, with a prevalence of 51.3%, followed by the poor metabolizer alleles CYP2D6*5 (7.2%) and a novel variant of CYP2D6*14. This novel *14B allele (2.0%) differs from the *14 allele by the absence of the C188T substitution and by the additional G1749C substitution. Furthermore, six duplication alleles of CYP2D6 were detected, including one duplication of the *10 allele (*10X2).

Conclusions: The CYP2D6 allele frequencies in mainland Chinese shows some genetic diversity compared with Chinese from other regions: a novel *14B allele, a slightly higher frequency of the *5 allele, and a slightly lower frequency of the *10 allele than in most other Chinese populations.
Urokinase Plasminogen Activator and Its Inhibitor, PAI-1, as Prognostic Markers in Breast Cancer: From Pilot to Level 1 Evidence Studies
Authors: Duffy Michael J
Published: January 2020

Background: For optimum management of patients with cancer, accurate assessment of prognosis is essential. The primary determinant of outcome in malignancy is the formation of distant metastases. Urokinase plasminogen activator (uPA) is a serine protease causally involved in invasion and metastasis.

Content: Data from model systems show that uPA is unequivocally involved in cancer dissemination. Consistent with its role in metastasis, multiple independent groups have shown that high uPA concentrations in primary breast cancers correlate with poor prognosis. For determining outcome, the prognostic impact of uPA was both independent of traditionally used factors and prognostic in patients with axillary node-negative disease. Paradoxically, high concentrations of plasminogen activator inhibitor (PAI-1), an endogenous inhibitor of uPA, also correlate with poor prognosis in patients with breast cancer, including the subgroup with node-negative disease. The prognostic value of uPA/PAI-1 in axillary node-negative breast cancer patients was recently confirmed in both a prospective randomized trial and a pooled analysis, i.e., two different level 1 evidence (LOE-1) studies.

Conclusions: uPA and PAI-1 are among the first biological prognostic factors to have their clinical value validated using LOE-1 evidence studies. Determination of these analytes may help identify low-risk node-negative breast cancer patients for whom adjuvant chemotherapy is unnecessary.
Use of Horseradish Peroxidase- and Fluorescein-modified Cisplatin Derivatives for Simultaneous Labeling of Nucleic Acids and Proteins
Authors: van Gijlswijk Rob PM , Talman Eduard G , Peekel Inge , Bloem Judith , van Velzen Marcel A , Heetebrij Rob J , Tanke Hans J
Published: January 2020

Background: Microarray platforms will change immunochemical and nucleic acid-based analysis of cell homogenates and body fluids compared with classic analyses. Microarrays use labeled target and immobilized probes, rather than fixed targets and labeled probes. We describe a method for simultaneous labeling of nucleic acids and proteins.

Methods: Horseradish peroxidase- and fluorescein-modified cisplatin derivatives were used for labeling of nucleic acids and proteins. These reagents, called the Universal Linkage System (ULS), bind to sulfur- and nitrogen-donor ligands present in amino acids and nucleotides. For automated screening of proteins and nucleic acids on microarrays, it is advantageous to label these biomolecules without pre- or postpurification procedures. The labeling of antibodies and nucleic acids in whole serum was therefore pursued.

Results: Immunoglobulins in nonpurified serum were labeled efficiently enough to be used for immunochemistry. To investigate whether protein-adapted labeling allowed nucleic acid labeling as well, 1 μg of plasmid DNA was added to 1 μL of serum. DNA and serum proteins were simultaneously labeled, and this labeled DNA could be used as a probe for direct fluorescence in situ hybridization.

Conclusion: ULS provides a direct labeling tool for the (simultaneous) modification of proteins and nucleic acids even in unpurified samples.
Rapid and Reliable Method for Cytochrome P450 2D6 Genotyping
Authors: Stamer Ulrike M , Bayerer Bettina , Wolf Stephanie , Hoeft Andreas , Stüber Frank
Published: January 2020

Background: Single-nucleotide polymorphisms and single-base deletions within the cytochrome P450 2D6 (CYP2D6) gene have been associated with a poor metabolizer (PM) phenotype and display a frequency of 7–10% in the Caucasian population.

Methods: We developed a reliable and rapid procedure to identify five major PM-associated mutations (CYP2D6*4, *7, and *8) and deletions (CYP2D6*3 and *6) by real-time PCR with subsequent fluorometric melting point analysis of the PCR product. These polymorphisms within the CYP2D6 gene were detected by use of two primer pairs and five different pairs of hybridization probes. DNA extracted from whole blood of 323 individuals was analyzed, and results were compared with genotypes obtained by allele-specific multiplex PCR. In case of uncertain results, additional sequence analysis was performed.

Results: Genotyping results by real-time PCR were 100% reliable, whereas conventional allele-specific multiplex PCR produced uncertain results for 12.1% of samples, as confirmed by sequence analysis. Costs for reagents and consumables were considerably higher for the real-time PCR technology, but labor time was reduced by 2 h compared with allele-specific PCR. The allele frequencies in the population investigated were 0.186 for allele *4, 0.026 for allele *5, 0.009 for allele *3, 0.031 for allele *6, and 0.002 for allele *8. The defective CYP2D6*7 allele was not found. In addition, three additional mutations were detected, one of them displaying a PM genotype.

Conclusion: Genotyping of CYP2D6 by real-time PCR with fluorometric melting point analysis is a rapid and reliable method.
Urinary 17-Ketosteroids in Various Endocrine Disorders
Authors: Zakharycheva A A
Published: January 2020

On the basis of all that has been said, it is possible to state that the clinical determination of the 17-ketosteroids and their fractions (αand β) is very valuable for the diagnosis of disorders of the endocrine glands. However, in evaluating the data from these analyses, it is necessary to take into consideration the clinical picture and the results of other studies: pneumoadenography, the peripheral blood picture, biochemical data, characteristic changes in metabolism, etc.
The Rapid Determination of Calcium in Biologic Material
Authors: Appleton Harold D , West Marcia , Mandel Mary , Sala Angelo M
Published: January 2020

A rapid procedure for the routine determination of calcium in serum and urine has been described. 0.1 ml. of serum is titrated directly with standard EDTA, using either a calcein-KCl mixture or a calcein-thymolphthalein-KCl mixture. The end point with either indicating mixture is sharp. Excellent agreement is shown with the oxalate isolation and permanganate titration procedure.
Determination of Magnesium and Calcium in Urine
Authors: Carubelli Raul , Smith William O , Hammarsten James F
Published: January 2020

A procedure for the determination of both calcium and magnesium in urine is described.

The method consists of the separation of calcium from magnesium and individual spectrophotometric titrations of each using EDTA as a complexometric reagent and murexide and eriochrome black T as indicators.

Recoveries have proven very satisfactory.
The Standardization of the Van Den Bergh Reaction for Serum Bilirubin
Authors: Carter Robert E , McGann Charles J
Published: January 2020

Using commercially available bilirubin preparations, calibration curves for the van den Bergh reaction were obtained that differed significantly from many previously reported, The bilirubin preparations used were not considered the cause. Rather the indicator properties of azobilirubin and the instability of bilirubin pigment in conventionally employed solvents appear to account for the differences noted.
A Volumetric Method for Serum Sodium
Authors: Oesper Peter , Pauerstein Carl J
Published: January 2020

A simple, rapid method for determining sodium in serum is described; it requires no special equipment and no special training. Ions normally present in serum do not interfere at any conceivable physiologic concentration. The method has been tested on approximately 30 samples of serum with known sodium contents ranging from 103-175 mEq/L; the deviation from the flame photometer value averages approximately 3%.
A Procedure for the Determination of Cerebro spinal Fluid Total Protein and Gamma Globulin in Neurologic Disorders
Authors: Papadopoulos N M , Hess W C , O'Doherty D , McLane J E
Published: January 2020

A procedure for determinations of both γ-globulin and total protein is described. Its application for the analysis of samples of cerebrospinal fluid from non-neurologic, multiple sclerosis, and other central nervous system disorders, revealed a characteristically high ratio of γ-globulin to total protein in multiple sclerosis. These findings suggest that this simple chemical test may be used routinely as an aid in the diagnosis of multiple sclerosis.
A Simplified Procedure for the Routine Determination of Uric Acid
Authors: Sambhi Mohinder P , Grollman Arthur
Published: January 2020

1. A method for the colorimetric determination of serum uric acid is described which involves the adsorption of uric acid by an ionexchange resin and the addition of N-ethylmaleimide to remove interfering chromogens.

2. The procedure is simple, accurate, and suitable for routine clinical use.

3. The clinical significance of serum uric acid levels is discussed.
Excretion of Taurine during Healing of Experimental Wounds
Authors: Williamson Martin B , Passmann John M
Published: January 2020

A comparison of the excretion of amino acids by normal and wounded rats on a high protein and protein-free diet was made. The nontaurine amino acid nitrogen excretion was found to be the same the control and wounded animals, although the rats on the high protein diet excreted considerably more amino acid nitrogen than did those on the protein-free diet. The wounded animals on the high protein diet had the same total nitrogen output but excreted significantly larger amounts of taurine than did the nonwounded controls. The wounded animals given the protein-free diet excreted the same amount of taurine but produced more total nitrogen than did the control rats. On the basis of the total nitrogen-taurine excretion ratios, data indicate that cystine is conserved by the wounded as compared to the normal animals.
Storage of Blood Samples on Kieselguhr and the Determination of Cholinesterase Alkaline Phosphatase and Urea
Authors: Simmonds R A
Published: January 2020

A method is outlined for the storage of small volumes of blood and serum on kieselguhr tablets, the preparation of which is described.

Cholinesterase, alkaline phosphatase, and urea in blood samples stored in this manner have been determined, and the effects of temperature and duration of storage of the samples examined.
Determination of Serum Butanol-Extractable Iodine with a Modified Alkaline Incineration Procedure
Authors: Klein Daniel , Chernaik Joseph M
Published: January 2020

1. A method has been presented for the determination of serum butanol-extractable iodine. As in other methods, the serum is acidified and extracted with n-butanol, and the butanol extract is treated with Blau reagent to remove interfering compounds.

2. Total iodine in the butanol is determined by a modified alkaline incineration procedure. After removal of the butanol, crystalline bovine albumin is added, and a total iodine determination is carried out.

3. Careful consideration is given to the details of the procedure, including those for the construction of the standard iodide curve.