bluuebunny/crossref_metadata_2025 huggingface dataset:
Triglyceride measurements: a review of methods and interferences
Authors: Klotzsch S G , McNamara J R
Published: January 2020

The National Cholesterol Education Program has emphasized the need to identify individuals at risk for coronary artery disease (CAD). Because increased triglycerides may be a risk factor for CAD and because triglycerides are used to estimate concentrations of low-density lipoprotein (LDL) cholesterol, which has definitely been shown to be a risk factor for CAD, it is important that reliable results be obtained. Many methods are available for measuring triglyceride concentrations in serum or plasma, but there is no definitive method that confirms the reliability of any of these procedures. Accuracy and precision guidelines are extremely difficult to determine, owing to broad biological variability both within and among individuals. Here, we review the major triglyceride quantification methods in the literature, some of the potential interference problems, and the limitations regarding standardization that should be addressed when establishing such guidelines.
Setting analytical goals for random analytical error in specific clinical monitoring situations
Authors: Fraser C G , Hyltoft Peterson P , Larsen M L
Published: January 2020

Strategies abound for the setting of analytical goals in clinical chemistry. Many, especially those more recently proposed for particular clinical situations, are concerned with tests used in diagnosis. We suggest a general theory for the setting of goals in situations that specifically involve the monitoring of individuals. Goals are calculated from the formula CVA less than [(delta c 2/2Z2)-CVB2]1/2, where CVA is the analytical imprecision (as coefficient of variation, CV); delta c is the percentage change in serial results that is considered clinically significant; Z is the Z-statistic, which depends only on the probability selected for statistical significance; and CVB is the average inherent within-subject biological variation (as CV). Examples given show applications in hematology and in monitoring diabetes mellitus, chronic renal failure, and hepatitis. The derived goals are for total random analytical error (imprecision and intermittent systematic variation), and provide objective criteria that should be achieved in practice. The effect of analytical variability on both variability in test results and the probability that a stated change can be considered significant should be calculated whether or not the goals are attained.
Concentrations of atrial natriuretic peptide in plasma and urine of kidney disease patients
Authors: Marumo F , Sakamoto H , Ando K , Ishigami T , Ishigama T
Published: January 2020

Concentrations of human atrial natriuretic peptide-like immunoreactivity (hANP-LI) were measured by a highly sensitive and specific radioimmunoassay (Biochem Biophys Res Commun 1986;137:231-6) in normal subjects and in renal disease patients without accompanying congestive heart failure, hypertension, edema, diabetes, or pregnancy. We attempted to clarify whether the hANP-LI concentration in plasma was increased by loss of renal mass. We found no correlation between the hANP-LI concentration in plasma and creatinine clearance (Ccr, 4.6-122.3 mL/min) in patients with renal disease (n = 63, r = -0.196), nor between hANP-LI concentrations in plasma and urine (n = 97, r = -0.207). The fractional excretion of hANP (FEhANP) correlated significantly with Ccr (n = 63, r = 0.520, P less than 0.01) and with FENa (n = 35, r = -0.503, P less than 0.01). Increased FEhANP in patients with chronic renal failure may have resulted because of an increase in single-nephron glomerular filtration rate similar to the FENa increase in these patients. The present data indicate that decreased renal function itself does not increase the concentration of hANP-LI in plasma.
Assay of creatine kinase isoenzyme MB in serum with time-resolved immunofluorometry
Authors: Lianidou E S , Christopoulos T K , Diamandis E P
Published: January 2020

We describe the first time-resolved immunofluorometric assay for creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB) in serum. The assay is based on the formation of the complex: solid-phase anti-CK-MB-CK-MB-biotinylated anti-CK-BB-streptavidin-BCPDA-Eu3+, where anti-CK-MB and anti-CK-BB are monoclonal antibodies against the CK isoenzymes MB and BB, respectively, and BCPDA is the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid. The solid-phase complex is fluorescent and is measured on the dry solid-phase (microtiter well) in a specially designed time-resolved fluorometer that uses laser excitation. The assay requires 25 microL of serum and is not affected by the presence of either CK-MM (up to 5000 micrograms/L) or CK-BB (up to 1000 micrograms/L) in the sample. Precision and accuracy indices for the assay were satisfactory.
Increase in lactate dehydrogenase isoenzyme-1 in plasma in pediatric malignancy
Authors: Sacks S S , Mulligan G
Published: January 2020

We investigated 28 cases of pediatric malignancy in which total lactate dehydrogenase (LD, EC 1.1.1.27) activities were increased and isoenzyme LD-1 exceeded LD-2 (flipped pattern). Of these, 11 had a flipped pattern at presentation and 17 showed a flipped pattern during chemotherapy. Those with flipped patterns at presentation were four with germ-cell tumors, one with acute lymphocytic leukemia, and six with nephroblastomas (Wilms tumor). Four of five nephroblastoma homogenates contained predominantly LD-1; one revealed a structurally normal LD-1 with normal kinetics. We conclude that an increase in LD with a flipped pattern is common in nephroblastoma and, in addition, may develop in cancer patients treated with chemotherapy.
Common and rare genotypes of human apolipoprotein E determined by specific restriction profiles of polymerase chain reaction-amplified DNA
Authors: Richard P , Thomas G , de Zulueta M P , De Gennes J L , Thomas M , Cassaigne A , Béréziat G , Iron A
Published: January 2020

The three common isoforms of human apolipoprotein E (apo E) differ at positions 112 and 158 and are named E3, E4, and E2 according to phenotyping by isoelectric focusing (IEF). The polymerase chain reaction (PCR) method allows the detection of common and several rare allelic apo E variants not detected by IEF. We propose a genotyping procedure for apo E that characterizes a given allele on the basis of amplification of specific sequences of the gene followed by the action of restriction endonucleases. When the nucleotide change does not lead to a restriction site, PCR-directed mutagenesis creates the discriminant site, and the differentiation of the three common alleles and five rare variants is possible. We present here profiles of common alleles and of three rare alleles, Weisgraber [Cys112/Asp127/Cys158], Christchurch [Cys112/Ser136/Arg158], and a new rare variant [Cys112/Leu142/Cys158].
Analytical evaluation of Kone Microlyte determination of ionized magnesium
Authors: van Ingen H E , Huijgen H J , Kok W T , Sanders G T
Published: January 2020

We performed an analytical evaluation of a commercially available instrument for determining ionized magnesium through use of a neutral carrier, liquid-membrane-based ion-selective electrode. Reproducibility (CV 2-4%), linearity (0.30-2.50 mmol/L), lower limit of detection (0.30 mmol/L), and absence of interference from Ca2+ indicate adequate performance for measuring ionized magnesium in plasma or serum samples in the normal to high-concentration range. Sodium in excess of 150 mmol/L caused a negative bias, which can be explained by ionic strength-induced changes in activity coefficients. The use of heparin as an anticoagulant must be restricted to concentrations < 15 units/mL because of the binding of magnesium to heparin. The mean +/- SD concentration of ionized magnesium and its fraction of total magnesium in 76 healthy volunteers were 0.56 +/- 0.05 mmol/L and 0.65 +/- 0.04, respectively.
Time-resolved immunofluorometric assay of total renin in plasma and follicular fluid
Authors: Matinlauri I , Eskola J U , Aalto M , Koskinen P , Irjala K
Published: January 2020

We developed a new time-resolved immunofluorometric assay (TR-IFMA) using two monoclonal antibodies for the total renin measurement in human plasma and follicular fluid. No conversion of prorenin to renin was needed because the assay detected both renin and prorenin. The detection limit of the assay was 10 ng/L and the linear range was 10-25,000 ng/L. Within-assay precision (CV) was 15-8% at renin concentrations of 50-12,200 ng/L. Between-assay precision was 19-3% at concentrations of 100-18,000 ng/L. Analytical recovery of added renin was 85-104% (n = 5) in plasma samples and 104-119% (n = 3) in follicular fluids. For plasma, the reference interval was 78-262 ng/L in men (n = 44) and 36-226 ng/L in women (n = 43).
Complex pattern of alternative splicing in the normal uroporphyrinogen decarboxylase gene: implications for diagnosis of familial porphyria cutanea tarda
Authors: McManus J F , Begley C G , Ratnaike S
Published: January 2020

We describe multiple alternative transcripts of uroporphyrinogen decarboxylase mRNA in normal individuals and patients with familial porphyria cutanea tarda. mRNA was reverse-transcribed, subjected to the polymerase chain reaction, and analyzed for nucleotide sequence. Seven different transcripts were characterized, and a cryptic splice acceptor site was identified in intron 1. In all mRNAs the exons abutted at previously defined exon boundaries. Characterization of the splice junctions in the genomic DNA showed that splice donor and acceptor sequences complied with the consensus sequences for these sites except for the splice acceptor sequences of exons 3 and 10. THese deviations were present in two normal individuals and one patient with familial porphyria cutanea tarda and were thus unable to explain the multiple aberrant uroporphyrinogen decarboxylase transcripts. We conclude that apparent deletions observed in transcripts derived from the uroporphyrinogen decarboxylase gene in patients with familial porphyria cutanea tarda should be interpreted with caution.
Indicators of Iron Status in Nonanemic Elderly Subjects: Influence of Sex and Age
Authors: Marie Bernadette , Cals Marie-Joséphe , De Jaeger Christophe , Lowenstein William , Durand Hervè , Ekindjian Ohvanesse G
Published: January 2020

Vol. 40:

p. 1780. In the Technical Brief by B. Marie, M.-J. Cals, C. De Jaeger, W. Lowenstein, H. Durand, and O. G. Ekindjian entitled "Indicators of iron status in nonanemic elderly subjects: influence of sex and age," 1994;40:1779-81, in the legend to Fig. 1, the concentration of ferritin should have been expressed in micrograms per liter (µg/L), not picograms per liter.
Association of pancreatic adenocarcinoma, mild lung disease, and delta F508 mutation in a cystic fibrosis patient
Authors: Tsongalis G J , Faber G , Dalldorf F G , Friedman K J , Silverman L M , Yankaskas J R
Published: January 2020

A case of adenocarcinoma of the pancreas and mild lung disease in a 39-year-old man homozygous for the delta F508 cystic fibrosis mutation is presented. Cystic fibrosis is the most common lethal genetic disease in Caucasians, and is most commonly associated with severe obstructive lung disease. To our knowledge, this is only the fifth case of adenocarcinoma of the pancreas in a CF patient to be reported and the first case for which molecular data are available. The rare incidence of this type of malignancy in the general population suggests a possible association of CF with this malignant disease.
Interference with clinical laboratory analyses
Authors: Kroll M H , Elin R J
Published: January 2020

Interference by endogenous and exogenous substances with assays for clinical analytes is a common problem in laboratory medicine. For this review, we defined interference as "the effect of a substance present in the sample that alters the correct value of the result, usually expressed as concentration or activity, for an analyte." There are four major endogenous compounds that consistently interfere with laboratory results: hemoglobin, bilirubin, lipids, and paraproteins. The major exogenous sources of interference are drugs prescribed for the patient; and there are several excellent compendia of the effect of drugs on clinical laboratory tests. We recommend determining whether the interference is dependent or independent of the analyte for the assay. Further, we propose an approach to the identification and resolution of an interference problem for the clinical laboratory and make recommendations to manufacturers.
Fully automated flow-injection system for quantifying 1,5-anhydro-D-glucitol in serum
Authors: Tanabe T , Umegae Y , Koyashiki Y , Kato Y , Fukahori K , Tajima S , Yabuuchi M
Published: January 2020

We have developed a flow-injection system with colorimetric detection to measure 1,5-anhydro-D-glucitol in serum. Serum samples are directly and serially injected into a clean-up column every 3.5 min to remove interferences before the enzymatic reaction. 1,5-Anhydro-D-glucitol, after being passed through the column, is oxidized by immobilized pyranose oxidase (EC 1.1.3.10), and the hydrogen peroxide produced reacts with the chromogen substrate in the presence of immobilized horseradish peroxidase (EC 1.11.1.7) to form Bindshedler's Green. The detection limit was 1.2 mumol/L (1.2 pmol). The correlation between results obtained with the present system (y) and gas chromatography-mass spectrometry (GC-MS) (x) in samples containing < 30 mumol/L 1,5-anhydro-D-glucitol, including many samples from patients with diabetes mellitus, was y = 0.975x-0.111 mumol/L (r = 0.997), which was superior to that obtained between the enzymatic and GC-MS methods. Our system needs only to be set up; it runs without any manual pretreatment, assays 17 samples/h, and shows imprecision (CV) of < 2%.
Measurement of human urinary prolactin as a noninvasive study tool
Authors: Keely E J , Faiman C
Published: January 2020

We used a time-resolved solid-phase fluoroimmunoassay with a sensitivity of 25 ng/L on 40-fold-concentrated urines to measure urine prolactin (PRL) excretion. The nature of the immunoreactive material was verified to be PRL by: (a) column chromatography showing a monomeric 23-kDa peak; (b) similarity between fluoroimmunoassay and bioassay (Nb2 lymphoma cell) results; and (c) Western blot identification. In 20 normal subjects [serum PRL 6.8 (3.8-14.0) micrograms/L, median (and range)], urine PRL excretion was 0.15 (0.07-0.23) ng/h and 0.24 (0.15-0.54) micrograms/mol of creatinine. Urine values in seven hyperprolactinemic patients were all greater than the upper limit of normal. The correlation between urinary excretion rate and serum values was highly significant (r = 0.979; P < 0.001). These results indicate that a monomeric, immunologically reactive, biologically active form of PRL can be measured in urine at concentrations approximately 0.0005 that in serum. This urine PRL method may provide a practical tool for the repetitive, noninvasive study of PRL dynamics in field studies and in patients with reproductive disorders.
Pancreatic lipase assays with triglycerides as substrate: contribution of each sequential reaction to product formation
Authors: Lykidis A , Mougios V , Arzoglou P
Published: January 2020

Human pancreatic lipase assays are usually performed in the presence of either emulsified triglycerides or diglycerides within the limits of their solubility. Two reactions are catalyzed in the presence of triglycerides: hydrolysis of triglycerides to diglycerides, and diglycerides to monoglycerides. The contribution of each reaction to the final result was determined after extensive kinetic studies on the appearance and/(or) accumulation of intermediates and/(or) products. Acylated glycerides were analyzed after extraction from the reaction mixture, separation of lipid classes by thin-layer chromatography, and quantification by capillary gas chromatography. The results show that after 10 min of reaction in the presence of high concentrations of triolein, 75% of the released fatty acids arise from the first reaction. Relative merits and disadvantages of each substrate (triglyceride or diglyceride) are discussed in terms of practicability.
Laboratory support for transplantation
Authors: Yatscoff R W
Published: January 2020

The clinical laboratory plays a major role in support of transplantation. Depending on the spectrum and number of transplants performed, such programs can consume up to 35% of all laboratory testing. This testing can be transplantation-nonspecific or -specific. The former constitutes routine laboratory procedures in various areas of laboratory medicine that are common to other categories of patients. Transplantation-specific testing includes tissue typing, procedures for assessment of graft viability/rejection, and measurement of immunosuppressive drugs. Because of the development of new immunosuppressive drugs such as FK506, cyclosporin G, and rapamycin, an increased burden will be placed on laboratories to establish new therapeutic monitoring programs. Preliminary guidelines for monitoring these drugs, similar to those previously established for cyclosporin A, are proposed herein. With the use of an increasing spectrum of immunosuppressive drug combinations, and the possible use of xenotransplantation on the horizon, new demands will be placed on the laboratory for the support of transplant programs.
Advances in diagnosis, prevention, and management of hepatic allograft rejection
Authors: Wiesner R H
Published: January 2020

Despite recent improvements in immunosuppressive therapy, hepatic allograft rejection remains a major cause of morbidity and late graft loss in patients undergoing liver transplantation. Although some biochemical tests suggest hepatic allograft damage, the gold standard for defining rejection remains based on morphologic findings. Acute cellular rejection usually occurs within the first 3 weeks posttransplantation and the incidence varies between 40% and 70%. Ductopenic rejection occurs in 5-10% of patients undergoing initial liver transplantation and usually occurs between 6 weeks and 6 months after the procedure. Induction and maintenance of immunosuppression with triple-drug therapy (cyclosporine, prednisone, and azathioprine) and other combinations that include anti-lymphocyte preparations have led to an overall decrease in the incidence of both cellular and ductopenic rejection. In addition, the availability of FK506 as a rescue therapy has saved grafts in some patients experiencing chronic (ductopenic) rejection. Overall, the correlation between the degree of biochemical liver dysfunction and the presence and severity of histologic rejection remains poor. Histologic severity of rejection, however, suggests which patients will require more immunosuppressive therapy and which patients may need antilymphocyte therapy to control the rejection episode. Some rejection episodes remain resistant to all known therapy and eventually lead to graft loss. New immunosuppressive agents and regimens are needed to improve graft and patient survival, decrease the incidence of cellular and ductopenic rejection, minimize drug-related side effects and complications, and reduce the high cost of immunosuppressive therapy.
External quality assessment in primary health care by using fresh whole blood
Authors: Raabo E , Odum L
Published: January 2020

Desktop analyzers and single-test meters used in primary healthcare are mainly calibrated to measure whole blood. To minimize matrix effects of control materials in external quality assessment schemes, we stabilized fresh human EDTA-treated blood with sodium iodoacetate (1.8 mmol/L). The whole-blood based control material was useful for control of hemoglobin and glucose, the two most common analyses in primary care, even after storage at room temperature for at least 10 days and at 5 degrees C for 3 weeks. The material was also useful for control of cholesterol and creatinine analyses for samples stored as long as 3 days at ambient temperature.
Determination of a T/G polymorphism at nucleotide 3206 of the apolipoprotein C III gene by amplification refractory mutation system
Authors: Tsai M Y , Hanson N Q , Copeland K R , Beheshti I , Garg U
Published: January 2020

We used the amplification refractory mutation system (ARMS)--a polymerase-chain-reaction-based method--to determine the 3206 T-to-G polymorphism on exon 4 of the apolipoprotein (apo) C III gene. Apo C III is an inhibitor of the enzyme lipoprotein lipase (EC 3.1.1.34). Previous studies have demonstrated that a polymorphism at nucleotide 3175 on exon 4 of this gene is associated with hypertriglyceridemia. We studied 45 hypertriglyceridemic and 46 age-matched controls for the 3206 T-to-G polymorphism. The results showed a significant difference in the distribution of the genotypes with respect to this allele between the hypertriglyceridemic and control individuals. We also determined the presence of the SacI site at nucleotide 3175 in these same individuals and found no significant difference in SacI genotypes between the two groups. This study reaffirms the usefulness of ARMS as a simple, reliable method for detecting mutations and polymorphisms in clinical and epidemiological studies.
Detection of alpha-thalassemias by multiplex polymerase chain reaction
Authors: Bowie L J , Reddy P L , Nagabhushan M , Sevigny P
Published: January 2020

Although alpha-thalassemia is the most common genetic abnormality in the world, there is currently no routine laboratory method to definitively identify individuals who are affected. We describe a rapid and simple method that utilizes deletion-sensitive primers to amplify normal DNA sequences. Deletions involving the regions responsible for most of the alpha-thalassemia cases in the US prevent amplification with these primers. In tests with DNA isolated from small amounts (10 microL) of whole blood, the deletion-sensitive primers gave rise to the expected 248- and 375-bp (base pair) amplification products in normal individuals. These primers, along with primers designed to bind to a nonaffected control sequence from the hemoglobin beta chain, could be amplified simultaneously (multiplex polymerase chain reaction). This made it possible to detect heterozygotes for alpha-thalassemia-2 (one alpha locus deleted) by determining the ratios of the 248- and 375-bp amplification products to the product of the control sequence (268 bp). The method is rapid and simple and can be performed in a routine clinical laboratory.
Mass screening for cystic fibrosis heterozygotes: two assay systems compared
Authors: Gilfillan A , Axton R , Brock D J
Published: January 2020

Two different assay systems were used to detect mutant cystic fibrosis alleles in mouthwash samples from pregnant women attending antenatal clinics. In the first phase of the study, comprising 3110 consecutive samples, we compared the performance of an in-house system and a commercial amplification refractory mutation system (ARMS). Successful analyses were completed at first attempt in approximately 90% of samples, and the patient resampling rate was 1.1%. There were no false positives and only one detected false negative. In the second phase, comprising 3333 consecutive samples, only ARMS was used. Again, there were no false positives and the patient resampling rate dropped to 0.5%. On the basis of convenience and reduced operator time, we have elected to use ARMS for further screening for cystic fibrosis heterozygotes.
Solid-phase extraction combined with radioimmunoassay for measurement of zalcitabine (2',3'-dideoxycytidine) in plasma and serum
Authors: Roberts W L , Buckley T J , Rainey P M , Jatlow P I
Published: January 2020

Of the antiviral agents that are currently in clinical use in the US for therapy for human immunodeficiency virus infections, zalcitabine (ddC) is the most potent and is effective at the lowest plasma concentrations. The two reported procedures for measuring these low concentrations involve a chromatographic technique coupled with mass spectrometry. We have developed a procedure combining solid-phase extraction with a strong cation-exchange resin and commercially available RIA reagents for the quantification of ddC in plasma or serum. The method demonstrates good linearity, specificity, and precision, with overall CVs of < 10% from 2-20 micrograms/L and 17% at 0.8 microgram/L (the lower limit of quantitation). No significant cross-reactivity with nucleoside analogs other than ddC analogs was noted. The major advantages of this assay are its efficiency and relative simplicity, which should facilitate its performance in many laboratories.
Estimating and minimizing effects of biologic sources of variation by relative range when measuring the mean of serum lipids and lipoproteins
Authors: Cooper G R , Smith S J , Myers G L , Sampson E J , Magid E
Published: January 2020

Biologic intraindividual variation (CVb) is a major source of inaccuracy in current lipid and lipoprotein measurements. Metaanalysis has been used to estimate the average CVb of serum total cholesterol (TC), high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDLC), and triglyceride (TG). These CVb values are larger than the National Cholesterol Education Program-accepted and -proposed analytic (CVa) goals. Measuring serial specimens reduces the error in determination of the mean concentration used in classification of the patient by cutoff points. We show (a) a convenient technique, based on the relative range, to qualitatively estimate and interpret biologic variation of TC, HDLC, LDLC, and TG, and (b) the number of serial specimens required to meet a total variation goal for measurements of mean lipid and lipoprotein values. A total variation goal has been selected that can be met by two serial specimens for a majority of individuals.
Plasma amino acids determined by liquid chromatography within 17 minutes
Authors: Terrlink T , van Leeuwen P A , Houdijk A
Published: January 2020

We present an HPLC method for the determination of amino acids in plasma. The method is based on automated precolumn derivatization of amino acids with o-phthalaldehyde, separation of the derivatives by reversed-phase chromatography, and quantification by fluorescence detection. Complete separation was achieved within 12 min. Total analysis time, including derivatization, chromatography, and reequilibration of the column, was 17 min. The assay was linear from 5 to 800 mumol/L for all amino acids. Recovery of amino acids added to plasma samples was 96-106%, except for tryptophan (89%). Within-run precision (CV) was 1.8-6.4%, and between-run precision was 2.1-7.2%. The method can be used for determining primary amino acids in plasma and cerebrospinal fluid. The simple sample preparation and short analysis time make the method particularly suitable for routine analysis of large series of samples.
Urinary and plasma catecholamines and urinary catecholamine metabolites in pheochromocytoma: diagnostic value in 19 cases
Authors: Gerlo E A , Sevens C
Published: January 2020

We review our data on the measurement of catecholamines and their metabolites in 19 patients with pheochromocytoma. All the assays were specific high-performance liquid chromatographic procedures with electrochemical detection. The assay of fractionated metanephrines was 100% sensitive. Normal values for both urinary norepinephrine and epinephrine were found in two asymptomatic patients with pheochromocytoma. Normal values for 3-methoxy-4-hydroxymandelic acid (VMA) were found in two patients with pure epinephrine-secreting tumors and in one patient with multiple endocrine neoplasia type II. Plasma catecholamines were usually less increased than their urinary counterparts. We recommend the specific measurement of norepinephrine and epinephrine as the initial test for patients with suggestive symptoms, and specific measurement of normetanephrine and metanephrine for patients in whom an adrenal mass is incidentally found. We argue against the use of total metanephrines, total catecholamines, and VMA because of their lack of diagnostic sensitivity.
Quantification of serum 1,5-anhydroglucitol in uremic and diabetic patients by liquid chromatography/mass spectrometry
Authors: Niwa T , Dewald L , Sone J , Miyazaki T , Kajita M
Published: January 2020

We developed a new method for measuring serum 1,5-anhydroglucitol (1,5-AG), using liquid chromatography/atmospheric pressure chemical-ionization mass spectrometry (LC/MS). With this method we measured serum 1,5-AG concentrations in uremic and diabetic patients and compared these values with those determined by an enzymatic method. Serum 1,5-AG concentrations were significantly less in the undialyzed and dialyzed uremic patients and in diabetic patients; the values in the dialyzed patients decreased after hemodialysis. In uremic patients with high concentrations of serum myo-inositol, the 1,5-AG values determined by LC/MS were significantly lower than those determined enzymatically. There was no significant correlation between 1,5-AG and fructosamine in the uremic patients. These results demonstrate that serum 1,5-AG cannot be used as an index for glycemic control in uremic patients and that the LC/MS method is indicated in uremic patients with high concentrations of myo-inositol.
Neuropsychopharmacologic challenge in biological psychiatry
Authors: Coccaro E F , Kavoussi R J
Published: January 2020

Psychopharmacologic challenge procedures offer many useful features for biological psychiatry research. First, challenge procedures acutely stimulate neurotransmitter and (or) hormonal systems in the brain and offer the chance to assess the function, and (or) reserve, of the brain neurotransmitter/hormone system of relevance. Second, these paradigms often offer functional information about specific areas of the brain (e.g., limbic-hypothalamus). Regardless of these advantages, the power of these procedures is limited to the specificity of the agent and of the functional anatomy of the "circuit" that leads to the outcome measure, which, in turn, is linked to the stimulation by the acute pharmacologic challenge. We describe here the development of the psychopharmacologic challenge methodology and the strengths and weaknesses of this approach. Finally, the application of this paradigm in the study of various neuropsychiatric disorders will be reviewed and critiqued.
Localized in situ amplification (LISA): a novel approach to in situ PCR
Authors: Tsongalis G J , McPhail A H , Lodge-Rigal R D , Chapman J F , Silverman L M
Published: January 2020

Amplification of specific gene target sequences has become a routine molecular procedure in a variety of laboratories. When coupled with either a direct or indirect method of detecting amplified product, in situ amplification offers an extremely powerful investigative tool. We describe a protocol for a localized in situ amplification (LISA) reaction that includes tissue-culture cloning rings and allows for the amplification of gene target sequences in specific regions of paraffin-embedded tissue sections. Digoxigenin-11-dUTP was added to the amplification reaction and thus incorporated into the amplified products, providing a mechanism by which direct nonisotopic detection could be performed. To demonstrate the approach, LISA was performed on known positive Pneumocystis carinii rat lung tissues, with primers specific for the P. carinii rRNA gene sequence.
Measuring swainsonine in serum of cancer patients: phase I clinical trial
Authors: Baptista J A , Goss P , Nghiem M , Krepinsky J J , Baker M , Dennis J W
Published: January 2020

Swainsonine, an indolizidine alkaloid and competitive inhibitor of Golgi alpha-mannosidase II (EC 3.2.1.114), reduces tumor growth and stimulates immune function in mice. On the basis of these observations, a phase I clinical trial was initiated to determine whether swainsonine could be administered safely to cancer patients. We describe a method for extraction, acetylation, and quantification of swainsonine in human serum samples. Methyl alpha-D-mannopyranoside and methyl beta-D-galactopyranoside were added to serum samples as internal standards and, after sequential extraction of lipids and proteins with chloroform and acetonitrile, respectively, samples were acetylated with acetic anhydride and 4-dimethylaminopyridine and separated by gas-liquid chromatography. The identity of swainsonine and the internal standards after their extraction from serum and acetylation was confirmed by gas chromatography/mass spectrometry. Swainsonine was recovered at an efficiency of 90%, relative to internal standards, and calibration graphs were rectilinear from 3 to 18 mg/L with a detection limit of approximately 0.1 mg/L. The CV for multiple samples was < or = 6.7%. In patients receiving swainsonine (50-550 micrograms/kg per day) continuously for 5 days by intravenous infusion, serum concentrations of the drug reached 3-11.8 mg/L, 100 to 400 times greater than the 50% inhibitory concentration for Golgi alpha-mannosidase II and lysosomal alpha-mannosidases. Accurate measurements of swainsonine in biological fluids with this method should facilitate further clinical studies with the drug.
Simple method for determining specific binding capacity of vitamin D-binding protein and its use to calculate the concentration of "free" 1,25-dihydroxyvitamin D
Authors: Vieth R
Published: January 2020

A quantitative method to measure the specific binding capacity for 25-hydroxyvitamin D (25D-binding capacity) is described that resembles the qualitative "T3-uptake" assay. Patient's serum or standard (10 microL) is mixed with 0.5 mL of reagent containing 0.5 mumol/L 25-hydroxyvitamin D [25(OH)D3] plus 3000 counts/min [3H]25(OH)D3. After 0.5 h at 37 degrees C, the samples are treated with dextran/charcoal on ice for 1 h and centrifuged. The radioactivity of the bound tracer in the supernate is counted. Calibration is linear to approximately 10 mumol/L. 25D-binding capacity in reference-group serum samples was 4.33 (0.58 SD) mumol/L. The relationship between the inverse of 25D-binding capacity and the free fraction of [3H]1,25-dihydroxyvitamin D3 [1,25(OH)2D3] measured by ultrafiltration isodialysis was essentially linear (r = 0.934, P < 0.0001). Given this relationship, the calculated free fraction of 1,25(OH)2D3 equals 4.88 x 10(-3)/25D-binding capacity. The 25D-binding capacity was significantly lower in newborn babies and in adults with liver disease, and was increased during pregnancy (P < 0.01 for each). This method is applicable to situations where the biologically available concentration of 1,25(OH)2D is of interest.
Microparticle-enhanced nephelometric immunoassay of anti-thyroid peroxidase autoantibodies in thyroid disorders
Authors: Harchali A A , Montagne P , Ruf J , Cuillière M L , Bene M C , Faure G , Duheille J
Published: January 2020

Crude thyroid peroxidase extracted from human thyroid microsomes was covalently bound onto polyacrylic and polyfunctional copolymerized microparticles. We observed agglutination of the thyroid peroxidase-microparticle conjugate with 13 monoclonal antibodies (mAbs) specific for epitopes on four different antigenic domains of human thyroid peroxidase (TPO; EC 1.11.1.7), after addition of anti-mouse immunoglobulins. We quantified agglutination by measuring with a specially designed nephelometer the light scattered by the conjugates. This allowed us to develop a microparticle-enhanced nephelometric immunoassay for human anti-TPO autoantibodies (aAbs) with defined epitopic specificity, based on the ability of aAbs to inhibit mAb-induced agglutination. Applied to patients with autoimmune thyroid diseases, this assay confirmed the polyclonality of anti-TPO aAbs and their preferential reactivity toward epitopes located on the A and B antigenic domains of the TPO molecule. The same specificities seem to be present in patients with Hashimoto thyroiditis or Graves disease.
Development and validation of a monoclonal antibody enzyme immunoassay for measuring progesterone in saliva
Authors: O'Rorke A , Kane M M , Gosling J P , Tallon D F , Fottrell P F
Published: January 2020

A nonextraction, competitive, solid-phase enzyme immunoassay with a monoclonal antibody was developed and validated for measuring progesterone in saliva. The antibody was raised against 11 alpha-hydroxyprogesterone hemisuccinate-bovine serum albumin conjugate and was indirectly immobilized to the walls of microtiter wells. The labeled analyte (progesterone-horseradish peroxidase conjugate) was homologous with the immunogen. The lower detection limit (concentration equivalent to B0-3 SD) was 38 pmol/L of saliva sample. We validated the assay with studies to establish the independence of the concentration determined from the volume of saliva assayed, quantitative recovery of progesterone added to saliva, interference from possible cross-reactants, and agreement with a similar assay that incorporated an extraction step. In addition, we determined luteal-phase concentrations of salivary progesterone in normal women and compared the results with those of an older assay involving a polyclonal antibody.
Evaluating test methods by estimating total error
Authors: Chinchilli V M , Miller W G
Published: January 2020

A common procedure for evaluating a test method by comparison with another, well-accepted method has been to use a repeated measurements design, in which several individual subjects' specimens are assayed with both methods. We propose the use of the intrasubject relative mean square error, which is a function of the intrasubject relative bias and the coefficient of variation of the test method, as a measure of total error. We construct for each individual subject a score that is based on how well an individual's estimate of total error compares with a maximum allowable value. If the individual's score is > 100%, then that individual's estimate of total error exceeds the maximum allowable value. We present a distribution-free statistical methodology for evaluating the sample of scores. This involves the construction of an upper tolerance limit to determine whether the test method yields values of the total error that are acceptable for most of the population with some level of confidence. Our definition of total error is very different from that defined in the National Cholesterol Education Program (NCEP) guidelines. The NCEP bound for total error has three main problems: (a) it incorrectly assumes that the standard error of the estimated relative bias is the test coefficient of variation; (b) it incorrectly assumes that the individual estimated relative biases follow gaussian distributions; (c) it is based on requiring the relative bias of the average individual in the population to lie within prescribed limits, whereas we believe it is more important to require the total error for most of the individuals in the population, say 95%, to lie within prescribed limits.
Total discrimination of peritoneal malignant ascites from cirrhosis- and hepatocarcinoma-associated ascites by assays of ascitic cholesterol and lactate dehydrogenase
Authors: Castaldo G , Oriani G , Cimino L , Topa M , Mostarda I , Castellano L , Del Vecchio-Blanco C , Budillon G , Salvatore F , Sacchetti L
Published: January 2020

No laboratory test completely distinguishes malignant ascites (MA) from ascites associated with cirrhosis and (or) hepatocellular carcinoma (A/C-HC). Ascitic cytology is highly specific but has a diagnostic sensitivity of only 40-60%. We determined 11 ascitic analytes and cytology in 58 patients with cirrhosis, 15 with hepatocellular carcinoma, and 21 with MA (10 ovarian cancers, 4 mesotheliomas, 6 gastrointestinal neoplasias, 1 leukemia). Ascitic total protein, cholesterol, pseudouridine, and lactate dehydrogenase (LD), and the ascitic:serum ratios of total protein and of LD showed the most significant differences between the two groups of patients. Stepwise multiple linear discriminant analysis (applying the Wilks' lambda criterion) of several variables, corroborated by the "jack-knife" reallocation procedure, showed that the ascitic cholesterol and ascitic LD association correctly identified 100% of MA and A/C-HC; cytology had a diagnostic specificity of 100%, but identified only 48% of MA. This association may represent a primary tool for the discrimination of ascites of unknown origin, particularly in the presence of negative cytology findings.
Chemiluminescence receptor assay for measuring vitamin B12 in serum evaluated
Authors: Wentworth S , McBride J A , Walker W H
Published: January 2020

We evaluated a chemiluminescence receptor assay for vitamin B12 in serum (Magic Lite; Ciba Corning Diagnostics), in which an acridinium ester label is used with magnetic particle separation. Within- and between-batch precisions were generally acceptable, except at low analyte concentrations. The reference range determined from 104 elective preoperative patients was 120-610 pmol/L, compared with 150-590 pmol/L for our in-house radioligand-binding assay. Magic Lite discriminated between normal and abnormal results as effectively as the in-house method when local reference ranges were applied. Magic Lite demonstrated a negative bias at low analyte concentrations and was unable to detect any vitamin B12 in two B12-deficient patients. Assay accuracy--judged from analytical recovery and comparisons with the in-house method and two other radioassay kits (Quantaphase, Bio-Rad Labs., and Immophase, Ciba Corning Diagnostics)--was poor at low B12 concentrations when the manufacturer's recommended two-point calibration was used. This problem was partially corrected by using a full set of calibrators.
Interference in ionized calcium measurements by heparin salts
Authors: Landt M , Hortin G L , Smith C H , McClellan A , Scott M G
Published: January 2020

We determined the suitability of various heparin salts used for anticoagulation of whole-blood specimens for measurement of ionized calcium (iCa), blood gases, and electrolytes. We were particularly interested in a new heparin product containing both zinc and lithium cations (CNLZ heparin), in which the binding sites with greatest affinity for divalent cations are bound with zinc and low-affinity sites with lithium. In initial experiments Li heparin decreased iCa concentrations 0.07 mmol/L at the lowest heparin concentration (3000 units/L) and progressively lowered them at higher concentrations. Zn heparin initially increased iCa concentrations 0.06 mmol/L but progressively lowered them as the heparin concentration was increased. Li heparin interfered even when present in amounts (9 units per 3-mL syringe) minimally effective in preventing coagulation. Use of CNLZ heparin (36 units per 3-mL syringe; Zn 63-78 g/kg of heparin) largely eliminated interference of heparin in iCa measurements. In studies that included the effects of concentration of heparin through partial filling of syringes, specimens anticoagulated with CNLZ heparin compared well with unheparinized controls in measurements of iCa, blood gases, and electrolytes. Blood gases and iCa results on CNLZ-heparinized specimens from intensive-care-unit patients also compared well with specimens anticoagulated with a preparation of heparin (EB heparin) in which calcium has been added to balance the calcium-binding capacity. However, the presence of calcium in EB heparin significantly increased measured total calcium concentrations, whereas the new CNLZ heparin did not interfere in total calcium determinations.
Effect of serum lipoprotein(a) on estimation of low-density lipoprotein cholesterol by the Friedewald formula
Authors: Li K M , Wilcken D E , Dudman N P
Published: January 2020

The calculation of serum low-density lipoprotein cholesterol (LDL-C) by the Friedewald formula does not account for the cholesterol associated with lipoprotein(a) [Lp(a)]. To quantify the contribution of Lp(a) cholesterol to total serum cholesterol, we measured concentrations of serum Lp(a) by an ELISA and concentrations of other serum lipids and lipoproteins by standard assays in 23 normolipemic women, ages 50-60 years. In measuring serum high-density lipoprotein we found that polyethylene glycol 6000 precipitated > 99.8% of all Lp(a). When serum Lp(a) concentrations were < or = 300 mg/L, 301-600 mg/L, and > 600 mg/L, the uncorrected serum LDL-C was overestimated, respectively, by a mean of 4.1% (n = 7), 8.5% (n = 8), and 21.4% (n = 8). Serum Lp(a) concentrations were positively correlated with percentage overestimation (P < 0.001), but were not correlated with either corrected or uncorrected serum LDL-C. We conclude that the Friedewald formula should be modified to take into account the contribution of Lp(a) cholesterol to total serum cholesterol.
Biological variability of cholesterol, triglyceride, low- and high-density lipoprotein cholesterol, lipoprotein(a), and apolipoproteins A-I and B
Authors: Marcovina S M , Gaur V P , Albers J J
Published: January 2020

Biological variability is a major contributor to the inaccuracy of cardiovascular risk assessments based on measurement of lipids, lipoproteins, or apolipoproteins. We obtained estimates of biological variation (CVb) for 20 healthy adults and calculated the percentiles of CVb as an expression of the variability of CVb among individuals for cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, apolipoprotein (apo) A-I, apo B, and lipoprotein(a) [Lp(a)] by four biweekly measurements of these analytes. The CVb for the group was approximately 6-7% for cholesterol, HDL cholesterol, apo A-I, and apo B; approximately 9% for LDL cholesterol; and 28% for triglyceride. However, for each analyte, there was a considerable variation of CVb among individuals. For all analytes except Lp(a), there was no relation between the individual's CVb and the analyte concentration. Lp(a) was inversely related to CVb, and there was a very wide variation in the CVb for Lp(a) among the participants, ranging from 1% to 51%. The number of independent analyses to perform to accurately assess an individual's risk for coronary artery disease should be determined on the basis of the individual CVb for a given analyte rather than the average CVb.
Decreased signal in Emit assays of drugs of abuse in urine after ingestion of aspirin: potential for false-negative results
Authors: Wagener R E , Linder M W , Valdes R
Published: January 2020

During routine drug analysis with the Syva d.a.u. Emit immunoassays we observed a high frequency of urines with lower rates of changes in absorbance (delta A R) than the rate for a drug-free urine calibrator. Many of these urines contained salicylates. Among 40 urines with apparent salicylate concentrations between 15 and 420 mg/dL tested for benzoylecgonine (BE), 20 had delta A R < -4 (range +2 to -28 mA/min). The rates decreased with increasing salicylate: delta A R = -0.057 x (salicylate, mg/dL) -0.22 mA/min (r = 0.85, n = 40, P < 0.01). Urines from 100 control subjects (no salicylate) had mean +/- SD delta A R values of -1.05 +/- 2.2 mA/min (range +3 to -7; only two were < -4 mA/min). Although direct addition of salicylic acid (200 mg/dL) to urine specimens did not reproduce the negative bias, ingestion of aspirin (acetylsalicylic acid) did by -0.09 mA/min per 1 mg/dL (72.4 mumol/L) salicylate. Negative biases observed for other Emit d.a.u. assays after salicylate ingestion lead us to conclude that ingestion of therapeutic doses of aspirin may cause false-negative results for drug screens in urines by this technology.