bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved amplification of cytomegalovirus DNA from urine after purification of DNA with glass beads
Authors: Buffone G J , Demmler G J , Schimbor C M , Greer J
Published: January 2020

Cytomegalovirus can be detected in a variety of specimens including leukocytes, urine, saliva, feces, and various tissues by polymerase chain reaction (PCR) amplification of viral DNA. Although methods for amplification are fairly standard, sample preparation is not well characterized, especially for tissue. Typically, preparation of samples for PCR amplification ranges from simple boiling to phenol/chloroform extraction and quantification before testing. Several reports have described inhibition of the PCR in some samples types. Here we show that reliable detection of cytomegalovirus DNA in urine is obtained only after some degree of DNA purification, presumably because of PCR inhibition by a yet unidentified component present in a few of the urine samples tested. Glass, in the form of fine beads, was used to adsorb DNA such that protein and other substances could be selectively eluted before the recovery of DNA for PCR amplification. Urine samples prepared by this method did not show inhibition, and results correlated well with those by tissue culture for detection of cytomegalovirus.
Multianalyte microspot immunoassay--microanalytical "compact disk" of the future
Authors: Ekins R P , Chu F W
Published: January 2020

Throughout the 1970s, controversy centered both on immunoassay "sensitivity" per se and on the relative sensitivities of labeled antibody (Ab) and labeled analyte methods. Our theoretical studies revealed that RIA sensitivities could be surpassed only by the use of very high-specificity nonisotopic labels in "noncompetitive" designs, preferably with monoclonal antibodies. The time-resolved fluorescence methodology known as DELFIA--developed in collaboration with LKB/Wallac--represented the first commercial "ultrasensitive" nonisotopic technique based on these theoretical insights, the same concepts being subsequently adopted in comparable methodologies relying on the use of chemiluminescent and enzyme labels. However, high-specific-activity labels also permit the development of "multianalyte" immunoassay systems combining ultrasensitivity with the simultaneous measurement of tens, hundreds, or thousands of analytes in a small biological sample. This possibility relies on simple, albeit hitherto-unexploited, physicochemical concepts. The first is that all immunoassays rely on the measurement of Ab occupancy by analyte. The second is that, provided the Ab concentration used is "vanishingly small," fractional Ab occupancy is independent of both Ab concentration and sample volume. This leads to the notion of "ratiometric" immunoassay, involving measurement of the ratio of signals (e.g., fluorescent signals) emitted by two labeled Abs, the first (a "sensor" Ab) deposited as a microspot on a solid support, the second (a "developing" Ab) directed against either occupied or unoccupied binding sites of the sensor Ab. Our preliminary studies of this approach have relied on a dual-channel scanning-laser confocal microscope, permitting microspots of area 100 microns 2 or less to be analyzed, and implying that an array of 10(6) Ab-containing microspots, each directed against a different analyte, could, in principle, be accommodated on an area of 1 cm2. Although measurement of such analyte numbers is unlikely ever to be required, the ability to analyze biological fluids for a wide spectrum of analystes is likely to transform immunodiagnostics in the next decade.
Albuminuria and Diabetic Nephropathy: an Evolving Story
Authors: Watts Nelson B
Published: January 2020

The lifetime risk of diabetic nephropathy is ˜45% for patients with Type I (insulin-dependent) diabetes (1) and at least 30-35% for patients with Type II (non-insulin-dependent) diabetes (2, 3). In Type I diabetes, the incidence of readily detectable nephropathy rises steeply 10 years after the onset of diabetes (1). The progression from the appearance of “clinical proteinuria” (a positive test for protein upon routine urinalysis) to end-stage renal failure requiring dialysis or kidney transplantation occurs over about five years. This progression is usually relentless (4), but the rate of deterioration may be slowed or stabilized with improved glucose control (5-7), by the use of angiotensin-converting enzyme inhibitors (8, 9), diltiazem (10) [but not nifedipine (11)], and dietary protein restriction (12). Early intervention, before the development of clinical proteinuria, appears to be much more effective than later treatment.
Free thyroxin measured in dried blood spots from normal, low-birth-weight, and hypothyroid neonates
Authors: Lemonnier Françoise , Masson Joëlle , Laroche Dominique , Travert Josette , Travert Georges
Published: January 2020

We have adapted a new radioimmunoassay for free thyroxin (FT4) measurement in dried blood spots for use in neonatal screening for hypothyroidism. The method is easy, fast, and cheap. Within-assay and between-assay CVs are respectively 9.6% and 13.2%. In 997 neonates three days postpartum with normal thyrotropin concentrations, the mean FT4 concentration was 27.2 pmol/L (SD 7.3 pmol/L). There was no significant difference in mean FT4 concentration between boys and girls. FT4 concentrations increased linearly with birth weight or with gestational age, as expressed by multiple linear regression: FT4 (pmol/L) = 0.0016 birth weight (g) + 0.6931 gestational age (weeks) − 4.8772. Only gestational age significantly affected the FT4 value. For five hypothyroid infants tested on day three postpartum, FT4 values were all below the 1st percentile of values from healthy neonates. Thus, when the neonatal concentration of thyrotropin is above normal, FT4 measured in the same sample can provide a reliable earlier diagnosis of hypothyroidism.
Frequencies of Immunoglobulin Classes Involved in Monoclonal Gammopathies in Guadeloupe
Authors: Pileire Bernard , Bangou Jacqueline
Published: January 2020

The distribution of immunoglobulin classes in 213 monoclonal gammopathies from a West Indian population was studied over a five-year period at the CHRU of Pointe-a-Pitre, Guadeloupe. Monoclonal gammopathies were detected by routine electrophoresis on cellulose acetate (Sebia, France). Immunoglobulin antisera were used according to a laser immunonephelometric method (Behring, France).
Accuracy, precision, and interferences of three modified EMIT procedures for determining serum phenobarbital, urine morphine, and urine cocaine metabolite with a Cobas-Fara
Authors: Ammann Hélène , Vinet Bernard
Published: January 2020

Sung and Neely‘s proposed cost-reductive EMITTM (Syva Co., Palo Alto, CA) procedure allowed a 20-fold increase in the number of tests that can be performed with a single EMIT kit (1). They extensively diluted both EMIT reagents and supplemented the reaction medium with more substrate (glucose 6-phosphate, G6P) and cofactor (NAD+) to saturate the enzyme activity. This principle has been successfully applied to the determination of serum theophylline (1), serum anticonvulsanta (2), urineopiates (3), and urine benzoylecgonine, a cocaine metabolite (4).
Stability of phencyclidine and Amphetamines in Urine Specimens
Authors: Hughes R , Hughes A , Levine B , Smith M L
Published: January 2020

Knowledge about the stability of drugs in biological specimens can be critical to the proper interpretation of results. Stability of drugs of abuse in biological specimens has been recently reviewed (1). Two drugs for which limited stability data have been published are phencyclidine (PCP) and amphetamine (AMP)/methamphetamine (MA). Therefore, we undertook a stability study of these drugs in urine specimens. Urine samples from five drug-free individuals were fortified with PCP, AMP, and MA at three concentrations. These specimens were divided into aliquots; one was stored at room temperature (25 °C), the other in the refrigerator (4 °C). Over a six-month period, aliquots of each specimen were quantified by gas chromatography-mass spectrometry (2). A known control was analyzed with each batch; results were within 10% of the expected value.
Urinary Excretion of Free Toluenediamines in a Patient with Polyurethane-Covered Breast Implants
Authors: Chan Slu C , Birdsell Dale C , Gradeen Claire Y
Published: January 2020

We report the detection of free 2,4-toluenediamine in urine of a patient implanted with polyurethane-covered breast implants. Samples were collected on several dates, ranging from 21 days to seven months after the insertion of the implants, and these samples all showed the presence of free 2,4-toluenediamine at a concentration of about 1 μg/L. The chemical was not found in a urine sample collected before implantation. This finding is important for risk assessment of cancer in patients with this type of breast implant because the chemical is a suspected carcinogen. Free 2,6-toluenediamine, an isomer, was also found in all samples from this patient.
Interference in assay of free triiodothyronine by triiodothyronine-binding antibodies
Authors: Tokmakjian Sonya D , Haines D S Milton , Edmonds Merril W
Published: January 2020

A 39-year-old woman with a 20-year history of hypothyroidism caused by Hashimoto thyroiditis had been managed adequately with oral thyroxun (T4), 200 mg/day, until a few months before referral, at which time she developed symptoms of hyperthyroidism. Her thyroid hormone proffle at the time of referral is shown in Table 1 (Sample 1). Her normal value for serum thyrotropin (TSH) concentration indicates that she was euthyreid but, because of the high serum triiodothyronune (T3) value, the daily oral doseof T4 was decreased to 100 mg. The serum thyroid tests were repeated four weeks later (Table 1, Sample 2): The decreased T4 and increased TSH show that the patient had become hypothyroid at this dosagé of T4 but the serum T3 concentration was still in the high normal range.
Increased Estradiol Concentration of Unknown Origin
Authors: Vasiliades John , Hilgers T , Gentrup B , Remsburg J
Published: January 2020

We report an interesting case of above-normal serum estradiol concentration of unknown origin. A 30-year-old white woman was seen for infertility problems. Hormonal evaluation revealed the following results. Postovulatory progesterone concentrations and profile (a plot of hormone concentration vs time of cycle) were within the normal range for our laboratory (Table 1). However, the post-ovulatory estradiol concentrations were extremely high, although the curve appeared normal in profile (Table 1).
Heterozygous hypobetalipoproteinemia with fasting chylomicronemia
Authors: Huet G , Dieu M C , Martin A , Grard G , Bard J M , Fossati P , Degand P
Published: January 2020

We describe a disorder in which low-density lipoprotein (LDL)-cholesterol and apolipoprotein B are in low concentration (0.47 mmol/L and 0.28 g/L, respectively) and chylomicrons are still present in plasma after an 18-h fast. The d less than 1.006 fraction was isolated by flotation ultracentrifugation and the apolipoproteins were analyzed by electrophoresis, immunoblotting with anti-apolipoprotein B-100 antiserum, and isoelectric focusing. In the d less than 1.006 fraction of the fasting serum, we found an apolipoprotein B form with the same apparent molecular mass as apolipoprotein B-48 and similar in amount to apolipoprotein B-100 (respective percentages, 46% and 54%). The monosialylated form of the apolipoprotein C-III was severely decreased. After an oral fat load, the repartition of the two species of apolipoprotein B did not change greatly (respective percentages, 60% and 40%), and the concentration of serum triglyceride increased only from 1.20 to 1.65 mmol/L.
Approaches to minimizing interference by cross-reacting molecules in immunoassays
Authors: Miller James J , Valdes Roland
Published: January 2020

Here we review techniques useful in eliminating or reducing interferences caused by molecules that cross-react in immunoassays. The biochemical rationale for using these techniques is discussed. Examples are taken from recent studies aimed at reducing interferences caused by endogenous molecules such as digoxin-like immunoreactive factors or steroid hormones. In this context the role of protein-binding of cross-reacting molecules is also considered. Immunoassay ligand selectivity can be inherently limited by the heterogeneity of the antigenic response or by the structural similarity of epitopes on multiple ligands. Certain empirical approaches have proved useful in maximizing the analytical specificity of immunoassays. These approaches include isolating the relevant ligands before immunoassay, adjusting the kinetic or equilibrium conditions used during the assays, and developing more specific antisera. The physicochemical properties of the cross-reacting molecule best dictate which technique(s) to use. The approaches discussed here are general and apply to minimizing interference caused by a wide variety of both endogenous and exogenous cross-reacting molecules.
Accumulation of quinolinic acid in uremic serum and its removal by hemodialysis
Authors: Niwa Toshlmitsu , Yoshizumi Hldeo , Emoto Yutaka , Miyazaki Takashl , Hashimoto Naosuml , Takeda Naohfto , Tatematsu Akira , Maeda Kenji
Published: January 2020

Quinolinic acid was first identified in uremic serum by use of gas chromatography/mass spectrometry. Quantification by selected ion monitoring revealed that the serum concentration of quinolinic acid was markedly increased in chronic hemodialysis patients, and that the acid could be removed by conventional hemodialysis. The serum concentration of quinolinic acid was weakly but significantly correlated with the serum uric acid concentration. Accumulation of quinolinic acid in uremic blood may be involved in the pathogenesis of anemia, suppressed immune system, and uremic encephalopathy.
Concentrations of salivary testosterone and plasma total, non-sex-hormone-binding globulin-bound, and free testosterone in normal and hirsute women during administration of dexamethasone/synthetic corticotropin
Authors: Swinkels L M J W , van Hoof H J C , Ross H A , Smals A G H , Benraad Th J
Published: January 2020

Preliminary data indicate that the concentration of circulating cortisol may affect the binding of testosterone to plasma proteins. This interdependency was evaluated by the assessment of plasma cortisol, salivary (ST) and plasma total testosterone (TT), testosterone not bound to sex-hormone-binding globulin (n-SHBGT), and free testosterone (FT) in normal and in hyperandrogenemic women during combined dexamethasone/synthetic corticotropin administration. The concentrations of TT, FT, and ST significantly increased (P less than 0.001) during this dynamic test both in the controls and in hirsute women. Remarkably, however, n-SHBGT remained virtually unchanged in normal women in the follicular phase, decreasing in the luteal phase, and decreasing even more markedly in the hirsute women. Both the normal and the hirsute women showed a statistically significant negative correlation between the percentage of n-SHBGT and plasma total cortisol (P less than 0.001). The apparent decrease of n-SHBGT was the result of displacement of T from corticosteroid-binding globulin (CBG) by the increase in cortisol after the infusion of synthetic corticotropin: the CBG-bound T was measured as n-SHBGT because CBG was not precipitated with 50% saturated ammonium sulfate. Our results indicate that ST or FT better represents the clinical status of androgenicity than does n-SHBGT, as assessed by ammonium sulfate precipitation, because n-SHBGT also depends on the concentration of cortisol.
Interlaboratory comparison of the measurement of albumin in urine
Authors: Mueller PatrIcIa W , MacNeil Mary LouIse , Smith S Jay , Miller Dayton T
Published: January 2020

Because of increased interest in the assay of albumin in urine and the sensitivity required to quantify concentrations associated with (a) increased risk of developing end-stage renal disease and cardiovascular disease among people with diabetes and (b) renal damage caused by exposure to nephrotoxic substances, we conducted a pilot study of the variation of these measurements within and among five laboratories that use various immunoassays. These assays included two different enzyme immunoassays, two different immunoturbidimetric assays, a fluorescent immunoassay, and a zone immunoelectrophoresis assay. The results indicate considerable variation both within and among laboratories for measurements at or near the normal range. Variability is equally attributable to the precision of individual immunoassays and to the variation of the mean values obtained by each laboratory. Individual laboratory CVs ranged from 5.8% to 18.2% for mid- and high-concentration samples treated with preservative and from 8.4% to 23.6% for mid- and high-concentration samples containing no preservative. The relative bias of individual laboratory means ranged from -56.4% to 20.5% for the two preserved materials and from -32.6% to 0.8% for the two materials containing no preservative. To reduce the chance of misdiagnosing the risk associated with above-normal albumin concentrations in urine, we need to address the problems contributing to imprecision and inaccuracy, particularly laboratory-to-laboratory variability.
Precipitation with polyethylene glycol and density-gradient ultracentrifugation compared for determining high-density lipoprotein subclasses HDL2 and HDL3
Authors: Widhalm Kurt , Pakosta Renate
Published: January 2020

The purpose of this study was to compare quantification of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 by precipitation with polyethylene glycol (PEG) with that by density-gradient ultracentrifugation. Fresh serum samples from 32 fasting, obese children were analyzed with precipitation reagent "Quantolip" (Immuno AG), and then fractionated with a Beckman TL 100 ultracentrifuge with a swinging-bucket rotor. After centrifugation we carefully removed the supernate with a syringe and measured the cholesterol from each fraction enzymatically with CHOD-PAP reagent (Boehringer Mannheim). The low-density lipoprotein (LDL-), HDL2-, and HDL3-cholesterol values measured by ultracentrifugation did not differ significantly from those obtained by precipitation; the correlation coefficients (r) between the two methods were 0.96 for LDL, 0.75 for HDL2, and 0.96 for HDL3. The relatively simple PEG precipitation method used in this study measures total HDL and its major subclasses HDL2 and HDL3 with accuracy and precision comparable with those of the well-established ultracentrifugation method.
Electrochemical enzyme immunoassay for phenytoin by flow-injection analysis incorporating a redox coupling agent
Authors: Tang Hua T , Halsall H BrIan , Heineman William R
Published: January 2020

Using phenytoin as a model analyte, we demonstrate an electrochemical enzyme immunoassay based on flow-injection analysis and incorporating 2,6-dichloroindophenol (DCIP) as a redox coupling agent. DCIP reacts with NADH to form NAD+ and DCIPH2, the reduced form of the coupling agent. The production of DCIPH2 is monitored at +250 mV vs Ag/AgCl. This low applied potential improves selectivity in the biological matrix, differentiating against components that are oxidizable at the more-positive potentials required for direct electrochemical detection of NADH. The kinetics-based assay also eliminates other common interferences, mainly from ascorbic acid and glutathione. This system does not require precolumns or analytical columns for isolation of the NADH response. Good agreement with a routine clinical laboratory procedure for phenytoin is obtained for clinical samples (r = 0.95), illustrating the feasibility of such an approach.
Individual differences in lutropin immunoreactivity revealed by monoclonal antibodies
Authors: Pettersson Kim S , Söderholm JuanIta R
Published: January 2020

We screened 11 monoclonal anti-lutropin (LH) antibodies for use in two-site immunometric combinations, with time-resolved fluorescence as the detection principle. Seven immunofluorometric assays (IFMA), six detecting only the intact hormone and one detecting both the intact hormone and free beta subunit, were compared with each other and with a conventional radioimmunoassay involving a polyclonal antiserum. Two monoclonal antibodies that exclusively reacted with intact LH showed restricted reactivity with LH in 25% of the samples tested. The LH of one individual was not detected at all by one of the antibodies. The existence of two distinct populations suggests the existence of a genetic variant of LH. A comparison of the results obtained by IFMAS with those by radioimmunoassay showed marked discrepancies in the low range, with IFMAS measuring much lower values. The excellent correlation between the various monoclonal IFMA strongly suggests that the discrepancies seen are caused by the insufficient sensitivity and matrix effects of the radioimmunoassay.
Quantifying bone and liver alkaline phosphatase by the resolution of two-component inactivation data obtained with a centrifugal analyzer
Authors: Forsman Rodney W , O'Brien John F
Published: January 2020

In this method we use a linear equation to resolve two-component decay data from the urea inactivation of mixtures of alkaline phosphatase (EC 3.1.3.1) of liver and bone origin. The specificity for the bone/liver isoenzyme is enhanced by including L-phenylalanine in the urea reaction to inhibit the intestinal and placental forms. Bone and liver fractions are each quantified from results of a single run on the centrifugal analyzer. Total activity and the L-phenylalanine-inhibited fraction are measured on separate runs. A simplification of this method has been used in our laboratory for 14 years in performing greater than 80,000 analyses of clinical specimens. Speed, accuracy, and precision are improved over previous methods by this mathematical solution. Data reduction is automated through the use of a personal computer interfaced to the analyzer. With such systems generally available, this method can now be suggested as suitable for routine use to separate these and perhaps other isoenzyme mixtures.
Individual probability for onset of full-blown disease in patients infected with human immunodeficiency virus type 1
Authors: Reibnegger Gilbert , Spira Thomas J , Fuchs Dietmar , Werner-Felmayer GabrIele , Dierich Manfied P , Wachter Helmut
Published: January 2020

Increased concentrations of neopterin, a marker for cell-mediated immune activation, and decreased numbers of CD4+ T cells, are predictors for progression of disease after infection with human immunodeficiency virus type 1. Previous studies have demonstrated different rates of onset of full-blown acquired immunodeficiency syndrome (AIDS) for groups of patients, defined by laboratory marker values, who were initially symptom-free. By re-analysis of one such study, we demonstrate how for an individual patient, the individual marker values, together with a prior risk estimate, can be converted into current or accumulated post-test probability of onset of AIDS at a certain time. We used a statistical technique suggested by Albert et al. (Clin Chem 1984;30:69-76), which allows incorporation of fixed and time-dependent covariates. Besides allowing individual projections, the method shows that the predictive abilities of CD4+ T cell numbers and of neopterin concentrations do not vary greatly with regard to time of observation; both laboratory markers independently modulate the underlying prior probability of AIDS onset, which is significantly increased with the passage of time.
Diagnostic and prognostic utility of phospholipase A activity in patients with acute pancreatitis: comparison with amylase and lipase
Authors: Kazmierczak Steven C , Van Lente Frederick , Hodges Edna D
Published: January 2020

We compared the diagnostic and prognostic utility of phospholipase A (PLA; EC 3.1.1.4) for acute pancreatitis with that of amylase and lipase by analysis of sera from 151 consecutive patients presenting with abdominal pain in whom assays of serum amylase and (or) lipase had been ordered. We determined the diagnostic accuracy for both the initial and the peak enzyme activities. Maximal diagnostic accuracy obtained for the initial activities of amylase, lipase, and PLA was 0.83, 0.83, and 0.76 at cutoff values of 650, 650, and 41 U/L, respectively. Use of peak enzyme activities showed maximal diagnostic accuracy of 0.85, 0.86, and 0.73 at cutoff values of 650, 1050, and 42 U/L, respectively. Receiver-operator characteristic curve analysis revealed the diagnostic performance of amylase and lipase to be similar, whereas that of PLA was almost random and not incremental. As with amylase and lipase, PLA activities in sera showed no relation to patients' survival; three patients who died after an attack of acute pancreatitis failed to demonstrate the dramatic increases in PLA activity previously described. We conclude that assessing the severity of acute pancreatitis by using enzyme activities still remains problematical. Measurements of amylase or lipase activities provide similar diagnostic discrimination when appropriate cutoff values are used and remain the methods of choice for diagnosis of acute pancreatitis.
Use of chicken antibodies in enzyme immunoassays to avoid interference by rheumatoid factors
Authors: Larsson Anders , Karlsson-Parra Alex , Sjöquist J
Published: January 2020

Rheumatoid factor (RF) is a major source of interference in many immunoassays. Most immunoassays use mammalian polyclonal or monoclonal antibodies, and RF can react with IgG from mammalian species, thus causing false-positive results. In this work we have studied RF interference in a sandwich ELISA, where RF in the sample may react with both the capture antibody and the detection antibody to give a false-positive reaction. We show that rheumatoid factors do not react with chicken IgY; if the capture antibody or detection antibody (or both) is of avian origin, the interference of RF or other anti-IgG antibodies in sandwich ELISA can be avoided.
DNA enzyme immunoassay: general method for detecting products of polymerase chain reaction
Authors: Mantero Giovanni , Zonaro Antoneil , Albertini Aiberto , Paola Alberto , Primi Daniele
Published: January 2020

We developed a new colorimetric method, DNA enzyme immunoassay (DEIA), for detecting specific hybrids of complementary nucleic acids and applied it to the detection of hepatitis B virus (HBV) DNA amplified from serum samples by means of the polymerase chain reaction (PCR) technique. The method is based on the ability of an anti-DNA monoclonal antibody to discriminate between single-stranded and double-stranded DNA. A solid phase was coated with a specific oligonucleotide probe, internal to the amplified region of HBV DNA, via an avidin-biotin bridge. The denatured PCR product was hybridized with the solid-phase probe, and the amplified DNA probe hybrid was then incubated with a monoclonal antibody specific for double- but not single-stranded DNA. Colorimetric detection of the DNA-antibody complex was achieved by adding an anti-mouse Ig antibody labeled with horseradish peroxidase. The combined use of DEIA and PCR can reveal a few HBV genome copies present in a serum sample. This method has several advantages: (a) the sensitivity is adequate for the detection of amplified DNA; (b) the signal is associated with the hybridization event, independently of modifications of the probe or of the amplification primers; and (c) the test is simple and rapid and, most importantly, requires only the standard facilities of a routine clinical laboratory.
Macromolecular creatine kinase type 1: a serum marker associated with disease
Authors: Laureys Muriȵl , Sion Jean-Pau , Slabbynck Hans , Steenssens Laurette , Cobbaert Chrlsta , Derde Marie-Paul , Gorus Frans K
Published: January 2020

The prevalence of circulating macromolecular creatine kinase type 1 (macro CK type 1 or CK-immunoglobulin complexes) is significantly higher in a patient population selected for CK isoenzyme assay than in age- and sex-matched blood donors (n = 1304). In greater than 8000 patients studied, 49 individuals with macro CK type 1 were identified, yielding an overall prevalence of 0.61%. Macro CK type 1 complexes occurred more frequently in women and in patients older than 70 years, and were often associated with complications of cardiovascular disease, life-threatening conditions, and poor outcome. These latter clinical associations could arise, at least partly, from the selection of patients for whom CK isoenzyme analysis was ordered.
Performance of techniques used to detect drugs of abuse in urine: study based on external quality assessment
Authors: Wilson J F , Williams J , Walker G , Toseland P A , Smith B L , Richens A , Burnett D
Published: January 2020

Twenty-five samples of lyophilized urine from the U.K. External Quality Assessment Scheme for Drugs of Abuse were analyzed by an average of 95 laboratories between April 1987 and December 1989. Samples contained mixtures of analytes and included replicated concentrations of morphine, methadone, amphetamine, and cocaine at 0, 1, 2, and 5 mg/L and of benzoylecgonine at 0, 0.4, 1, 2, and 4 mg/L. Some chromatographic techniques were inadequate for detecting morphine, amphetamine, cocaine, and benzoylecgonine at the lower concentrations of analytes studied: gas-liquid chromatography was least sensitive for morphine; in-house thin-layer chromatography (TLC) was least sensitive for the other analytes. Few significant differences in specificity were detected between techniques, although significant interference from structurally related compounds was demonstrated in assays of morphine, methadone, and amphetamine. Exceptions were the Boehringer (BCL) test for opiates and TLC applied to amphetamine and benzoylecgonine, which demonstrated considerable lack of specificity.
Genetic screening of newborns for sickle cell disease: correlation of DNA analysis with hemoglobin electrophoresis
Authors: Skogerboe KrIsten J , West Sheyla F , Murillo Magdalena D , Glass Michael W , Shaunak Santosh , Tait Jonathan F
Published: January 2020

Although DNA analysis based on the polymerase chain reaction (PCR) offers potential advantages for screening newborns for sickle cell disease, few data are available concerning the reliability of PCR-based tests for such screening. We describe a protocol for detecting the A, S, and C alleles of the beta-globin gene in dried blood from phenylketonuria screening cards. This method is based on PCR and detection with allele-specific oligonucleotide probes. Results of a blind comparison of PCR analysis of the dried blood with hemoglobin electrophoresis of whole-blood samples agreed for 80 of 81 samples. The single discrepancy is probably not attributable to a failure of the PCR method, but rather to limitations of the electrophoresis method. The PCR method should be a highly accurate means of detecting beta-globin alleles in routine genetic screening with dried blood already collected for (e.g.) phenylketonuria screening.
Development of a radioligand receptor assay for measuring follitropin in serum: application to premature ovarian failure
Authors: Schneyer Alan L , Sluss Patrick M , Whitcomb Randall W , Hall Janet E , Crowley William F , Freeman Ruth G
Published: January 2020

We have developed a radioligand receptor assay (RRA) with sufficient sensitivity and specificity for quantifying follitropin (FSH) in unextracted serum samples. Standard curves prepared by adding pituitary FSH to either buffer or gonadotropin-free serum were parallel and statistically indistinguishable in this assay, whereas gonadotropin-free serum alone had no activity. Cross-reactivity with related pituitary hormones was negligible. Pituitary FSH was calibrated with commonly used reference preparations so that RRA results could be compared with RIA results for identical standards. The patterns in daily blood samples in six normal menstrual cycles were similar by both methods. The mean RIA:RIA ratio in both the follicular and luteal phases was between 0.6 and 0.7, and at mid-cycle decreased to 0.48, suggesting an alteration of isohormone composition at mid-cycle. In 27 women with premature ovarian failure, RRA:RIA ratios ranged from below the RRA minimum detectable dose to 4.6, suggesting that immunoreactive FSH might not be capable of binding to the FSH receptor in some patients, whereas in patients with high RRA:RIA ratios, circulating inhibitors of FSH receptor binding might be present and perhaps contributing to the observed ovarian failure. Use of this RRA in conjunction with RIA and in vitro bioassays may better define the relative contribution of FSH isohormones, autocrine or paracrine modulators of FSH bioactivity, and FSH-receptor binding competitors to the "total FSH biological signal" as detected by the gonadal FSH receptor.
GCDFP-70 protein in cyst fluid identified as albumin and used to classify cysts in women with breast gross cystic disease
Authors: Balbín Milagros , Vizoso Francisco , Sánchez Luis M , Venta Rafael , Ruibal Alvaro , Fueyo Antonio , López-Otín Carlos
Published: January 2020

We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to study cyst fluids from women with breast gross cystic disease. The subjects could be classified into two categories according to the concentrations of protein GCDFP-70 in the cyst fluid: those with Type I cysts had a very low content of this protein; those with Type II cysts had very high concentrations. Analysis of the amino acid sequence of GCDFP-70 from both cyst fluid types confirmed that this protein is human plasma albumin. The average concentration of albumin found in Type I cyst fluids was 0.32 g/L and that corresponding to Type II was 10.16 g/L. Thus, albumin quantification from cyst fluids or analysis by either polyacrylamide or agarose gel electrophoresis provides a simple procedure for classifying these fluids, yielding results that correlate well with previous classifications based on other measurements such as sodium, potassium, and chloride concentrations. This albumin-based quantification method may improve the classification of breast cysts and might be useful in further studies on functional changes in the cysts and their relationship to breast cancer.
The spot test is not a reliable screening procedure for mucopolysaccharidoses
Authors: de Jong J G N , Hasselman J J F , van Landeghem A A J , Vader H L , Wevers R A
Published: January 2020

To check the reliability of the Ames MPS paper spot test, which is based on the Azure A dye, we sent a series of urine samples to three laboratories where the spot test is part of the metabolic screening for mucopolysaccharidoses. In these laboratories false-negative results ranged between 19% and 35% and false-positive results ranged between 12% and 29% of all samples submitted. In contrast, the quantitative dimethylmethylene blue test (Clin Chem 1989;35:1472-7) detected an increased glycosaminoglycan content in all urine samples from mucopolysaccharidosis patients and gave no false-positive results. In the latter procedure, glycosaminoglycan content is expressed per millimole of creatinine, and age-dependent reference values are used. We conclude that the Ames spot test and other spot tests are unreliable as a screening procedure for mucopolysaccharidoses and should not be used to screen for these diseases.
The biotin-(strept)avidin system: principles and applications in biotechnology
Authors: Diamandis E P , Christopoulos T K
Published: January 2020

The biotin-(strept)avidin system has been used for many years in a variety of different applications. Here we present a general overview of the system, describe its components and advantages, and show how the system is used in various applications, with emphasis on immunological and nucleic acid hybridization assays. This system is now considered a versatile independent technology with broad applications in many branches of biotechnology. Clearly, its use will continue to grow in the years to come.
Carcinoembryonic antigen in serum of patients with colorectal polyps: correlation with histology and smoking status
Authors: Shahangian S , Fritsche H A , Hughes J I
Published: January 2020

To measure the concentration of carcinoembryonic antigen (CEA) in serial serum specimens of 47 healthy subjects and in serial sera of 59 patients with colorectal polyps of confirmed histology, obtained before or after (sometimes both) polypectomy, we used the Roche EIA and the Hybritech Tandem-E double monoclonal immunoenzymometric assays. Both methods gave comparable results for the healthy subjects but discordant values for some polyp patients. The Tandem-E test gave increased CEA results more frequently for two groups of polyp patients, the group of ex-smokers with neoplastic polyps (n = 14) and the group of smokers (n = 20). Smokers had significantly greater (P less than 0.025) mean concentrations of serum CEA than did nonsmokers, irrespective of whether they were healthy or had colorectal polyps. Smokers with recurrent polyps demonstrated significantly greater (P less than 0.050) mean concentrations of serum CEA than did smokers with a first occurrence of polyps. Polypectomy was followed by statistically significant decreases (P less than 0.050) of mean concentrations of serum CEA, primarily in polyp patients who were smokers. However, although CEA increases occurred frequently in polyp patients who smoked, these abnormalities did not fully correlate with polyp histology.
Development and validation of an immunoradiometric assay of parathyrin-related protein in unextracted plasma
Authors: Ratcliffe W A , Norbury S , Heath D A , Ratcliffe J G
Published: January 2020

This two-site immunoradiometric assay for human parathyrin-related protein 1-86 (PTHRP1-86) in plasma uses a mouse monoclonal antibody to PTHRP1-34 coupled to cellulose particles for immunoextraction of N-terminal immunoreactivity, and a rabbit antiserum to PTHRP37-67 that is indirectly labeled with 125I-labeled PTHRP37-67 for quantifying the bound analyte. The detection limit of the assay is 0.23 pmol/L, corresponding to 0.4 pg (0.04 fmol) per tube, for a sample volume of 200 microL. Recovery of PTHRP1-86 added to serum is essentially quantitative, and within- and between-batch precision is 4.4% and 11.1%, respectively. PTH1-84, PTHRP18-34, PTHRP9-34, PTHRP1-34, and PTHRP37-67 do not cross-react in the assay at concentrations up to 2 nmol/L. Plasma concentrations of PTHRP1-86 were below or close to the detection limit of the assay in normal subjects and in patients with primary hyperparathyroidism, hypoparathyroidism, chronic renal failure, and normocalcemic malignancy. In 37 hypercalcemic patients with various malignancies, we found detectable PTHRP1-86 concentrations in 35 (95%, mean 7.4 pmol/L, range 0.46-24.7). The data support the proposed humoral role of PTHRP in cancer-associated hypercalcemia and suggest that the assay has clinical utility in the differential diagnosis of hypercalcemia.
Quantifying intermediary metabolites in whole blood after a simple deproteinization step with sulfosalicylic acid
Authors: Khan K , Blaak E , Elia M
Published: January 2020

We assessed the reproducibility, recovery, and stability of several circulating metabolites--glucose, pyruvate, lactate, alanine, glutamate, glutamine, 3-hydroxybutyrate, acetoacetate, and glycerol--in the presence of sulfosalicylic acid (SSA), which was used to deproteinize blood. The assays, which involved reactions linked to NADH/NAD+, were carried out at 37 degrees C and measured at 355 nm with a Cobas-Bio centrifugal analyzer. The intra- and interbatch CVs were less than 2.1%, except for the interbatch CV for 3-hydroxybutyrate at low concentration (15-30 mumol/L), which was 5.4%. Analytical recovery of metabolites added to blood ranged from 96.4% to 103.0%. Of the metabolites studied, all were stable at -20 degrees C for 90 days in the SSA-blood extract, except for glutamine and acetoacetate, which progressively decreased with time. We conclude that these nine circulating metabolites can be satisfactorily measured after a single deproteinizing step with SSA. This single-step procedure has several advantages over many of the currently used methods.
Improved preparation of hepatic microsomes for in vitro diagnosis of inherited disorders of the glucose-6-phosphatase system
Authors: Coughtrie M W , Blair J N , Hume R , Burchell A
Published: January 2020

Disruption of microsomal membranes after freezing liver samples can undermine the reliability of in vitro enzymatic diagnosis of the type 1 glycogen storage diseases. However, freezing of biopsy material is necessary if biopsy samples are to be safely transported to the place of assay. We have therefore examined several different methods (each of which could easily be carried out in routine hospital laboratories) of preparing and freezing liver tissue before analysis for glucose-6-phosphatase (EC 3.1.3.9) enzyme activity, and determination of microsomal intactness. Our study showed that homogenizing fresh liver, and centrifuging the homogenate at 10,000 x g for 10 min at 4 degrees C, followed by freezing the resulting supernatant material at -80 degrees C, provided the optimum source of material for subsequent preparation of microsomes for analysis of glucose-6-phosphatase activity. We also demonstrated that 1-naphthol UDP glucuronosyltransferase (EC 2.4.1.17) activity could be used to assess microsomal intactness in cases of type 1a glycogen storage disease, where mannose-6 phosphatase activity cannot be used.
Choriogonadotropin-mediated thyrotoxicosis in a man
Authors: Cain Heather J , Pannall Peter R , Kotasek Dusan , Norman Robert J
Published: January 2020

A 38-year-old man with a metastatic gonadotropin-secreting tumor of unknown primary origin presented with both clinical and biochemical findings of hyperthyroidism in association with markedly increased concentrations of human choriogonadotropin (hCG) in plasma. After chemotherapy, the concentrations of both hCG and free thyroxin decreased and the patient became euthyroid. We discuss the rare occurrence of this presumably hCG-driven hyperthyroidism in men and compare it with the relatively more common eumetabolic hyperthyroidism associated with choriocarcinoma in women.
Quantitative fluorometric screening test for fecal porphyrins
Authors: Pudek Morris R , Schreiber William E , Jamani Azim
Published: January 2020

We describe a fluorometric method for screening and quantifying porphyrins in stool. A small sample of stool is extracted with concentrated HCI and is diluted 200-fold in 3 mol/L HCI before analysis. An excitation scan is done from 350 to 450 nm, monitoring emission at 603 nm. Total porphyrin is estimated at the isosbestic point for coproporphyrin and protoporphyrin (402.5 nm). Monitoring emission at 603 nm eliminates interference from chlorophyll, obviating the need for extraction with ether. The position of the excitation peak gives some indication of the nature of the porphyrins in the stool. The acid extract can be injected directly into an HPLC system for fractionation studies. Our method correlates well with the spectrophotometric method developed by Lockwood et al. (Clin Chem 1985;31:1163-7). However, in our method, the sample is easier to process and the assay has higher sensitivity than their assay. The reference interval for porphyrin in healthy individuals by the fluorometric method is less than 300 nmol/g dry weight. We can detect as little as 1 nmol of porphyrin per gram (dry weight) of stool. Results of the method vary linearly with stool porphyrin concentrations as great as 4000 nmol/g dry weight. The within-run imprecision of the method is 3%.
Single-spin density-gradient ultracentrifugation vs gradient gel electrophoresis: two methods for detecting low-density-lipoprotein heterogeneity compared
Authors: Dormans Tom P J , Swinkels Dorine W , de Graaf Jacqueline , Hendriks Jan C M , Stalenhoef Anton F H , Demacker Pierre N M
Published: January 2020

Single-spin density-gradient ultracentrifugation (DUC) has proven to be a reproducible method for detection of low-density-lipoprotein (LDL) heterogeneity. Recently another method has been described for this: gradient gel electrophoresis (GGE) of serum, a method that might be more suitable for screening. To gain insight into the relationship of GGE to DUC and into their reproducibility, we determined LDL heterogeneity by DUC and GGE in 41 healthy individuals. In 90.2% (n = 37) of the subjects, the number of LDL subfractions found by both methods agreed. In addition, the density and the relative migration distance of the predominant LDL subfraction observed with the respective methods showed a strong correlation (Pearson correlation, r = 0.85, P less than 0.0001). Although it was not possible to compare for all aspects of LDL heterogeneity, these data suggest that GGE is a valid method of analysis for LDL heterogeneity. In screening programs, it may be necessary to store samples. Therefore, we studied in 24 sera the influence of storage at -80 degrees C for one, four, and 12 weeks on the LDL subfraction distribution detected by each method. LDL heterogeneity was maintained during storage under these conditions.
Five treatment procedures evaluated for the elimination of ascorbate interference in the enzymatic determination of urinary oxalate
Authors: Inamdar Kalpana V , Raghavan Keddy G , Pradhan Dinkar S
Published: January 2020

We evaluated the efficacies of five treatment procedures for eliminating ascorbate interference in the enzymatic determination of urinary oxalate. Aliquots of urine samples, containing different amounts of added ascorbate and oxalate, were individually subjected to ferric chloride, sodium nitrite, sodium periodate, charcoal, or ascorbate oxidase treatment to eliminate ascorbate interference. Oxalate contents of the urine samples were then determined by a banana oxalate oxidase-horseradish peroxidase-linked assay with 3-methyl-2-benzothiazolinone hydrazone and 3-(dimethylamino)benzoic acid as chromogens. Only those urine samples treated with ascorbate oxidase or charcoal consistently gave recovery of oxalate close to 100%. Treatment with other reagents, though improving the recovery of oxalate, gave inconsistent results. On the basis of these data, we describe procedures for simply and reliably assaying oxalate by using banana oxalate oxidase.
Age- and sex-related differences for the urinary excretion of norepinephrine, epinephrine, and dopamine in adults
Authors: Gerlo Erik A M , Schoors Danny F , Dupont Alain G
Published: January 2020

Using a specific high-performance liquid-chromatographic method, we measured norepinephrine, epinephrine, and dopamine in 24-h urine collections from 459 men and 497 women, ages 17 to 88 years. We found a significant negative correlation between age and the 24-h excretion of dopamine in men and women (P less than 0.001). Epinephrine excretion decreased with age in men (P less than 0.001). No age dependence was observed for norepinephrine (P greater than 0.2). The excretion of all three catecholamines, expressed in nmol/24 h, was significantly greater in men than in women. The differences, however, were small. With data expressed in nmol/g of creatinine, only epinephrine excretion was greater in men; norepinephrine and dopamine excretions were slightly greater in women. Also, expressed in these units, urinary excretion of norepinephrine in both sexes and of epinephrine in women was significantly positively related with age; urinary excretion of dopamine was significantly inversely related to age in women, but not in men. Reference values are provided for age-independent variables in both sexes.