bluuebunny/crossref_metadata_2025 huggingface dataset:
Is there a practical alternative to therapeutic drug monitoring in therapy with tricyclic antidepressants?
Authors: Dawling S
Published: January 2020

Optimization of tricyclic antidepressant (TCA) therapy by dosage adjustments made in response to inappropriate concentrations in plasma or side effects can be extremely slow owing to the long half-lives of these drugs. I examine the practicality of alternative methods of arriving quickly and reliably at an adequate starting dosage. The clearance of a single test dose from plasma has been used to select individualized dosages before commencing therapy, but this takes several days and requires computer-assisted calculation of clearance. A simpler technique is to measure the concentration in a single timed plasma sample as an index of metabolism, and to infer the required dosage directly from a nomogram. Ideally, the nomograms should be interchangeable between patient populations and independent of the analytical method used, and the drug must have linear kinetics. Furthermore, TCAs are metabolized by common routes--demethylation and hydroxylation--so one might apply a single tolerance test for the entire class of drugs. Hydroxylation of TCAs can also be correlated with that of debrisoquine. The debrisoquine clearance test is non-invasive, faster, and analytically less demanding than TCA measurements. In the absence of rigid therapeutic ranges, tests that identify abnormally slow metabolizers may well be invaluable in preventing iatrogenic poisoning. Despite the usefulness of these methods in establishing effective initial dosages, their continued success depends upon good compliance, the maintenance of the patient's concurrent drug therapy, and a stable physical condition. In the non-ideal world, therefore, TDM cannot be dispensed with, but must be seen as an essential part of effective TCA treatment, based ultimately of course on sound clinical judgement.
Measurement of antidepressants by liquid chromatography: a review of current methodology.
Authors: Wong S H
Published: January 2020

Antidepressant measurement by liquid chromatography (LC) has enhanced the therapy of patients who are being treated with some of the first generation tricyclic antidepressants; the merits of routinely monitoring the other antidepressants await further study. Currently, the role of LC is changing from dominant to complementary as a result of the recent availability of monoclonal antibody immunoassays with increased specificity. For successful application of LC, considerations would include designing the sampling protocol and matching assay that together are uniquely suitable for a particular laboratory. The assay should be simple, the sample preparation manual (liquid-liquid, solid-phase extraction columns), semi-automated, or automated. Normal- or reversed-phase columns with functionalities such as C-18, CN, C-8, and phenyl are used. Other useful LC variables include particle size, capping, ion-pairing, and recycling. This survey of LC methods includes the first and second generations, and new antidepressants such as alprazolam, amoxapine, bupropion, maprotiline, trazodone, and selected metabolites. Potential chromatographic interference by (e.g.) benzodiazepines and neuroleptics is addressed, followed by proposed guidelines for their resolution. Future developments are discussed.
Effect of blood collection and processing on radioimmunoassay results for apolipoprotein A-I in plasma.
Authors: Brown S A , Rhodes C E , Dunn K , Gotto A M , Patsch W
Published: January 2020

We studied the effects of different procedures of blood collection and processing on quantification of apolipoprotein A-I (apoA-I) by radioimmunoassay. ApoA-II and apolipoproteins of low- and very-low-density lipoproteins did not cross react in the assay. Analytical recovery of apoA-I at different doses was complete. ApoA-I concentration in pooled human plasma was stable for as long as a year stored at -70 degrees C. Inter- and intra-assay CVs averaged 7% and 5%, respectively. We collected blood from 20 subjects into tubes containing EDTA alone or EDTA with antiproteolytic agents, then separated the plasma either immediately or after 3 h at 4 degrees C. We tested various formulations of antibacterial, antiproteolytic, and anti-oxidant agents added to plasma, measuring apoA-I concentrations either within 24 h of blood collection or after storage of plasma for 6 weeks at -70 degrees C. No significant difference in the concentrations of apoA-I was found in these specimens, regardless of the conditions studied. We conclude that addition of protective agents other than EDTA is not necessary during blood collection or specimen processing for reliable quantification of apoA-I in fresh or frozen human plasma.
Urinary alanine aminopeptidase assay improved as result of multivariate response-surface analysis.
Authors: Flandrois C , Lahet C , Feldmann D , Gabastou J M , Gonnon A , Maire I
Published: January 2020

Optimization of determination of alanine aminopeptidase in urine by univariate study led to a method involving pretreatment of urine with Sephadex G50. Re-examination of the optimization by multivariate study led us to recommend higher optimal concentrations: 5.8 mmol/L for the substrate and 300 mmol/L for the Tris buffer. Under these new conditions, pretreatment of urine was no longer necessary and the assay could be completely automated.
Excretion of endogenous digoxin-like immunoreactive factors in human urine is a function of urine flow rate.
Authors: Siegfried B A , Valdes R
Published: January 2020

We studied the effect of varying water and salt intake on the renal excretion of endogenous digoxin-like immunoreactive factors (DLIF). DLIF were measured in human urine and serum by competitive displacement of 125I-labeled digoxin from anti-digoxin antibodies. Diuresis was selectively induced in normal healthy humans by acute water ingestion, and natriuresis was preferentially induced by acute saline ingestion. We found the amount of endogenous immunoreactivity excreted in urine to be correlated with urine flow rate but not with urinary sodium excretion. Urinary excretion of DLIF, normalized to creatinine, was 3.6-fold greater at a urine flow rate of 5.5 mL/min than at 0.5 mL/min. On the other hand, saline intake increased urine flow rate 1.9-fold and increased sodium excretion threefold, but did not affect urinary excretion of DLIF. Fractional excretion of DLIF was linearly related to fractional excretion of water. This study demonstrates that normalization of DLIF values to urinary creatinine does not make DLIF excretion independent of urine flow rate and underscores the need for information on urine flow rate when DLIF measurements in urine are being interpreted.
A user evaluation of four Kodak Ektachem slide assays.
Authors: Bissell M G , Hussain S , Sanghavi P , Ward E , Shaw S T
Published: January 2020

Evaluations of magnesium, theophylline, creatinine, and creatine kinase isoenzyme MB assays by the Kodak Ektachem multilayer-film technique are reported.
Liquid-chromatographic determination of free and total 3,4-dihydroxyphenylglycol and 3-methoxy-4-hydroxyphenylglycol in urine.
Authors: Julien C , Rodriguez C , Sacquet J , Cuisinaud G , Sassard J
Published: January 2020

We describe the conjoint measurement in urine of the alcoholic catecholamine metabolites 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG). The method involves extraction with ethyl acetate and back-extraction into acetic acid (MHPG), followed by adsorption onto alumina (DHPG). The separation is performed by reversed-phase liquid chromatography with column switching and amperometric detection. Total (free plus conjugated) compounds are determined after a 24-h enzymatic hydrolysis. Twelve-hour excretion values for normal and hypertensive subjects are given, together with results obtained for patients with pheochromocytoma, chemodectoma, and congenital dopamine-beta-hydroxylase deficiency. The assay is sensitive, reproducible, and free of interferences, and may lead to a better understanding of overall catecholamine metabolism in health and disease.
Development and evaluation of an automated dye-binding assay for protein in cerebrospinal fluid.
Authors: Huang C M
Published: January 2020

Addition of sodium dodecyl sulfate (SDS) to Coomassie Brilliant Blue reagent equalizes the binding variability of the dye to various proteins and markedly improves the accuracy of quantification of protein in cerebrospinal fluid. In the presence of SDS, the absorption spectrum and the absorption maximum are affected by reaction time and temperature, age of the dye preparation, and protein constituent. I automated this procedure, to optimize precision, enable use of a smaller sample, decrease hands-on time, maintain consistency in the time of reading, and avoid carryover. The results (y) compared well with those of the aca (x), with a Deming de-biased regression equation of y = 0.991x + 14.1 mg/L, Sy.x = 33.4 mg/L. The within-run and between-run precision (CV) was less than 2.5% and less than 4.5%, respectively. Commonly used antibiotics, flucytosine, or amphotericin B do not interfere. This automated procedure is fast and accurate and requires only 10 microL of sample.
Concentrations of chromium, cesium, and tin in cerebrospinal fluid of patients with brain neoplasms, leukemia or other noncerebral malignancies, and neurological diseases.
Authors: el-Yazigi A , Martin C R , Siqueira E B
Published: January 2020

We measured the concentrations of chromium, cesium, and tin in the cerebrospinal fluid (CSF) of 29 patients with brain tumors [21 benign (BBT) and eight malignant (MBT)], 28 leukemic patients, 14 patients with lymphoma or noncerebral solid tumors (NLCT), and 32 control patients (15 with neurological disorders and 17 with noneurological conditions) by use of flameless atomic absorption spectrophotometry. We detected chromium in 94% of the patients, tin in 79%, and cesium in 50%. The mean (and SEM) concentrations (micrograms/L) of these metals in the control group were 4.7 (1.1) for chromium, 3.8 (1.6) for cesium, and 6.4 (1) for tin. We observed significant differences (P less than 0.05) in the concentration of chromium in CSF between the MBT group and all other tumor groups; the ratios for the mean CSF concentration of chromium in patients with BBT, leukemia, or NLCT to that in patients with MBT were 2.6, 2.1, or 4.4, respectively. We saw no significant differences in the concentrations of cesium or tin among the various groups investigated.
Evaluation of a new strategy for detection of thyroid dysfunction in the routine laboratory.
Authors: John R , Henley R , Lloyd G , Elder G H
Published: January 2020

We assessed the use of a new strategy for detecting thyroid disorders, utilizing a sensitive assay for concentrations of thyrotropin (TSH) and free thyroid hormone in serum as follow-up tests. Of 1279 patients who were not on thyroxin (T4) replacement treatment, 82% could be classified as euthyroid and would require no further tests. In patients who were on T4 replacement, 41% fell into the euthyroid category and would require no further tests. Using this strategy to replace our existing strategy of free thyroxin as a "first-line" test would reduce the proportion of patients who would require one or more follow-up tests from 49% to 24%.
Rapid sample preparation for determination of iron in tissue by closed-vessel digestion and microwave energy.
Authors: Van Wyck D B , Schifman R B , Stivelman J C , Ruiz J , Martin D
Published: January 2020

We developed a rapid acid-digestion method for preparing tissue samples for iron determination. Specimens were digested in nitric acid and hydrogen peroxide under high temperature and pressure in closed Teflon vessels, with microwave energy. Analysis for iron in 25- to 250-mg portions of digested bovine liver powder (National Bureau of Standards Certified Reference Material no. 1577a) showed excellent linearity ([predicted] = 1.007[actual] - 0.166 micrograms per sample) and analytical recovery (98%). Precision (CV) was 5.4% when iron content was 10 micrograms per sample. Assaying split samples of mouse tissues, we found a close correlation between iron concentrations obtained with closed vs open vessels ([closed] = 0.878[open] + 68 micrograms/g, r = 0.994, range 400-4600 micrograms/g dry weight). In contrast to time-consuming conventional procedures for tissue dissolution, closed-vessel digestion with microwave energy dramatically shortens time for tissue preparation, minimizes use of caustic acid, reduces risk of sample loss or contamination, and yields accurate and reproducible results.
IgG2 subclass deficiency: IgG subclass assays and IgG2 concentrations among 8015 blood donors.
Authors: Madassery J V , Kwon O H , Lee S Y , Nahm M H
Published: January 2020

Among the four IgG subclasses in humans, IgG2 is preferentially expressed in antibodies to carbohydrate antigens whereas IgG1 subclass is commonly associated with antibodies to protein antigens. Because of this association with carbohydrate antigens, values for IgG2 in serum are often used as an index of immunocompetence against carbohydrate antigens. To investigate the value of IgG2 measurements in a general population, we have developed a convenient IgG subclass assay, using monoclonal antibodies and particle concentration fluorescence immunoassay. Our assay is specific, precise, convenient, and accurate. When IgG2 concentrations were determined in the serum samples from 8015 adult blood donors, there were more individuals with low IgG2 concentrations than predicted by the log-normal distribution. The observed distribution suggested the presence of a subpopulation with low IgG2 concentration. Because apparently healthy individuals in a general population have low IgG2 concentrations, IgG2 measurements alone may have a limited clinical usefulness as an index of immune function against carbohydrate antigens.
The SimulTRAC FT4/TSH assay evaluated as a first-line thyroid-function test.
Authors: Desai R K , Deppe W M , Norman R J , Govender T , Joubert S M
Published: January 2020

We evaluated the SimulTRAC FT4 57Co/TSH 125I dual-isotope assay for the simultaneous measurement of free thyroxin (FT4) by radioimmunoassay analog techniques and of thyrotropin (TSH) by immunoradiometry. Inter- and intra-assay CVs were less than 10% over the entire range tested except for 15.9% at the lowest FT4 concentration. Results obtained by the SimulTRAC assay allowed complete differentiation of 85 hyperthyroid patients and 35 hypothyroid patients from normal subjects. However, such estimations of FT4 or TSH concentrations occasionally were misleading for assessing thyroid status in various clinical conditions. We conclude that the SimulTRAC assay has the same inherent disadvantages possessed by FT4 analog and TSH immunoradiometric assays; however, where results of one of the simultaneous assays may be misleading, the results provided by the other may indicate the underlying pathology without requiring an additional assay.
Familial occurrence of diminished pancreatic amylase in serum--a "silent" Amy-2 allelic variant?
Authors: Brock A , Mortensen P B , Mortensen B B , Røge H R
Published: January 2020

We describe a family of 32 subjects, including two healthy siblings without detectable pancreatic amylase in serum and five healthy relatives (one of them a sister) exhibiting subnormal pancreatic amylase activities. Comparisons of immunoreactive amylase concentrations with the corresponding amylase activities may justify the assumption of a "silent" Amy-2 allelic variant.
Three methods compared for isoamylase separation in tissue homogenates.
Authors: Whitten R O , Chandler W L , Thomas M G , Clayson K J , Fine J S
Published: January 2020

Using homogenates of autopsy tissue, we compared three widely available techniques for separating amylase isoenzymes: wheat-germ inhibition (WI), and electrophoresis on cellulose acetate (CA) or agarose (AG). WI separated amylase into two isoforms, CA into seven (three pancreatic and four salivary), and AG into nine (five pancreatic and four salivary). CA and WI had similar isoamylase detection limits (8-10 U/L) and similar imprecision in measuring percent S-type vs P-type isoamylase (within-run SD 1-2%), and they demonstrated a linear response to added S or P isoamylase. In contrast, the AG method had higher detection limits (10-15 U/L), greater imprecision (within-run SD 3%), and showed a nonlinear response to added S or P isomylase. We conclude that CA and WI have essentially equivalent assay attributes, superior to AG, but that CA resolves more amylase isoforms than WI.
A sensitive liquid-chromatographic method for determination of 3'-azido-3'-deoxythymidine (AZT) in plasma and urine.
Authors: Hedaya M A , Sawchuk R J
Published: January 2020

We describe a liquid-chromatographic assay for AZT in human plasma and urine. This assay involves the use of two internal standards, allowing reference of AZT peaks to the appropriate internal standard, the choice depending on the range of concentrations encountered. This method is isocratic, specific, sensitive enough to allow quantification of AZT in concentrations observed clinically, and requires only 13 min of chromatographic time. We saw no interference from various over-the-counter and prescription drugs often used in treating the infectious complications of AIDS.
Nontransferability of C-peptide measurements with various commercial radioimmunoassay reagents.
Authors: Koskinen P
Published: January 2020

I evaluated the between-method variation of C-peptide determined with C-peptide radioimmunoassay kit reagents from Mallinckrodt, Behring, Diagnostic Products Corp., and Immuno Nuclear Corp., and antisera from Novo (M1230, M1221, and K6) and Cambridge Medical Diagnostics (299-029P). C-peptide concentrations corresponding to 0.6 nmol/L (as determined with M1230) ranged from 0.54 (by Immuno Nuclear Corp.) to 1.06 nmol/L (by M1221). Furthermore, pretreating plasma specimens with Polyethylene Glycol 6000 variably decreased the results. Several factors may contribute to the between-method variation of C-peptide analyses: differences in the specificity of antisera, the type of C-peptide preparation used as standard and tracer, liability of the immunoassay to nonspecific interference, and possibly the heterogeneity of C-peptide immunoreactivity in the specimens.
Monoclonal-antibody-based noncompetitive avidin-biotin assay for lutropin in urine.
Authors: Vilja P , Wichmann L , Isola J , Tuohimaa P
Published: January 2020

This noncompetitive avidin-biotin assay for lutropin (LH) in urine is based on the use of solid-phase, monoclonal antibodies, avidin-biotin binding, and enzyme label. The detection limit for LH is 0.5 int. unit/L. Performance time is 2 h. Intra- and interassay CVs were 4.2% and 6.2%, respectively. The assay is more specific for intact LH than are RIAs, and the results are unambiguous as compared with those by RIAs. LH is stable for a week in refrigerated urine; thus urine is a convenient sample material in follow-up studies for clinical ovulation timing. This rapid and sensitive nonisotopic LH assay avoids the disadvantages of blood samples. The assay provides clinically relevant predictions of ovulation timing from the amount of hormone measured in a 3-h urine specimen.
Measurement of rimantadine in plasma by capillary gas chromatography/mass spectrometry with a deuterium-labeled internal standard.
Authors: Herold D A , Anonick P K , Kinter M , Hayden F G
Published: January 2020

Rimantadine is a synthetic antiviral agent used in prophylaxis and in treating the early stages of uncomplicated influenza A illness. We describe a stable isotope-dilution assay involving capillary gas chromatography/mass spectrometry. We used 200 ng of d3-rimantadine, added to 1 mL of plasma, as the internal standard. The rimantadine was extracted from the plasma with a Bond-Elut CN column, the column was washed with water, and the rimantadine was eluted with methanol, dried, and treated to form the t-butyldimethylsilyl derivative. The mass spectrometer was operated in the selected ion monitoring mode. Ions at m/z 236 and m/z 239 were monitored, corresponding to the loss of C4H9 from the rimantadine derivative and d3-rimantadine, respectively. Within-run precision (CVs) ranged from 8.9% at 29 micrograms/L to 3.2% at 1666 micrograms/L. Corresponding data for between-run precision were 5.4% and 1.7%. Treated volunteers (n = 86) provided plasma samples with a concentration range of 153 to 1127 micrograms/L. This simplified method allows rapid, precise assay of rimantadine in plasma.
Simple, rapid determination of zinc and acid phosphatase in seminal plasma with an ABA-100 bichromatic analyzer.
Authors: Gavella M
Published: January 2020

I describe an automated assay for zinc and acid phosphatase in seminal plasma. These, which are markers of the function of the prostate, were assayed bichromatically with an Abbott ABA-100 analyzer. As many as 25 samples of human seminal plasma can be analyzed sequentially with CVs of 3.1% for zinc and 1.5% for acid phosphatase. The sensitivity, specificity, and speed of this assay system make it practicable for use in investigation of male infertility.
Age- and sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer.
Authors: Lockitch G , Halstead A C , Albersheim S , MacCallum C , Quigley G
Published: January 2020

Using the Ektachem-700 multilayer film analyzer, we defined age- and sex-specific reference intervals for 20 analytes in sera from a healthy population of neonates and children ages one to 19 years. Upper and lower normal reference intervals for each analyte were determined by nonparametric methods as the 0.975 and 0.025 fractiles, respectively. Newborns have lower concentrations of total protein and albumin, and higher concentrations of phosphate, bilirubin, and enzymes in serum than older children do. Concentrations of urea, glucose, calcium, phosphate, and bilirubin change rapidly postnatally. Outside the neonatal period, no significant age- or sex-related difference was found for plasma glucose, serum amylase, conjugated or unconjugated bilirubin, or lipase. There was no sex-related difference in reference intervals for albumin, total protein, calcium, phosphate, or urea. However, concentrations of uric acid and creatine kinase are much higher in postpubertal boys than in girls. Alkaline phosphatase values peak later in boys. Except for lactate dehydrogenase and gamma-glutamyltransferase, the reference intervals defined here do not differ strikingly from data derived with use of other analyzers. The age- and sex-related trends are independent of method. However, each laboratory should determine the degree to which these reference ranges can be directly applied to analyses performed with another analyzer.
Detection of protein binding abnormalities in euthyroid hyperthyroxinemia.
Authors: George P M , Sheat J M , Palmer B N
Published: January 2020

This is a procedure for rapidly identifying the three common abnormalities in binding of thyroxin by protein. After incubation with [125I]thyroxin, serum proteins are separated by electrophoresis on agarose gel and binding of thyroxin to the various protein fractions is determined after autoradiography. Quantitatively abnormal binding to albumin or prealbumin and thyroxin autoantibodies is easily demonstrated by this technique. Normally, less than 6% is bound to albumin, and no binding by prealbumin is detected. In dysalbuminemic hyperthyroxinemia, about 30% of the serum thyroxin is bound to albumin; in prealbumin-associated hyperthyroxinemia, 7% is bound to prealbumin. With this procedure these protein-binding abnormalities can be simply identified, and it may be useful when results of a thyroxin assay are not consistent with results of a sensitive thyrotropin assay or the patient's clinical examination.
Choriogonadotropin and its beta subunit separated by hydrophobic-interaction chromatography and quantified in serum during pregnancy by time-resolved immunofluorometric assays.
Authors: Alfthan H , Schröder J , Fraser R , Koskimies A , Halila H , Stenman U H
Published: January 2020

Concentrations of human choriogonadotropin (hCG) and its free beta subunit (beta hCG) were measured in serum by highly sensitive and specific time-resolved immunofluorometric assays (IFMAS). The results were confirmed by completely separating beta hCG and hCG by a novel method based on hydrophobic-interaction chromatography. We used three monoclonal antibodies in two different combinations. In both assays an antibody reacting with both free beta hCG and with intact hCG was immobilized onto the wall of a microtiter strip well. For assay of intact hCG we used as the indicator antibody an antibody against the alpha subunit, labeled with a europium chelate. For assay of beta hCG we used an indicator antibody that reacted only with the free beta subunit. hCG cross-reacted in the assay of beta hCG by 0.6%. Quantifying hCG in serum after in vitro fertilization showed that, seven to eight days after embryo transfer, the hCG concentration started to increase, thereafter increasing with a doubling time of 1.9 days during the following three weeks. hCG concentrations in serum peaked six to 10 weeks later, corresponding to eight to 12 weeks after the last menstrual period. Throughout pregnancy, measurable amounts of beta hCG were present in serum. The highest beta hCG/hCG ratio (maximum 7.3%, median 3.0%) was observed during early gestation. During the fourth to 13th weeks after the last menstrual period the ratio of beta hCG/hCG decreased gradually, being 1.0% during the second and third trimesters.
Nonisotopic "sandwich" immunoassay of thyroglobulin in serum by the biotin-streptavidin technique: evaluation and comparison with an immunoradiometric assay.
Authors: Preissner C M , Klee G G , Krco C J
Published: January 2020

In this sequential assay, the thyroglobulin in serum binds to polystyrene beads coated with two mouse monoclonal antibodies. These beads then react with a rabbit polyclonal antibody, biotinylated sheep anti-rabbit IgG, streptavidin-horseradish peroxidase, and a peroxidase substrate to yield a colored product that is measured spectrophotometrically at 492 nm. The range of the standard curve is 1.6 to 100 micrograms/L. The detection limit of the assay is 1.2 micrograms/L. The interassay coefficient of variation is 14.0% at 6.2 micrograms/L, 5.7% at 32.7 micrograms/L; the intra-assay CVs range from 7.8% to 14.8%. The reference intervals are 2.7 to 42.1 micrograms/L for euthyroid persons and less than or equal to 5 micrograms/L for athyreotic patients not on thyroxin replacement therapy. Some autoantibodies to thyroglobulin cause thyroglobulin values to be falsely low. The concentration of autoantibodies is not correlated with the analytical recovery of human thyroglobulin. The coefficient of correlation between the measurement of thyroglobulin by this assay and by an immunoradiometric assay was 0.969 for 186 autoantibody-negative samples, 0.963 for 37 autoantibody-positive samples.
New one-step enzyme immunoassay for free thyroxin.
Authors: Kunst A , Seidenschwarz E , Bürk H , Schauer S , Haug H , Lehmann P , Ehrhardt V
Published: January 2020

This enzyme immunoassay for free thyroxin (FT4) involves simultaneous incubation of sample and thyroxin conjugated to horseradish peroxidase (EC 1.11.1.7) in antibody-coated tubes at room temperature. The test can be performed in 90 min. It is not influenced by variations in concentrations of thyroxin-binding globulin (TBG), thyroxin-binding prealbumin, or albumin. With increased concentrations of non-esterified fatty acids, the measured FT4 equivalent concentrations increase immediately, indicating displacement of endogenous thyroxin from its binding sites. FT4 values obtained with this assay for 110 samples, including sera with low albumin and high TBG as well as sera of patients treated with heparin, agreed well with those measured by a two-step radioimmunoassay and with the T4/TBG ratio. The measurable range of FT4 with our assay is 2.5 to 65 ng/L. Intra-assay CVs range from 3.4% to 2.2%, interassay CVs from 6.6% to 2.6% at concentrations of 9 to 45 ng of FT4 equivalent per liter.
Solid-phase enzymoimmunoassay of estrone in serum.
Authors: Folan J , Gosling J P , Fottrell P F
Published: January 2020

This rapid solid-phase enzymoimmunoassay for estrone in serum or plasma is done on microtiter plates after the serum is extracted with diethyl ether. No chromatographic or centrifugation steps are involved. The detection limit of the assay is 380 fg per well (28 pmol/L). Intra- and interassay coefficients of variation for the assay of low, medium, and high concentrations of estrone in plasma were respectively 4.4, 9.3; 2.3, 9.1; and 2.0, 6.3 percent. There was a good correlation (correlation coefficient 0.95) between estrone concentrations measured with this assay and with a commercial radioimmunoassay. The analytical procedure is simple, and one person can assay 80 serum samples per working day. We conclude that the assay is very suitable for serum estrone measurements and is more convenient than published radioimmunoassays.
Ten electrophoretic methods compared with a selected method for quantifying lactate dehydrogenase isoenzymes in serum.
Authors: Moses G C , Ross M L , Henderson A R
Published: January 2020

Using the Selected Method of McKenzie and Henderson (Selected Methods Clin Chem 1983;10:59-67) as a reference method, we compared the performance of 10 commercially available methods for determination of lactate dehydrogenase (LD, EC 1.1.1.27) isoenzymes. Results were expressed as percentage of total LD activity, as determined with two different types of densitometers shown to have an average difference less than 1.4% for each isoenzyme. All methods gave generally comparable results, as judged by Bland-Altman plots and correlation analyses. However, in general, estimates by the commercial methods for LD-1, LD-2, and LD-3 were lower, and for LD-4 and LD-5 were higher than with the Selected Method. The overall CV was less than 20% for all methods and isoenzymes, except for LD-4 and LD-5 by the Beckman Paragon, Helena LD-VIS, Gel LDH, Gel PC, and Iso Dot, Gelman LDH Isozyme, and Sebia Hydragel assays, for which it was greater than 20%. Overall, accuracy was best with the Helena Iso Dot and LD-VIS assays, followed by the Corning LD Flur assay; accuracy was poorest with the Gelman LDH Isozyme, Sebia Hydragel, and Beckman Paragon assays.
Abbott's fluorescence polarization immunoassay for cyclosporine and metabolites compared with the Sandoz "Sandimmune" RIA.
Authors: Sanghvi A , Diven W , Seltman H , Starzl T
Published: January 2020

A new procedure for measuring cyclosporine in plasma has been introduced by Abbott Laboratories, involving their TDx instrumentation and fluorescence polarization immunoassay. Radioimmunoassay (RIA) and high-performance liquid chromatography are currently the conventional methods for measuring cyclosporine in plasma and whole blood. In an effort to find a method that will decrease the radioactive hazard, the reagent and supply cost, and the labor requirements associated with RIA procedures, we used specimens from transplantation patients to compare the Abbott assay with the Sandoz Sandimmune assay. We believe that the Abbott assay offers some advantages over the Sandimmune RIA procedure, providing a reliable but simpler and less hazardous technology.
Direct and indirect techniques for free thyroxin compared in patients with nonthyroidal illness. I. Effect of free fatty acids.
Authors: Csako G , Zweig M H , Glickman J , Kestner J , Ruddel M
Published: January 2020

We examined the effect of endogenous free fatty acids (FFA) on the measurement of free thyroxin (FT4) by five different methodologies represented in 16 different assays in a large number of patients with nonthyroidal illness (NTI). Some, but not all, one-step (analog) FT4 RIAs negatively correlated with FFA concentration. All two-step FT4 RIAs, equilibrium dialysis FT4, and the dialyzable (free) fraction of T4 positively correlated. In contrast, a binding-rate-based FT4 RIA, FT4 indices based on T3 macroaggregated albumin uptake, and T4/TBG ratios did not correlate. We also analyzed the FT4-FFA relationship with a second, more sensitive approach by correlating test results with FFA/albumin molar ratio as an estimate of the "excess" (nonalbumin bound) FFA. We found that all FT4 RIAs, equilibrium dialysis FT4, FT4 indices based on T3 uptake, the dialyzable fraction of labeled T4 in equilibrium dialysis, the fraction of labeled T4 bound to solid phase antibody in the binding-rate-based RIA, and T3 uptake correlated with the FFA/albumin molar ratio. This FFA dependency was comparable among all the various techniques and was relatively small. Thus, increases or decreases in FT4 results due to varying FFA (and albumin) concentrations are highly likely with most currently available methods (only the T4/TBG ratio did not reveal FFA-dependency), but the magnitude of changes varies with the "excess" FFA.
Four nonisotopic immunoassays of free thyroxin evaluated.
Authors: Perdrisot R , Bounaud M P , Bounaud J Y , Jallet P
Published: January 2020

We evaluated four new nonisotopic immunoassays of free thyroxin (FT4)--Amerlite FT4 (Amersham International), Magic Lite FT4 (Ciba Corning Diagnostics), Stratus FT4 (Dade--Baxter Travenol), and FT4 Enzelsa (Compagnie ORIS Industrie)--by comparison with two FT4 radioimmunoassays: Amerlex and Sclavo. Inter- and intra-assay coefficients of variation were less than 10% in the working range and there was no significant interaction between the tracer conjugate and albumin. All methods except Enzelsa gave results equivalent to those obtained with the Sclavo chromatographic technique. In a population consisting of 325 euthyroid subjects and 111 hyperthyroid and 61 hypothyroid patients without any treatment, we observed slight overlaps between the hyperthyroid and euthyroid populations and diagnostic sensitivities were approximately 0.95 and 0.80 for hyperthyroidism and hypothyroidism. In specific populations (69 patients with chronic renal failure, 20 patients in intensive-care, 17 patients treated with heparin, and 27 pregnant women), results were quantitatively similar to those obtained by the Sclavo assay. We conclude that these nonisotopic methods are a valid alternative to current radioisotopic commercial methods.
Measurement of cyclosporine concentrations in whole blood: HPLC and radioimmunoassay with a specific monoclonal antibody and 3H- or 125I-labeled ligand compared.
Authors: Wolf B A , Daft M C , Koenig J W , Flye M W , Turk J W , Scott M G
Published: January 2020

We compared cyclosporine concentrations in whole blood as measured by HPLC and by RIA with a monoclonal antibody specific for cyclosporine with 3H- or 125I-labeled cyclosporine ligand. The 3H-RIA kit slightly underestimated cyclosporine concentrations (greater than 600 micrograms/L) in comparison with HPLC. Over a wide range of concentrations, cyclosporine measured with the 125I-RIA kit correlated well with HPLC (slope = 0.99, n = 301, r = 0.98), observed for samples from recipients of kidney, heart, or liver allografts (respective slopes: 1.01, 0.93, and 1.00). The 125I-RIA standard curve was linear to 1000 micrograms of cyclosporine per liter. Inter- and intra-assay CVs for 125I-RIA measurements of cyclosporine were less than or equal to 7%. Evidently, the 125I-RIA kit involving a monoclonal antibody specific for cyclosporine is equivalent to the HPLC assay and can replace it for therapeutic drug monitoring of cyclosporine therapy.
Evaluation of the IFCC reference method for alanine aminotransferase: spurious blank ALT activity due to contamination of D-alanine with L-alanine, and recommendations for a correction.
Authors: Okorodudu A O , Pelletier P R , Valcour A A , Bowers G N , McComb R B
Published: January 2020

During an evaluation of the IFCC reference method for alanine aminotransferase (ALT, EC 2.6.1.2), we noted that the specimen blank activity reaction was markedly increased. Experience with five different lots of D-alanine from four commercial sources indicated that substantial and varying negative bias (up to -10%) could be introduced into the blank-corrected ALT activity, depending on the lot of D-alanine used. Although the IFCC procedure for ALT mentions the possibility of this L-alanine contamination, we believe that the degree of contamination in commercial reagents is underestimated. Analyzing the five lots of D-alanine for L-alanine, we found the magnitude of negative bias to be correlated directly with L-alanine contamination. Here, we describe a quick, sensitive assay based on coupled reactions of L-amino acid oxidase/peroxidase for quantifying L-alanine in the concentration range of 0-15 mmol/L without a sample-dilution step. Results by this alternative L-alanine assay agreed well with those recommended in the IFCC ALT procedure. Further examination suggested an even simpler solution to the L-alanine contamination problem, because we found no difference in the blank-corrected ALT activity determined in Tris HCl buffer, with or without D-alanine (free of L-alanine). We therefore propose that D-alanine be omitted from the IFCC reference ALT procedure.
Tumoral calcinosis: seasonal biochemical studies and chemical studies of eyelid lesion.
Authors: Bruns D E , Boskey A L , Lieb W , Conway B P , Savory J , Wills M R
Published: January 2020

We recently described (Arch Ophthalmol 1988; 106:725-6) the presence of unique calcific lesions in the eyelids of a young woman with a history of hyperphosphatemic tumoral calcinosis. Here we document that no immediate family members showed similar lesions and that none was hyperphosphatemic. Dental roentgenography revealed characteristic abnormalities in the patient that confirmed the clinical diagnosis of tumoral calcinosis. Seasonal biochemical studies demonstrated persistently increased concentrations of phosphorus and 1,25-dihydroxyvitamin D in her serum. A calcific eyelid excrescence removed from the patient, studied by x-ray diffraction, was found to consist of crystals of hydroxyapatite. Microprobe analysis indicated the major elements in the deposit to be Ca, P, S, and Cl, just as in the periarticular deposits found in tumoral calcinosis. The Ca concentration in the patient's tear fluid, measured by atomic absorption spectrometry, was within the range found in tears of healthy volunteers. Phosphorus was undetectable (less than 30 mumol/L) in tears of the patient and the volunteers. These findings suggest that the eyelid lesions represent a new manifestation of the pathological process that produces the characteristic periarticular calcific masses of tumoral calcinosis.
Increased concentrations of heparin cofactor II in diabetic patients, and possible effects on thrombin inhibition assay of antithrombin III.
Authors: Gram J , Jespersen J
Published: January 2020

We compared concentrations of antithrombin III (AT-III) in plasma, as determined by an immunological method and by a functional thrombin inhibition method, in the presence of heparin in 160 blood samples from Type I diabetics. Although the correlation was highly significant (P less than 0.001) between the results obtained by the two methods, our data demonstrated that results by the thrombin inhibition assay, 121 (SD 15)%, expressed as percentages of the results for a normal plasma pool, were significantly (P less than 0.001) higher than by the immunoreactive method, 104 (SD 15)%, indicating an overestimation of functionally active AT-III. Concentrations of functionally active AT-III determined by a factor Xa inhibition assay, 105 (SD 13)%, were in the same range as immunoreactive AT-III. Addition of IgG antiserum to normal pooled plasma quenched only about 90% of the AT-III activity determined by the thrombin inhibition assay, but all of the AT-III activity determined by a factor Xa inhibition assay. These results demonstrate that the factor Xa inhibition assay is more specific for the determination of AT-III than the thrombin inhibition assay. We suggest that the high concentrations of heparin cofactor II, 117 (SD 17)%, might have caused an overestimation of AT III in this group of patients with diabetes Type I, and should not be overlooked in other clinical situations.
Automated determination of cholinesterase activity in plasma and erythrocytes by flow-injection analysis, and application to identify subjects sensitive to succinylcholine.
Authors: Lainé-Cessac P , Turcant A , Allain P
Published: January 2020

This automated spectrophotometric method for determination of cholinesterase activity in erythrocytes and plasma is based on measurement of the choline produced at 30 degrees C by the hydrolysis of acetyl-, butyryl-, or succinylcholine. Blanks, standards, and samples are prepared by a Gilson robotic unit. Use of flow-injection analysis for detection allows use of smaller volumes of reagent and sample. We applied this method to the study of 91 healthy subjects and members of two families with succinylcholine sensitivity. Results with use of the three different substrates for determination of activity in plasma correlated well (r greater than 0.94). Results for plasma and erythrocytes from healthy subjects are lower in women less than 50 years old than in women greater than 50 years or men. Values for plasma obtained with succinylcholine substrate (range: 31 to 100 U/L) allow detection of very sensitive subjects--AA phenotypes (less than 10 U/L)--but do not distinguish the UA from the UU phenotype.
Two-site immunoenzymometric assay for the 52-kDa cathepsin D in cytosols of breast cancer tissues.
Authors: Rogier H , Freiss G , Besse M G , Cavalié-Barthez G , Garcia M , Pau B , Rochefort H , Paolucci F
Published: January 2020

We developed a solid-phase two-site immunoenzymometric assay (IEMA) of the estrogen-induced 52-kDa cathepsin D (EC 3.4.23.5) and its processed forms (48-kDa and 34-kDa proteins) in cytosols of breast cancer tissues, using two monoclonal antibodies directed against two different epitopes of these antigens. The first antibody is bound to a polystyrene microtiter well; the second is labeled with alkaline phosphatase. The assay involves a simultaneous incubation of the antigen with both antibodies, because we observed signal loss during sequential incubations. Alkaline phosphatase was chosen because other enzymes (peroxidase, beta-galactosidase) were inhibited by cytosol extraction buffers. The measurable range of 52-kDa-related proteins is from 0.3 to 6 nmol/L with precision (CVs) within and between runs of 3.9% and 15.8%, respectively. The sensitivity, accuracy, and rapid turnaround time of the two-site IEMA should facilitate the clinical evaluation of this new marker in oncology.
Critical analytical and clinical aspects of ionized calcium in neonates.
Authors: Wandrup J
Published: January 2020

Clinical diagnosis in neonates is often based primarily on biochemical data, because clinical symptoms are difficult to assess and inconclusive. A knowledge of the validity of the data would therefore seem very important. Practical technical assessments lead me to conclude that measurement of ionized calcium at actual pH in anaerobic capillary samples of whole blood, currently practicable, is an easy and optimal laboratory analysis when biologically significant data are required for assessment of acute disturbances in calcium homeostasis of neonates. Values are given for ionized calcium, describing the relations in cord and maternal blood in uncomplicated pregnancies at term. Recent published cross-sectional and longitudinal reference values for ionized calcium, measured during the first weeks in healthy full-term and preterm neonates, seem to provide a new basis for the diagnosis and management of early neonatal hypocalcemia.
Simple enzymatic method for determining 1,5-anhydro-D-glucitol in plasma for diagnosis of diabetes mellitus.
Authors: Yabuuchi M , Masuda M , Katoh K , Nakamura T , Akanuma H
Published: January 2020

We have developed a simple method for determination of 1,5-anhydro-D-glucitol in plasma, based on use of pyranose oxidase (EC 1.1.3.10), an enzyme with specificity toward pyranoid compounds such as 1,5-anhydro-D-glucitol and glucose. Plasma samples deproteinized with trichloroacetic acid are passed through a two-layer mini-column packed with strongly basic anion (OH- form, the upper layer) and strongly acidic cation (H+ form, the lower layer) exchange resins. 1,5-Anhydro-D-glucitol is efficiently recovered in the flow-through fraction, which is almost devoid of other sugars that are sensitive to pyranose oxidase. The hydrogen peroxide formed in the enzymatic oxidation of 1,5-anhydro-D-glucitol is detected by a standard method utilizing an enzymatic color-developing system. The overall assay system is highly specific for 1,5-anhydro-D-glucitol. The correlation between results obtained in the present method (x) and in the gas-liquid chromatographic (GLC) method (y) was: y = 1.062x-0.293 mg/L (r = 0.997, n = 49, Sxy = 10.78 mg/L). Compared with GLC, our method is simpler in the sample treatment step and quicker in the measuring step. The precisions of the two methods are comparable.
Distribution of thiobarbituric acid-reactive substances in lipoproteins and proteins in serum.
Authors: Bonnefont D , Legrand A , Peynet J , Emerit J , Delattre J , Galli A
Published: January 2020

We assessed the distribution of malondialdehyde (MDA) in lipoproteins and proteins in serum after using two procedures to separate the lipoproteins: sequential ultracentrifugation or selective precipitation with a sodium phosphotungstate and magnesium chloride reagent followed by ultracentrifugation of the supernate. MDA concentrations were determined by the thiobarbituric acid reaction and quantified by fluorometry. We found that 43% of the thiobarbituric acid-reactive substances (TBARS) was bound to the lipoproteins--27% to very-low- and low-density lipoproteins (VLDL-LDL) and 16% to high-density lipoproteins (HDL)--and from 11.5% to 15.8% to proteins, depending on the separation procedure. Residual unbound TBARS were located in the ultracentrifugation layers that contained no lipoproteins or proteins. The TBARS concentration in serum lipoproteins containing apolipoprotein B (i.e., VLDL-LDL) was the same after ultracentrifugation or selective precipitation. We therefore consider the precipitation method more suitable for routine TBARS determination in these lipoproteins, because it is easier to handle and faster. However, for determination of TBARS in HDL, selective precipitation requires subsequent ultracentrifugation at a density of 1.21 kg/L.
An interlaboratory comparison of control materials for use with hematofluorometers.
Authors: Parsons P J , Stanton N V , Gunter E W , Huff D , Meola J R , Reilly A A
Published: January 2020

This interlaboratory study was conducted to examine four erythrocyte protoporphyrin control materials from Aviv Biomedical, Helena Laboratories, Kaulson Laboratories, and the New York State Department of Health for use with hematofluorometers. Our principal aims were to monitor the stability of these materials at three different storage temperatures (room, refrigerator, freezer) and, where appropriate, to validate the manufacturer's target values. Measurements for the study were generated in three reference laboratories that used a total of five hematofluorometers, three from Environmental Science Associates and two from Aviv Biomedical. Each instrument was calibrated against a consensus acetic acid-ethyl acetate extraction procedure. We found the materials from Aviv to be the most stable, followed by the New York State material. However, the target values assigned by Aviv were not within the acceptable range determined by consensus. The target values assigned by Kaulson Laboratories for their materials did fall within the acceptable consensus range, but they were the least stable of the materials evaluated. The materials from Helena Laboratories were originally designed for use as calibrators with Helena's "ProtoFluor Z" hematofluorometer, which reports in different units. They were deemed unsuitable for use as control materials with the Aviv or Environmental Science Associates hematofluorometers because of the narrow range of values and the wide scatter of results.