bluuebunny/crossref_metadata_2025 huggingface dataset:
A new automated turbidimetric immunoassay for quantifying alpha 1-antitrypsin in serum.
Authors: Viedma J A , de la Iglesia A , Parera M , López M T
Published: January 2020

This rapid, sensitive equilibrium turbidimetric immunoassay for quantification of alpha 1-antitrypsin involves a monospecific antibody, polyethylene glycol 6000 to accelerate and enhance the immunoprecipitation reaction, and Tween 20 surfactant to decrease and stabilize the sample-blank values. Turbidity at 334 nm is measured by an automated discrete analyzer. Grossly lipemic, icteric, or hemolyzed samples can be assayed. Correlation with results by radial immunodiffusion (RID) was excellent (r = 0.97, n = 84). Analytical recovery averaged 97.7 (SD 2.9)%. Within-run CVs ranged from 1.6 to 1.9%, between-day CVs from 2.0 to 3.5%. Reference values for healthy adults (n = 147) were determined by parametric estimation (for an assumed normal distribution of untransformed data). The lower limit (g/L) with its 0.90 confidence interval is 1.23 (range 1.18-1.28), the upper limit is 2.15 (2.10-2.20), and the mean is 1.69 g/L.
Liquid-chromatographic determination of urinary pentachlorophenol.
Authors: Chou P P , Bailey J L
Published: January 2020

We developed and evaluated a simple liquid-chromatographic method for urinary pentachlorophenol (PCIP) determination with ultraviolet detection. PCIP and the internal standard (2,3,5,6-tetrachlorophenol, TCP) were extracted with a solid-phase extraction column, and 50 microL of the collected eluate was injected into the chromatograph. The intra-assay and interassay CVs were 6.6% (n = 20) and 7.9% (n = 10), respectively. The analytical recoveries varied from 89% to 96%, with absolute recoveries ranging from 82% to 88%. Thirty urine specimens collected from 30 unexposed individuals were analyzed by the current method, and results were all less than the minimum detection limit, 0.25 mg/L. Results for five specimens from workers who had been exposed to PCIP ranged from 7.0 to 12.4 mg/L. The standard curve was linear to 300 mg/L. This procedure is adaptable to automated analysis, and therefore suitable for monitoring industrial exposure of workers.
Increased lactate dehydrogenase isoenzyme 1 in serum and tumor tissue of a patient with small-cell carcinoma.
Authors: Christenson R H , Scroggs M W , Odom J D
Published: January 2020

Histological examination of supraclavicular lymph node tissue obtained at biopsy from a 63-year-old man disclosed metastatic small-cell carcinoma. On admission and for four days subsequently, total lactate dehydrogenase (LD; EC 1.1.1.27) activity in serum was 6.5 times normal; studies of LD isoenzyme showed persistently increased LD-1, with LD-1 greater than LD-2. Isoenzyme electrophoresis of tissue homogenates prepared from the patient's tumor also showed the LD-1 greater than LD-2 pattern. Isoenzyme studies for supraclavicular lymph node tissue from five control subjects showed contrasting isoenzyme patterns as compared with the patients in whom LD-2, LD-3, and LD-4 predominated. Because these abnormalities were persistent, they differ from the temporal sequence for LD usually seen in myocardial infarction. This emphasizes the importance of repetitive sampling for clinical interpretation of data on this enzyme.
A simple, direct radioimmunoassay for salivary progesterone determination during the menstrual cycle.
Authors: Bourque J , Sulon J , Demey-Ponsart E , Sodoyez J C , Gaspard U
Published: January 2020

We describe a direct radioimmunoassay for progesterone in saliva. Results for extracted and unextracted samples agree well, showing that extraction with petroleum ether is unnecessary. The direct assay is specific and accurate, and detects as little as 2 pg of progesterone per tube (12 pmol/L). Intra- and inter-assay CVs are less than 10%. The correlation between concentrations of progesterone in saliva and plasma is good during the luteal phase of the menstrual cycle (r = 0.78, p less than 0.001, n = 76) but not during the follicular phase. We present mean concentrations of progesterone in saliva and plasma for the whole cycle in 14 normal women. Although citric acid is an effective salivary-flow stimulant, its deleterious effect on the direct radioimmunoassay precludes its use with this assay for monitoring ovulation.
Immunoturbidimetric assays of apolipoproteins A, AI, AII, and B in serum.
Authors: Rifai N , King M E
Published: January 2020

In these immunoturbidimetric assays developed for apolipoproteins A, AI, AII, and B we used the Cobas-Bio centrifugal analyzer and commercially available antisera and calibrators. Within-day and within-run precision ranged from 1.0 to 5.8% for the four assays. The addition of bilirubin, up to 150 mg/L, or hemoglobin, 50 g/L, to sera did not interfere with the measurement of these apolipoproteins. Comparison of results of the immunoturbidimetric assays with those of radial immunodiffusion assays yielded slopes of 1.002, 0.822, and 0.957 and intercepts of -0.02, 0.47, and 0.03 g/L for apolipoproteins B, A, and AI, respectively. Reference intervals for the four apolipoproteins were determined by using sera from 193 apparently healthy individuals.
Rapid measurement of serum water to assess pseudohyponatremia.
Authors: Faye S , Payne R B
Published: January 2020

Pseudohyponatremia is caused by an increased serum protein or lipid concentration producing a "space-occupying lesion" in serum water. Its presence and magnitude must be assessed in hyponatremic patients with, for example, paraproteinemia or hyperlipemic diabetic coma. In the absence of a direct-reading ion-selective electrode system, a method for measuring the water content of serum is required. We describe two rapid methods for measuring the diffusible water of serum: osmometry before and after dilution and chloride measurement before and after ultrafiltration. Either of these methods allows the true sodium status of a patient's serum to be determined.
A rapid enzymatic procedure for "fingerprinting" bacteria by using pattern recognition of two-dimensional fluorescence data.
Authors: Pau C P , Patonay G , Moss C W , Carlone G M , Rossi T M , Warner I M
Published: January 2020

We describe a rapid fluorescence technique for bacterial "fingerprinting," based on the differences in enzyme content and activity of various bacteria. Bacterial cells are incubated with a mixture of carefully chosen fluorogenic enzyme substrates to produce fluorophores with unique emission and excitation properties. The resulting two-dimensional fluorescence spectrum of the product mixture produces a pattern characteristic of the bacterium. A Fourier-transform-based pattern recognition algorithm is used for spectral matching and for differentiating visually similar spectra. This sensitive technique is potentially applicable to differentiation and identification of bacteria in clinical laboratories.
Relative contribution of various expressions of cAMP excretion to other indices of parathyroid function, as tested by discriminant multivariate linear regression analysis.
Authors: Valsamis J , Van Peborgh J , Brauman H
Published: January 2020

We evaluated the relative contribution to the diagnosis of hyperparathyroid disease from current laboratory indices of parathyroid function--plasma calcium (I), phosphate (II), carboxy-terminal (III) and predominantly amino-terminal (IV) radioimmunoassays of parathyrin, the urinary excretion ratios of cyclic adenosine monophosphate (cAMP) to creatinine (V) or to glomerular filtrate (VI), and the ratio of the nephrogenous fraction of cAMP to glomerular filtrate (VII)--in 224 subjects: 40 with surgically proven hyperparathyroid disease, the others normoparathyroid. The decreasing order of sensitivity was I greater than VI greater than VII greater than V greater than III greater than IV greater than II; all these indices differed significantly between normoparathyroid and hyperparathyroid patients. The decreasing order of specificity was VII, III greater than I greater than IV greater than V, II greater than VI. Discriminant multivariate linear regression analysis was performed in a subset of 58 subjects (17 hyper- and 41 normoparathyroid) from the population studied here, chosen because all of the laboratory indices were determined for each subject. The classification accuracy was 98.3% for combining I, VII, and III (r = 0.908), or I and V (r = 0.893), or I and VII (r = 0.889). The other variables did not add to the precision of classification.
Detection of apolipoprotein E variants by an oligonucleotide "melting" procedure.
Authors: Funke H , Rust S , Assmann G
Published: January 2020

An oligonucleotide "melting" procedure was developed whereby we can monitor for a sequence heterogeneity in the gene for apolipoprotein E production. Two oligonucleotides were synthesized, one recognizing the common epsilon 3 allele but not the common epsilon 2 allele, the other recognizing the epsilon 2 allele but not the epsilon 3 allele. Samples from 15 subjects with different apolipoprotein E phenotypes as classified by isoelectric focusing were analyzed by this method for the presence of an Arg----Cys substitution in position 158 of the apolipoprotein E amino acid sequence. In 14 of these subjects the genotype determined by oligonucleotide melting agreed with the phenotype identified by isoelectric focusing. In one patient, however, whose phenotype was E2/2 by isoelectric focusing, the DNA hybridized with both oligonucleotides. We conclude that this patient has a mutation in one of his alleles for apolipoprotein E that differs from the frequently seen Arg----Cys change. The apolipoprotein E gene may thus be more heterogeneous than previously anticipated.
Laser-excited immunofluorometric assay of prolactin, with use of antibodies coupled to lanthanide-labeled diethylenetriaminepentaacetic acid.
Authors: Déchaud H , Bador R , Claustrat F , Desuzinges C
Published: January 2020

We describe an immunofluorometric assay for prolactin based on lanthanide labeling of a monoclonal antibody and measuring time-resolved fluorescence. In this "sandwich"-type assay, the label (Eu3+) was bound to the second antibody by means of a simple, rapid method involving the anhydride of diethylenetriaminepentaacetic acid. To measure the photoluminescence of europium (or other lanthanides), we have developed a time-resolved fluorometer with a nitrogen laser as the pulsed excitation source. During the assay, the solid-phase antibody immobilized inside a polystyrene tube is incubated with the plasma sample and the second antibody in a one-step procedure. Results for 67 human plasmas correlated well (r = 0.98) with those by an immunoradiometric method.
Rapid enzymatic pretreatment of samples before determining chromium in serum or plasma.
Authors: Offenbacher E G , Dowling H J , Rinko C J , Pi-Sunyer F X
Published: January 2020

A simplified method for preparing blood serum and plasma for Zeeman electrothermal atomic absorption spectrometry is described and applied to the measurement of chromium in human serum and plasma. This enzymatic degradation with bacterial protease (EC 3.4.21.3) requires little laboratory apparatus, decreases the work and time of sample preparation, and obviates some potential sources of contamination. We used bovine reference serum (USDA No. 7292) to validate Cr concentration. There was less Cr in serum than in plasma, whether sodium heparin or sodium citrate was used as anticoagulant. For six human subjects, Cr in serum averaged 0.15 (SD 0.02) micrograms/L, 0.26 (SD 0.03) micrograms/L in heparinized plasma, and 0.28 (SD 0.02) micrograms/L in citrated plasma. We postulate that the Cr concentration is lower in serum because some of the Cr binds to proteins complexed with the clot in the coagulation process.
Use of a quality-control plasma sample to decrease interassay variation in radioimmunoassays of apolipoprotein A-I.
Authors: Au Y P , Hallaway B J , Kottke B A
Published: January 2020

The apolipoprotein A-I (apo A-I) radioimmunoassay established in our laboratory involves use of purified apo A-I as the primary standard for quantifying apo A-I in plasma and a pooled plasma (quality-control sample) as a secondary standard to decrease interassay variation. The measured values for apo A-I increased over time as the labeled antigen degraded. We observed these results in two separate studies: (a) apo A-I in plasma from 16 subjects was measured 12 times during four months, and (b) apo A-I in a single pooled plasma was measured 48 times during 10 months. We show that use of a quality-control plasma sample as a secondary standard decreased interassay variation, which was, in part, ascribable to degradation of the labeled antigen.
Transient hyperphosphatasemia in a child with autosomal recessive vitamin D dependency.
Authors: Cole D E
Published: January 2020

The case of a 4.5-year-old girl with autosomal recessive vitamin D dependency is described. Although she had been effectively treated since one month postpartum with 1 alpha-hydroxycholecalciferol [1 alpha(OH)D3, alfacalcidol], her mean alkaline phosphatase (EC 3.1.3.1) activity in serum increased to 3680 U/L from a stable value [335 (SD 50) U/L; n = 12] within three weeks, then returned to baseline over the ensuing four months. Transient hyperphosphatasemia was diagnosed. Extensive investigation of an isolated episodic increase in alkaline phosphatase activity is as superfluous in the child with adequately treated metabolic bone disease as it is in other healthy and asymptomatic children.
Kinetic fluorometric determination of aluminum in serum.
Authors: Ioannou P C , Piperaki E A
Published: January 2020

We describe a simple fluorometric method for determining aluminum in serum samples by monitoring the rate of reaction of 2-hydroxy-1-naphthaldehyde-p-methoxybenzoylhydrazone with aluminum ions. The emission of the resulting fluorescent metal-chelate formed is measured at 475 nm. Aluminum was measured in the supernate of serum after proteins were removed by precipitation with concentrated nitric acid, and calculations were based on the technique of standard additions. Within-run precision (CV) was 7.8% and 4.8% at mean aluminum concentrations of 7.7 and 60.7 micrograms/L, respectively (n = 10); between-run precision (CV) was 8.9% and 5.7% at mean aluminum concentrations of 23.3 and 46.8 micrograms/L, respectively (n = 10). The standard curve for the method is linear over the range of 0-250 micrograms of aluminum per liter. Samples from 49 patients were analyzed for aluminum by the proposed method (y) and by electrothermal atomic absorption spectroscopy (x). Linear regression analysis of the results yielded the equation y = 0.98x + 2.3 (r = 0.989, Syx = 6.7). The proposed method is comparable in sensitivity to the well-accepted atomic absorption spectrometric method but is simpler and less expensive.
Methods for eliminating interferences in digoxin immunoassays caused by digoxin-like factors.
Authors: Graves S W , Sharma K , Chandler A B
Published: January 2020

Endogenous, circulating digoxin-like immunoreactive factors (DLIF) are known to cross react with many antisera used in digoxin assays, complicating the quantification of serum digoxin. We have explored ways to decrease or remove the interference from DLIF in such assays. Prolonging the assay incubation from 30 to 60 min decreased apparent digoxin concentrations (attributable to DLIF) by an average of 68% in the digoxin radioimmunoassays studied. The serum protein-binding of DLIF was also exploited to remove them from serum. Ultrafiltration, performed as part of a simple centrifugation step, removed approximately 90% of the DLIF. Analytical-recovery studies with true digoxin also demonstrated ultrafiltration to be an adequate method (greater than 95% of digoxin was recovered) for routine clinical use. Heat- or acid-precipitation of protein removed DLIF less effectively. Appropriate incubation times and ultrafiltration can dramatically minimize or eliminate DLIF interference in digoxin immunoassays.
Paraprotein interference with colorimetry of phosphate in serum of some patients with multiple myeloma.
Authors: Sonnenblick M , Eylath U , Brisk R , Eldad C , Hershko C
Published: January 2020

In untreated serum of three patients with multiple myeloma, concentrations of inorganic phosphate ranged from 130 to 270 mg/L as measured with a chromogenic assay based on the interaction of phosphate ion with ammonium molybdate in the presence of ferrous sulfate. There were no clinical features of hyperphosphatemia, and values for total calcium concentration in serum remained within normal limits throughout. Subsequent investigations demonstrated that this hyperphosphatemia was spurious and was caused by high concentrations of the paraprotein interfering with the chromogenic assay. Because this type of assay, adapted for automated systems, is now widely used in clinical laboratories, we call attention to this limitation to avoid confusion in clinical evaluations of patients with multiple myeloma.
Urinary protein as measured with a pyrogallol red-molybdate complex, manually and in a Hitachi 726 automated analyzer.
Authors: Watanabe N , Kamei S , Ohkubo A , Yamanaka M , Ohsawa S , Makino K , Tokuda K
Published: January 2020

In this new method for determining urinary protein, the reaction is complete within 10 min at 37 degrees C. This method is applicable to automated as well as manual measurements. Protein concentration and absorbance at 600 nm are linearly related throughout a wide range of concentrations, 10 to 16 000 mg/L. However, the chromogenicity of the gamma-globulins in this method is 70% of that of albumin, as estimated from results by a biuret method. Within-run CVs were less than 3.3%; the day-to-day CV was 2.9%. Errors due to interfering components in urine are less than 2%. The normal range for urinary protein as measured by this method was from 28 to 141 mg/day. Results by this method (y) and by a trichloroacetic acid-biuret method (x) correlated well (n = 80, r = 0.995; y = 0.99x - 2.9).
Decreased activity of carnosinase in serum of patients with chronic liver disorders.
Authors: Bando K , Ichihara K , Toyoshima H , Shimotuji T , Koda K , Hayashi C , Miyai K
Published: January 2020

We measured the activity of carnosinase, a prominent hepatic peptidase, in sera from 69 patients with liver disorders. Mean values (and SDs) for those with liver cirrhosis (17 cases) and hepatoma (seven cases) were 0.51 (0.28) and 0.68 (0.21) mumol/mL per hour, respectively--clearly less than for normal adults: 4.19 (0.95) mumol/mL per hour. Samples from 17 cases of chronic hepatitis also showed moderately decreased activity, 1.41 (0.97) mumol/mL per hour. In contrast, 14 cases of acute hepatitis generally showed values falling within the normal limits: 3.41 (1.97) mumol/mL per hour. Our results for carnosinase correlated with those for cholinesterase (r = 0.70) and with the concentration of albumin in serum (r = 0.59), but not with the activity of either creatine kinase, aspartate aminotransferase, or alanine aminotransferase in serum. Carnosinase values differed more among groups of disorders than did the values for cholinesterase or albumin. Measurement of serum carnosinase activity may be of clinical value in assessing the severity of chronic liver-cell damage, but not in differentiating liver disease from nutritional, muscle, or endocrine disorders.
Lactate dehydrogenase isoenzymes in serum during unstable angina.
Authors: Rotenberg Z , Weinberger I , Sagie A , Fuchs J , Sperling O , Agmon J
Published: January 2020

Values for total lactate dehydrogenase (LD, EC 1.1.1.27) activity in serum, LD isoenzymes 1 and 2, and the LD 1:2 ratio in 25 patients with unstable angina were compared with the same variables in 25 patients whose angina was stable 24, 48, and 72 h after admission. Mean total LD activity and mean LD-2 activity were found to be within the normal range, both in the unstable angina and stable angina groups of patients. In the unstable angina group the mean LD-1 was significantly higher (p less than 0.01) than in the stable angina group at each time studied. The mean LD 1:2 ratio was also significantly different (p less than 0.001) between the two groups of patients. In the unstable angina group of patients the ratio was increased (0.85, SD 0.09), as in patients with acute myocardial infarction, whereas in the stable angina group of patients the ratio was normal (0.60, SD 0.06). We conclude that a high LD 1:2 ratio, even in the presence of normal total LD activity, may indicate myocardial damage in some patients with unstable angina and could therefore help in the clinical and functional evaluation of patients with unstable angina.
Substrate-leash amplification with ribonuclease S-peptide and S-protein.
Authors: Ehrat M , Cecchini D J , Giese R W
Published: January 2020

The S-peptide and S-protein fragments of ribonuclease S (RNase S, no EC no. assigned) have been immobilized onto separate Sepharose gels via a "leash" of polycytidylic acid substrate. Each of these gels releases its RNase fragment when treated with the complementary enzyme fragment or with RNase A (EC 3.1.27.5), and the released fragments recombine to give RNase S activity. Thus this system provides substrate-leash amplification (SLA), such that more enzymatic activity is eluted from the system than is applied. For example, 100 pg of RNase applied to the S-peptide gel is amplified by 1.9 X 10(4) to the equivalent of 1.9 micrograms of activity in 20 h, when followed by combination of the released S-peptide with excess S-protein. We also tested a three-stage amplification system, with a pair of S-peptide and S-protein gels at each stage. In this system the cumulative amplification of the initial 1-ng dose of RNase A is 4.9, 52, and 25-fold after each stage, respectively. Only 2 mg of each SLA gel is used per stage in these experiments, reflecting the magnitude of their production of RNase S activity.
Strand displacement applied to assays with nucleic acid probes.
Authors: Ellwood M S , Collins M , Fritsch E F , Williams J I , Diamond S E , Brewen J G
Published: January 2020

This novel method for the detection of specific nucleic acid sequences has potential applications to clinical diagnosis. During hybridization, a signal-bearing nucleic acid strand is displaced by the target nucleic acid from a partially single-stranded complementary probe strand of nucleic acid. The probe:signal strand complex is prepared by hybridizing single-stranded probe that is entirely complementary to the target nucleic acid with a shorter signal sequence that is complementary to a portion of the probe strand. The sample nucleic acid is added to this hybrid complex under hybridization conditions. The target sequence, if present in the sample, will hybridize first to the unoccupied probe sequences, and then will displace the labeled strand by branch migration. By this "strand displacement" the signal strands are freed in solution, where they may be separated from those still hybridized; the quantity of label measured is directly proportional to the amount of analyte sequences in the sample. This method, demonstrated here for model and synthetic DNAs, can easily be adapted for the detection of any RNA or DNA sequence and obviates the need for immobilization of sample. A wide variety of labeling techniques can be used, and the displacement can be performed in solution or with the hybrid complex attached to a solid support. This assay circumvents nonspecific binding of label to the filter matrix and the laborious washing steps inherent in other assays involving nucleic acid probes.
The "intelligent workstation" for cell-surface phenotyping based on principles of pattern recognition and image analysis.
Authors: Goin J E , Craven T M , Gilbert J R , Melnicoff M J , Tomaszewski J E , Wilding P
Published: January 2020

Technical advancements in computer hardware and the associated computerized architectures allow the design and fabrication of dedicated, modestly expensive computerized workstations to aid humans in complex decision-making tasks. We refer to these as "Intelligent Workstations" because they accommodate the hypothesis-driven reasoning ability of the user into the system. Concomitant with the rapid developments in computers, diagnostic and therapeutic technologies related to cell analysis have undergone growth. Combining these technologies requires careful formulation and analysis of the diagnostic model involving image cytometry. Here we present a model of the intelligent workstation for image cytometry and illustrate its advantages in cell-surface phenotyping. Combining cell morphology with cell-surface phenotyping has clear diagnostic utility that no other widely available methods can supply. Although the reported results are based on samples of peripheral blood, cell suspensions of disaggregated cells can also be analyzed.
Homogeneous, micelle quenching fluoroimmunoassay for detecting amphetamines in urine.
Authors: Halfman C J , Jay D W
Published: January 2020

We developed a homogeneous fluoroimmunoassay for detecting amphetamines in urine. Only fluorescence intensity need be measured because the emission of non-protein-bound fluorescein-labeled amphetamine is preferentially quenched by detergent micelles. In a previous reported prototype assay system for measuring gentamicin in serum we used fluorescein and dodecyl sulfate (Anal Chem 1985; 57:1928-30). We have found that favorable hydrophobic and (or) ionic character of the analyte and unfavorable polar and (or) ionic character of the fluor are important determinants of the desired interactions. An anionic detergent and fluorescein, therefore, should be appropriate for apolar of cationic analytes, such as gentamicin and amphetamines. A greater [H+] at the anionic micelle surface is important for quenching emission from the fluor moiety. Millimolar concentrations of dodecyl sulfate rapidly denature immunoglobulin unless hapten is bound with sufficiently high affinity. Affinity was sufficiently high for the antibody used in the prototype gentamicin assay but not for the amphetamine antibody. Thus for the amphetamine assay, we used a non-denaturing detergent, dodecyl(oxyethylene)12 sulfate. The assay requires 30 microL of specimen in 2 mL of total assay volume. Amphetamine(d-,dl-, and meth-), at a concentration of 1 mg per liter of urine, is readily detected.
Nonisotopic detection methods for strand displacement assays of nucleic acids.
Authors: Vary C P , McMahon F J , Barbone F P , Diamond S E
Published: January 2020

Using the enzymes terminal deoxyribonucleotidyltransferase (EC 2.7.7.31) and polynucleotide phosphorylase (EC 2.7.7.8), we constructed polyriboadenylic acid tracts, approximately 8000 AMP residues long, attached to the 3'-terminus of a synthetic deoxynucleotide. The polyadenylated DNA, termed the "signal strand", was used in a displacement-type nucleic acid probe assay (see pp 1631-6, this issue). A probe-signal strand complex was made by hybridizing the signal strand to a deoxycytidylate-terminal probe DNA. The probe-signal strand complex was immobilized on an oligo (dG)-cellulose support and subsequently displaced from the immobilized hybrid complex with various amounts of analyte DNA. After the displacement procedure, the polyadenylate tracts were converted to ATP by the combined action of polynucleotide phosphorylase and pyruvate kinase. ATP was quantified by a bioluminescence assay with luciferase from Photinus pyralis. Displacement events were also quantified with biotinylated signal strand bound to avidin-conjugated horseradish peroxidase. Such enzyme-amplified assays offer considerable versatility: they may be coupled to a variety of detection systems including colorimetry, fluorimetry, and luminometry.
Application of the EXPERT consultation system to accelerated laboratory testing and interpretation.
Authors: Van Lente F , Castellani W , Chou D , Matzen R N , Galen R S
Published: January 2020

The EXPERT consultation system-building tool, a knowledge-based artificial intelligence program developed at Rutgers University, has been applied to the development of a laboratory consultation system facilitating sequential laboratory testing and interpretation. Depending on the results of a basic panel of laboratory tests, the system requests that specific secondary tests be performed. Input of these secondary findings can result in requests for tertiary testing, to complete the database necessary for interpretation. Interpretation of all results is based upon final inferences from the collected findings through a series of rules, a hierarchical network that yields an efficient production system not easily obtained through conventional programming. The rules included in this model are based upon initial results for total protein, calcium, glucose, total bilirubin, alkaline phosphatase, lactate dehydrogenase, aspartate aminotransferase, thyroxin, hemoglobin, mean corpuscular volume, and the concentrations of four drugs. Pertinent clinical history items included are jaundice, diabetes, thyroid disease, medications, and ethanol. Implementing this system in a laboratory-based accelerated testing program involving outpatients maximized the effective use of laboratory resources, eliminated useless testing, and provided the patient with low-cost laboratory information.
Immunoenzymometric determination of trypsin/alpha 1-protease inhibitor complex in plasma of rats with experimental pancreatitis.
Authors: Fletcher T S , Tsukamoto H , Largman C
Published: January 2020

The currently available radioimmunoassays of trypsin measure total immunoreactive trypsin (EC 3.1.1.7), which includes both trypsinogen and alpha 1-protease inhibitor-bound trypsin. Hitherto, the only way to differentiate these two forms of trypsin has been to fractionate them on a gel-filtration column. We describe here a solid-phase immunoenzymometric assay that rapidly measures the amount of cationic trypsin bound to alpha 1-protease inhibitor in the plasma of rats with experimental pancreatitis. The assay specifically measures this complex within the range from 0.2 to 5.0 ng without interference by high concentrations of free alpha 1-protease inhibitor. The high correlation (r = 0.985) of the values obtained by size fractionation and by this assay demonstrates the accuracy of the assay, which is the first single-tube method for determining this form of activated cationic trypsin in plasma.
Radioimmunoassay for amatoxins by use of a rapid, 125I-tracer-based system.
Authors: Andres R Y , Frei W , Gautschi K , Vonderschmitt D J
Published: January 2020

In this new radioimmunoassay system for determination of amatoxins in urine and plasma, a novel chemical approach is used for antigen and 125I-tracer production, based on a detoxified alpha-amanitin derivative (aldoamanitin). Total assay time, including data processing, is less than 100 min. The lowest detectable concentration is 1 microgram/L for urine, 0.1 microgram/L for plasma. In the clinically significant range, within-run CVs are less than 8%. This new 125I-based assay is a significant improvement over existing 3H technology in terms of speed, precision, and freedom from interference.
Rate of synthesis of IgG within the blood-brain barrier and the IgG index compared in the diagnosis of multiple sclerosis.
Authors: Hische E A , van der Helm H J
Published: January 2020

Receiver-operating characteristic curves of the IgG synthesis rate and the IgG index show that the IgG index has better discriminating properties with respect to multiple sclerosis. This, added to the fact that the precision of determining the index is much higher than that of the synthesis rate, makes the IgG index the parameter of choice in the diagnosis of multiple sclerosis.
Criteria for evaluating nonquantitative assays: application to serum choriogonadotropin.
Authors: Delfert D M , Rea M R , Kessler G , Siegfried B A , Valdes R
Published: January 2020

We present guidelines for assessing the performance of nonquantitative (qualitative) assay methods. Criteria to be evaluated include analytical sensitivity, imprecision near limits of detection, analytical specificity, accuracy over a wide range of analyte concentrations, potential interferents, and technical ease of performance. A protocol was developed to evaluate several nonquantitative assay kits for detection of human choriogonadotropin (hCG) in serum. These include Tandem Icon HCG (Hybritech), Quest Pregnancy Test (Quidel), Concep-7 beta hCG (Leeco), and Beta Quik V (Pacific Biotech). Quantitative measurement of beta-hCG by RIA (Immophase beta hCG, Corning Medical) was used as the reference method. Results of this evaluation are discussed. The guidelines established and utilized in this report are adaptable to the evaluation of assay kits that measure other analytes by qualitative techniques.
Electrochemical determination of hemoglobin, hematocrit, and hemolysis.
Authors: Kobos R K , Abbott S D , Levin H W , Kilkson H , Peterson D R , Dickinson J W
Published: January 2020

Novel electrochemical methods have been developed for determination of total hemoglobin, hematocrit, and detection of hemolysis in whole blood. Hemoglobin is measured through its peroxidase activity, a fluoride ion-selective electrode being used to monitor the rate of fluoride ion production from the oxidation of an organofluorine compound. Results agree well with those obtained with the cyanmethemoglobin method (r = 0.970). Hematocrit is determined from the ratio of the sodium ion concentrations measured with an ion-selective electrode before and after lysis of the erythrocytes. Results by this and the microhematocrit method correlated well (r = 0.987). Hemolysis in a whole-blood sample is detected by using an oxygen electrode to measure the oxygen released when hemoglobin in plasma is oxidized.
Enzyme immunoassay of free triiodothyronine in serum.
Authors: Hata N , Miyai K , Endo Y , Iijima Y , Doi Y , Amino N , Ichihara K
Published: January 2020

In this new solid-phase antibody enzyme immunoassay for free triiodothyronine (FT3) in serum, beta-D-galactosidase conjugated to triiodothyronine is used. Results are uninfluenced by physiological concentrations of thyroxin-binding globulin or albumin. Results correlate well with those determined by equilibrium dialysis (r = 0.95). The mean CVs within and between assays were 6.1 and 9.5%, respectively. The measurable range of FT3 in serum is 0.7 to 26 ng/L; the normal reference interval is 1.9 to 8.9 ng/L. Concentrations of FT3 in serum of patients with hyperthyroidism were high; those of patients with hypothyroidism were within normal limits or low, and those of patients with congenitally decreased or increased TBG were within the normal range. In normal pregnant women, concentrations of FT3 as determined by radioimmunoassay correlated with those of albumin, declining as pregnancy progressed, but FT3 values determined by the proposed method or equilibrium dialysis were within the normal range and did not change during pregnancy.
Improved 63Ni radiochemical assay of free fatty acids in plasma.
Authors: Barash H , Akov S
Published: January 2020

We have modified the 63Ni radiochemical method of Ho (Anal Biochem 1970;36:105) for determination of free fatty acids (FFA) in plasma. Extracting 1 or 0.1 mL of plasma with Dole's mixture (J Biol Chem 1960;235:2595) and washing the "heptane" layer with two volumes of isopropanol/water/dilute (0.5 mol/L) H2SO4 (25/25/1, by vol) removes about 90% of the lipid phosphorus from the "heptane" layer without removing any FFA and is more convenient than treatment with silicic acid. The following modifications decrease background radioactivity and improve separation of the organic phase from the water phase containing the uncomplexed 63Ni: (a) use glassware instead of plastic test tubes; (b) evaporate the organic phase to dryness before adding the 63Ni (this removes the isopropanol, which interferes with the 63Ni assay); and (c) add anhydrous sodium sulfate before the final centrifugation step.
Potential of monoclonal antibodies to improve therapeutic monitoring of cyclosporine.
Authors: Quesniaux V , Tees R , Schreier M H , Maurer G , van Regenmortel M H
Published: January 2020

We show that monitoring of cyclosporine by immunoassay could be improved by using monoclonal antibodies (MAbs) of restricted specificity instead of polyclonal antisera that recognize both unmodified cyclosporine and its metabolites. MAbs with high affinity for cyclosporine have been prepared and characterized. We tested their ability to discriminate between native cyclosporine and its metabolites in indirect solid-phase enzyme immunoassay with a set of cyclosporine metabolites modified at residues 1, 4, 6, and 9 (corresponding to the six known sites of metabolism of cyclosporine). All the metabolites tested were detected by MAb1 at least 15- to 1000-fold less well than unmodified cyclosporine. A second MAb recognized unmodified cyclosporine and most of its metabolites equally well. Both MAbs retained their activity when coupled to alkaline phosphatase and could therefore be used in a direct solid-phase enzyme immunoassay.
Europium and samarium as labels in time-resolved immunofluorometric assay of follitropin.
Authors: Bador R , Déchaud H , Claustrat F , Desuzinges C
Published: January 2020

This time-resolved immunofluorometric assay for human follitropin involves use of europium- or samarium-labeled monoclonal antibodies, with an average incorporation ratio of 3 mol of Eu3+ or Sm3+ per mole of antibody. These lanthanide ions are bound to the antibody molecules by means of the anhydride of diethylenetriaminepentaacetic acid. The solid-phase antibody is immobilized inside polystyrene tubes in which plasma samples were assayed in a one-step procedure. After incubation, the fluorescence intensity of Eu3+ or Sm3+ label is measured by time-resolved fluorometry, with a nitrogen laser as the pulsed excitation source. The sensitivity of the assay is largely better with Eu3+ than with Sm3+ because of the difference in their intrinsic luminescence properties. Results obtained with the proposed methods correlated well with those by an immunoradiometric method.
The clinical usefulness of glycated hemoglobin in monitoring diabetes mellitus: a long-term study.
Authors: Lenzi S , Giampietro O , Giovannitti G , Sampietro T , Miccoli R , Navalesi R
Published: January 2020

To assess the long-term clinical usefulness of measuring glycated hemoglobin (Hb A1), we carried out a two-year longitudinal study involving 234 diabetic patients (116 males, 118 females; 139 with type I diabetes mellitus, 95 with type II). Hb A1 values correlated significantly (p less than 0.001) with a score index based on plasma glucose in a specimen collected after overnight fasting, and urinary glucose, and ketones in a 24-h specimen. However, we found that one of every three well-controlled patients (both type I and II subjects) had high values for Hb A1. Among poorly controlled patients, only those with "brittle" diabetes had good values for Hb A1.
Free and total insulin as determined after precipitation with polyethylene glycol: analytical characteristics and effects of sample handling and storage.
Authors: Arnqvist H , Olsson P O , von Schenck H
Published: January 2020

We evaluated results of radioimmunoassays of free and total insulin after precipitation of endogenous antibodies with polyethylene glycol (PEG), and we investigated the influence of collection time, temperature, and storage in heparin- cr EDTA-treated plasma or serum on results for free insulin. Analytical recovery of free insulin was 99.3%, of total insulin 96.4%. For free insulin, assay precision (CV) was 4.0-13.0% (intra-assay) and 7.8-10.7% (inter-assay); for total insulin, 3.6-9.5% and 6.6-11.7%, respectively. Free insulin decreased in plasma (p less than 0.05) and serum (p less than 0.01) at room temperature after 3 h and in promptly analyzed serum (p less than 0.01). Storage of samples at -20 degrees C increased the concentration of free insulin in plasma (p less than 0.025) and serum (p less than 0.005), whereas the free insulin content of supernates after PEG precipitation was stable, except for a slight decrease in serum samples (p less than 0.02). We conclude that, for radioimmunoassay of free and total insulin, plasma should be used, treated with PEG without delay; supernates then are analytically stable for as long as 26 weeks at -20 degrees C.
Human IgG subclass measurements in the clinical laboratory.
Authors: Hamilton R G
Published: January 2020

Complement activation, cell surface-receptor binding, blocking activity, and possibly placental transfer are among the biologically important functional differences that have been detected between the four human IgG subclasses by use of polyclonal antisera. In 1985, a IUIS/WHO panel of immunologists, using eight immunological methods, documented the specificity of select monoclonal antibodies for the IgG subclasses. Clinical assays have been developed involving these monoclonal antibodies that allow quantification of the concentration of IgG subclass protein and distribution of the IgG subclass antibodies in human immune responses. This review addresses issues of concern to investigators who are evaluating and (or) developing quantitative human IgG subclass assays in the clinical laboratory. Unique physical (structural) and biological (functional) properties of human IgG subclasses are summarized, with a focus on aspects pertinent to their clinical importance and in vitro quantification. The HP-series monoclonal antibodies with documented specificity are examined within the context of their application to several immunological methods. I describe unique technical aspects of total and antigen-specific IgG-subclass immunoassays involving these monoclonal antibodies. Finally, this report outlines clinical applications and indications for IgG-subclass measurements in the study of human health and disease.
Use of restriction fragment length polymorphism analysis for detecting carriers of "fragile X" syndrome.
Authors: Thibodeau S N
Published: January 2020

Recombinant DNA technology promises to play an increasingly important role in the future of medicine. Application of this technology to the study of human disease will help us to define and clarify the molecular pathology of many clinical disorders, provide new diagnostic tools and approaches, and, finally, will provide new therapeutic agents including gene-replacement therapy. We have begun to exploit these powerful new techniques to aid in the laboratory diagnosis of several genetic disorders for which reliable assays are currently not available, such as "Fragile X" syndrome.
Determination of conjugated and total bilirubin in serum of neonates, with use of bilirubin oxidase.
Authors: Mullon C J , Langer R
Published: January 2020

A new, simple, and rapid assay for conjugated bilirubin that does not require serum-matrix standards was developed by using the enzyme bilirubin oxidase (EC 1.3.3.5). This procedure can also be modified to measure total bilirubin. Measurements from these assays were compared with results obtained with the Sigma 605 (Jendrassik-Gróf method), Sigma 550/551 (Walters-Gerarde method), and Kodak Ektachem (BuBc) bilirubin assays. The one-year study involved serum specimens from 283 infants younger than 30 days. Linear-regression analysis of data for conjugated bilirubin collected by this assay and by the Kodak Ektachem assay yielded a slope of 0.975, an intercept of 0.088, an Syx of 1.47 mg/L, and a correlation coefficient of 0.94 for a total of 49 specimens. Correlation was also good (r = 0.95) between results for total bilirubin by this assay and both the Sigma 605 and the Kodak Ektachem methods.
Ascorbic and dehydroascorbic acids simultaneously quantified in biological fluids by liquid chromatography with fluorescence detection, and comparison with a colorimetric assay.
Authors: Lopez-Anaya A , Mayersohn M
Published: January 2020

We describe a "high-performance" liquid-chromatographic method for separating and quantifying ascorbic acid (AA) and dehydroascorbic acid (DHA) in plasma and urine. We used a reversed-phase C18 column with an ion-pair reagent and detected the analytes by post-column reaction with 4,5-dimethyl-o-phenylenediamine to form a fluorescent derivative (measured at excitation and emission wavelengths of 365 and 440 nm, respectively). Isoascorbic acid (IA) is the internal standard. Retention times for DHA, AA, and IA are 5.6, 15.5, and 19.9 min, respectively. Between-day CVs for AA in plasma in concentrations of 8 and 20 mg/L were 9% and 7%, respectively. The limit of detection is 10 and 4 ng for AA and DHA, respectively. Results by the present method and the methoxyaniline colorimetric method for AA are comparably accurate.
Simplified sodium dodecyl sulfate-polyacrylamide gel electrophoresis of unconcentrated urine with enhanced resolution and detection sensitivity.
Authors: Marshall T , Williams K M
Published: January 2020

We applied a simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis method to urine. The method, developed for serum protein analysis (Clin Chem 1984;30:475-9), has a high sample throughput and gives excellent resolution with unconcentrated urine. It clearly distinguishes and characterizes proteinuric urine (7.5 microL) by Coomassie Blue staining and gives complex silver-stained patterns with nonproteinuric urine (2 microL). The former is recommended for routine clinical screening, the latter for research purposes.
Glycated proteins in serum: effect of their relative proportions on their alkaline reducing activity in the fructosamine test.
Authors: Zoppi F , Mosca A , Granata S , Montalbetti N
Published: January 2020

The fructosamine test is considered clinically useful for assessing short-term integrated control of blood glucose, but there are few published data to support this hypothesis. We fractionated glycated and nonglycated proteins by affinity chromatography on phenylboronate columns and, with specific immunochemical methods, determined in the eluted fractions the following proteins, selected according to their biological half-lives and relative concentrations in serum: albumin, IgA, IgG, IgM, apolipoprotein B, haptoglobin, transferrin, alpha 1-antitrypsin, and alpha 2-macroglobulin. We found the following correlations between fructosamine (mmol/L) and, respectively, glycated albumin, IgG, and (albumin + IgG) (each in grams per liter): r = 0.901, 0.702, 0.878. IgM had the highest percentage of glycated molecules (range 11.1-37.5%, mean 22.4%), haptoglobin and alpha 1-antitrypsin the least. This result was almost independent of the proteins' molecular masses and fractional catabolic rate. Albumin evidently contributes most to results of the fructosamine test, confirming conclusions obtained in different ways by others.
Prostate-specific antigen as a marker for prostatic cancer: a monoclonal and a polyclonal immunoassay compared.
Authors: Chan D W , Bruzek D J , Oesterling J E , Rock R C , Walsh P C
Published: January 2020

We evaluated the clinical utility of prostate-specific antigen (PSA) in diagnosis and management of prostatic cancer, using a monoclonal immunoradiometric assay (M-PSA) and a polyclonal radioimmunoassay (P-PSA). Assay CVs ranged from 1.3% to 4.9% (M-PSA) and from 5.1% to 8.0% (P-PSA). Detection limits were 0.1 microgram/L (M-PSA) and 0.2 microgram/L (P-PSA). Reference intervals established for healthy men were: 0-2.8 micrograms/L (M-PSA) and 0-4.2 micrograms/L (P-PSA). The regression equation for the PSA concentrations (0-100 micrograms/L) measured in normal men, men with primary prostatic cancer, and men with benign prostatic hypertrophy (BPH) was: P-PSA = 1.603 M-PSA - 0.120 microgram/L (r = 0.9923, n = 201). The P-PSA calibrators yielded one-half their assigned values when analyzed by the M-PSA assay. With either assay there were proportional increases in PSA values with advancing cancer. The proportion of PSA values exceeding the reference interval was 50% for patients with stage A prostatic cancer, 80% (stage B), 100% (stages C and D), and 65% for men with BPH. After radical prostatectomy, the follow-up PSA values for most patients were within the reference intervals for women (0-0.2 microgram/L for M-PSA, 0-0.8 microgram/L for P-PSA). Most patients with above-normal PSA values have clinically detectable disease.