bluuebunny/crossref_metadata_2025 huggingface dataset:
Colorimetric determination of serum 5'-nucleotidase without deproteinization.
Authors: Wood R J , Williams D G
Published: January 2020

We present a sensitive colorimetric determination of 5'-nucleotidase based on the measurement of liberated phosphate by reaction with stannous chloride/molybdenum blue. No protein precipitation is required. The method is simple, sensitive, economical, and easy to use, and involves relatively stable reagents.
Measurement of hemoglobin A1 by liquid chromatography and by agar gel electrophoresis compared.
Authors: Hayes E J , Gleason R E , Soeldner J S , Wacks M , Blankstein L
Published: January 2020

We compare measurement of total fast hemoglobin (HbA1) by "high-performance" liquid chromatography and by electrophoresis on agar gel. Blood samples were obtained from a diverse population (n = 222): offspring of two diabetic parents, diabetic patients with and without retinopathy, diabetic and non-diabetic pregnant women, patients in the coronary-care unit, and normal persons. Precision studies with a normal and an above-normal A1 sample resulted in overall CVs of 9.0% and 4.6% for the electrophoretic method and 4.4% and 2% for the chromatographic method. Linear regression analysis of values for total fast hemoglobin for the complete sample population and for each subgroup showed results of the electrophoretic method to be in excellent agreement with those by the chromatographic method. We conclude that the agar gel electrophoretic method offers a reproducible means for HbA1 determination that is comparable to the HPLC method in terms of accuracy and is highly suited for routine laboratory use.
Extended clinical trial and evaluation of urea nitrogen determination with the Ektachem GLU/BUN analyzer.
Authors: Bandi Z L , Fuller J B , Bee D E , James G P
Published: January 2020

We followed the "abbreviated precision protocol" of the National Committee for Clinical Laboratory Standards for the evaluation of precision, accuracy, and carryover in analysis for urea nitrogen with the multilayer film analysis system ("Ektachem"). We analyzed 456 clinical samples with this instrument, by the manual urease/glucose dehydrogenase method, and with the Beckman System I GLU/BUN Analyzer. Precision and accuracy were estimated for 50, 220, 270, and 500 mg/L urea nitrogen concentrations in 100, 30, or 20 microL of serum. Potential interference of 15 compounds was evaluated. Random error (defined as 1.965 X SD) was 7, 10, 12, and 18 mg/L. Systematic error was 3, 4, 5, and 15 mg/L. Total analytical error was 11, 14, 17, and 34 mg/L for analysis of 100 microL of serum at the above-stated urea nitrogen concentrations. The greatest interference (6 mg/L) was caused by ethanol (300 mg/L) and by hemoglobin (500 mg/L) in the urea nitrogen (at 260 mg/L) determination. Urea nitrogen concentration, as determined with the Ektachem was linearly related to the expected concentration, at least up to 1187 mg/L. Carryover was not statistically significant.
Screening for the Tay-Sachs carrier: a compromise program.
Authors: Potter J L , Robinson H B
Published: January 2020

A modified program designed to screen for the Tay-Sachs carrier is presented in which testing is limited to one or both partners, as needed, in an Ashkenazi Jewish (Jews of central and eastern Europe ancestry) mating when there is a definite commitment toward having a child, or in the early stages of the pregnancy. Testing of unmarried individuals is discouraged. The approach maximizes individualization of both the medical and laboratory aspects of the program and promotes a positive and beneficial relationship between physician and clinical chemist. There is little involvement of the lay population or clergy, and no special sources of funding are required. Whereas most mass screening programs for the Tay-Sachs carrier have attempted to educate large numbers of the lay public to bring pressure upon the medical community, we have reversed this approach and taken the much easier course of educating small numbers of physicians to better care for their patients. The program has been used successfully in a moderate-size city in which communication lines between laboratory and physician are easily established.
Quantitative determination of apolipoproteins C-I and C-II in human plasma by separate electroimmunoassays.
Authors: Curry M D , McConathy W J , Fesmire J D , Alaupovic P
Published: January 2020

Separate electroimmunoassays are described for measuring human plasma apolipoproteins C-I and C-II. Purified apolipoproteins C-I and CII were used in preparing monospecific antisera and as the primary standards. These assays are sensitive (maximal sensitivity, 20 ng), specific, rapid, precise (the within- and between-assay coefficients of variation for both assays were 5 and 8%, respectively), and accurate (accuracy was based on comparison of calculated and measured C-I, C-II, and C-III contents of an ApoC-containing column-eluent fraction) and are applicable to measurement of C-I and C-II polypeptides in whole plasma and density classes. However, plasma samples with triglyceride (triacylglycerol) concentrations greater than 6000 mg/L must be delipidized before analysis for C-II, as must those with greater than 12 000 mg/L before analysis for C-I polypeptide. Mean concentrations (and SD) of C-I in plasma of normolipidemic subjects and hyperlipoproteinemic phenotypes IIa, IIb, IV, and V were 60 (15), 70 (20), 100 (20), 100 (20), and 260 (94) mg/L, respectively. The corresponding C-II values were 40 (20), 43 (20), 68 (20), 65 (20), and 210 (70), respectively. C-I and C-II concentrations in patients with phenotypes IIb v, IV, or V significantly (p less than 0.001) exceeded those in normal persons or phenotype IIa. The observed correlations (r = 0.92 and r = 0.94) between triglyceride and C-I and C-II values suggest that these two polypeptides, like C-III, are excellent plasma markers for assessing the state of triglyceride metabolism.
Characterization of amylases produced by tumors.
Authors: Takeuchi T , Fujiki H , Kameya T
Published: January 2020

Amylases were purified and characterized from three amylase-producing human tumors. The relative molecular mass of the amylases was estimated to be 54 000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, different from human salivary amylase (61 000 and 64 000) and human pancreatic amylase (60 000). The tumor amylases had completely identical antigenicities with human salivary and pancreatic amylases against antibody to human salivary amylase, while the intensity of the tumor amylases was less than 20% of that of human salivary and pancreatic amylases on single radial immunodiffusion. On isoelectric focusing, two of the three tumor amylases showed a major peak at pH 6.4, which corresponded to a major peak of human salivary amylase, the other showed a major peak at pH 6.4, which corresponded to a minor peak of human salivary amylase. The three tumor amylases showed similar amino acid composition, different from those of human salivary and pancreatic amylases. These findings suggest that tumor amylases have a tertiary structure similar to that of normal human amylases, but differ from them in amino acid composition.
Determination of Cu, Mn, Fe, and Ca in six regions of normal human brain, by atomic absorption spectroscopy.
Authors: Goldberg W J , Allen N
Published: January 2020

A method is reported for measuring copper, manganese, iron, and calcium in brain tissue. We solubilized 100-mg samples of brain tissue with Soluene-350 and diluted the mixtures with methyl isobutyl ketone. Copper and manganese concentrations were measured by using a carbon rod atomizer, iron and calcium concentrations by using an air/acetylene and nitrous oxide/acetylene flame, respectively. The basal ganglia (caudate, putamen, globus pallidus, and substantia nigra) contained high concentrations of iron (10 to 19 mumol/g dry wt) as compared to concentrations found in the cortex (4 mumol/g) or the hippocampus (2.8 mumol/g). Copper exhibited a similar distribution pattern among the regions studied; 526 to 1140 mumol/g (dry wt) in the basal ganglia vs 496 mumol/g in the cortex and 426 mumol/g in the hippocampus. Manganese and calcium were uniformly distributed in the regions examined.
125I radioimmunoassay of delta-9-tetrahydrocannabinol in blood and plasma with a solid-phase second-antibody separation method.
Authors: Owens S M , McBay A J , Reisner H M , Perez-Reyes M
Published: January 2020

In this sensitive and specific radioimmunoassay for delta-9-tetrahydrocannabinol, an iodinated tracer with high specific activity and a solid-phase separation are used. Within-run coefficients of variation for 5.0 and 30.0 microgram/L concentrations were 7.8 and 4.2% for plasma and 14 and 10.6% for hemolyzed blood specimens, respectively. Day-to-day coefficients of variations ranged from 7.3 to 13.6% (for 7.6 to 33.0 microgram/L concentrations) for plasma and 13.4 to 18.1% (3.0 to 52.1 microgram/L) for hemolyzed blood specimens. Data for time after start of smoking of a standard THC-containing cigarette vs the concentration of delta-9-tetrahydrocannabinol in the plasma were similar to those obtained by others. Positive plasma specimens from the smoking study were analyzed by gas chromatography-mass spectrometry and by our radioimmunoassay. Nonparametric statistical comparison and linear regression (r2 = 0.972) showed that results by the two methods of analysis correlate well. The sensitivity of the assay was at least 0.3 microgram/L for plasma, 1.1 microgram/L for hemolyzed blood.
Modified heparin-Sepharose procedure for determination of plasma lipolytic activities of normolipidemic and hyperlipidemic subjects after injection of heparin.
Authors: Wang C S , Bass H B , Downs D , Whitmer R K
Published: January 2020

A modified heparin-Sepharose affinity chromatography procedure (Boberg et al., J. Lipid Res. 18:544-547, 1977) was developed to determine two different triglyceride lipase activities in human post-heparin plasma: hepatic triglyceride lipase (I) and lipoprotein lipase (II). With this procedure, lipoproteins were separated from the eluted lipases. The total lipolytic activity of II was eluted from heparin-Sepharose by heparin. The use of heparin as eluting agent prevents the partial inhibition of II, in contrast to the procedure based on elution of II with a high concentration of NaCl. In a comparative study with the modified heparin-Sepharose affinity column chromatography, the immunochemical and protamine sulfate inhibition procedures, the results indicated that these three procedures are equally suitable for the determination of I and II from normolipidemic subjects. However, because of possible interference by plasma, the column-chromatographic procedure is the preferred method for measuring lipase concentrations in post-heparin plasma of hyperlipidemic patients. The II activity of post-heparin plasma from normolipidemic subjects was not significantly age-(20-39 and 40-60 years) or sex-related. I activity was also not significantly different with respect to age, but was significantly greater in men than in women.
Use of a dried blood spot in immunoreactive-trypsin assay for detection of cystic fibrosis in infants.
Authors: Kirby L T , Applegarth D A , Davidson A G , Wong L T , Hardwick D F
Published: January 2020

We assayed more than 5000 blood spots dried on filter paper and approximately 1000 serum samples for immunoreactive trypsin, with commercial reagents (Behring and Sorin). The assay procedures were modified so that newborn screening is technically feasible. Both kits are satisfactory for serum assay, but the Sorin materials are better adapted for blood spot analysis. Immunoreactive trypsin in blood spots rapidly decreases with specimen age, but is stable in frozen serum. Values for premature infants do not differ significantly from those for full-term babies. Children with cystic fibrosis were readily distinguished from those without, up to at least one year of age.
Radioimmunoassay for pteroylglutamic acid.
Authors: Hendel J
Published: January 2020

I describe a radioimmunoassay for specific determination of pteroylglutamate in plasma, erythrocytes, and urine. Antiserum was raised in rabbits immunized with a conjugate of pteroylglutamic acid and methylated bovine serum albumin. The lowest detection limit of the assay was 0.1 microgram/L; the coefficient of variation ranged from 4.01 to 14.63%. No significant interference was demonstrated from tetrahydrofolic acid, 5-methyl-tetrahydrofolic acid, or methotrexate. Under normal sampling and storing conditions dihydrofolic acid was oxidized to pteroylglutamic acid and estimated as such in the assay. The clinical applicability of the assay is demonstrated by measurements of pteroylglutamate absorption rates after oral doses of pteroylglutamic acid or pteroyltriglutamic acid and by the estimation of the accumulation of pteroylglutamate and dihydrofolate in plasma during therapy with methotrexate.
The 16-hour-standing test and lipoprotein electrophoresis compared for detection of chylomicrons in plasma.
Authors: McNeely S , Seatter K , Yuhaniak J , Kashyap M L
Published: January 2020

We compared the 16-hour-standing plasma test to lipoprotein electrophoresis on agarose gel for detection of chylomicrons in 129 patients' samples with triglyceride values greater than or equal to 4.00 g/L. Chylomicrons were observed in 12 samples (9.3%) by use of the standing-plasma test and in 58 samples (45.0%) by use of agarose-gel electrophoresis. Thus the standing-plasma test did not detect chylomicrons in 46 samples where they were observed by electrophoresis, or 79.3% of all cases where chylomicrons were present. Chylomicronemia was missed in the presence of lower triglyceride concentrations as well as at very high ones. We recommend that lipoprotein electrophoresis be routinely performed on plasma of patients with triglycerides concentrations greater than 4.00 g/L to distinguish between types IV and V hyperlipoproteinemia, as well as to detect failure of a patient to fast before sample collection.
Cryoprotected Lactobacillus casei: an approach to standardization of microbiological assay of folic acid in serum.
Authors: Grossowicz N , Waxman S , Schreiber C
Published: January 2020

Folate-depleted, cryoprotected preparations of Lactobacillus casei are stable for at least eight months at -18 degrees C, capable of reproducible growth and suitable as a ready source of inoculum for measurement of folates in physiological fluids. Cryoprotected microorganisms can be a commercially available laboratory reagent, thus simplifying and further standardizing the microbiological assay of various nutriments. Standard folate growth curves of cryoprotected L. casei, prepared at intervals over eight months, are superimposable, have a low blank, and should eliminate the variations encountered with the continuous passages of microorganisms required for the classic microbiological assays. Serum folate values obtained by use of the cryoprotected L. casei fall into the same diagnostic groups as determined by the classic microbiological assay.
Automated Jendrassik-Grof method for measurement of bilirubin in serum with the Greiner Selective Analyzer (GSA II D), and comparison with the method involving diazotized 2,4-dichloroaniline.
Authors: Cherian A G , Soldin S J , Hill J G
Published: January 2020

We automated the Jendrassik-Grof method for measurement of bilirubin with the Greiner Selective Analyzer GSA II D. Comparison with the manual Jendrassik-Grof method showed a highly significant correlation coefficient of 0.994 for total and 0.980 for "direct" bilirubin measurement. The automated method showed good precision and was linear to 667 mu mol/L, the highest concentration tested. Addition of hemolysate caused a greater effect on the measurement of direct bilirubin than on total bilirubin. The inhibitory effect on measurement of total bilirubin, though small, was statistically significant at hemoglobin concentrations greater than 2 g/L. Comparison with the method involving diazotized 2,4-dichloroaniline showed the Jendrassik-Grof method to be relatively less affected by hemolysis and turbidity in the measurement of total bilirubin.
Suitability of control materials. General principles and methods of investigation.
Authors: Bretaudiere J P , Dumont G , Rej R , Bailly M
Published: January 2020

We propose methods for characterizing the behavior of quality-control specimens. Candidate quality-control specimens and authentic patients' specimens were analyzed by various methods. Patients' specimens were chosen to be fully representative of those encountered, including subsets from persons who were healthy, had defined disease states, were in therapy, or whose specimens were lipemic, icteric, etc. The analytical methods chosen include those most commonly used as well as reference analytical methods. Procedures for characterizing the behavior of patients' specimens and candidate quality-control specimens are proposed and their applicability is demonstrated. The linear ratio method is a univariate graphical approach in which differences in accuracy among methods for any specimen or group of specimens are each displayed on a linear scale. Correspondence analysis is a descriptive multivariate statistical technique that allows both the specimens and the analytical methods to be characterized. The statistical techniques, in our application, allow the behavior of quality-control specimens to be assessed with respect to authentic patients' specimens without influencing the assessment process. Correspondence analysis provides a graphic representation by projecting both the specimens and the analytical methods on factorial planes. The appropriateness of te behavior of a quality-control specimen may be inferred from its position relative to those of authentic patients' specimens. These statistical techniques also provide some information regarding the specificity of analytical methods.
Automation of plasma antithrombin III assays.
Authors: Holmes E W , Fareed J , Bermes E W
Published: January 2020

We have developed automated assays for antithrombin III for use with the Gilford 3500 and the Multistat Microcentrifugal Analyzer. The assays are two-stage kinetic-rate methods and involve use of human thrombin and the chromogenic peptide substrate S-2238. The reagents are prepared in-house from commercially available components. Both automated methods have good precision and results correlate well with both an established chromogenic substrate assay and an immunological assay. The automated assays offer the advantages of speed, sensitivity, high throughput, and low reagent consumption and are useful for both routine work and high-volume testing in clinical studies and screening programs.
Separation of serum high-density lipoprotein for cholesterol determination: ultracentrifugation vs precipitation with sodium phosphotungstate and magnesium chloride.
Authors: Seigler L , Wu W T
Published: January 2020

We compared two methods for separation of serum high-density lipoprotein: selective precipitation with sodium phosphotungstate and magnesium chloride, and ultracentrifugation in sodium chloride solution (relative density 1.063). When the cholesterol content (determined enzymically with a centrifugal analyzer) of fractions obtained by each method was compared (ultracentrifugation = x), the correlation coefficient was 0.97; y = 1.01x - 11.2 mg/L; p less than 0.05; n = 54. The within-day and between-day coefficients of variation for this method were 1.1 and 4.0%, respectively. Reference intervals for high-density lipoprotein cholesterol in subpopulations categorized by age and sex were based on data obtained from volunteer blood donors.
Bile acid assays as an index of cholestasis.
Authors: Woodman D D , Maile P A
Published: January 2020

Four bile acid profiles, bilirubin, alkaline phosphatase, alanine aminotransferase, glutamate dehydrogenase, and isocitrate dehydrogenase were measured in rats in which normal bile flow had been disturbed either by administration of alpha-naphthylisothiocyanate or by surgical ligation of the bile duct. The most sensitive index to the early onset of cholestasis was an increase in total cholate. This increase was measurable before morphological changes could clearly be identified.
Liquid-chromatographic analysis for common tricyclic antidepressant drugs and their metabolites in serum or plasma with the technicon "FAST-LC" system.
Authors: Bannister S J , van der Wal S , Dolan J W , Snyder L R
Published: January 2020

We describe a single procedure for assay of seven tricyclic antidepressant drugs and metabolites in serum or plasma: protriptyline, nortriptyline, amitriptyline, desmethyldoxepin, doxepin, desipramine, and imipramine. With the Technicon "FAST-LC" system, samples are aspirated directly into the unit and pretreated via double extraction; the concentration of each drug is then determined by "high-performance" liquid chromatography. Final chromatograms are monitored at 205 nm, at analysis rates of 7.5 samples/h. Concentration and absorbance are linearly related for each drug from 0 to 1400 micrograms/L. Day-to-day CVs averaged 5 to 6% for each drug, and there is good correlation of FAST-LC values with those obtained by gas-chromatographic methods. Total sample volume is 750 microliters.
Comparison of concentrations of some trace, bulk, and toxic metals in the hair of normal and dyslexic children.
Authors: Capel I D , Pinnock M H , Dorrell H M , Williams D C , Grant E C
Published: January 2020

Hair from dyslexic children, analyzed by flameless atomic absorption spectrometry, showed significantly higher concentrations of magnesium and copper than did hair from control subjects. The hair from dyslexic children also contained significantly higher concentrations of aluminum and cadmium than that from control children; the cadmium concentration exceeded the normal acceptable range. There were no significant differences in the case of lead, calcium, selenium, or mercury. Our results indicate that excessive cadmium burden could be implicated in this form of learning disorder.
Kinetic immunoturbidimetry of human choriomammotropin in serum.
Authors: Price C P , Spencer K
Published: January 2020

The sensitivity of kinetic immunoturbidimetry as a method for the measurement of specific proteins has been shown previously to be equivalent to 1 mg of analyte per liter. We confirm this in the method for human choriomammotropin described here. A relatively large sample volume is required, which results in other components of serum influencing the immunoprecipitin reaction. The technique is shown to be precise, and results compare well with those by a commonly available radioimmunoassay. The method performs well, as judged from results obtained for assayed quality control materials and in an external quality assurance program. The method is rapid (instrument assay time 120 s per sample), automated, and economical for routine use.
Enzyme immunoassay for specific determination of the synthetic estrogen, ethynyl estradiol, in plasma.
Authors: Turkes A , Dyas J , Read G F , Riad-Fahmy D
Published: January 2020

In this enzyme immunoassay for ethynyl estradiol, a conjugate of the 3-(O-carboxymethyl)ether with horseradish peroxidase is used as the label and a conjugate of the 6-(O-carboxymethyl)oxime with bovine serum albumin as the immunogen. A solid-phase antibody procedure is used in separating antibody-bound and free steroid. The lower limit of sensitivity of the assay is 2 pg per assay tube. Interference by synthetic progestagens was minimized by extracting the sample with an anti-estrogen serum before assay. Results obtained with a comparison radioimmunoassay and this procedure agreed well (r greater than 0.98).
Steroid radioimmunoassay--effect of shortened incubation time on specificity.
Authors: Vining R F , Compton P , McGinley R
Published: January 2020

Use of a briefer incubation interval in several steroid radioimmunoassays markedly increases cross reactions with some closely related steroids. The dissociation rates of the various steroid/antibody complexes play a critical role in determining the specificity of the antiserum, and the maximum specificity of an antiserum will be exploited only if it is incubated to equilibrium. The commonly used method for estimating the time required to reach equilibrium--i.e., the time required for the %(B0/T) value to "plateau"--grossly underestimates the true interval required, and we suggest that the minimum incubation period for steroid radioimmunoassay should be based on a knowledge of both the dissociation and the association rate.
Optimization of a mechanized amidolytic factor-X assay; influence of reaction conditions and reagents.
Authors: van Wijk E M , Kahlé L H , Wiebosch M , ten Cate J W
Published: January 2020

We describe the effect of pH and NaCl concentration on the activation of factor X in plasma by Russell's viper venom and on the amidolytic activity of the activated enzyme towards factor Xa-sensitive chromogenic substrates. An increasing NaCl concentration results in a decrease in the activation rate of factor X by Russell's viper venom, whereas in this step no pH effect is observed. Increasing NaCl concentrations decrease the Km and Vmax for both the factor Xa-sensitive chromogenic substrates, S 2222 and S 2337. Km values were lowest between pH 7.8 and 8.6; Vmax increased with increasing pH. Comparison of NaCl and KCl in the activation step as well as in the amidolytic step shows that the observed effect is specific for Na+, not just an effect of ionic strength. No difference was detected between activation by the crude venom or its purified factor X-activating enzyme. Also, values with the substrates S 2222 or S 2337 were the same in the mechanized amidolytic factor X assay.
Fluorescence polarization immunoassay. I. Monitoring aminoglycoside antibiotics in serum and plasma.
Authors: Jolley M E , Stroupe S D , Wang C H , Panas H N , Keegan C L , Schmidt R L , Schwenzer K S
Published: January 2020

Fluorescence polarization immunoassays of the aminoglycoside antibiotics gentamicin, tobramycin, and amikacin in plasma and serum are described and shown to be clinically useful. The aminoglycoside tracers were prepared by reacting the parent compounds with 5-[(4,6-dichlorotriazin-2-yl)-amino] fluorescein. Antisera specific for the compounds were raised in rabbits by conventional procedures. Tracer, sample, and diluted antiserum are combined and, after a 15-min incubation at ambient temperature, the polarization of the fluorescence of the tracer is determined in a specially designed fluorometer. The assays are designed to give accurate trough (i.e., minimum during therapy) values and to be free of matrix effects. Severely icteric samples may interfere, but this can be overcome by blank subtraction. The performance of the assays with clinical specimens compared favorably with that of some commercially available assays.
Differential assay for urinary catecholamines by use of liquid chromatography with fluorescence detection.
Authors: Jackman G P
Published: January 2020

I describe a relatively simple and rapid procedure for quantitative analysis for unconjugated urinary norepinephrine, epinephrine, and dopamine. Catecholamines are isolated from urine on a carboxylic acid ion-exchange resin (Bio-Rex 70) and eluted with 0.8 mol/L boric acid. The eluates are assayed for the individual catecholamines by "high-performance" liquid chromatography with an octadecylsilane reversed-phase column and 10 mmol/L perchloric acid-acetonitrile (99/1) as the mobile phase. The catecholamines are detected by measuring their intrinsic fluorescence on excitation at 200 nm. The method is highly reproducible, the coefficient of variation for normal concentrations in urine being 4.4% for norepinephrine, 8.8% for epinephrine, and 4.8% for dopamine. Moreover, it appears to be practically free of interference by drugs or endogenous compounds in urine. The range of catecholamine concentrations found in 143 hypertensive patients agrees well with previous studies.
Radioimmunoassay for serum and urinary lysozyme.
Authors: Thomas M J , Russo A , Craswell P , Ward M , Steinhardt I
Published: January 2020

We describe a radioimmunoassay for lysozyme (EC 3.2.1.17, mucopeptide N-acetylmuramohydrolase) in plasma and urine. It involves use of an antibody, raised in rabbits, to lysozyme from urine from a patient with monocytic leukemia. This antiserum, diluted 4000-fold, is incubated with radiolabeled lysozyme for 2 h and antibody-bound lysozyme is separated from free lysozyme with dextran-coated charcoal. Validation of the assay included precision and parallelism studies with serum and urine samples from patients with monocytic leukemia and analgesic nephropathy. Results of the radioimmunoassay and those obtained with a Micrococcus lysodeikticus lytic assay correlated well (r = 0.94). Sensitivity of the assay was 3.5 micrograms/L for both serum and urine. The main advantages of the assay are its good precision and reproducibility and its high sensitivity.
Thin-layer chromatography, with fluorescence detection, of benzodiazepines and tricyclic antidepressants in serum from emergency-room patients.
Authors: Meola J M , Rosano T G , Swift T
Published: January 2020

We describe a rapid, sensitive procedure for detecting benzodiazepines and tricyclic antidepressants in serum. After the compounds are adsorbed on charcoal and eluted with an organic solvent mixture, the drugs are thin-layer chromatographed and detected by their fluorescence. Because it is so fast and easy (turn-around time of about 1 h), the method can be used for emergency toxicology screening. An analysis requires 1.0 mL of serum, and the limit of detection for each of the drugs is about 0.75 mg/L. The procedure is sensitive enough to allow the use of serum instead of urine for the detection of these drugs, because serum has certain advantages over urine as the sample and better reflects the state of the patient's toxicity.
Labile glycosylated hemoglobin contributes to hemoglobin A1 as measured by liquid chromatography or electrophoresis.
Authors: Nathan D M
Published: January 2020

The utility of glycosylated hemoglobin (HbA1) measurement as an index of chronic control in diabetes can be adversely affected by interference from a labile glycosylated fraction that changes rapidly with acute changes in blood glucose concentration. I used a "high-pressure" liquid-chromatographic assay and a newly developed electrophoretic assay to quantitate the contribution of this labile fraction. If erythrocytes are incubated at 22 degrees C in isotonic saline for 12 h before hemolysis, the labile fraction is eliminated. Its contribution is similar as measured by both assays: 2-3.5% of total HbA1 in normal cells and 7-9.5% in diabetic cells. A 3-h incubation of erythrocytes with glucose produces acute changes in apparent HbA1 concentrations by both assays, but such changes can be eliminated by the incubation in saline. Although current methods are time consuming, the labile glycosylated hemoglobin must be removed when the sample is prepared for HbA1 measurement by liquid chromatography and electrophoresis if results are not to be factitiously high.
Urinary metanephrines as measured by liquid chromatography with an on-line post-column reaction detector.
Authors: Flood J G , McComb R B
Published: January 2020

In this method for measuring metanephrine and normetanephrine in urine, they are freed from their conjugates by hydrolysis in acid, neutralized, and isolated on a cation-exchange resin. Both metanephrines are co-eluted with ammoniacal methanol and the eluate is concentrated 10-fold with respect to the original urine volume by evaporation and reconstitution. The recovered metanephrines ae separated by reversed-phase (C18) "high-pressure" liquid chromatography with use of a mobile phase consisting of 10 mmol/L trichloroacetate, pH 2.7, containing 30 mL of acetonitrile per liter. The metanephrines are detected at 365 nm after being converted to vanillin in a post-column reaction with alkaline periodate. Total metanephrine values for most patients were appreciably lower than values obtained by the comparison procedure, that of Pisano (Clin. Chim. Acta 5: 406, 1970); however, results obtained by the two methods for two patients with pheochromocytomas agreed well.
Three commercial immunoradiometric "kit" assays for serum ferritin evaluated.
Authors: Dunn C D , Boden D J
Published: January 2020

We evaluated three commercially available immunoradiometric assays for serum ferritin, with particular emphasis on the statistical validity of the results. The data show that it was unusual (a) for dose/response relationships of the standards to be linear over the whole concentration range suggested by the manufacturers and (b) for dose/response relationships of test sera to parallel those of the standards. These findings cast doubt on the ability of any of the assays reliably to discriminate small differences in ferritin concentrations. Nevertheless, all three methods gave reproducible results, but this reflects technical expertise rather than accuracy of the results per se. The method based on liver ferritin as tracer detected less of the iron-binding protein than those based on spleen ferritin, despite significant cross reactivity of the two antibodies.
Feasibility of measuring free and total bilirubin electrochemically in serum.
Authors: Koch T R , Akingbe O O
Published: January 2020

Bilirubin undergoes irreversible reduction at the hanging-mercury-drop electrode in tris(hydroxymethyl)methylamine buffer (0.1 mol/L, pH 8.5). Peak current is linearly related to concentration in the range 0-1 mg/L. The half-wave potential of -1.32 V vs the saturated calomel electrode is little affected by pH, whereas peak current increases with pH to pH 9.5, then decreases rapidly. Exposing the test solution to light causes the peak current to decline at a rate proportional to concentration. Albumin-bound bilirubin is electrochemically inactive, so free bilirubin can be measured in the presence of bilirubin-saturated albumin. Serum total bilirubin can be measured after much of the protein is precipitated by adding methanol. Results for serum bilirubin as measured electrochemically (y) in 20 methanolic filtrates of serum compared well with the Jendrassik-Grof Method (x) in preliminary studies: y = 0.981x -0.311; r = 0.9716. We discuss the possible mechanism of the reduction process and potential clinical applications. Polarographic measurement of bilirubin and bilirubin-binding capacity is feasible and may be advantageous as compared with existing techniques.
Charcoal hemoperfusion for treatment of carbamazepine poisoning.
Authors: Chan K M , Aguanno J J , Jansen R , Dietzler D N
Published: January 2020

We report a case of carbamazepine (Tegretol) poisoning that was treated with charcoal hemoperfusion. When admitted, the patient was comatose and required ventilatory support. Hemoperfusion with coated activated charcoal successfully decreased her serum carbamazepine concentration from 60 mg/L to 36 mg/L within 2 h, with subsequent clinical improvement. Approximately 39 h would have been required to obtain this decrease via the patient's inherent elimination rate. Activated charcoal hemoperfusion may be useful as an adjunctive therapy for patients in advanced comatose states with associated respiratory depression and potentially fatal outcome.
Proteins of human semen. I. Two-dimensional mapping of human seminal fluid.
Authors: Edwards J J , Tollaksen S L , Anderson N G
Published: January 2020

The proteins in human seminal plasma were mapped by high-resolution two-dimensional electrophoresis (ISO-DALT and BASO-DALT systems). When analyzed under dissociating conditions, samples from normal fertile males revealed a pattern of over 200 proteins, ranging in mass from 10 000 to 100 000 daltons. Comparison of the mapped proteins from these males and those who had undergone vasectomy allowed us to identify one series of glycoproteins as missing from the semen from vasectomized individuals. Glycoproteins isolated by affinity chromatography with use of concanavalin A were also mapped. Some of the protein spots were identified either by co-electrophoresis with purified proteins or by the electrophoretic transfer of proteins to nitrocellulose sheets and subsequent detection by immunological procedures. The proteins identified include a number of serum proteins as well as prostatic acid phosphatase and creatine kinase. Proteolytic events shown to occur during the liquefaction of semen that occurs early after collection indicate the importance of carefully controlled collection and preparation methods for clinical evaluation of seminal plasma. Ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride inhibit this proteolysis.
Liquid-chromatographic determination of cyclosporin A in blood and plasma.
Authors: Sawchuk R J , Cartier L L
Published: January 2020

We describe a liquid-chromatographic assay for cyclosporin A in blood and plasma. The method is sensitive enough to allow quantitation of the drug at concentrations observed clinically, is isocratic, required no derivatization, and takes only 10 min of analysis time.
Protein clearances and selectivity determinations in childhood nephrosis: a reappraisal.
Authors: Ellis D , Buffone G J
Published: January 2020

To critically evaluate the clinical utility of determining specific proteins in patients with extensive proteinuria, we used immunonephelometric methods to measure albumin, transferrin, IgG, and alpha 2-macroglobulin in serum and in 24-h urine specimens from 37 children with idiopathic nephrotic syndrome. Renal biopsy demonstrated minimal change disease (I) in 15, focal glomerulosclerosis (II) in 15, and membranoproliferative glomerulonephritis (III) in seven patients. A three-group nonparametric rank test and three-group discriminant function analysis of the protein excretion and clearances of the four proteins we measured revealed significant differences in the excretion of IgG and the clearance of alpha 2-macroglobulin among the three groups of patients (p less than 0.05). Only patients with III had low serum complement C3 concentrations. Patients with I or II were best discriminated by differences in the excretion of transferrin and IgG, the clearance of alpha 2-macroglobulin, and the selectivity index (the clearance ratio of IgG/transferrin). These data indicate that measurement of specific urinary proteins and selectivity determinations may be helpful in predicting the type of histopathology and the prognosis of nephrotic children who have normal complement concentrations.
Sedimentation of lung-derived phospholipid during low-speed centrifugation of amniotic fluid.
Authors: Duck-Chong C G , Brown L M , Hensley W J
Published: January 2020

In most methods proposed for the assessment of fetal lung maturity, amniotic fluid is subjected to a preliminary low-speed centrifugation in an attempt to separate whole cells and cell debris from lung-derived surfactant phospholipid (lamellar body phospholipid). However, because lamellar body phospholipid is present in amniotic fluid in a membranous or particulate form, it is also partly sedimented by this procedure. The sedimentation of total phospholipid and lamellar body phospholipid by low-speed centrifugation has been quantitated for 49 samples of amniotic fluid from pregnancies of 30--41 weeks gestation. Isopycnic density-gradient centrifugation in a small air-driven ultracentrifuge was used to isolate lamellar body fractions from whole and centrifuged amniotic fluid. Centrifugation for 5 min at 1000 x g removed 0--70% of total phospholipid or lamellar body phospholipid, the mean values being 34 or 29%, respectively. There was an appreciable increase in lamellar body phospholipid relative to total phospholipid as a result of centrifugation in only 51% of the samples. In general, the effects of centrifugation were not related to gestational age of the fetus or the state of maturity of its lungs.
Early (chemical) diagnosis of bacterial meningitis--cerebrospinal fluid glucose, lactate, and lactate dehydrogenase compared.
Authors: Knight J A , Dudek S M , Haymond R E
Published: January 2020

Both lactate and lactate dehydrogenase are more sensitive as early indicators of bacterial meningitis than is glucose, and both appear to help differentiate aseptic from bacterial meningitis. In selected cases, lactate dehydrogenase may be more sensitive than lactate. We also give reference intervals for cerebrospinal fluid cell count, glucose, lactate, and lactate dehydrogenase.
Determination of sodium with ion-selective electrodes.
Authors: Levy G B
Published: January 2020

The advent of ion-selective electrodes made possible the potentiometry of sodium in serum and plasma. These methods were based on dilution of serum, as done in flame photometry, and the results were identical. Analysis of whole blood precludes dilution and so "direct" potentiometry was developed. Results by this technique are variable but tend to compensate for the spurious hyponatremias found by the "indirect" dilution methods due to displacement of volume by lipids and protein. However, there is no unambiguous theoretical basis on which to choose between the various direct ion-selective-electrode techniques and instruments. As an alternative, I propose use of current indirect methods, with numerical correction for the shift in normal sodium values in the presence of abnormal lipid and (or) protein. A table was constructed for making such corrections.
Adaptation of Prostatic-Group-Label Homogeneous Immunoassay to reagent-strip format.
Authors: Tyhach R J , Rupchock P A , Pendergrass J H , Skjold A C , Smith P J , Johnson R D , Albarella J P , Profitt J A
Published: January 2020

The Prostatic-Group-Label Immunoassay (PGLIA) technique has been incorporated into a reagent-strip format. We report use of flavin N6-(N'-2,4-dinitrophenyl-6-aminohexyl)adenine dinucleotide (DNP-FAD) as the prosthetic group derivative and 6-N-(2,4-dinitrophenyl)aminohexanoic acid (DNP-caproate) as the competing ligand. DNP-FAD not bound by antibody combines with glucose oxidase apoenzyme, which then reacts with glucose and oxygen, and gives color through a peroxidase-linked system. The rate of color generation is thus a function of the DNP-caproate concentration. PGLIA reagent strips are prepared by sequential impregnations of filter paper with an acetone solution of indicator (3,3',5,5'-tetramethylbenzidine); an aqueous solution containing glucose oxidase apoenzyme, the rest of the color generation system, stabilizers, and antibody to DNP; and a solution of DNP-FAD in n-propanol. This preparation permits effective antibody binding, and prevents premature interaction of immunoassay components. A quantitative color response to concentrations of DNP-caproate in the range of 1 to 8 mumol/L was demonstrated with these reagent strips. Prototype PGLIA reagent strips for theophylline and phenytoin have been successfully developed by substituting the appropriate FAD derivative and antibody for the corresponding reagents in the DNP model system.
A flexible, computer-controlled video microscope capable of quantitative spatial, temporal, and spectral measurements.
Authors: Rich E S , Wampler J E
Published: January 2020

A video microscope system has been constructed and tested that incorporates computer-controlled video cameras for high-resolution and low-light microscopy. The low-light camera system involves a dual microchannel plate-image intensifier capable of photon gain as high as 500 000 and a gated silicone-intensified target vidicon to achieve usable photon sensitivity with a noise equivalent signal of only 2 photons (500 nm) per pixel per second. We have compared the limitations and capabilities of this camera system with those of a high-resolution video camera and conventional photomicroscopy. Uses of the low-light camera coupled to a computer system include image acquisition of weak-light images from self-luminous specimens, fluorescence microscopy with weak exciting light, kinetic resolution of calcium-mediated events as monitored by the calcium-sensitive bioluminescence of aequorin, and spatially resolved spectroscopic measurements. Flexible use of this system in these various applications is possible because it allows operation with illumination intensities over a dynamic range of 100 000:1.
Fluorescence polarization immunoassay. iii. an automated system for therapeutic drug determination.
Authors: Jolley M E , Stroupe S D , Schwenzer K S , Wang C J , Lu-Steffes M , Hill H D , Popelka S R , Holen J T , Kelso D M
Published: January 2020

A fully automated system for performing fluorescence polarization immunoassay has been developed. Reagents for each assay are contained in coded reagent packs, and no reagent reconstitution is required. A common buffer is used for all assays, minimizing changeover and set-up times for each assay. A single sample may be assayed in 5 min, or 20 samples in 10 min. A single-tube blank subtraction for each sample results in highly precise polarization values and obviates sample interferences. We have used this method for assays of gentamicin, theophylline, phenytoin, and phenobarbital. CVs are 1-4%, and the results correlate well with those by other methods. Because of the instrument design and the stability of the reagents, daily calibration is not required; samples may therefore be run immediately upon receipt or batched as desired.
The hybridoma-an immunochemical laser.
Authors: David G S , Wang R , Bartholomew R , Sevier E D , Adams T H , Greene H E
Published: January 2020

The advent of hybridoma technology has provided the immunotechnologist a finely tunable instrument that should permit a marked advance in the immunologic sciences. The ability to choose the precise antibody required and the virtually unlimited availability of easily purified antibodies have already resulted in the simultaneous immunometric assay and potential reagents for immunoscintigraphy and immunotherapy. Using an antibody with a specific affinity and recognition for a specific antigenc determinant can greatly influence the shape and range of a radioimmunometric calibration curve. With monoclonal antibodies, assay systems based on immune complexes (e.g., turbidimetric assays and counterimmunoelectrophoresis) can be made more precise, thus permitting study of the basic physicochemical principles underlying the antigen-antibody reaction and the development of greatly improved quantitative assays. On the other hand, the ability to select an antibody exhibiting specific characteristics implies the necessity to select. One no longer has the luxury of using a mixture of antibodies, hoping to take advantage of the fact that some will have desirable secondary characteristics such as electrophoretic mobility or the ability to absorb to plastics.
Use of monoclonal antibodies for quantitative analysis of antigens in normal and neoplastic tissues.
Authors: Brown J P , Hellström K E , Hellström I
Published: January 2020

We describe two methods of analyzing tissues for cell-surface antigens identified by monoclonal antibodies. The first method is a binding assay, in which a membrane fraction prepared by differential centrifugation of a tissue homogenate is incubated with 125I-labeled antibody. The specificity of the observed binding is established by competition with unlabeled antibody. The second method, double-determinant immunoassay, involves two monoclonal antibodies that recognize two distinct epitopes of the antigen molecule. An immunoabsorbent prepared from one of the antibodies is incubated with a detergent lysate of the tissue. The immunoabsorbent is then washed and incubated with the 125I-labeled second antibody. In both assays the amount of radiolabeled antibody bound is proportional to the amount of antigen in the test sample, and the limit of detection is approximately 10 pg of antibody, corresponding to less than 1 fmol of antigen.