bluuebunny/crossref_metadata_2025 huggingface dataset:
Interference by hemolysis with various methods for total calcium and its correction by trichloroacetic acid precipitation.
Authors: Chan K M , Arriaga C , Landt M , Smith C H , Ng R H
Published: January 2020

We evaluated the influence of hemolysis on total serum calcium as determined with the Du Pont aca, SMA 12/60, Ektachem 400, Corning 940 EGTA titrator, and the Beckman Astra 8, comparing results with those obtained by atomic absorption spectroscopy. We find that hemoglobin does not influence calcium measurements with the SMA 12/60, Ektachem 400, or Beckman Astra 8. The presence of hemoglobin exceeding 2 to 3 g/L caused falsely high results with the aca and falsely low results with the Corning 940 titrator. Similar interferences may be observed with other titrating or colorimetric procedures that involve direct reaction of the sample with o-cresolphthalein complexone or calcein. Upon removal of the hemoglobin by precipitation with trichloroacetic acid, the values obtained with the aca and the Corning 940 EGTA titrator were similar to those measured by atomic absorption. With nonhemolyzed serum samples the acid treatment had little or no effect on the aca procedure but resulted in a positive bias of approximately 10% with the EGTA procedure. Thus we recommend this trichloroacetic acid procedure for measuring calcium in hemolyzed samples with the aca and, with certain reservations, with the EGTA titrator.
An inter- and intralaboratory quality-control survey of radioimmunoassay of insulin, thyroxin, thyrotropin, cortisol, digoxin, gastrin, beta 2-microglobulin, and IgE in Japan with commercially available kits.
Authors: Shishiba Y , Irie M , Yamada H , Kinoshita F
Published: January 2020

We report results of a quality-control survey of radioimmunoassay (RIA) of eight analytes, participated in by 249 laboratories of various types throughout Japan. For the purpose of the external control survey, lyophilized specimens containing two different concentrations of each analyte were sent to the participating laboratories, which supplied both their assay values and their raw data for the standard curves and for the samples. The individual data for the standard curve were analyzed by the RIA data-processing program described by Faden and Rodbard. The precision profile obtained for the individual assay was used as the estimate of the quality of each assay. Although there was a wide scatter in assay values, the quality of the assay by the individual laboratories was not the major contributor to this scattering, because the values they reported for the assays and precision profiles of the standard curves were almost identical with those obtained from the kit manufacturers. When the reported assay values were analyzed according to the brand of kits, by analysis of variance, the treatment mean square due to difference in the make of the kits significantly exceeded the error mean square due to "within-kit" variation in the assay of insulin, thyroxin, thyrotropin, cortisol, gastrin, and digoxin. The CVs for "between-kit," "within-kit," "between-assay" and "within-assay" variations (the latter two aspects were studied in a representative laboratory, for convenience of comparison) were about 20, 15, 13, and 7%, respectively. Because the observed within-kit variation is of almost the same magnitude as that expected from the combination of within-assay and between-assay variation, the skill of the users probably was equal and the accuracy and precision of the kits probably had the greatest impact on assay variation. This survey also emphasizes the criteria for commercially available RIA kits.
Free triiodothyronine and free thyroxin in sera of pregnant women and subjects with congenitally increased or decreased thyroxin-binding globulin.
Authors: Franklyn J A , Sheppard M C , Ramsden D B , Hoffenberg R
Published: January 2020

Concentrations of free thyroxin (T4) and free triiodothyronine (T3) were measured in sera from pregnant women, in subjects with congenitally increased or decreased thyroxin-binding globulin (TBG), and in euthyroid controls. Measured free hormone concentrations were compared with calculated values for free hormone derived from concentrations of total T4, total T3, and binding proteins. Measured and calculated concentrations of free T4 and free T3 were below normal in the pregnant subjects but were normal in subjects with congenital increases of TBG. The low concentrations of free hormone in pregnancy are at variance with the euthyroid status of these subjects. Measured free T4 was normal, but concentrations of free T3 were less in the euthyroid congenitally low-TBG group.
Development of a screening system for cystic fibrosis.
Authors: Coury A J , Fogt E J , Norenberg M S , Untereker D F
Published: January 2020

We have developed a simple method for detecting high concentrations of chloride in sweat from ambulatory subjects, a measurement useful in the detection of cystic fibrosis. The method is based on the standard approach of stimulating sweat generation through iontophoresis of pilocarpine nitrate into the skin, followed by collection and analysis of the sweat for chloride concentration. The sweat-stimulating reagents are contained in polymeric gel pads, which are used in conjunction with a small battery-powered stimulator. The chloride analysis is subsequently done on the stimulated site by use of a thin test patch that picks up a fixed amount of sweat and changes color if the chloride concentration is higher than a predetermined value. The successful completion of a test is indicated by a fill tab, which changes color when the appropriate amount of sweat has been picked up by the chloride test patch.
Homogeneous immunoassay based on chemiluminescence energy transfer.
Authors: Patel A , Campbell A K
Published: January 2020

The chemiluminescent compound aminobutylethyl-isoluminol (ABEI) and its isothiocyanate derivative have been coupled to a range of haptens (progesterone, cyclic AMP, cyclic GMP) and protein antigens (IgG, C9). All the derivatives were chemiluminescent, immunologically active, and stable for more than six months. When the ABEI-labeled antigens bind to their respective fluorescein-labeled antibodies, there is a shift in the ratio of chemiluminescence at 460 nm (blue) to that at 525 nm (green). This nonradiative energy transfer was used to establish homogeneous immunoassays, which require no separation step. These assays were at least as sensitive as the conventional radioimmunoassays and could accurately measure substances in serum (i.e., for IgG, results correlated by r = 0.96 relative to those by 125I assay) and tissue extracts (cyclic AMP, r = 0.91 relative to 3H assay), and also were used to evaluate the kinetics of antibody-antigen binding. Chemiluminescence energy transfer provides a new method for quantifying ligand-ligand interactions in the 10(-15) to 10(-18) mol range without first separating bound and free ligand. This provides a unique opportunity to investigate chemical events in single cells and intact cells.
Trace metal analysis by laser-enhanced ionization in flames.
Authors: Travis J C , Turk G C , DeVoe J R
Published: January 2020

We review atomic flame spectrometry of metals based on the selective laser-enhancement of the rate of atomic ionization. A discussion of basic principles, prior literature, and instrumentation is followed by an overview of analytical figures of merit for the technique, and a look at its present status and areas for future development.
Stabilities of some constituents of marmoset (Callithrix jacchus) plasma under various conditions of storage.
Authors: Davy C W , Jackson M R , Walker J M
Published: January 2020

The stability of various marmoset (Callithrix jacchus) plasma constituents was investigated after storage at room temperature, 4 degrees C, and -20 degrees C. The method of sequential analysis ensured that the between-run bias of the methods of analysis used was drastically reduced, and the definitions of stability were linked to the imprecision of these methods. Optimal conditions for storage for as long as 48 h depended on the analyte being measured. Room temperature was optimal for cholinesterase and acetylcholinesterase; 4 degrees C for protein, albumin, alanine aminotransferase, isocitrate dehydrogenase, sorbitol dehydrogenase, lactate dehydrogenase, and glutamate dehydrogenase; and -20 degrees C for glutathione reductase and alkaline phosphatase. For aspartate amino-transferase and gamma-glutamyltransferase, either 4 degrees C or -20 degrees C would be suitable. Reasons are advanced for some conflicting reports in the published work, and we emphasize the need to investigate each analyte and species separately.
A screening method for biotinidase deficiency in newborns.
Authors: Heard G S , Secor McVoy J R , Wolf B
Published: January 2020

We describe a method for neonatal screening for biotinidase (EC 3.5.1.12) deficiency. Biotinidase activity is assessed colorimetrically from dried samples of whole blood spotted on the same filter papers as used in the neonatal screening for phenylketonuria. After the reaction, samples from normal infants are characteristically purple, whereas those from affected individuals are straw-colored. To confirm the deficiency, the enzyme is quantitatively assayed in additional blood spots or serum. A pilot study has been initiated with samples obtained by the Commonwealth of Virginia for phenylketonuria testing.
Clinical usefulness of estriol assay for predicting "light-for-dates" infants.
Authors: Evans J J , Wilkinson A R , Aickin D R
Published: January 2020

A method was developed for determining clinical usefulness of a test for predicting "light-for-dates" infants before birth. The method was applied in a prospective study of assay of maternal estriol at 35-36 weeks of gestation as a means of identifying such infants. With the tenth centile for estriol values as the cutoff, low estriol values identified less than a third of all light-for-dates infants, and less than a third of pregnancies with low estriol values were associated with births of light-for-dates infants. Our reanalysis of data from three other published studies, using the same method of assessment, gave similar results. When laboratory tests are expected to provide clinically relevant information, we urge that data should be appropriately collected, analyzed, and reported, so clinical usefulness can be evaluated.
Influence of substrate concentration on the activation of transketolase, aspartate aminotransferase, and glutathione reductase by coenzymes.
Authors: Hafkenscheid J C , van Dijk C M
Published: January 2020

We investigated the mechanism by which the three most commonly measured enzymes in erythrocytes are activated by their respective coenzymes by determining the catalytic activity concentrations of transketolase (EC 2.2.1.1), aspartate aminotransferase (EC 2.6.1.1), and glutathione reductase (EC 1.6.4.2) in relation to various substrate concentrations. We conclude that the underlying mechanisms by which the enzymes are activated are not the same.
Results of a nationwide survey of analyses for creatine kinase and creatine kinase isoenzymes.
Authors: Boone D J , Duncan P H , MacNeil M L , Smith B F , Houston B , Hearn T L
Published: January 2020

More than 300 laboratories participated in an interlaboratory survey of creatine kinase (CK, EC 2.7.3.2) determinations in which they analyzed seven lyophilized samples for total CK and CK isoenzymes and furnished information about their methodology. The samples were not necessarily intended to mimic typical patients' specimens but rather to determine the analytical ability of the laboratories to distinguish isoenzyme fraction CK-MB from CK-BB and to detect small but abnormal amounts of CK-BB. For total CK measurement, most laboratories used an NADP+ reduction method monitored at 340 nm (89%), and reported results in units per liter (U/L) (99%) at either 30 degrees C (34%) or 37 degrees C (60%). Despite the variety of analytical conditions, most laboratories (89%) correctly reported results within their normal range for all samples. The 287 laboratories that reported isoenzyme distributions in the samples used either cellulose acetate (37%) or agarose (44%) electrophoresis, ion-exchange chromatography (9%), or immunoinhibition (7%). Results from laboratories that used nonspecific CK-MB immunoinhibition techniques were biased when a significant amount of CK-BB isoenzyme was present.
On the interpretation of serial laboratory measurements in acute myocardial infarction.
Authors: Albert A , Harris E K , Chapelle J P , Heusghem C , Kulbertus H E
Published: January 2020

Serial laboratory determinations are now routinely performed on patients admitted to intensive-care units. Adequate interpretation of such cumulative information for clinical decision-making purposes is a challenging problem. We describe a statistical method for predicting--sequentially as the data become available--the patient's outcome, death or survival. Thus, the method goes beyond previously reported techniques that base such prediction on only a single multivariate observation. The method has been applied to daily measurements of serum urea and lactate dehydrogenase, performed during one week on patients hospitalized in the coronary-care unit with acute myocardial infarction. Two baseline variables were also included in the dynamic risk index so derived: the age of the patient and the number of previous myocardial infarctions recorded on admission. We also discuss the problems of selecting the most-predictive laboratory tests and of determining for each test the amount of past data needed to achieve satisfactory prediction. We distinguish between global evaluation of the dynamic risk index obtained (in terms of specificity and sensitivity) and individual interpretation (in terms of posterior/prior probability ratio) of a given risk score for a particular patient. The approach described may contribute to more effective use of results of repeated laboratory tests on critically ill patients.
Quantification of alprazolam in serum or plasma by liquid chromatography.
Authors: McCormick S R , Nielsen J , Jatlow P
Published: January 2020

A reversed-phase "high-performance" liquid-chromatographic assay for the quantification of alprazolam in serum or plasma is described. Serum or plasma is extracted with toluene/isoamyl alcohol (99/1 by vol), evaporated, and reconstituted in the mobile phase. The latter is washed with hexane, then subjected to reversed-phase liquid chromatography and ultraviolet detection at 202 nm. Either U-31485, an alprazolam analog, or lorazepam, a 3-hydroxybenzodiazepine, is satisfactory as internal standards. Major alprazolam metabolites and various other commonly used drugs do not interfere. The useful lower limit of sensitivity for quantification is 2.5 micrograms/L. Peak height and alprazolam concentration are linearly related from 2.5 to 100 micrograms/L. For 10 and 20 micrograms/L concentrations, within-run CVs were 1.4% and 0.9% and the between-runs CVs 4.8% and 3.2%. Steady-state serum concentrations ranged from 25 to 55 micrograms/L in patients taking 1.5 to 6.0 mg per day, orally. Preliminary data suggest the method is also suitable for analysis of the structurally similar triazolobenzodiazepine, triazolam.
Isolation and characterization of cancer-associated galactosyltransferase isoenzyme.
Authors: Ram B P , Munjal D D
Published: January 2020

We isolated galactosyltransferase (EC 2.4.1.22) from pleural effusions of a lung cancer patient and a patient with cirrhosis by precipitation with ammonium sulfate, followed by gel filtration on Sepharose 6B, and affinity chromatography on columns of alpha-lactalbumin-agarose and protein A-Sepharose. By this procedure the enzyme from both sources was purified 40 000-fold with approximate yields of 37% and 60%, respectively, and did not contain immunoglobulin. Electrophoresis on polyacrylamide gel of the enzyme from the cancer patient (slower moving) and from the non-cancer patient (faster moving) gave one sharp band for each. Their respective relative molecular masses, 74 131 and 107 151, were estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and gel filtration, respectively. The isoenzymes were active between pH 5 and 8, most active at 7, and showed no activity below pH 4 or above pH 9. Activity was greatest at temperatures between 37 and 40 degrees C. At 30 degrees C or 50 degrees C the activity was more than halved, and was lost completely above 60 degrees C. The isoenzymes had an absolute requirement for Mn2+. Omitting the surfactant Triton X-100 from the buffer resulted in considerable loss in activity of both isoenzymes. Glucose can be used as an acceptor for these isoenzymes if alpha-lactalbumin is present in the assay mixture. These isoenzymes had different Km values for UDP-galactose, N-acetylglucosamine, and Mn2+.
Serum copper concentration significantly less in abnormal pregnancies.
Authors: Buamah P K , Russell M , Milford-Ward A , Taylor P , Roberts D F
Published: January 2020

We estimated copper concentration in maternal serum during 244 normal and 15 abnormal pregnancies. Values were lower in the abnormal pregnancies than in the normal ones, and did not vary with gestational age between 15 to 18 weeks in normal pregnancies.
Automated continuous-flow radioimmunoassay for salivary estriol.
Authors: Truran P L , Read G F
Published: January 2020

We have automated a radioimmunoassay for salivary estriol by using the "Southmead" continuous-flow system. The assay, based on a well-validated manual assay, involves a Sepharose-coupled antiserum and an 125I-radioligand. The antibody-bound and free radioligand fractions are separated by on-line filtration. Performance of the automated assay has been reliable during six months of regular use, and compares favorably with that of the manual assay. Drift (0.5 nmol/L per 100 samples) and sample carryover (3%) are acceptable, and the throughput rate is 55 samples per hour. The system is suitable for the assay of the large numbers of samples generated by daily saliva specimen collection in the third trimester of pregnancy.
Aberrant creatine kinase and lactate dehydrogenase response in a myocardial infarct patient.
Authors: Smalley D L , Womack B , Handorf C , Acchiardo S
Published: January 2020

A 65-year-old woman failed to develop increased creatine kinase or lactate dehydrogenase activity after a myocardial infarction. She had no measurable creatine kinase MB isoenzyme and no detectable patterns of normal LD isoenzyme activity. These determinations five months after the infarction showed normal values for total activity and isoenzyme patterns.
Biclonal IgA and IgM gammopathy in lymphocytic lymphoma.
Authors: Weinstein S , Jain A , Bhagavan N V , Scottolini A G
Published: January 2020

We report the case of a 76-year-old white man with a diffuse, well-differentiated lymphocytic lymphoma at the base of his tongue. Although serum electrophoresis, immunoelectrophoresis, and immunofixation showed he had a biclonal IgA kappa and IgM kappa gammopathy, biopsy of the tumor showed a positive immunoperoxidase response only for IgM kappa. The biclonal pattern persisted after chemotherapy, despite shrinkage of the tumor mass. The association of IgA and IgM appears to be the least frequent combination of separate biclonal immunoglobulins, the clinical course of such patients being more often that of lymphoma or macroglobulinemia than of myeloma. However, the symptomatology can be highly variable, as our case uniquely demonstrates.
Operational research and cost containment: a general mathematical model of a workstation.
Authors: Winkel P
Published: January 2020

To facilitate planning and management, I have derived a mathematical model describing the dynamics of a workstation that receives a mixture of routine and emergency specimens. The model parameters include the maximal specimen-processing rate (to be defined by personnel representatives) and the longest delay allowed for emergency specimens at the workstation. Based on the model, a computer-simulation technique has been developed to maximize the length of time (planned pauses) during which the workstation could be closed without causing undue delay of routine and emergency specimen results. The application of this technique is illustrated with real data, in which more than 50% of the specimens were emergency specimens. Three pauses, constituting 35% of working hours, could be introduced with a negligible impact on the turnaround time of emergency specimens (mean increase = 8 min). The model may also be used to derive, as a function of time of day, the largest extra workload that may be presented to a workstation without creating overwork. The workload could be increased by 45%, provided that all additional specimens arrived before noon.
Determination of free thyroxin in serum by ultrafiltration: validation of a method and preliminary results.
Authors: Shannon N , Woolf P D
Published: January 2020

We describe the determination of concentrations of free thyroxin in serum by ultrafiltration based on the Amicon micropartition system. The assay is straightforward and requires about 4 h for 25 samples. Our results demonstrate a correlation coefficient of 0.81 compared with equilibrium dialysis, and of 0.88 compared with radioimmunoassay. For sera from 49 normal, 25 hyperthyroid, 24 hypothyroid, 50 euthyroid sick, and 13 pregnant subjects, the means (and SD) were respectively: 19.3 (4.9), 40.1 (13.6), 5.0 (2.9), 25.9 (10.5), and 16.1 (3.2) ng/L. The interassay CV was 9.9% and the intra-assay CV 7.2%. We conclude that this procedure is useful in the diagnosis of thyroid disease and offers advantages of simplicity, rapidity, and economy.
Automated calmagite compleximetric measurement of magnesium in serum, with sequential addition of EDTA to eliminate endogenous interference.
Authors: Liedtke R J , Kroon G
Published: January 2020

We describe an automated method for magnesium assay, for use with the Cobas-Bio centrifugal analyzer. Magnesium is reacted with calmagite, a dye, and the absorbance of the magnesium-dye complex at 520 nm is measured. EDTA is then added to break up the complex and the absorbance at 520 nm is re-measured to correct for sample color and turbidity. Including ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and triethanolamine in the dye reagent eliminates interference by calcium and iron. Within-run CVs were less than 2.0% for concentrations of magnesium ranging from 18 to 40 mg/L, and less than 4% for a magnesium concentration of 9.4 mg/L. Day-to-day precision data, determined over five months were: mean = 21.0 mg/L, CV = 2.7%; mean = 43.0 mg/L, CV = 3.2% (n = 550 for both). Comparison of the Cobas-Bio method (y) with an atomic absorption spectrometric method (x) gave the following results: y = 0.968x + 0.448, r = 0.986, Sy/x = 0.34 mg/L, mean x = 19.8 mg/L, mean y = 19.6 mg/L, n = 44. Hemoglobin, bilirubin, and turbidity do not interfere. The standard curve is linear up to a magnesium concentration of 97 mg/L.
Combined immunochemical and electrophoretic determinations of proteins in paired serum and cerebrospinal fluid samples.
Authors: Papadopoulos N M , Costello R , Kay A D , Cutler N R , Rapoport S I
Published: January 2020

Albumin and immunoglobulin G were simultaneously determined in paired serum and cerebrospinal fluid samples from healthy volunteers. The albumin quotient and the IgG index were calculated to establish normal values. High-resolution electrophoresis was also performed with the same samples and the electrophoretic patterns were correlated with the numerical values to improve the reliability of the test results. Representative examples from abnormal conditions are also shown, to illustrate the increased accuracy and useful applications of this combined analysis in the diagnosis of neurological conditions.
Acid-base-induced changes in the distribution of water between plasma and erythrocytes, as measured with a sodium-ion-selective electrode.
Authors: Fogh-Andersen N , Siggaard-Andersen O
Published: January 2020

aNa+ measured in whole blood changes with the pH of plasma, and the effect depends on the erythrocyte volume fraction (phi E). The relative change in aNa+ with pH, dlnaNa+/dpH, was -0.01 at phi E = 0 and -0.11 at phi E = 0.76. The observations agreed with the combined predicted effects of changes in phi E, in concentration of the NaHCO3 complex, and in liquid-junction potential. The pH-induced osmotic transfer of water between plasma and erythrocytes will affect the concentration of all components confined to plasma. Although the clinical importance of this may be limited, blood samples for research should either be dealt with immediately or stored anaerobically at 4 degrees C until separation, to avoid bias.
A new technique for studying the relationship between maternal diabetes and the sialic acid content of fetal pulmonary surfactant.
Authors: Delahunty T
Published: January 2020

By adapting a standard method for precipitation of high-density lipoprotein cholesterol with phosphotungstic acid (PTA) and Mg2+, fetal pulmonary surfactant can be rapidly isolated from human amniotic fluid, 97% of the total disaturated phosphatidylcholine being precipitated from the sample. The lecithin/sphingomyelin ratio for 17 separate specimens correlated reasonably well (r = 0.76) with the concentration of disaturated phosphatidylcholine in the PTA precipitate. Using thiobarbituric acid as the chromophore, I measured sialic acid in the PTA precipitate after overnight treatment with neuraminidase. The sialic acid/protein ratio for the PTA precipitate was identical to that for the surfactant, as isolated by ultracentrifugation. The concentrations of insulin and C-peptide were significantly greater in specimens of amniotic fluid from mothers with diabetes than from non-diabetic mothers (p less than 0.001). When the specimens were segregated according to a C-peptide cutoff value of 4 micrograms/L, there was a small, significant decrease in PTA-precipitated concentrations of sialic acid in the samples with C-peptide greater than 4 micrograms/L. The results suggest a possible mechanism for the increased incidence of respiratory distress among infants born to diabetic mothers.
An evaluation of kits for measuring thyrotropin in newborns.
Authors: Pierson E A , Therrell B L , Franz S F , Rudenstein A J , Dziuk P E
Published: January 2020

Several commercial radioimmunoassay kits are now marketed specifically for determination of thyrotropin (TSH) from whole-blood specimens collected on filter paper in neonatal screening programs. We have compared five kits in use in such screening programs in the United States. The reagent kits from Becton Dickinson, Neometrics, and Nuclear Medical Systems gave similar satisfactory results. That from Pharmacia was somewhat more difficult to use and gave greater coefficients of variation. Diagnostic Products' kit appeared to perform satisfactorily, but the analytical values obtained were significantly low, which may suggest erroneous calibrator values within the kit.
Reactivation of serum creatine kinase isoenzyme BB in patients with malignancies.
Authors: Abbott L B , Lott J A
Published: January 2020

Reactivation of serum creatine kinase isoenzyme BB (CK-BB) with 2-mercaptoethanol and EDTA increased the electrophoretic detection rate of CK-BB from 34% to 78% in 58 hospitalized patients with various malignancies. Patients with solid tumors showed the largest and patients with hematologic malignancies the smallest percentage increase in CK-BB after reactivation. For serum from 50 hospitalized patients without cancer, reactivation resulted in detectable CK-BB in two patients; the CK-BB band was never seen in 15 healthy adults. For reasons unknown, five of eight patients with senile dementia of the Alzheimer's type showed CK-BB in serum after reactivation, as did two of five patients suspected of having this disorder. Serum CK-BB may be a useful tumor marker if reactivation with a thiol and EDTA is used immediately after collection.
Particle-enhanced turbidimetric inhibition immunoassay for theophylline evaluated with the Du Pont aca.
Authors: Opheim K E , Glick M R , Ou C N , Ryder K W , Hood L C , Ainardi V , Collymore L A , DeArmas W , Frawley V L , Hutchinson J
Published: January 2020

We evaluated the Du Pont Particle-Enhanced Turbidimetric Inhibition Immunoassay (PETINIA) for theophylline. The imprecision (CV) of the assay was less than 4.7% between-run and less than 3.6% within-run for theophylline concentrations between 5 and 30 mg/L. Standard curves for the assay were linear for theophylline concentrations from 0 to 46 mg/L and were stable throughout the study (i.e., for at least three months). The monoclonal antibody against theophylline used in this assay increases specificity; of the possibly interfering drugs, metabolites, and anticoagulants tested, only 1,3-dimethyluric acid and EDTA showed measurable effects. Bilirubin (less than 300 mg/L), hemoglobin (less than 6 g/L), or lipemia (triglycerides less than 6 g/L) does affect the quality of the assay. Analytical recovery of theophylline added to serum (5 to 40 mg/L) averaged 98% (range 93% to 112%). Comparison of results for patients' sera by the PETINIA method with those by enzyme immunoassay (EMIT) and by "high-performance" liquid chromatography yielded slopes and intercepts not significantly different from 1.0 and 0.0, respectively, and correlation coefficients ranging from 0.986 to 0.995.
Two-dimensional electrophoretic analysis of cytosols from human breast tumors: optimal migration conditions.
Authors: Motté P , Bidart J M , Delarue J C , Comoy E , Moingeon P , Bohuon C
Published: January 2020

In an examination of cytosols from human breast tumors, we performed two-dimensional electrophoretic analysis. Several migration conditions were tried in a search for a homogeneous repartition of cytosolic proteins. The most workable gels were obtained with a 4.40 to 8.05 pH gradient in the first dimension and a homogeneous 125 g/L acrylamide/sodium dodecyl sulfate gel, 1 mm in thickness, in the second dimension. The practicability of this method is discussed.
Protein-pattern changes and morphological effects due to methionine starvation or treatment with 5-azacytidine of the phorbol-ester-sensitive cell lines HL-60, CCL-119, and U-937.
Authors: Anderson N L , Gemmell M A
Published: January 2020

Methionine starvation causes changes in the protein pattern of HL-60 promyelocytic leukemia cells as observed by two-dimensional electrophoresis. One group of proteins is apparently modified, appearing in new positions. A further series of proteins, including several principal nuclear polypeptides, is substantially diminished. The morphology of a fraction of the cells in the culture changes concomitantly, with condensation and fragmentation of the nucleus and eventual remolding of the cell to a "grape-cluster" appearance. Similar effects are produced by a DNA methylation inhibitor, 5-azacytidine, but not by various other toxic agents tested. A defect in DNA methylation, either by depletion of S-adenosyl-L-methionine (the methyl donor) or by inactivation of the relevant enzyme, may be responsible. The T-lymphoblastoid line CCL-119 and the histiocytic lymphoma line U-937 also show these effects, but most fibroblast, epithelial, and lymphoblastoid lines do not. These changes can be largely prevented in each of the three susceptible lines by prior treatment with the tumor promoter, phorbol myristate acetate (PMA), an agent known to cause differentiation in at least two of the lines. The results thus suggest interesting relationships between methionine metabolism, protein and structural changes in the cell nucleus, and PMA-induced cell differentiation.
Spot identification in two-dimensional patterns by a least-squares template matching.
Authors: Pardowitz I , Zimmer H G , Neuhoff V
Published: January 2020

The coordinates of the center of measured spots are nonlinearly transformed to get an optimal match between the transformed coordinates and the given coordinates of a reference pattern. The parameters of the transformation are determined by the minimum of a function of squared distances between all spots of the sample and of the reference pattern. The algorithm requires a priori defined correspondences between some pivot points in the sample and the reference and treats their distances differently from the others. The parameters of the transformation are the solutions of a system of nonlinear equations; their numerical values are obtained by iteration.
Computerized comparison of two autoradiographs.
Authors: Hruschka W R
Published: January 2020

Our laboratory previously reported (Anal Chem 55: 2345-2348, 1983) the development of a digitizer and FORTRAN program for display and quantification of digitized autoradiographic images. We have since added several capabilities to the program. Coarse "rubber sheeting" (i.e., transforming coordinates to superimpose images) of the image by a quadratic transformation of pixel coordinates is developed from interactive identification of a few matching spots. The program determines thresholds to display a given proportion of the total protein in the image, or a given number of proteins, and it creates a protein list containing the spatial coordinates of each protein with a peak value above this threshold, together with the amount of the protein as measured by the ratio of its integrated intensity above its half-maximum to the total integrated intensity of the image. The lists can be stored on a disk and are used as the primary data for fine adjustments to superimpose images, quantitative comparison of images, and abstract display of proteins. Methods are discussed for presenting comparisons between gels in cases where spots for many proteins change in intensity.
Two-dimensional electrophoresis used to differentiate the causal agents of American tegumentary leishmaniasis.
Authors: Saravia N G , Gemmell M A , Nance S L , Anderson N L
Published: January 2020

American tegumentary leishmaniasis is caused by a diverse group of Leishmania classified within two species complexes: L. mexicana and L. braziliensis. Because distinct disease forms are associated with certain species or subspecies, prognosis requires taxonomic identification of the pathogen. Biological criteria allow identification only to the species level; thus, biochemical and immunological markers are eagerly sought. We have used two-dimensional electrophoresis to examine the relationships among reference strains of New World Leishmania and stocks isolated from Colombian patients. The L. mexicana and L. braziliensis species complexes are shown to be extremely disparate, and the relative affinities of the subspecies reference strains and Colombian Leishmania stocks are documented. The latter displayed a variety of patterns--some clearly identifiable with a particular L. braziliensis subspecies reference strain, others intermediate between those of L.b. panamensis and L.b. guyanensis. Such comparisons are useful both in establishing relatedness and identifying subspecies and variant marker proteins, which may have biological significance.
A "high-performance" 2D gel scanner.
Authors: Kronberg H , Zimmer H G , Neuhoff V
Published: January 2020

We describe the design principles of a photometric flatbed scanner with a scan area of 250 X 250 mm2, a dynamic range of 4000 gray levels, a signal/noise ratio of 2000/1, a step size of 0.1 mm, and a step frequency of 2000 steps per second. It is controlled by a microcomputer and used to acquire data for quantitative evaluation of gels or chromatograms. The performance of the instrument is demonstrated by the data obtained by scanning gratings, gray step filters, and high-resolution electropherograms.
Myosin light chain phosphorylation and tension development in stretch-activated arterial smooth muscle.
Authors: Ledvora R F , Bárány M , Bárány K
Published: January 2020

Phosphorylation of the 20 000-Da light chain of myosin in functionally different porcine carotid arteries was determined, with use of two-dimensional gel electrophoresis. Stretching arteries to 1.7 times their resting length resulted in maximal phosphorylation. Intracellular Ca2+ was mobilized for stretch-induced light chain phosphorylation. Releasing the stretch from the arteries produced active tension spontaneously, without the participation of any exogenous stimulating agent. Prolonged treatment of arteries with a chelating agent (EGTA) not only abolished stretch-induced phosphorylation, it also prevented the development of active tension when the stretch was released. However, when the EGTA was washed out and the strips were restretched and again released, the stretch-induced phosphorylation and the stretch-release-induced active tension were restored. Evidently, arteries must contain phosphorylated light chain if they are to produce active tension. The myosin light chain became partly dephosphorylated in arteries that developed active tension when the stretch was released, but more than half of the light chains remained phosphorylated. This result suggests that phosphorylation of light chain is involved, not in the generation of active tension, but rather in activation of smooth muscle.
Molecular characterization of an abnormal fibrinogen by two-dimensional electrophoresis.
Authors: Polack B , Valiron O , Concord E , Freyssinet J M , Hudry-Clergeon G
Published: January 2020

We examined normal and abnormal fibrinogen (fibrinogen "Grenoble") by two-dimensional gel electrophoresis to obtain data on possible defects at the molecular level. Fibrinogen Grenoble is characterized by an abnormal rate of fibrin monomer aggregation. The electrophoretic analysis revealed the presence of abnormal gamma chains. Two kinds of gamma chains can be detected in fibrinogen Grenoble: (a) normal gamma chains and (b) gamma chains Grenoble (gamma G) with a greater molecular mass but no modification in isoelectric point. The latter chain can be detected in whole plasma by two-dimensional gel electrophoresis. Metrological analysis was performed in an attempt to quantify observed differences between normal fibrinogen and fibrinogen Grenoble. On use of gels stained either with Coomassie Brilliant Blue or with silver, the partly qualified evaluation gives about 60% normal gamma chain and 40% gamma chain Grenoble.
Roche RIA and Abbott EIA carcinoembryonic antigen assays compared.
Authors: Fleisher M , Nisselbaum J S , Loftin L , Smith C , Schwartz M K
Published: January 2020

We have evaluated Roche Diagnostics' RIA-CEA and Abbott Diagnostics' EIA-CEA methods for precision, normal reference interval, concordance, and correlation of malignant disease with increase in carcinoembryonic antigen (CEA) in plasma. In examining concordance, we used data on 138 patients with primary carcinomas of the breast, colon, lung, or pancreas, each further classified by extent of dissemination. We find the two methods to be comparably precise. The respective upper reference limits of normal for the Roche and Abbott methods were determined to be 5.0 micrograms/L and 4.6 micrograms/L. The regression equation for a log transformation of the 177 data points is y = 0.966x + 0.03, where x = Roche and y = Abbott, with a correlation coefficient of 0.948. According to the criteria we used, the concordance was 78.7%. The largest discordance was observed in widely disseminated disease states and in cancers of the colon and pancreas. Paired data analysis of discordance indicated greater increases in apparent CEA by the Abbott method in most colon cancers with liver involvement; six of the eight discordant pancreatic cancers had higher Roche-CEA values. CEA heterogeneity and the role of the liver in CEA metabolism appear to contribute to the observed differences. We show why the two methods should not be used interchangeably, and that baseline values for CEA must be established for each method.
A family with a high serum aminopeptidase (microsomal) activity: properties of the enzyme from serum of the propositus.
Authors: Hiwada K , Tokioka-Terao M , Nishimura K , Kokubu T
Published: January 2020

We found a family with a high activity for hydrolyzing L-alanyl-beta-naphthylamide in their serum. This enzyme was confirmed to be aminopeptidase (microsomal) (EC 3.4.11.2) by means of immunological experiments involving anti-human kidney aminopeptidase (microsomal) antibody. We could not find the cause of the increased activity from the results of clinical and laboratory examinations. The enzyme from the propositus resembled that from the serum of normal subjects in molecular mass (240 000 Da), Km value (87 mumol/L), and degree of inhibition by antiserum to human kidney aminopeptidase (microsomal). It differed from the normal enzyme with respect to electrophoretic mobility (R1 value, 0.58; normal, 0.51), isoelectric point (pI, pH 3.4; normal, pH 3.8), and heat stability (more labile than normal). From information on this family and another one reported in the Japanese literature, we suggest that the mode of inheritance of a high activity of serum aminopeptidase (microsomal) may be autosomal dominant.
Reduction of organic mercury in water, urine, and blood by sodium borohydride for direct determination of total mercury content.
Authors: Margel S , Hirsh J
Published: January 2020

We have established the optimal conditions for use of sodium borohydride as the reducing agent before the direct determination of mercury in water, urine, and blood by atomic absorption spectroscopy. We evaluated the effects of pH, temperature, and cupric sulfate concentration on the direct determination of both organic and inorganic compounds of mercury. Accurate and precise quantification of mercury requires that the pH be between 9.3 and 9.5, the reaction temperature above 25 degrees C, the reaction time longer than 1 min, and, for urine samples only, the cupric sulfate concentration 10 mumol/L. The detection limit of the method is 1 to 2 ng and the precision (CV) is 3.8% for blood and 4.0% for urine.
An evaluation of the Technicon RA-1000 random-access analyzer.
Authors: Bromberg I L , Pollard A , Cheng J , Romaschin A
Published: January 2020

We evaluated the Technicon RA-1000 analyzer, with emphasis on its potential for user-defined method development. Optical linearity and sample pipetting linearity were good. The reagent pipetting system delivered slightly less than the nominal amount, owing to the volumetric effect of added mixing bubbles. Carryover of aqueous solutions was negligible. The instrument had good adaptability for user-defined methods and performed well in method comparisons. The observed dynamic range for enzymes (0-3000 U/L) was excellent. The worklisting software performed its intended functions well, but has limitations. We believe that the RA-1000 represents a significant contribution to the practice of clinical chemistry. A sophisticated benchtop machine, it includes several innovations, along with a few problems that are peculiar to its technology.
How to improve estimates of imprecision.
Authors: Krouwer J S , Rabinowitz R
Published: January 2020

Authors sometimes report total imprecision as being less than within-run imprecision. This leads one to question how well the underlying statistical concepts are understood. This article explains statistical concepts relevant to imprecision studies and reviews proper use of the relevant statistical terms. Calculation of total imprecision and its components is illustrated by a numerical example. Two of the most common calculation mistakes are pointed out. Finally, we recommend improvements in experimental design that will result in more precise estimates.
Use of a reference standard to improve the accuracy and precision of seven kits for determination of ferritin in serum.
Authors: Iacobello C , Ghielmi S , Belloli S , Arosio P , Albertini A
Published: January 2020

We assessed the accuracy and precision of seven commercial kits for serum ferritin. The concentration of ferritin as determined by these assays for a liver ferritin reference standard, a human heart ferritin standard, and normal sera correlated highly, but the absolute values varied widely. Use of an international reference standard for ferritin to prepare the standard curve greatly diminished the variability, but did not eliminate it. Although the seven kits differed in specificity for various human isoferritins, this did not appear substantially to affect accuracy. Six of the seven kits appeared sufficiently precise for clinical use over a wide range of ferritin concentrations.
Liquid-chromatographic evaluation of age-related changes in the urinary excretion of free catecholamines in pediatric patients.
Authors: Rosano T G
Published: January 2020

Pediatric patients (to age 18) without neuroblastoma show an age-related decrease in urinary excretion of each of the catecholamines--epinephrine, norepinephrine, and dopamine--in relation to creatinine excretion. From these data, I have developed reference intervals for pediatric age groups. Application of these ranges to seven patients with neuroblastoma and ganglioneuroblastoma indicated a high clinical sensitivity for the urinary dopamine determination but significant false-negative results for epinephrine and norepinephrine.
Plasma enzyme activities in primary hypothyroidism.
Authors: White G H , Walmsley R N
Published: January 2020

Results of clinical chemistry tests are often initially interpreted by relatively inexperienced junior medical staff, and therefore a major role of clinical chemists is to ensure that their laboratory tests are utilized and interpreted appropriately. The cases described below demonstrate the interaction of a diagnostic problem and clinical inexperience in an area where clinical chemists can, and should be, of professional assistance.
Influence of incomplete cholesteryl ester hydrolysis on enzymic measurements of cholesterol.
Authors: Wiebe D A , Bernert J T
Published: January 2020

Esterolytic activity of three enzymic cholesterol reagents was evaluated with use of human serum-based reference materials. Primary cholesterol standards were used to calibrate the enzymic methods, and incomplete cholesteryl ester hydrolysis was measured as the bias between the enzymic cholesterol values and corresponding reference values. The average biases observed with eight reference specimens were 0.15, -1.16, and -7.24%, respectively, for a commercial enzymic reagent with microbial cholesterol esterase, a commercial reagent with an animal source of esterase, and a Centers for Disease Control (CDC) reagent preparation with a microbial cholesterol esterase. By liquid chromatography, we examined the respective specificities of the three esterase reagent preparations and found that the preparations differ in the rate and specificity of catalysis of cholesteryl ester hydrolysis. The CDC reagent was only partly active towards cholesteryl arachidonate, which probably accounts for most of the negative bias of this reagent. Esterolytic activity of the various enzymic reagents is related to the source and matrix of the cholesterol esterase and must be considered with respect to proper selection of calibration materials for serum cholesterol measurement.
Multicomponent analysis of hemoglobin derivatives with reversed-optics spectrophotometer.
Authors: Zwart A , Buursma A , van Kampen E J , Zijlstra W G
Published: January 2020

We describe a method for the simultaneous determination of the five clinically relevant hemoglobin derivatives (Hb, HbO2, HbCO, Hi, SHb) in a blood sample by means of a reversed-optics spectrophotometer (Hewlett-Packard HP8450 A UV/Vis). A built-in computer program is used for multicomponent analysis in an overdetermined system, i.e., a system in which the number of independent equations used exceeds the number of unknowns to be determined. First, the spectra of the five hemoglobin derivatives are measured in a series of different human blood samples. Thereafter, the multicomponent method for the simultaneous determination of the five hemoglobin derivatives is tested by comparison with conventional methods for the separate determination of oxygen saturation, HbCO, Hi, and SHb fractions. The multicomponent (multiwavelength) method is sufficiently reliable, accurate, and easy to justify its use in physiological chemical research as well as its routine application in the clinical chemical laboratory.