bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluating the clinical effectiveness of amniotic fluid assays in predicting respiratory distress syndrome in the neonate.
Authors: Amenta J S , Brocher S C , Serenko-Aber A L
Published: January 2020

Amniotic fluid phospholipids from 346 patients' specimens were quantified and evaluated against the clinical outcome (i.e., respiratory distress syndrome or normal respiratory function). Concentrations of lecithin, sphingomyelin, phosphatidylglycerol, and the lecithin/sphingomyelin reflectance ratio were evaluated by ordered frequency distribution and stepwise discriminant function analysis. The lecithin/sphingomyelin ratio was the best single test for discriminating between respiratory distress syndrome and normal pulmonary function in the fetus, slightly superior to lecithin assay alone. A combination of lecithin/sphingomyelin ratio and lecithin concentration, however, appeared to optimize the discriminant function, although the clinical significance of this test combination remained marginal. High concentrations of phosphatidylglycerol were correlated with high concentrations of lecithin, and virtually ruled out respiratory distress syndrome. Absence of phosphatidylglycerol was not diagnostic. High concentrations of sphingomyelin increased the probability of respiratory distress syndrome. We suggest the following stepwise series of tests to optimize diagnosis: phosphatidylglycerol concentration, sphingomyelin concentration, and finally lecithin-sphingomyelin ratio.
Increased lactate dehydrogenase in serum in measles infection.
Authors: Sugaya N , Takeuchi Y , Kanno T
Published: January 2020

We determined lactate dehydrogenase (LD; EC 1.1.1.27) in serum from 75 sequential measles patients and 105 control patients. In the measles patients, LD was increased to 1147.9 +/- 43.8 (mean +/- SE) U/L, markedly more than in the control group (517.2 +/- 15.0 U/L, p less than 0.001). Both LD-3 and LD-4 isoenzymes were increased, but the concentration of LD-5 was normal. Patients with high LD values (greater than 1500 U/L) had longer hospital stays than did those with lower values (p less than 0.01). Our results suggest that increased LD in serum is a common finding in measles infection and presumably originates from lymphocytes.
Immunoassay of rheumatoid factor by latex particle counting.
Authors: Borque L A , Cambiaso C L , Masson P L
Published: January 2020

We assayed rheumatoid factor by instrumental latex particle counting. The calibration curve ranged from 12.5 to 500 int. units/mL. Maximal within- and between-assay CVs were 5 and 11%, respectively. Analytical recoveries ranged from 92.4 to 108%, and the relation between results and dilutions was linear in the range of 15 to 400 int. units/mL. Correlation with an enzyme-linked immunoassay (ELISA, Cordia kit) was r = 0.934 (n = 58), with turbidimetry r = 0.825 (n = 100), and with the Waaler-Rose test r = 0.834 (n = 73). Of 260 blood donors, 95% gave a value less than 10 kilo-int. units/L, which was taken as the upper normal limit. In a population of patients with rheumatoid arthritis (n = 47), 87.2% had rheumatoid factor greater than 10 kilo-int. units/L.
D-mannose as a preservative of glucose in blood samples.
Authors: Nakashima K , Takei H , Nasu Y , Andoh Y
Published: January 2020

We evaluated whether any monosaccharides inhibit glycolysis in erythrocytes and discovered that D-mannose does. In the presence of D-mannose, glucose can be accurately measured by either the hexokinase procedure or the glucose oxidase procedure. In comparison studies with other glucose preservatives, we found that after 2 h at room temperature glucose decreased by 21 (SD 13) mg/L in D-mannose-treated blood, 93 (SD 10) mg/L in sodium fluoride-treated blood, 28 (SD 21) mg/L in ice-cooled blood, and 144 (SD 28) mg/L in control blood (no preservative treatment). Because D-mannose acted in the early phase of glycolysis, it was a more effective preservative than sodium fluoride; moreover, its use did not preclude measurement of sodium and potassium in the blood samples. D-Mannose did not interfere with other routine chemical tests except for the assay of creatine kinase involving coupled enzymes hexokinase/glucose-6-phosphate dehydrogenase. Creatine kinase could be correctly assayed in the presence of D-mannose by using glucokinase instead of hexokinase. D-Mannose can be used with or without anticoagulant and is compatible with most types of multi-channel automated analyzers.
New immunochemical method for rapid detection of human choriogonadotropin in urine.
Authors: Shoham Z , Katz E , Blickstein I , Katz Z , Zosmer A , Dgani R , Lancet M
Published: January 2020

We describe a visual assay for rapid detection of choriogonadotropin (hGC) in human urine, and evaluate a pregnancy test kit that is based on this assay and designed for use by the general public. The assay involves the formation of an antibody-antigen complex between the anti-hGC antibodies coated on the membrane protein of Staphylococcus aureus bacteria, prestained with hematoxylin, and the hGC concentrated on a column of Sepharose-concanavalin A. The test was calibrated to detect as little hGC as 0.9 (SD 0.15) int. unit/mL. The kit, clinically tested with 448 urine samples, was 99.6% accurate. Simple to perform, the test gives highly reliable results as early as five days after the missed menstrual period.
Effect of tetrasodium EDTA on enzymatic determinations of urinary oxalate.
Authors: Thode J , Amyx C , Valdes R , Kessler G
Published: January 2020

We studied the effects of pretreating urine samples with tetrasodium EDTA (TEDTA) before measuring urinary oxalate with an enzymatic kit (Sigma). Mean analytical recovery of added oxalic acid was only 49% (SD +/- 13%) when the assay was performed as recommended by the manufacturer, but treating samples with TEDTA improved recoveries (96 +/- 10%). In 20 unselected 24-h urine samples assayed with and without TEDTA treatment, the mean oxalate concentrations were significantly (P less than 0.001) different: 15.6 +/- 8.7 and 12.2 +/- 7.9 mg/L, respectively. TEDTA-treated urine samples stored for 14 days at -20 degrees C lost 20% of their oxalate concentration. Use of TEDTA simplifies sample preparation by eliminating the alkalinizing step needed to dissolve EDTA or disodium EDTA.
Obviating interferences in the assay of urinary oxalate.
Authors: Barlow I M
Published: January 2020

Evaluation of the Sigma Kit for determination of urinary oxalate highlighted two major limitations of which users must be aware: the pH of urine samples before extraction is critical, and ascorbic acid interferes positively. I give details, and describe simple modifications to the manufacturer's protocol that overcome these problems.
Total cholesterol in serum determined by isotope dilution/mass spectrometry, with liquid-chromatographic separation.
Authors: Takatsu A , Nishi S
Published: January 2020

We describe an accurate, precise method for determination of total serum cholesterol by isotope dilution/mass spectrometry (IDMS) with liquid-chromatographic separation. After adding [3,4-13C]cholesterol to serum and hydrolyzing the cholesterol esters, we extract the total cholesterol. "High-performance" liquid chromatography (HPLC) is used to separate the extracted cholesterol for measurement by electron-impact mass spectrometry with use of a direct-insertion device. To evaluate the specificity and the accuracy of this method, we also studied the conventional IDMS method, which involves converting cholesterol to the trimethylsilyl ether and assay by gas chromatography-mass spectrometry with use of a capillary column. The coefficient of variation for the HPLC method was a little larger than that for the conventional method, but mean values by each method agreed within 1% for all sera tested.
A model for assessing interference.
Authors: Kroll M H , Ruddel M , Blank D W , Elin R J
Published: January 2020

Most studies of analytical interference indicate the magnitude but not the type of interference. We developed a model for interference that assesses the magnitude of the following types of interference: independent of analyte concentration, dependent on analyte concentration, and a combination of the two. The experimental design for the model is an orthogonally arranged matrix with progressively increasing concentrations of analyte and interfering agent. Multiple regression with these independent variables (concentration of analyte, concentration of interfering agent, and the product of the two) is used to determine the magnitude, direction, and significance of each type of interference. Applying the model to study interference by hemoglobin with determinations of creatinine, direct bilirubin, and total bilirubin showed that hemoglobin interferes with determination of creatinine independent of the analyte concentration, interferes with total bilirubin dependent on the analyte concentration, and interferes with direct bilirubin by a combination of these two.
Mechanism of interference with the Jaffé reaction for creatinine.
Authors: Kroll M H , Roach N A , Poe B , Elin R J
Published: January 2020

We investigated the mechanism of the Jaffé reaction for determination of creatinine by studying the spectrophotometric, kinetic, and equilibrium properties of the reaction of picrate with creatinine and with cyclic and aliphatic ketones. Absorbance spectra for the reaction products of picrate with all the ketones were superimposable with that of creatinine (Amax, 490 nm). Cyclic ketones not containing nitrogen had a molar absorptivity less than half that of creatinine and equilibrium constants approximately 0.01 that of creatinine. Aliphatic ketones, except for benzylacetone, had molar absorptivities similar to that of creatinine, but all of these compounds had equilibrium constants approximately a tenth or less that of creatinine. The common structure for all of the compounds reacting with picrate is the carbonyl group. The variable magnitude of interference for aliphatic and cyclic ketones is ascribable to the different rate constants, molar absorptivities, and equilibrium constants as compared with creatinine. Structures adjacent to the carbonyl group significantly affect the absorptivity and equilibrium constant, but steric hindrance is the major factor affecting the rate of reaction. We postulate that the carbonyl group is required for the Jaffé reaction, and we suggest a mechanism for the reaction.
Plasma protein glycation as measured by fructosamine assay.
Authors: Mosca A , Carenini A , Zoppi F , Carpinelli A , Banfi G , Ceriotti F , Bonini P , Pozza G
Published: January 2020

The fructosamine test for assessing control of glucose in blood has been extensively evaluated, but some questions remain regarding its validity. From the analytical and clinical evaluation we present here, we conclude that: the test is sensitive to variations in the composition of the sample protein; the fructosamine reaction is almost completely unaffected by labile fractions; the concentrations of fructosamine correlate well with the degree of glycation of total serum proteins, especially with glycated albumins and glycated immunoglobulins, as determined by affinity chromatography; the correlation with glycated hemoglobin (Hb A1c), measured as the stable fraction, is very poor, in diabetics treated with insulin (r = 0.373), or with oral hypoglycemic agents (r = 0.390); and (e) fructosamine and Hb A1c are, in fact, expressions of different periods of metabolic control and therefore have different clinical meanings.
A monoclonal antibody that specifically inhibits human salivary alpha-amylase.
Authors: Gerber M , Naujoks K , Lenz H , Wulff K
Published: January 2020

Our monoclonal antibody 88E8 specifically binds to and inhibits human salivary alpha-amylase (EC 3.2.1.1) and cross reacts negligibly with the pancreatic isoenzyme, inhibiting it by less than 1%, as compared with about 90% for the salivary isoenzyme. The antibody binds the S1 and S2 types of salivary alpha-amylase, but no pancreatic alpha-amylase isoenzyme forms. A pancreatic alpha-amylase assay involving 88E8 is under development, with alpha-glucosidase as auxiliary enzyme and p-nitrophenyl-maltoheptaoside as substrate; we give preliminary data on this assay. The assay has to be done by substrate start, because the antibody interacts very slowly with the enzyme in the presence of substrate. Assay results for pancreatic alpha-amylase correlate well with those for isoamylase assayed with use of an inhibitor from wheat-germ.
Interpreting results of coulometry and immunoprecipitation in diagnosing iron disorders.
Authors: Rettmer R L , Labbé R F , Huebers H A , Brown W P , Josephson B M , Finch C A
Published: January 2020

Concentration of iron in plasma, total iron-binding capacity (TIBC), and transferrin saturation are often determined by standard spectrophotometric methods, but iron concentration may be quantified by immunoprecipitation or, electrochemically, by controlled-potential coulometry. Because these iron assays do not all measure the same form(s) of iron, we studied subjects in various states of iron nutriture: normal adults, iron-deficient patients, thalassemia patients with unsaturated transferrin or oversaturated transferrin, and patients with idiopathic hemochromatosis. The spectrophotometric and coulometric methods detected essentially all non-heme iron in plasma; results correlated well but showed a negative bias toward the coulometric method. Results by an immunoprecipitation procedure, which measures only transferrin-bound iron, correlated well with those obtained coulometrically but were slightly higher than the latter. The characteristics of the various methods for iron must be understood by the clinical laboratory if diagnosis of iron disorders is to be accurate.
Three commercial kits and one liquid-chromatographic method evaluated for determining 25-hydroxyvitamin D3 in serum.
Authors: Lindbäck B , Berlin T , Björkhem I
Published: January 2020

The accuracy of three commercial kit methods and one liquid-chromatographic (HPLC) method for determining 25-hydroxyvitamin D3 in serum was evaluated by using isotope dilution-mass spectrometry as a reference technique. The kit methods were based on radioimmunoassay and radioreceptor assay. Serum samples were analyzed from male and female volunteers, some of whom had been exposed to ultraviolet B light or given vitamin D3 orally. Results obtained with the three commercial kits were less accurate than those by HPLC. The agreement between the HPLC method and the comparison method was relatively good: r = 0.99; slope = 1.23; intercept = -2.9 micrograms/L. We conclude that methods based on HPLC should be preferred in routine clinical work and that the accuracy of the three commercial kits tested is not sufficient for their intended use.
Quality-control scheme for blood glucose measured outside the laboratory.
Authors: Forest J C , Rousseau F , Carrier R , Hivon P , Gosselin M
Published: January 2020

We propose a quality-control (QC) scheme to evaluate the performance of blood glucose estimates made with a reflectometer in hospital wards or at home. We constructed a chart illustrating the comparison by regression analysis of the results of 254 reflectometer readings (y) and simultaneous capillary blood glucose analyses in the laboratory (x). Arbitrary acceptance limits around the regression line (y = 0.5 + 0.87x; r = 0.97, Sy/x = 0.8 mmol/L) were established, based on the mean +/- 2 SD of the reflectometer readings of a series of classes of 1.1 mmol/L interval, made with the results from the laboratory analyzer. Each ward using a reflectometer is provided with the QC chart, and the first blood glucose estimation with the reflectometer on each working shift is controlled by re-assay in the laboratory. If the reflectometer reading is outside the limits, the next glucose estimation is also re-assayed. If two consecutive readings are outside the limits, the reflectometer procedure is considered to be failing, the instrument's readings are not used for medical decisions, and remedial action is taken. The chart can also be used as a teaching tool for training the patients or the nurses and to evaluate individual performance.
Melatonin secretion in humans assessed by measuring its metabolite, 6-sulfatoxymelatonin.
Authors: Bojkowski C J , Arendt J , Shih M C , Markey S P
Published: January 2020

Comparing a direct radioimmunoassay for 6-sulfatoxymelatonin (aMT6s) with an established gas chromatographic/mass spectrometric method for 6-hydroxymelatonin, we found a good correlation r = 0.94 (P less than 0.001, n = 100). aMT6s was stable, both in urine and plasma samples, without preservative, for at least two years at -20 degrees C and for five days at room temperature. Urinary excretion of aMT6s showed considerable inter-individual differences; however, the aMT6s excretion of any one individual was consistent over a four-day period, as assessed by continuous collection from 18 normal volunteers. Total 24-h urinary excretion of aMT6s was significantly correlated with the area under the curve of the respective profiles for plasma melatonin (r = 0.75, P = 0.0002) and plasma aMT6s (r = 0.70, P = 0.0005) for 22 healthy volunteers. At 24:00 h and 03:00 h, sampling plasma at 30-s intervals provided no evidence for episodic secretion (in short pulses) of either melatonin or aMT6s.
Highly sensitive two-site immunoradiometric assay of parathyrin, and its clinical utility in evaluating patients with hypercalcemia.
Authors: Nussbaum S R , Zahradnik R J , Lavigne J R , Brennan G L , Nozawa-Ung K , Kim L Y , Keutmann H T , Wang C A , Potts J T , Segre G V
Published: January 2020

We have developed a highly sensitive, two-site immunoradiometric assay (IRMA) for human parathyrin (PTH) that is specific for the intact, secreted, biologically active 84-amino-acid peptide. This assay has several technical advantages: it does not detect even high concentrations of inactive carboxyl-terminal fragments, results are available within 24 h, and the detection limit for intact hormone is low (1 ng/L). The assay readily measures concentrations of PTH in all healthy subjects and distinguishes these values from low or undetectable PTH values observed in clinical situations in which PTH secretion is expected to be suppressed. We found complete separation of results from 37 patients with surgically proven hyperparathyroidism and those from 23 patients with hypercalcemia associated with malignancy, the latter having PTH values at or below the lower limits of normal for this assay. The sensitivity, specificity, and rapid turnaround time of this two-site IRMA should advance the laboratory evaluation of patients with disorders of calcium metabolism.
Specificity of sensitive assays of thyrotropin (TSH) used to screen for thyroid disease in hospitalized patients.
Authors: Spencer C , Eigen A , Shen D , Duda M , Qualls S , Weiss S , Nicoloff J
Published: January 2020

Thyrotropin (TSH) concentrations were measured in 1580 hospitalized patients and 109 normal persons. Using the mean +/- 3 SD limits of the log values for the controls (0.35-6.7 milli-int. units/L), the proportion of abnormal TSH results in the hospitalized patients was 17.2%. TSH was undetectable (less than 0.1 milli-int. unit/L) in 3.1% of patients, suggesting hyperthyroidism, and high (greater than 20 milli-int. units/L) in 1.6%, suggesting hypothyroidism. On follow-up of 329 patients, 62% with abnormal TSH (less than 0.35 or greater than 6.7 milli-int. units/L) and 38% with normal TSH concentrations, only 24% of those with undetectable TSH had thyroid disease: 36% of them were being treated with glucocorticoids and 40% had nonthyroidal illness (NTI). Although half the patients with TSH greater than 20 milli-int. units/L had thyroid disease, 45% of patients had high TSH values associated with NTI. TSH concentrations usually returned towards normal when patients' therapy with glucocorticoids was discontinued or they recovered from NTI. TSH test sensitivity appeared good when the mean +/- 3 SD limits of the reference population were used, i.e., no cases of hyper- or hypothyroidism, as identified by free thyroxin index (FT4I), were missed. However, TSH test specificity was inferior to that of the FT4I test (90.7% vs 92.3%), although specificity could be improved to 97.0% if the wider TSH reference limits of 0.1 to 20 milli-int. units/L were used--limits considered pathological if applied to outpatients. Evidently, different reference intervals for TSH are needed for hospitalized and nonhospitalized patients. We conclude that a "sensitive TSH assay" is not a cost-effective thyroid screening test for hospitalized patients as compared with the FT4I.
Isotope dilution/mass spectrometry of serum cholesterol with [3,4-13C]cholesterol: proposed definitive method.
Authors: Pelletier O , Wright L A , Breckenridge W C
Published: January 2020

We describe a new gas-chromatographic/mass-spectrometric (GC/MS) isotope-dilution method for determination of serum cholesterol. The method has been fully optimized and documented to provide the high accuracy and precision expected for a Definitive Method. In the presence of [3,4-13C]cholesterol, cholesteryl esters in serum are hydrolyzed under optimum conditions and the entire cholesterol pool is extracted and derivatized to silyl ethers. The cholesterol derivatives are resolved from other sterols by gas-liquid chromatography on a fused silica column, and selected ions characteristic of cholesterol and the [3,4-13C]cholesterol are monitored with a GC/MS quandrupole system. We estimated the cholesterol content of samples by bracketing each sample with standards of comparable cholesterol concentration that also contained the [3,4-13C]cholesterol. The procedure was highly reproducible (CV less than 0.5%), better accuracy and precision being obtained with [3,4-13C]cholesterol than with heptadeuterated cholesterol. Mean values per gram of dry serum for one serum pool assayed by this method and that of the National Bureau of Standards differed by 0.5%. We conclude that the method satisfies the criteria for a Definitive Method.
Deproteinizing methods evaluated for determination of uric acid in serum by reversed-phase liquid chromatography with ultraviolet detection.
Authors: Sakuma R , Nishina T , Kitamura M
Published: January 2020

We evaluated six deproteinizing methods for determination of uric acid in serum by "high-performance" liquid chromatography with ultraviolet detection: those involving zinc hydroxide, sodium tungstate, trichloroacetic acid, perchloric acid, acetonitrile, and centrifugal ultrafiltration (with Amicon MPS-1 devices). We used a Toyosoda ODS-120A reversed-phase column. The mobile phase was sodium phosphate buffer (40 mmol/L, pH 2.2) containing 20 mL of methanol per liter. Absorbance of the eluate was monitored at 284 nm. The precipitation method with perchloric acid gave high recoveries of uric acid and good precision, and results agreed with those by the uricase-catalase method of Kageyama (Clin Chim Acta 1971;31:421-6).
Improved procedure for determining glutathione in plasma as an index of myocardial oxidative stress.
Authors: Curello S , Ceconi C , Cargnoni A , Cornacchiari A , Ferrari R , Albertini A
Published: January 2020

Glutathione plays an important role in the detoxification processes of electrophilic metabolites of xenobiotics and oxygen free radicals, such that release of reduced and oxidized glutathione into the plasma is considered a reliable index of oxidative stress. However, reduced glutathione in plasma undergoes spontaneous autoxidation, with mixed disulfide formation. We developed a new, simple, quick method to overcome this problem by treating the blood, immediately after collection, with thiol reagents. We add 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide to the blood before determination of total and oxidized glutathione, respectively. We find the proposed assay useful for investigating oxidative stress in clinical situations.
Improved liquid-chromatographic assay of labetalol in plasma.
Authors: Luke D R , Matzke G R , Clarkson J T , Awni W M
Published: January 2020

This is an assay for labetalol in plasma by "high-performance" liquid chromatography, with 5-(2-[4-(4-chlorophenyl)ethyl]) salicylamide hemihydrate as the internal standard. Plasma samples (500 microL) are extracted with acetonitrile, evaporated under nitrogen, reconstituted in the mobile phase, and injected onto a PRP-1 (Hamilton) column packed with particles of poly(styrene-divinylbenzene) copolymer. Fluorescence, enhanced by post-column introduction of NH4OH, was measured in the effluent (excitation wavelength 340 nm, emission wavelength 418 nm). Retention times for labetalol and the internal standard were 1.99 and 3.32 min, respectively. Inter- and intraday CVs for high and low concentrations of the drug were less than 7.5%. The assay standard curve is linear from 1 to 250 micrograms/L. Some commonly co-administered drugs were tested and did not interfere.
Effect of two anticoagulants on leukocyte yield and function, and on lysosomal enzyme activity.
Authors: Branum E , Cummins L , Bartilson M , Hopper M , Pruett S , O'Brien J F
Published: January 2020

We compared acid citrate-dextrose (ACD-B) and heparin to determine which anticoagulant better preserves leukocytes for lysosomal enzyme assays if processing was done immediately or delayed for 24 h or more. Twenty normal subjects had blood drawn into tubes containing either ACD-B or heparin. The leukocytes were isolated by sedimentation in dextran (50 g/L) less than 2, 24, 48, and 72 h later. The most apparent difference was that cell counts indicated a 30% reduction in the number of leukocytes for ACD-B and a 95% reduction for heparin-treated cells at 48 h. The neutrophil function assay indicated that leukocyte processing must be done in less than 24 h regardless of the anticoagulant used, and that heparin is to be preferred. A comparison of heparin and ACD-B for maintenance of the activity of arylsulfatase A (EC 3.2.6.1) and hexosaminidase (EC 3.2.1.50) indicates that there is no effect of anticoagulant. However, at 48 h after venipuncture, there is an 80% reduction in the number of heparin-treated samples that are suitable for use in the assay. Those laboratories doing lysosomal enzyme tests on mailed specimens, which are most often greater than 24 h old, should use ACD-B as anticoagulant.
One-point recalibration of heterogeneous enzyme immunoassays with non-linear calibration curves.
Authors: Meyer H D , Keller H
Published: January 2020

We have investigated one-point recalibration of heterogeneous enzyme immunoassays involving nonlinear calibration curves. By using a factor determined from a single standard we updated calibration curves obtained earlier by means of multiple-point determination. The prerequisite for this method is a reproducible working procedure, i.e., as ensured by a fully mechanized system. In our investigations with Enzymun-Test system ES 600 (Boehringer Mannheim) we determined alpha 1-fetoprotein, carcinoembryonic antigen, digoxin, ferritin, thyroxin-binding globulin, triiodothyronine, and thyroxin in human serum pools over a period of two weeks with both full calibration and one-point recalibration. Comparisons of the results thereby obtained show that the means of the concentrations and the CVs for the alternative calibration procedures differed negligibly, even if we used reagents exposed to different temperatures for different periods of time. With the one-point recalibration procedure presented here, therefore, one can considerably reduce the time and effort spent on calibration.
Improved determination of selenium in serum by Zeeman atomic absorption spectrometry.
Authors: Morisi G , Patriarca M , Menotti A
Published: January 2020

We have improved Zeeman atomic absorption spectrometric determination of selenium in serum by using an acidic solution of Ag + Cu + Mg as a matrix modifier and performing the charring step in flowing O2. Under these conditions, we could calibrate serum results with selenium standards in bovine albumin solutions. Mean analytical recovery was 98%, and the CV was 1.8% within runs, 2.9% between runs. Analysis of Reference Materials from the U.S. National Bureau of Standards (SRM 909 and RM 8419) yielded the values of 106 (SD 2.4) and 15 (SD 1.6) microgram/L, respectively, in good agreement with the expected values (106 and 16, respectively). The method--being reliable and relatively simple and rapid--is suitable for use in epidemiological screenings. Mean selenium concentrations in serum sampled from 274 adults in central Italy were 90 (SD 15) microgram/L. For 11-year-old children, these values were lower and showed a tendency to sex-related difference: 82 (SD 9.9) microgram/L for 97 boys, 78 (SD 9.3) microgram/L for 90 girls.
Two dry-reagent systems evaluated for determinations of enzyme activities.
Authors: Hafkenscheid J C , van der Ven-Jongekrijg J
Published: January 2020

We evaluated the Kodak Ektachem DT60/DTSC and the Boehringer Mannheim Reflotron for measuring activity concentrations of six enzymes. The Ektachem CVs for low concentrations of aspartate aminotransferase and alanine aminotransferase were high. As compared with the Ektachem and a routine "wet-chemistry" system, values for aspartate aminotransferase and alanine aminotransferase were lower as measured by the Reflotron, because no pyridoxal 5'-phosphate is included in the Reflotron slides. Activity concentrations of gamma-glutamyltransferase and creatine kinase measured with the Ektachem and with the routine procedure did not agree well, possibly because the Ektachem gave too-high results for the enzyme activities. Assays of several commercial test sera indicated that test results by the dry-chemistry and routine procedures are not interconvertible. This contrasts with our previous experience, in which between-test agreement for several analytes was acceptable.
Albumin Cooperstown: a serum albumin variant with the same (313 Lys----Asn) mutation found in albumins in Italy and New Zealand.
Authors: Huss K , Putnam F W , Takahashi N , Takahashi Y , Weaver G A , Peters T
Published: January 2020

Albumin Cooperstown is an electrophoretically fast genetic variant (alloalbumin) inherited in a family in New York State. To determine the structural change, we separated the alloalbumin from normal albumin A by "high-performance" liquid chromatography, reduced and carboxymethylated it, and cleaved it with CNBr. Isoelectric focusing showed that the substitution was in fragment CB4 (residues 299 to 329). The variant CNBr fragment from the albumin of two siblings was purified by liquid chromatography. Automated sequence analysis established the substitution of lysine-313 by asparagine (313 Lys----Asn), which corresponds to mutation of a single nucleotide base. The same substitution has recently been reported in albumin variants in Italy and New Zealand. This provides the first evidence for independent mutations at a single site within the structure of the mature albumin molecule. Because of the significance of albumin for study of protein evolution, clinical chemists are asked to be on the alert for cases of bisalbuminemia.
Heterophilic antibodies: a problem for all immunoassays.
Authors: Boscato L M , Stuart M C
Published: January 2020

We verified that antibody-binding substances in serum that interfere in two-site immunoassays involving murine antibodies are heterophilic antibodies. Incubation of serum containing heterophilic antibodies and a murine monoclonal antibody to human choriogonadotropin (hCG) leads to formation of a series of soluble immune complexes. We investigated the recognition of hCG by reagent antibody in the presence of heterophilic antibodies and found this recognition to be diminished. Consequently, about 30% of serum samples containing heterophilic antibodies falsely appear to contain increased concentrations of hCG. The effect on analyte recognition probably results from steric inhibition of hCG binding to complexed antibody. Heterophilic antibodies detected with a murine antibody also bound immunoglobulin from several other species but did not bind all of those tested.
Effect of lot-to-lot variability in filter paper on the quantification of thyroxin, thyrotropin, and phenylalanine in dried-blood specimens.
Authors: Slazyk W E , Phillips D L , Therrell B L , Hannon W H
Published: January 2020

We prepared whole-blood pools to contain various concentrations of phenylalanine (Phe), thyroxin (T4), and thyrotropin (TSH) and applied them to six different lots of Schleicher & Schuell Grade 903 filter paper, two of which represented extremes for serum-absorbancy. Individual measured T4 values showed minimal overlap among all pools for each individual filter-paper lot and for all lots combined, but Phe values overlapped considerably among the high-concentration pools within and among lots. Individual TSH values also showed considerable overlap among the high-concentration pools for all lots combined, but little overlap within each lot. Maximum differences in mean observed values among lots ranged from 6% to 36% for all analytes. Assay results from hemolyzed blood specimens generally were lower than from intact-cell blood specimens for T4 and TSH, but slightly higher for Phe. Maximum among-lot differences in mean values ranged from 13% to 29% for all analytes when each tested lot was used for assay calibration. Lot-to-lot differences in measured values were not strongly related to serum absorbancy values. We conclude that routinely encountered within- and among-lot filter paper variability, as measured by serum-absorbancy, is not alone sufficient to cause gross quantification errors in neonatal screening programs.
Rate and delta checks compared for selected chemistry tests.
Authors: Lacher D A , Connelly D P
Published: January 2020

We compared delta and "rate" check methods for 12 selected chemistry tests. Rate checks were determined by dividing delta checks by inter-specimen interval time. The delta and rate check methods were based on differences and percent change of untransformed and absolute value-transformed values. The distribution of delta differences was not symmetrical for calcium, alkaline phosphatase, aspartate aminotransferase, or phosphorus, which led to different check limits between untransformed and absolute value-transformed methods. The dispersion of rate checks was large. The interval time between two consecutive tests was multimodal, which probably reflected adherence to fixed testing protocols.
Enzyme-linked immunosorbent assay of CA-125 in serum of patients with endometriosis: efficacy in diagnosis.
Authors: Kang J O , Hudak W A , Keller N , Criswell B S
Published: January 2020

We used an enzyme-linked immunosorbent assay to quantify CA-125, a cancer antigen, in serum of patients with endometriosis. Concentrations of CA-125 in 103 patients with untreated endometriosis significantly (P less than 0.005) exceeded those for 31 controls with no evidence of disease: 9.1 (SD 12.3) vs 2.2 (SD 3.2) arb. units/mL. Values for patients with treated endometriosis were lower (2.3, SD 2.6, arb. units/mL, n = 9) than those for patients with untreated endometriosis. The mean concentrations of CA-125 in different stages of untreated endometriosis were 4.8 (SD 4.5), 7.1 (SD 9.9), 9.8 (SD 13.2), and 10.8 (SD 13.0) arb. units/mL for Stages I, II, III, and IV, respectively. The differences between controls and each stage were statistically significant (for all stages: P less than 0.05). We also assayed serum sampled from controls and patients with untreated endometriosis during menstruation (Group A) and during nonmenstruation (Group B). The efficacy of the present assay for concentrations of CA-125 in serum of patients with endometriosis was greater for Group B than for Group A. Evidently, determination of CA-125 in serum by the present assay may assist in such diagnosis.
Electroimmuno- and immunonephelometric assays of apolipoprotein A-I by using a mixture of monoclonal antibodies.
Authors: Pruvot I , Fievet C , Durieux C , Vu Dac N , Fruchart J C
Published: January 2020

The use of mixtures of well-defined monoclonal antibodies may represent a step forward in the standardization of immunochemical assays. We developed and optimized working conditions for using such a mixture to determine apolipoprotein A-I in human sera by two independent techniques (electroimmuno- and immunonephelometric-assays). Six monoclonal antibodies, each addressed to distinct epitopes located at the surface of apolipoprotein A-I, were used in combination to permit a reproducible measurement of the protein, without prior delipidation of samples. Parallel standard curves for a high-density lipoprotein subfraction (HDL3, the primary standard) and a reference serum (the secondary standard) were obtained. Within- and between-run coefficients of variation were acceptable for both methods. Apolipoprotein A-I concentrations, as measured in 60 subjects selected to present a large range of apolipoprotein content by electroimmunoassay (y1) and immunonephelometric assay (y2) with monoclonal antibodies, compared well with those measured by the same techniques but with polyclonal antibodies (x): r1 = 0.96, r2 = 0.99; y1 = 1.19x - 0.11 g/L, y2 = 0.98x. Comparison of results obtained by electroimmunoassay and immunonephelometric assay performed with monoclonal antibodies was also good: r = 0.96; y2 = 1.08y1 + 0.13 g/L.
Laboratory and clinical evaluation of an assay of thrombin-antithrombin III complexes in plasma.
Authors: Hoek J A , Sturk A , ten Cate J W , Lamping R J , Berends F , Borm J J
Published: January 2020

We evaluated a recently developed commercial assay for quantifying thrombin-antithrombin III (TAT) complexes in human plasma. The assay is precise (within-assay CV less than 10%, between-assay CV less than 13%), and sensitive (detection limit 0.7 micrograms of TAT per liter of plasma). Measurements for healthy volunteers yielded a normal reference (95 percentile) interval of 0.8 to 5.0 micrograms/L (n = 50, mean 2.1 micrograms/L, range 1.1 to 7.5 micrograms/L). TAT concentrations were increased in 25 of the 41 patients who fulfilled the clinical criteria of disseminated intravascular coagulation (DIC, overall mean 15.8 micrograms/L) and in 30 of the 35 patients with deep-vein thrombosis of the leg (overall mean 9.4 micrograms/L). We assessed the accuracy of the TAT assay by comparison with established criteria for the laboratory diagnosis of DIC involving various cutoff values for antithrombin III, factor V, fibrinogen, platelet count, fibrin/fibrinogen degradation products, and activated partial thromboplastin time. The low specificity of the TAT assay with regard to some of these criteria indicates that the latter are probably insensitive.
Intra-individual variation of serum thyroxin and triiodothyronine in pregnancy.
Authors: Hölzel W G , Deschner W
Published: January 2020

The mean concentrations of triiodothyronine (T3) and thyroxin (T4) in serum were increased in pregnancy, the increases for individuals remaining stable for week 16 to week 40 of gestation. For this period biological intra-individual variations of T3 and T4 in serum were estimated and compared with those of non-pregnant women. The average biological intra-individual CVs for T3 and T4 were of the same order for pregnant and non-pregnant women (6.9-8.4%). The ratios of the biological intra-individual CVs to the biological group CVs were 0.5 to 0.6. Individual values were normally distributed. There was no increase of the intra-individual variation with the lapse of time between two consecutively observed values. The estimated average biological intra-individual CVs were used to derive decision-making criteria in monitoring thyroid function during the 2nd and 3rd trimester of gestation.
Estradiol-17 beta quantified in serum by isotope dilution-gas chromatography-mass spectrometry: reversed-phase C18 high-performance liquid chromatography compared with immuno-affinity chromatography for sample pretreatment.
Authors: Thienpont L M , Verhaeghe P G , Van Brussel K A , De Leenheer A P
Published: January 2020

Here, isotope dilution-gas chromatography-mass spectometry is used as a reference technique for determining the concentration of estradiol-17 beta in candidate human serum Reference Material. The accuracy of assigned concentrations in biologic materials is not only determined by instrumental performance, it also depends greatly on the selectivity of the procedure for isolating the analyte from the biological matrix, an issue which we consider insufficiently addressed in the literature. We introduced reversed-phase C18 high-performance liquid chromatography as a fractionation procedure in addition to the commonly used solvent extraction and column chromatography on Sephadex LH-20. The validity of this approach as part of a Reference Method for measurement of estradiol-17 beta by isotope dilution-gas chromatography-mass spectrometry was investigated by comparison with immuno-affinity chromatography, which on theoretical grounds is generally considered as highly selective.
An alternative method for assaying cerebrospinal fluid protein in the presence of methotrexate.
Authors: Kasper L M , Moorehead W R , Oei T O , Markanich M
Published: January 2020

Therapeutic concentrations of methotrexate can cause significant positive interference in cerebrospinal fluid (CSF) protein values when assayed in the Du Pont aca. Conversely, our modified turbidimetric method, in which trichloroacetic acid (TCA) plus a sample blank containing dilute hydrochloric acid is used in place of TCA, exhibits little or no interference from methotrexate. This was verified by assaying solutions that contained a constant amount of protein (approximately 430 mg/L) and various amounts of methotrexate (0.0-2.3 x 10(-4) mol/L) by both the Du Pont aca and the manual turbidimetric method. As expected, the aca results showed increasing protein values with increasing methotrexate, whereas the manual method gave results approximating the expected protein value irrespective of the methotrexate concentration.
Falsely high high-density lipoprotein triglyceride values by the heparin-manganese precipitation method.
Authors: Simo I E , Kiss Z , Ooi T C
Published: January 2020

Recent evidence indicates that high-density lipoprotein triglyceride (HDL-Tg) may be a predictor of coronary artery disease. We examined three methods for HDL-Tg measurement, comparing results obtained by measurement of Tg in the supernate after heparin-manganese chloride (heparin-Mn) precipitation of EDTA-treated plasma (I) with results obtained after preparative ultracentrifugation (II and III). In II, we used heparin-Mn precipitation of low-density lipoprotein (LDL) from the infranate after ultracentrifugation at d 1.006 to remove very-low-density lipoprotein (VLDL). In III, we performed sequential flotation ultracentrifugation at d 1.006 and 1.063, then measured Tg in the d greater than 1.063 fraction. Method I gave significantly higher HDL-Tg results than II and III, which gave essentially identical results. The difference in results between I and II was not caused by the presence of heparin or manganese chloride, because these were used in both methods. Prior removal of VLDL in II and III resulted in lower HDL-Tg values, and subsequent removal of LDL by precipitation or ultracentrifugation did not alter final HDL-Tg values. The higher values obtained in I were the result of the presence of VLDL-rich unsedimented precipitate in the supernate.
Lidocaine metabolite and creatinine measurements in the Ektachem 700: steps to minimize its impact on patient care.
Authors: Sena S F , Syed D , Romeo R , Krzymowski G A , McComb R B
Published: January 2020

The single-slide method used for creatinine in the Kodak "Ektachem" analyzer is far more precise than the two-slide method. We confirm that sera from patients on intravenous therapy with lidocaine exhibit a positive bias in results for creatinine but that lidocaine itself does not interfere. Instead, N-ethylglycine, a metabolite of lidocaine with a structure similar to that of sarcosine, is probably the cause. A method that allows N-ethyglycine to be measured directly is described. We followed the degree of this interference through five generations of the slide. Our investigations include two detailed comparison studies between the Kodak Ektachem 700 and the Beckman Astra analyzers. Creatinine determinations on lidocaine-treated patients when first-generation slides were used averaged 4.6 mg/L higher than determinations on these same specimens performed in the Astra. Serum creatinine results from patients not on lidocaine showed no significant difference between the two instruments. The average difference in generation 2, 3, and 4 slides was 0.24, 0.22, and 2.5 mg/L, respectively. No more than 2% of our creatinine results had a clinically significant lidocaine-related bias. We show how to identify and correct this small proportion of results that are biased because of lidocaine therapy.
Disposable-column radioimmunoassay for serum digoxin with less interference from metabolites and endogenous digitalis-like factors.
Authors: Longerich L , Vasdev S , Johnson E , Gault M H
Published: January 2020

A preparative extraction step using disposable C18 low-pressure chromatography columns greatly improved the specificity of a commercially prepared digoxin radioimmunoassay (RIA). Elution solvents were isopropanol/water (15/85 by vol), which extracted most immunoreactive digitalis-like factors and metabolites, and methanol, which extracted digoxin for RIA. Many different digoxin RIA kits could be used. The coefficients of variation for replicates and duplicates were 4.6% and 5.2%. Analytical recoveries of digoxin standards in serum of 1.0, 0.5, and 0.1 microgram/L were 96%, 95%, and 88%, respectively. Serum digoxin was assayed by this method in 200 patients, 47 of whom were studied by HPLC-RIA. Values correlated better with "true digoxin" by HPLC-RIA (r = 0.93) than did values found by direct assay (r = 0.63). The mean for the isopropanol fraction as a percentage of the mean direct RIA value was higher for the 21 dialysis-dependent patients than was that found for the 179 nondialysis patients (P less than 0.004). The method is suggested as being most useful when metabolites or digitalis-like factors are known to be often high and values for digoxin are disproportionate to the dose.
Determination of vanillylmandelic acid, vanillactic acid, and homovanillic acid in dried urine on filter-paper discs by high-performance liquid chromatography with coulometric electrochemical detection for neuroblastoma screening.
Authors: Kinoshita Y , Yamada S , Haraguchi K , Takayanagi T , Mori Y , Takahashi T , Haruki E
Published: January 2020

We report a method for determination of vanillylmandelic acid, vanillactic acid, and homovanillic acid by high-performance liquid chromatography (HPLC), with coulometric electrochemical detection, for mass screening of neuroblastoma. Urine samples were collected on filter paper, dried, and then pretreated. The chromatographic procedure is reliable and fast, allowing for a large sample throughput for routine screening. Intricate extraction procedures and centrifugal separation are unnecessary. Screening for neuroblastoma by HPLC is rapidly gaining acceptance in Japan, and our method is being used at many screening centers. Of 26,571 infants screened in one year in Yokohama City, our method detected five with neuroblastoma.
Liquid-chromatographic measurement of p-aminobenzoic acid and its metabolites in serum.
Authors: Yung-Jato L L , Durie P R , Soldin S J
Published: January 2020

This is a high-performance liquid-chromatographic method for measuring p-aminobenzoic acid (PABA) and its metabolites in plasma or serum. Samples are deproteinized, then extracted with organic solvents before chromatography. For quantification, the peak height of the individual compound is compared with that of the internal standard. Analytical recoveries ranged from 41% to 100%, depending on the compound studied. Comparison of patients' samples after oral administration of either N-benzoyl-L-tyrosyl-p-aminobenzoic acid or free PABA revealed that PABA is extensively metabolized and conjugated to either p-acetamidobenzoic acid, p-aminohippuric acid, or p-acetamidohippuric acid. PABA concentrations in serum as measured with the Bratton-Marshall ultraviolet spectrophotometric procedure would appear predominantly to reflect measurements of metabolites, with only a minor contribution from PABA itself.