bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein C in human plasma determined by homogeneous enzyme immunoassay with use of a centrifugal analyzer.
Authors: Shimamoto M , Yoshimura T , Kimura N , Kita M , Hoshino N , Motoi S , Suehisa E , Nishimura K , Hayashi C , Miyai K
Published: January 2020

We describe the simple and rapid enzyme immunoassay of protein C in human plasma with use of a Cobas Fara centrifugal analyzer. The antibody, labeled with horseradish peroxidase, is reacted with antigen (protein C) for 15 min. The peroxidase activity of the resulting antigen-antibody conjugate is measured at 500 nm for 5 min in the presence of excess H2O2, phenol, and 4-aminoantipyrine, as compared with that of free conjugates. Results are calculated from a stored standard curve and expressed as a percentage of the value determined for a pooled specimen of normal adult plasma. The standard curve is linear from 0% to 200%. The CV is generally less than 4% for different concentrations of protein C. In liver cirrhosis, hepatocellular carcinoma, therapy with warfarin, thrombosis, and disseminated intravascular coagulation, protein C concentrations are about 40-70% of normal. Results obtained with the present homogeneous enzyme immunoassay correlated well with those by enzyme-labeled immunosorbent assay (r = 0.97).
Use of computer modeling to predict the magnitude of intralaboratory error tolerated by proposed CDC interlaboratory proficiency testing performance criteria.
Authors: Laessig R H , Ehrmeyer S S
Published: January 2020

In December 1987, the Centers for Disease Control (CDC) proposed to the Health Care Financing Administration revised criteria for evaluating participating laboratories' performance in proficiency-testing programs. If these criteria are accepted, they will become the minimum standard for all regulatory proficiency-testing programs. To evaluate a laboratory's performance in a clinical chemistry proficiency-testing program, the CDC proposed a combination of the use of fixed limits, multiples of the interlaboratory group standard deviations, and absolute values. In addition, laboratories would be required to meet 70% of the most recent proficiency-testing challenges. Because the purpose of regulatory proficiency testing is to identify poorly performing laboratories, it is essential that regulators be aware of the relationship between the regulatory criteria and the actual magnitude of intralaboratory error they tolerate. Through computer simulation, we determined for 21 chemistry analytes the amount of intralaboratory error tolerated by the CDC-proposed criteria. We evaluated the effectiveness of the proposed criteria by comparing the levels of total intralaboratory error permitted by a proficiency-testing program by using the CDC criteria with actual currently achievable levels of performance and defined medical usefulness needs. The proposed CDC criteria were too lenient for six analytes, about correct for seven, and too stringent on two; no medical usefulness limits were available for six.
Improved specificity of the CA 125 enzyme immunoassay for ovarian carcinomas by use of the ratio of CA 125 to carcinoembryonic antigen.
Authors: Wu J T , Miya T , Knight J A , Knight D P
Published: January 2020

We found that ovarian cyst fluids contained carcinoembryonic antigen (CEA) and CA 19-9 and CA 125 tumor markers. However, only the ratio of CA 125 to CEA concentrations provided sufficient specificity to differentiate serous from mucinous cysts. For CEA measurement, our results suggested the use of a monoclonal CEA kit. When CEA was determined with a Hybritech monoclonal CEA kit, all ratios in mucinous ovarian cysts were less than 10 and most of the ratios were greater than 1000 in serous ovarian cysts. We also found that the ratio of CA 125 to CEA in serum could be used to differentiate ovarian from nonovarian malignant diseases when both sera contain increased CA 125 concentrations. The nonovarian malignancies consisted of colorectal, breast, lung, and pancreatic carcinomas. The mean ratio for serum from patients with nonovarian cancers was 0.94 (n = 19); for ovarian-cancer patients (n = 45), 916. Therefore, determining this ratio will greatly improve the specificity of the CA 125 test for ovarian cancer.
Automated enzymatic method for determining ammonia in plasma, with 14-day reagent stability.
Authors: Neeley W E , Phillipson J
Published: January 2020

This is an automated, cost-efficient enzymatic method for determining ammonia in plasma. In most assays for ammonia the reagents are stable for only one or two days at 4 degrees C, but the reagents in our method are stable for at least 14 days. Our method was developed for use with a Cobas-Fara analyzer but can be easily adapted to other automated analyzers. It is essential to use closed sample vials. Our results, obtained at much less cost, correlate well with those by the Du Pont aca method. Precision studies gave the following results: within-run (n = 20 each), for x = 46 mumol/L, SD = 0.7 mumol/L, CV = 1.6%, and for x = 593 mumol/L, SD = 4.0 mumol/L, and CV = 0.7%; day-to-day precision during a year: for x = 47.7 mumol/L, SD = 2.4 mumol/L, CV = 5.0% (n = 360); and for x = 643 mumol/L, SD = 20.5 mumol/L, and CV = 3.2% (n = 365). The standard curve was linear to 1000 mumol of NH3 per liter.
Standardization for four protein analytes with the Behring Nephelometer.
Authors: Schotters S B , McBride J H , Rodgerson D O , Higgins S , Pisa M
Published: January 2020

Owing to the generally higher values observed in the initial establishment of immunoglobulins (Ig) G, A, and M assays with the Behring Nephelometer, we elected to verify five commercial protein calibrators. This initial verification was performed by standardizing the Behring Nephelometer with the World Health Organization (WHO) International Reference Preparation for Human Serum Immunoglobulins G, A, and M. The instrument was also standardized for immunoglobulins and transferrin with use of the Reference Preparation for Serum Proteins (RPSP II). Analytical recoveries of the commercial calibrators varied. Also, assigned protein concentrations in both the WHO and RPSP II preparations made them unacceptable to us as benchmark calibrators for the Behring Nephelometer. Individual proteins (IgG, IgA, IgM, and transferrin) were obtained and primary standards prepared. Both transferrin and IgG were successfully standardized. However, IgA and IgM primary standards lacked 100% antigenicity, requiring removal of nonreactive IgA and IgM or reassignment of the correct value. The problem remains to find appropriate purified materials for IgA and IgM standardization.
Improved method for calculating calcium fractions in plasma: reference values and effect of menopause.
Authors: Nordin B E , Need A G , Hartley T F , Philcox J C , Wilcox M , Thomas D W
Published: January 2020

Multiple linear regression analysis showed that total calcium was significantly and positively related to albumin, globulin, anion gap, and bicarbonate in plasma from 556 normal postmenopausal women. The residual constant of 1.21 mmol/L approximated the normal mean for ionized calcium in plasma. The coefficients derived from the multiple regression analysis were used to calculate ionized calcium from total calcium and ligand concentrations in a series of 105 clinical cases in whom ionized calcium was measured with the calcium electrode. The regression equation was y = 1.00x -0.0039 mmol/L, where x is measured and y calculated Ca2+ (r = 0.78, P less than 0.001). The protein-bound, ultrafiltrable, and complexed fractions of the calcium were also calculated for plasma from 69 normal young men, 66 normal young women, and 305 normal postmenopausal women. The increased plasma calcium in the postmenopausal group was accounted for by an increase in the complexed fraction, due to increases in plasma bicarbonate and anion gap.
Turbidimetric latex immunoassay of placental lactogen on microtiter plates.
Authors: Collet-Cassart D , Limet J N , Van Krieken L , De Hertogh R
Published: January 2020

In this latex immunoassay for human placental lactogen, microtiter plates are used as the reaction vessel and the absorbance at 405 nm is measured to quantify the reactions. This 30-min assay necessitates only one serum dilution and two pipetting steps. The calibration curve extends from 0.5 to 15 mg/L. CVs range from 4.2% to 7.0% for within-run determination and from 7.0% to 11.2% for between-run determinations. A correlation coefficient of 0.949 was obtained for 84 sera when the method was compared with a commercial radioimmunoassay.
Fibronectin is unsuitable as a tumor marker in pleural effusions.
Authors: Delpuech P , Desch G , Fructus F
Published: January 2020

We studied 75 patients with nonmalignant pleural effusions (50 with pneumopathy, 16 with pulmonary tuberculosis, and nine with congestive heart failure) and 33 patients with malignant pleural effusions. We selected 105 mg/L as the most suitable cutoff concentration of fibronectin for distinguishing between the two groups. We found high concentrations of fibronectin in 21 of the 33 patients with malignant pleural fluid but also in 37 of the 75 patients with nonmalignant pleural fluid. Evidently, measuring fibronectin in pleural fluid will not help in differentiating nonmalignant from malignant pleural fluids (diagnostic accuracy: 55%).
Salivary progesterone excellently reflects free and total progesterone in plasma during pregnancy.
Authors: Meulenberg P M , Hofman J A
Published: January 2020

To see if saliva is a valid substitute for plasma in assay of progesterone even when concentrations of hormone and binding proteins are fluctuating, we determined the concentrations of total and free progesterone in plasma and salivary progesterone in specimens from 36 women volunteers during the course of pregnancy and six weeks postpartum, using a highly specific RIA for total progesterone after extraction and chromatographic purification of the steroid. The free fraction in plasma was determined via equilibrium dialysis, followed by the same RIA analysis for progesterone in the dialysate. Despite the dramatic increases in concentrations of total progesterone and binding proteins in plasma during pregnancy, we found highly significant correlations between total and free progesterone in plasma and salivary progesterone in the group as a whole as well as individuals (P less than 0.001 in almost all cases). The proportion of free progesterone in plasma and of salivary progesterone relative to total progesterone in plasma remained constant at approximately 1% and 0.5%, respectively, whereas during the postpartum period there was much more variance. Evidently salivary progesterone is a very good alternative to plasma as a sample for use in follow-up during pregnancy.
Streptokinase binds to lactate dehydrogenase subunit-M, which shares an epitope with plasminogen.
Authors: Podlasek S J , McPherson R A
Published: January 2020

The bacterial thrombolytic agent streptokinase binds to human, porcine, and chicken lactate dehydrogenase (EC 1.1.1.27; LD) isoenzyme subunit M, but not to the H or C subunits. There is amino acid sequence homology between LD and the streptokinase binding site on plasminogen to account for this interaction that results in the formation of high-molecular-mass complexes in serum that contain LD activity. Binding of highly immunogenic streptokinase with LD may lead to induction of anti-LD autoantibodies, known to occur in some patients after therapeutic administration of streptokinase for treatment of acute myocardial infarction. This interaction may also be a general mechanism for inducing autoimmunity against other proteins that share the streptokinase binding epitope.
An immunoassay of human band 5 ("tartrate-resistant") acid phosphatase that involves the use of anti-porcine uteroferrin antibodies.
Authors: Whitaker K B , Cox T M , Moss D W
Published: January 2020

We describe an immunoassay for human band-5 acid phosphatase in which antibodies to porcine uteroferrin, immobilized on Sepharose particles, are used. Band-5 acid phosphatase is the tartrate-resistant isoenzyme normally expressed in tissue macrophages such as osteoclasts and alveolar macrophages. The immunoassay is similar in reproducibility and sensitivity to assays based on inhibition by d-tartrate. However, compared with the latter, the greater specificity of the immunoassay makes it markedly less susceptible to errors arising from the presence of non-band-5 acid phosphatases, e.g., from prostate.
Specific binding of cardiac glycoside drugs and endogenous digitalis-like substances to particulate membrane fractions from human placenta.
Authors: Paci A , Cocci F , Piras F , Ciarimboli G , Clerico A
Published: January 2020

We studied the characteristics of binding of cardiac glycosides to particulate membrane fractions from human placenta, to demonstrate that placental tissue is a suitable source of receptors for digitalis drugs. Moreover, we performed preliminary experiments with 125I-labeled digoxin and placental particulates to develop a radioreceptor assay for measurement of endogenous substances with activity similar to cardiac glycoside drugs (EDLS). Placental membrane fractions were incubated with [3H]ouabain (10 nmol/L) or 125I-labeled digoxin (50 pmol/L). With both ligands, binding followed a pseudo-first-order reaction kinetics and was saturable. Scatchard analysis revealed a single class of sites [for ouabain, KD = 20.2 +/- 5.8 nmol/L (mean +/- SEM), Bmax = 3.1 +/- 0.9 nmol per gram of protein; for digoxin, KD = 29.7 +/- 1.9 nmol/L, Bmax = 24.3 +/- 1.1 nmol per gram of protein]. As expected, digoxin was less potent than ouabain in displacing both tracers from digitalis drugs receptors; progesterone, cortisone, digitoxose, furosemide, bumetanide, and propranolol had no or little effect. Specific 125I-labeled digoxin binding was competitively inhibited by plasma and (or) urine extracts from newborns, adults, pregnant women, and patients with renal insufficiency. Inhibition of binding and volume of plasma and urine assayed were linearly related. These findings support the hypothesis that cardiac glycosides and EDLS can interact with the human placenta and suggest placental tissue to be a suitable source of receptors for cardiac glycosides.
Empirically determined lead-poisoning screening cutoff for the Protofluor-Z hematofluorometer.
Authors: Stanton N V , Gunter E W , Parsons P J , Field P H
Published: January 2020

A recently introduced hematofluorometer, the "Protofluor-Z" (Helena Laboratories, Beaumont, TX), has several novel features, most notably reporting units expressed as the molar ratio of zinc protoporphyrin (ZPP) to heme, i.e., micromoles of ZPP per mole of heme. We analyzed human blood specimens on the Protofluor-Z and by an ethyl acetate/acetic acid extraction procedure. Data from three laboratories were pooled and used to provide a comparison of the two methods. Results indicate that, with the Protofluor-Z, a value of 70 mumol of ZPP per mole of heme is approximately equivalent to the recommended screening cutoff of 35 micrograms of erythrocyte protoporphyrin per 100 mL of whole blood used in programs for pediatric lead-poisoning prevention. This empirically determined value is slightly lower than either that recommended by the manufacturer or a theoretical cutoff value that was determined mathematically.
Analysis for alpha-amylase isoenzymes by automated isoelectric focusing.
Authors: Lessard F , Dion R
Published: January 2020

We describe the fractionation of alpha-amylase (EC 3.2.1.1) isoenzymes by use of the Pharmacia "Phastsystem" electrophoresis apparatus. The separation is done by isoelectric focusing on "Phastgel IEF 5-8." A complete run, including staining, takes 4 h and can easily resolve eight isoenzymes. Samples can be analyzed in a routine laboratory with use of conventional reagents.
Total protein determined in human breast milk by use of coomassie brilliant blue and centrifugal analysis.
Authors: Bergqvist Y , Karlsson L , Fohlin L
Published: January 2020

This simple method of centrifugal analysis for total protein in human breast milk is based on the change in the wavelength of the absorbance maximum of Coomassie Brilliant Blue G-250 when the dye is bound to protein. Within-run and between-day CVs were 3.8% and 4.8%, respectively. Compared with a micro-Kjeldahl method for determination of total nitrogen, the coefficient of correlation was 0.99.
Autoantibodies to lactate dehydrogenase in serum identified by use of immobilized protein G and immobilized jacalin, a jackfruit lectin.
Authors: Backer E T , Harff G A
Published: January 2020

In this method for identifying autoantibodies to lactate dehydrogenase (anti-LDs) in serum, we used immobilized Protein G to bind IgG-complexed LD and immobilized jacalin to bind IgA-complexed LD, leaving non-complexed LD in solution. The non-complexed LD and total LD were kinetically measured. We report results as LD bound to immobilized Protein G and LD bound to immobilized jacalin. Using sera demonstrating IgG and IgA anti-LDs by immunoelectrophoresis (IEP), respectively, we optimized the method for incubation time and concentration of binding agents. We demonstrated concomitant binding of LD and greater than or equal to 98% of IgG and of LD and greater than or equal to 92% of IgA. For LD bound to immunobilized Protein G the detection limit was 10 U/L, within- and between-run CVs ranged from 2.9% to 9.1%, and values for normal sera were less than or equal to 3% of total LD. Results for LD bound to immobilized jacalin were similar. We tested 10 sera displaying aberrant LD electrophoretograms: In seven, LD bound to immobilized Protein G was increased (range: 26-99% of total LD), indicating IgG-complexed LD. This was confirmed by IEP, demonstrating IgG1,2, or IgG3 anti-LDs in these sera. In the other three sera, LD bound to immobilized jacalin was increased (range: 38-72% of total LD), indicating IgA-complexed LD. This was confirmed by IEP, demonstrating IgA anti-LDs in these sera. Evidently this method is an alternative to IEP for identifying anti-LDs in serum.
Effect of glycation of low-density lipoprotein on the immunological determination of apolipoprotein B.
Authors: Press R D , Wilding P
Published: January 2020

Non-enzymatic glycation of low-density lipoprotein (LDL) may contribute to the premature atherogenesis of patients with diabetes mellitus. To assess whether glycation of apolipoprotein B, the predominant protein of LDL, interferes with the ability to immunologically quantify this protein, we prepared and purified glycated LDL by incubating normal plasma samples with high concentrations of glucose. Although both the plasma and the LDL specimens incubated with glucose contained significantly more glycated protein than control specimens, the quantitative interaction of an apolipoprotein B-specific antibody with glycated vs nonglycated LDL was not significantly different. We conclude that apolipoprotein B can be accurately quantified immunologically despite the presence of clinically excessive degrees of LDL glycation.
Studies on methylmalonic acid in humans. I. Concentrations in serum and urinary excretion in normal subjects after feeding and during fasting, and after loading with protein, fat, sugar, isoleucine, and valine.
Authors: Rasmussen K
Published: January 2020

Determination of methylmalonic acid (MMA) in serum or urine for evaluation of tissue cobalamin (vitamin B12) deficiency is becoming an important diagnostic procedure. Here I present the first investigation of dietary influence on concentrations of MMA in serum and urine. Everyday meals caused an increase in urinary excretion, whereas the concentration in serum was not increased significantly. It is difficult to prime the accumulation of MMA in normal subjects by stressing the metabolic pathway; after loading subjects with 100 mmol of isoleucine or valine, the absolute amount of MMA excreted increased by only about 3 mumol. Its concentration in serum tended to decrease and its urinary excretion declined after lack of protein intake for more than 15 h. Although a linear relationship was demonstrated, for the first time, between concentrations in serum and urinary excretion, my results indicate that patients with early evidence of cobalamin deficiency and normal subjects may best be differentiated by measurements in serum, especially in the case of nonfasting (i.e., ambulatory) patients.
Studies on methylmalonic acid in humans. II. Relationship between concentrations in serum and urinary excretion, and the correlation between serum cobalamin and accumulation of methylmalonic acid.
Authors: Rasmussen K , Moelby L , Jensen M K
Published: January 2020

Methylmalonic acid (MMA) concentrations are increased in cobalamin (vitamin B12) deficiency, but the relative diagnostic usefulness of determination of MMA in serum vs urine has not yet been assessed. We obtained urine collections and matched serum samples from 28 healthy volunteers and from 20 consecutive patients admitted for clinical and hematological evaluation because of low cobalamin concentrations in serum. Increased concentrations of MMA in serum were found in 12 patients, in all of whom a clinical diagnosis of cobalamin deficiency was established. By contrast, cobalamin deficiency was excluded in seven of the eight remaining patients, who all had normal MMA concentrations. Here we report that linear relationships exist between MMA concentrations in serum (investigated range: 0.05-34.2 mumol/L) and MMA concentrations in urine (r = 0.74), concentrations relative to creatinine (r = 0.98), and MMA excretion rates (r = 0.97) (P less than 0.001 in each instance). Our data are consistent with glomerular filtration and passive reabsorption of MMA by the tubules. We demonstrate, for the first time, a negative correlation between concentrations of cobalamin and MMA in serum in clinical cobalamin deficiency (r = -0.69; P less than 0.01; n = 12); when the values for MMA were log transformed, the correlation with cobalamin was much better (r = -0.84; P less than 0.0005).
Simultaneous liquid-chromatographic determination of vitamin K1 and vitamin E in serum.
Authors: Cham B E , Roeser H P , Kamst T W
Published: January 2020

We describe a high-performance liquid chromatographic procedure for the simultaneous measurement of vitamins K1 and E in human serum. Delipidated human serum (free of vitamins K1 and E) was used to make standard solutions of these vitamins, and cetyl naphthoate and alpha-tocopheryl acetate were the internal standards for vitamin K1 and vitamin E, respectively. A simple, novel separation method utilizing liquid-liquid partition chromatography was used as a preparative "clean-up" procedure. Cetyl naphthoate and vitamin K1 (after post-column reduction) were detected by fluorescence, alpha-tocopheryl acetate and vitamin E by ultraviolet absorption. Sensitivity (detection limit) of the assay was 30 pg for vitamin K1 and 5 ng for vitamin E per injection. The method is specific, precise, and more rapid than previously described procedures. Within- and between-assay CVs were 8.1% and 12.9%, respectively, for vitamin K1; 3.5% and 6.0%, respectively, for vitamin E. Analytical recoveries of vitamins K1 and E were 80% and 93%, respectively, from serum and from delipidated serum (standards). The average neonatal serum concentration of vitamin K1 was 83 ng/L, 2.5 mg/L for vitamin E; for normolipidemic adults, the values were 343 ng/L and 7.9 mg/L, respectively, and for hyperlipidemic adults, 541 ng/L and 11.1 mg/L, respectively.
Rapid enzymatic determination of urinary oxalate.
Authors: Li M G , Madappally M M
Published: January 2020

This new reagent kit for the quantitative measurement of oxalate in urine is a modification of an earlier Sigma oxalate assay procedure (procedure no. 590), a coupled enzyme assay involving oxalate oxidase and horseradish peroxidase. The new analytical procedure includes methods for processing urine specimens to eliminate interference with oxalate color development at 590 nm by ascorbic acid, divalent cations, and other urinary constituents. The reaction is complete in less than 5 min, and results are linearly related to oxalate concentration up to at least 1 mmol/L. Assay sensitivity and within-run and between-run precision were within the limits acceptable for other urinary oxalate procedures. Analytical recovery of added oxalate was close to 100%. This specific, simple, rapid procedure is suitable for routine clinical use.
Greater than expected alanine aminotransferase activities in plasma and in hearts of patients with acute myocardial infarction.
Authors: Giesen P L , Peltenburg H G , de Zwaan C , Janson P C , Flendrig J G , Hermens W T
Published: January 2020

Early increases in the activity of alanine aminotransferase (ALT, EC 2.6.1.2) in plasma are observed in about 7% of patients with acute myocardial infarction (AMI), of whom about half die. Some type of liver injury, secondary to AMI, could be responsible for this phenomenon. However, quantitative analysis shows that the release of ALT in most of these patients conforms to the myocardial release pattern. Moreover, extra release of hepatic aspartate aminotransferase (EC 2.6.1.1) is not observed. These findings suggest that the heart may occasionally contain a high ALT activity. This hypothesis was verified by determination of enzyme activities in 10 hearts obtained from patients who died after AMI. The mean ALT activity in these hearts, 21 (SD 12) U per gram wet weight, significantly (P less than 0.01) exceeds the value of 7.7 (SD 4.9) U/g found for seven control hearts and may reflect increased amino acid metabolism in the energy-depleted heart muscle, as described earlier for skeletal muscle.
Simultaneous determination of pentoxifylline and three metabolites in biological fluids by liquid chromatography.
Authors: Lambert W E , Yousouf M A , Van Liedekerke B M , De Roose J E , De Leenheer A P
Published: January 2020

We describe a sensitive and specific liquid-chromatographic assay for pentoxifylline and three of its metabolites in human plasma and urine. Addition of hydrochloric acid to the sample before extraction, and incorporation of acetic acid in the chromatographic eluent, allow the simultaneous determination of the four compounds plus an internal standard in one chromatographic run. Unlike gas-chromatographic procedures, this method does not involve derivatization no similar analysis of serum or plasma samples has been described before now. The method has been applied successfully to routine analysis and to pharmacokinetic studies.
Alpha-amylase determination with the Reflotron reagent carrier system: use of whole blood, plasma, and serum, and effect of isoenzymes.
Authors: Bais R , Badenoch J , Bayer P M , Foo Y , Keller H , Koller P U , Leinberger R , Weidemann G , Rosalki S B
Published: January 2020

In the Reflotron Amylase dry-reagent carrier system (Boehringer Mannheim GmbH) a new substrate is used for determining total amylase (EC 3.2.1.1) activity:indolyl-alpha-D-maltoheptaoside. The procedure shows low imprecision (median CV less than 3.2%), and results for sera, plasma, and capillary and venous blood (y) correlate well with those of a conventional alpha-amylase method involving p-nitrophenyl (PNP)-maltoheptaoside substrate (x) (for 209 blood samples: y = 0.981x + 9.7; r = 0.994). Correlation was also excellent with a method involving maltotetraose as substrate (r = 0.987). Attachment of an indoxyl residue rather than a PNP group to the maltoheptaoside did not affect the substrate response to pancreatic or salivary isoenzyme activity. Therefore, the relative proportion of these isoenzymes did not affect the correlation between the Reflotron Amylase reagent carrier and the alpha-amylase PNP-maltoheptaoside method. With a reaction time of less than 3 min, this system is especially suitable for amylase determination in situations where a prompt result is required.
IgG heavy-chain subclass typing of myeloma paraproteins by isoelectric focusing immunoblot analysis.
Authors: Fasullo F J , Fritsche H A , Liu F J , Hamilton R G
Published: January 2020

The objective of this study was to develop a clinical laboratory method for subclass typing of human immunoglobulin G (IgG) paraproteins. Serum proteins were isoelectrically focused (IEF) in a mini-gel and passively blotted by capillary diffusion onto untreated nitrocellulose. Unreacted sites on the nitrocellulose were blocked with bovine serum albumin and the bound IgG was detected with peroxidase-conjugated anti-human IgG1-4 monoclonal antibodies from WHO/IUIS clones. The IEF immunoblot specificity was demonstrated by analysis of documented IgG, IgA, and IgM myeloma proteins of known subclass and light-chain composition. IEF immunoblots of sera from 18 myeloma patients who had an above-normal total IgG concentration produced IEF immunoblot patterns composed of five to 10 discrete bands (pI range 6.0 to 8.4). In contrast, no detectable IgG bands were observed with sera containing IgA and IgM paraproteins. The observed subclass frequencies of IgG paraproteins were 56% IgG1 (10/18), 28% IgG2 (5/18), 11% IgG3 (2/18), and 5% IgG4 (1/18). IEF immunoblot analysis permits the monitoring of changes in the pI and subclass of an IgG paraprotein over the course of a myeloma patient's therapy program.
Short-term and long-term variability of indices related to nutritional status. I: Ca, Cu, Fe, Mg, and Zn.
Authors: Gallagher S K , Johnson L K , Milne D B
Published: January 2020

Five free-living women (ages 28-38 y) and five women (ages 23-44 y) residing in a metabolic unit and eating a constant diet were assessed for variation in indices related to mineral nutrition. Blood was sampled once a month for five months, once a week for five weeks, and once a day for five days to assess analytical and biological variability. Analytical variability was determined by using concurrently run duplicate control samples prepared from plasma or serum pools. Of the measured indices, serum ferritin varied most, with intra-individual variance of 4.72% to 18.0%. Much of this variance may have been because of changes in iron status or in the analytical technique used. Intra-individual month-to-month variance for other indices ranged from 17% for superoxide dismutase (EC 1.15.1.1) to 1.5% for calcium. Correction for long-term analytical variation indicated that most of the variance was associated with the biological component. The higher biological variabilities of some indices, including ferritin or superoxide dismutase, need to be considered when nutritional status is being evaluated or when serial observations are made over a protracted period in clinical studies or trials.
Diagnostic test for mucopolysaccharidosis. I. Direct method for quantifying excessive urinary glycosaminoglycan excretion.
Authors: Whitley C B , Ridnour M D , Draper K A , Dutton C M , Neglia J P
Published: January 2020

This direct method for quantifying excessive urinary glycosaminoglycan excretion exploits the specific binding of 1,9-dimethylmethylene blue (DMB). The procedure obviates cumbersome and labor-intensive procedures for separating glycosaminoglycans from other constituents of urine. Pediatric pharmaceutical formulations (except heparin), in concentrations expected in urine, do not interfere with spectrophotometry, nor does protein. Results can be expressed in terms of urinary creatinine; thus the test is applicable to very small urine specimens (0.1 mL), such as those obtainable from neonates. In a pilot study, results of the direct DMB test for 48 urine specimens agreed with the clinical diagnosis, and quantitative measurements correlated moderately (r = 0.76) with results of a commonly used procedure (carbazole-borate reactivity after precipitation with cetylpyridinium chloride). The present method was also used to assess metabolic correction in a patient with Hurler's syndrome after treatment by bone-marrow transplantation. This quantitative method surmounts the major technical problems of developing mass screening programs for infants, thus offering the potential for earlier diagnosis and treatment of mucopolysaccharidosis diseases.
Lactate dehydrogenase isoenzyme pattern in sera of patients with malignant diseases.
Authors: Giannoulaki E E , Kalpaxis D L , Tentas C , Fessas P
Published: January 2020

Total lactate dehydrogenase (LDH; EC 1.1.1.27) activity and the percentage distribution of LDH isoenzymes were determined in 127 patients with malignant diseases. A shift in the isoenzyme patterns was observed toward the M-type, with an increase in the percentage of LDH-4 and LDH-5 isoenzymes and a slight increase in total LDH activity of all patients. Serum samples from 68 of the patients contained an abnormal isoenzyme of LDH, "LDH-1 ex," that, on agarose gel electrophoresis at pH 8.6, migrated between albumin and LDH-1 isoenzyme. Chemotherapy, radiotherapy, or surgical removal of the tumor was accompanied by disappearance of this abnormal isoenzyme. The heat stability of LDH-1 ex isoenzyme appears to be similar to that of LDH-1 but greater than that of the other LDH isoenzymes. Statistical analysis of these data demonstrated a significant correlation between malignancy and the appearance of LDH-1 ex isoenzyme (P less than 0.001). In contrast, the relationship between LDH-1 ex isoenzyme and metastasis or anatomical location of the malignancy is not statistically important (P less than 0.1).
Pancreatic oncofetal antigen and carbohydrate antigen 19-9 in sera of patients with cancer of the pancreas.
Authors: Shahangian S , Fritsche H A , Hughes J I , Gelder F B
Published: January 2020

Pancreatic oncofetal antigen (POA) and carbohydrate antigen 19-9 (CA 19-9) were measured in the sera of 23 patients with cancer of the pancreas to determine the true positive rates of these cancer markers. In one group of unselected pancreatic cancer patients (n = 9), both tests showed above-normal results in three patients and both gave values that were within reference limits in three other patients. Two of the three remaining patients had increased CA 19-9 but normal POA values, and one patient had increased POA but normal CA 19-9 concentrations in serum. In another group of 14 pancreatic cancer patients, selected on the basis of increased concentrations of POA in serum, the CA 19-9 values were increased in eight. In four patients who had progressive disease, the concentrations of both markers increased with time in one patient, only POA in one, and only CA 19-9 concentration in another. (The fourth patient had increased but stable concentrations of POA and CA 19-9 in serum.) These data suggest that serum POA and CA 19-9 measurements should be used in combination in the evaluation of patients with cancer of the pancreas.
Standardization of a commercial (Boehringer Mannheim diagnostics) enzymic method for cholesterol.
Authors: Warnick G R , Spain M , Kloepfer H , Volke T M
Published: January 2020

The Laboratory Standardization Panel of the National Cholesterol Education Program recommends that cholesterol method accuracy ideally be within 3% of the true value determined by the Abell-Kendall Reference Method, a component of the National Reference System for Cholesterol. As one of the Abell-Kendall network laboratories established to facilitate cholesterol standardization, the approach we recommend for determining accuracy involves a comparison analysis on patients' specimens by the method in question and by the Abell-Kendall method. Use of fresh specimens precludes matrix interactions that may influence enzymic measurement. Using this approach, we assessed an enzymic method for cholesterol with two instruments (Boehringer Mannheim/Hitachi 717 and 737), with BMD reagent, controls, and calibrator. Fresh and frozen sera were analyzed with both instruments over three days. The Abell-Kendall method was used at the Northwest Lipid Research Center on frozen aliquots of the same sera. Both instruments demonstrated good agreement with the Reference Method, as determined by linear regression; overall bias averaged less than -2% for the Hitachi 717 and -1% for the Hitachi 737 at 2000 mg/L--i.e., within the accuracy recommendation. We observed a difference in bias for fresh and frozen specimens; with the Hitachi 717, fresh specimens exhibited -3% bias at 2000 mg/L, but there was virtually no bias of determinations of frozen specimens.
Diagnosis of acute myocardial infarction from two measurements of creatine kinase isoenzyme MB with use of nonparametric probability estimation.
Authors: Bernstein L H , Good I J , Holtzman G I , Deaton M L , Babb J
Published: January 2020

By using bivariate probability estimation for the diagnosis of acute myocardial infarction (AMI) we show how to overcome the difficulties encountered for patients whose clinical presentation is atypical and those encountered when multiple isoenzyme determinations are treated by univariate methods. We use the values for creatine kinase isoenzyme MB measured at the time of admission and 12 h later to estimate the Bayes factors in favor of AMI. The Bayes factors are compiled into a table that the clinician can use to estimate the posterior probability that a patient has AMI. The table of Bayes factors is based on data for a sample of 802 non-AMI patients and 180 AMI patients. Further to validate the method, we randomly chose 200 of the non-AMI and 50 of the AMI patients as an evaluation sample, then used the remaining 602 non-AMI and 130 AMI patients to recompute the Bayes factors. These Bayes factors were used to find the probability of AMI for each of the 250 patients in the evaluation sample. The method resulted in only one false positive and no false negatives. For the misclassified patient the measurements at admission and 12 h later were 1 and 11 U/L; the posterior odds were 15 to 1 in favor of AMI, but in fact the patient was non-AMI.
Automated determination of drugs in serum by column-switching high-performance liquid chromatography. IV. Separation of tricyclic and tetracyclic antidepressants and their metabolites.
Authors: Matsumoto K , Kanba S , Kubo H , Yagi G , Iri H , Yuki H
Published: January 2020

We describe automated column-switching high-performance liquid chromatography for determining nine tricyclic and tetracyclic antidepressants (TCAs) and their metabolites in human serum. TSKgel ODS-80TM and TSKprecolumn PW (Tosoh Co., Tokyo) are used in the analytical column and the precolumn, respectively. A 200-microL serum sample is directly injected onto the precolumn. After washing the serum proteins from the precolumn with potassium phosphate buffer, the precolumn connection is switched to introduce the retained substances onto the analytical column. The drugs are then eluted within 30 min with an acetonitrile/potassium phosphate buffer mixture containing sodium 1-heptanesulfonate. The analytical recoveries (95-104%), reproducibilities (within-run CV less than 3%), and detection limits (10 micrograms/L) indicate that this HPLC system is suited for therapeutic drug monitoring. Correlations were good between the TCA concentrations in serum and administered dose (r = 0.713, n = 41), and between 10-hydroxynortriptyline and nortriptyline in serum (r = 0.691, n = 24).
Monoclonal immunoglobulins: affinity blotting for low concentrations in serum.
Authors: McLachlan R
Published: January 2020

I describe a simple, economical technique for identifying low concentrations of monoclonal immunoglobulins in the presence of excessive amounts of immunoglobulins of other classes. The technique involves binding of specific antibody to nitrocellulose, separating proteins by isoelectric focusing or zone electrophoresis in agarose gels, using capillary transfer to bind proteins to the nitrocellulose via their antibody affinity, and then detecting transferred proteins with enzyme-labeled antibody. A monoclonal immunoglobulin can be completely characterized in 2 h. No expensive equipment is required. Affinity blotting is about 10-fold as sensitive as native blotting, 100-fold as sensitive as silver staining.
An algorithm for testing and reporting serum choriogonadotropin at clinically significant decision levels with use of "pregnancy test" reagents.
Authors: Bandi Z L , Schoen I , Waters M
Published: January 2020

We present an algorithm for monitoring the concentration of human choriogonadotropin (hCG) in serum at various clinical decision levels with use of fast, simple, and cost-effective qualitative pregnancy test reagents for hCG in serum. Based on correlation between laboratory data and clinical observations described in the literature, the following concentrations of hCG in serum can be considered as clinically significant decision levels: 5 int. units/L to exclude or "rule out" the presence of increased hCG; 25 int. units/L for "confirming pregnancy" or confirming the presence of increased hCG from sources other than the trophoblast; and 6500 and 82 500 int. units/L to enhance the sonographic diagnoses of ectopic pregnancies and molar pregnancies, respectively. We used Tandem Icon II (Hybritech) pregnancy test reagents and evaluated the reagents for possible "false-positive" findings at the 25 int. units/L limit of hCG detectability by analyzing 100 post-menopausal and more than 4000 premenopausal serum specimens. The performance of the reagents was validated also at 5 int. units/L and at limits of hCG detectability greater than 25 int. units/L.
Cyclosporine concentrations in blood after liver transplantation: correlation of immunoassay results with clinical events.
Authors: Haven M C , Sobeski L M , Earl R A , Markin R S
Published: January 2020

To investigate the clinical utility of immunoassays for cyclosporine and metabolites in plasma and whole blood, we monitored 25 patients after orthotopic liver transplantation. We compared cyclosporine as measured by TDx fluorescence polarization immunoassay (of both plasma and whole blood) and by two polyclonal radioimmunoassays (from Sandoz and INCSTAR). We found considerable differences in measured cyclosporine concentrations, which were dependent on method, matrix, and clinical condition. Correlation coefficients between results by the various methods for samples from individual patients ranged from 0.825 to 0.996. The three methods used for monitoring cyclosporine in whole blood gave proportional results (Sandoz less than INCSTAR less than TDx) in individual patients, but results for the two methods for plasma sometimes differed by more than 100%. In some cases, ratios of plasma cyclosporine concentration (result by TDx/result by Sandoz) were correlated with disturbances in hepatic excretory function or kidney function. This ratio may be useful in monitoring for nephrotoxicity.
A system for toxicological screening by capillary gas chromatography with use of a drug retention index based on nitrogen-containing reference compounds.
Authors: Manca D , Ferron L , Weber J P
Published: January 2020

Capillary gas chromatography with nitrogen-specific detection allows rapid screening of numerous drugs of toxicological interest. However, for accurate identification of individual peaks, the system must be well calibrated, e.g., through the use of retention indices (RI). To overcome problems associated with the use of RI's based on homologous series determined with nitrogen-specific detectors, we have developed an RI reference system based on molecular masses and retention times of nitrogen-containing compounds. The standards chosen are easily available in highly purified form and can be detected by the unmodified nitrogen-specific detector. By using temperature programming, we can obtain a linear relationship between the molecular masses of standards and their retention times. Used in conjunction with microcomputer data handling, this screening system is rugged and reliable, operating 22 h per day. In the past two years, we have screened greater than 3000 samples (blood, serum, urine, gastric lavage) without major problems.
Determination of cyclosporine in plasma: specific radioimmunoassay with a monoclonal antibody and liquid chromatography compared.
Authors: Vernillet L , Keller H P , Le Bigot J F , Humbert H
Published: January 2020

A radioimmunoassay of cyclosporine (Sandimmune) involving use of a mouse monoclonal antibody was tested to monitor specifically the parent drug in plasma. The cyclosporine concentrations obtained by RIA were compared with those obtained by the HPLC method. For the RIA method, the within- and between-assay CVs are less than 6%, the limit of detection is about 10 micrograms/L for a 50-microL sample of plasma. For the HPLC method, the within- and between-assay CVs are less than 20%, the limit of detection is about 15 micrograms/L for a 1-mL sample of plasma. The concentrations by RIA correlated well with those by HPLC in samples from patients receiving bone-marrow (n = 39), heart (n = 52), or liver (n = 51) transplants. In all indications, the ratio of values by RIA to those by HPLC for these samples remained stable and close to 1 during the drug-monitoring period, i.e., for up to 202 days. Therefore, the specific RIA can be used instead of HPLC to measure the parent drug in plasma.
Methods compared for determining total amylase activity and amylase isoenzymes in serum.
Authors: Badenoch J L , Bals R
Published: January 2020

We evaluated two kinetic methods for determining total amylase activity and isoenzyme composition in serum. Stability studies of reagents for measuring total activity indicate that reagents containing 4-nitrophenyl-alpha-glucosides or enzyme-linked reagents can be stored only for seven days at 4 degrees C. Methods based on 4-nitrophenyl-alpha-glucoside substrates cannot be used if the reagent absorbance at 405 nm exceeds 2. However, in the alpha-amylase EPS method (Boehringer Mannheim) an ethylidene-protected 4-nitrophenyl-alpha-D-maltoheptaoside substrate is stable for up to 28 days after reconstitution. Further studies indicated that the Amylase-DS (Beckman) and the alpha-Amylase EPS standard curves are linear to at least six times the upper limit of the reference interval. Within-batch imprecision (CV less than 1.1%) and between-batch imprecision (CV less than 3.3%) for these two methods are comparable with those for other kinetic methods, and there is excellent correlation (r2 = 0.983) between the two methods. The reference interval, determined by use of samples from 90 healthy blood donors, is 31 to 141 U/L for the amylase-DS method, 22 to 92 U/L for the alpha-Amylase EPS method. We also used these two methods to measure amylase isoenzymes after inhibiting the salivary isoenzyme with either a lectin or a monoclonal antibody. We found the monoclonal antibody method more specific than the lectin inhibition method for determining the isoenzymes.
Serum creatine kinase isoenzyme BB is a poor index to the size of various brain lesions.
Authors: Schwartz J G , Bazan C , Gage C L , Prihoda T J , Gillham S L
Published: January 2020

We divided patients with brain lesions into three groups: (a) patients with primary or metastatic brain cancer, (b) brain infarctions, and (c) brain contusion(s). We analyzed each patient's sera for creatine kinase isoenzyme BB (CK-BB), using a monoclonal antibody kit (Impres-BB; International Immunoassay Laboratories). Computerized axial tomography (CAT) scans were performed on each patient. The size of the various lesions was measured from the CAT scan and recorded in milliliters. Total CK, CK-BB, and their ratios were compared with the volume of damaged brain tissue. We found no correlation between any of the variables and the various brain lesions. We attribute this lack of correlation to an intact blood-brain barrier, the rapid elimination or inactivation of CK-BB, or some combination of these factors.