bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultraviolet-absorbing organic anions in uremic serum separated by capillary zone electrophoresis, and quantification of hippuric acid
Authors: Schoots A C , Verheggen T P , De Vries P M , Everaerts F M
Published: January 2020

Organic anions accumulated in blood serum of patients with chronic renal failure were separated by a novel technique: closed-system capillary zone electrophoresis (CZE) in a pH6 carrier-electrolyte system. Hippuric acid (HA), p-hydroxyhippuric acid, and uric acid were identified by their co-elution with standards prepared in ultrafiltered normal serum and by comparison with the corresponding ultraviolet-detected peaks positively identified in the HPLC analyses. Analysis time for the entire profile is 8 min. Repeatabilities (CVs) of CZE migration times and peak areas of the three acids in serum samples were about 0.7% and 6%, respectively. We quantified HA in 10 ultrafiltered uremic serum samples and compared results with those by a previously described HPLC procedure. The very good agreement further supports the identification of hippuric acid. Accuracy and precision of the CZE method were similar to those for the HPLC gradient-elution method, but analysis time for HA (8 min) is much less than by HPLC (90 min). Our technique is very suitable for selective, rapid analysis for (ultraviolet-absorbing) anionic constituents in ultrafiltered uremic serum, without any sample pretreatment.
Rapid, robust method for measuring low concentrations of albumin in urine
Authors: Medcalf E A , Newman D J , Gorman E G , Price C P
Published: January 2020

We describe a rapid particle-enhanced turbidimetric immunoassay for albumin in urine. Intra- and interassay CVs were less than 5% and less than 10%, respectively, the detection limit is 2 mg/L, and the working range extends to 200 mg/L. Mean analytical recovery of albumin added to centrifuged urines was 100% (SD 10.6%), and, when results were compared with those by the Pharmacia RIA, the correlation coefficient was 0.99. The working reagents are stable for at least six months; thus this assay is suited for both batch and urgent analysis.
Immunoadsorption of IgG onto second antibody covalently attached to Amino-Dylark beads for radioimmunoassays
Authors: Kakabakos S E , Evangelatos G P , Ithakissios D S
Published: January 2020

We present a solid-phase immobilization method for radioligand assays, using an immunoadsorption coating procedure of anti-triiodothyronine rabbit IgG (anti-T3 IgG) onto second antibody (sheep anti-rabbit IgG) covalently bound to Amino-Dylark beads. The second antibody was in excess, compared with the first antibody, thus eliminating reproducibility problems between immunoadsorptions. Beads coated with second antibody can be used to immobilize a variety of antigen-specific first antibodies. The amount of anti-T3 antibody required for solid-phase T3 radioimmunoassay (RIA) was only 10% more, per assay tube, than that utilized in liquid-phase T3 RIA, in which polyethylene glycol solution was the separation reagent; characteristics of assay performance were comparable. The immobilization procedure requires high-titer antisera or antigen-specific IgG and seems advantageous because of the decrease in antibody requirements without significant modification of antibody functionality.
Sensitive time-resolved fluorescence immunoassay of somatotropin in serum
Authors: Kahan I , Papanastasiou-Diamandi A , Ellis G , Makela S K , McLaurin J , D'Costa M , Diamandis E P
Published: January 2020

We describe a new "sandwich"-type non-isotopic immunoassay for human somatotropin (GH, growth hormone) in serum. In the assay, GH is captured by a monoclonal antibody immobilized in a white microtiter well and simultaneously reacted with a second biotinylated monoclonal antibody. The degree of binding of biotinylated antibody, which increases with increasing amount of GH in the sample, is quantified by adding streptavidin labeled with the europium chelate of 4.7 - bis(chlorosulfophenyl) - 1.10 - phenanthroline - 2.9 - dicarboxylic acid. The fluorescent complex on the solid phase is then measured by excitation at 337.1 nm (nitrogen laser) and monitoring the emission at 615 nm in a gated fluorometer/analyzer. The proposed procedure has short incubation times (less than 4 h protocol), uses only 25 microL of serum per microtiter well, and gives precise and accurate results. The method was clinically evaluated with samples obtained from pediatric patients undergoing investigation for growth abnormalities and from a patient with acromegaly.
Sample evaporation and its impact on the operating performance of an automated selective-access analytical system
Authors: Burtis C A
Published: January 2020

In a series of gravimetric and analytical experiments I measured how much analytical error is ascribable to sample evaporation during routine operation of the Dimension analytical system. This error, a time-dependent function, is related to the volume of liquid placed in either a 0.5- or 2.0-mL cup and whether the cup is capped. For the 0.5-mL cups, analytical error at a 4-h residence time ranged from 12% to 16% (uncovered) to 8% to 13% (covered), whereas errors for the 2.0-mL cup ranged from 6% to 10% (uncovered) to 2% to 5% (covered). To minimize evaporative loss, I recommend (a) protecting the surface areas of the samples, (b) using larger samples, and (c) minimizing the time that a sample is in its cup before and during analysis.
Alpha 1-acid glycoprotein decreases recovery of total protein in urine when trichloroacetic acid is used to precipitate the proteins
Authors: Beilby J P , O'Leary B A
Published: January 2020

Total urine protein was measured in 132 samples by an automated benzethonium chloride method and the Ponceau-S/trichloroacetic acid (PS/TCA) method. Of these, 27% gave a result 0.1 g/L or more higher by the benzethonium chloride method. Of this 27%, most contained an abnormally high concentration of the acute-phase reactant, alpha 1-acid glycoprotein. By assaying urine containing added alpha 1-acid glycoprotein and albumin, we found that alpha 1-acid glycoprotein causes the PS/TCA method to underestimate the total urine protein concentration, whereas the benzethonium chloride method is unaffected. Not all urinary albumin was precipitated by TCA when alpha 1-acid glycoprotein was present. Therefore, protein methods in which trichloroacetic acid is used as a concentrating step before the assay will underestimate total urine protein when the concentration of alpha 1-acid glycoprotein is high.
Blood cholesterol concentration: fingerstick plasma vs venous serum sampling
Authors: Greenland P , Bowley N L , Meiklejohn B , Doane K L , Sparks C E
Published: January 2020

To assess whether venous and fingerstick blood samples yield similar cholesterol concentrations, we obtained both types of samples simultaneously in 108 volunteers participating in a cholesterol screening program. All samples were analyzed by the same enzymatic method in a standardized laboratory, and pairs of simultaneous samples were measured in the same laboratory run. Cholesterol concentrations in fingerstick-derived plasma were consistently higher than in the venous serum (P less than 0.0001), by a positive bias averaging 3.6%. Cholesterol values in fingerstick plasma also were higher than cholesterol results for venous serum placed in a capillary collection tube (average bias +2.4%). The positive bias of fingerstick plasma vs venous serum results appears to be at least partly due to specimen handling, although a true physiological difference between venous and fingerstick cholesterol concentrations is probably also involved. If a positive bias of this magnitude from fingerstick blood sampling is left unadjusted, substantial numbers of people will be labeled "at risk" and referred to physicians when their true values were actually within the acceptable range.
Urinary hydroxyproline in infants with and without fractures/rickets
Authors: Koo W W , Krug Wispe S K , Succop P , Champlin A , Sherman R , Berry H
Published: January 2020

Molar ratios of peptide-bound and free hydroxyproline:creatinine (OHPr:Cr) in urine were measured at 3, 6, 9, and 12 months of age in two groups of very-low-birthweight (VLBW less than or equal to 1500 g) infants. Group A (15 infants) had radiographically confirmed fractures and (or) rickets (F/R); Group B (17 infants) did not. The urinary peptide-bound OHPr:Cr ratio varied widely within groups and was greatest at three months in both groups: A = 0.81 +/- 0.45 and B = 0.55 +/- 0.32 (mean +/- SD). The ratio decreased with increasing postnatal age for each group but was not statistically different between groups throughout the study. The urinary free OHPr:Cr ratio also was greatest at age three months (A = 0.32 +/- 0.15 and B = 0.53 +/- 0.46), rapidly decreasing afterwards, and was not statistically different between groups throughout the study. We conclude that, in VLBW infants, bone turnover as indicated by the urinary peptide-bound OHPr:Cr ratio is highest during early infancy; however, the wide range of values for this ratio suggests that its use alone is not sufficient for detection of F/R in VLBW infants. The rapid decrease in free OHPr:Cr ratio is presumably related to the maturation of renal tubular function.
Competitive heterogeneous enzyme immunoassay for theophylline by flow-injection analysis with electrochemical detection of p-aminophenol
Authors: Gil E P , Tang H T , Halsall H B , Heineman W R , Misiego A S
Published: January 2020

A competitive enzyme-linked immunoabsorbent assay based on the flow-injection amperometric detection of p-aminophenol has been investigated with use of the materials and general procedure of a commercial kit for the determination of theophylline in human serum. The antibody is immobilized on glass beads, and the enzyme label is alkaline phosphatase (EC 3.1.3.1). The high currents generated during the electrochemical detection allowed a rapid (35 min) and simple determination of theophylline throughout its therapeutic range (10-20 mg/L) and also in the subtherapeutic range (detection limit of about 80 micrograms/L).
Dichlorobenzoquinone chloroimine colorimetry of uric acid in urine
Authors: Miller P , Oberholzer V
Published: January 2020

In this nonenzymatic colorimetric method, dichlorobenzoquinone chloroimine is used as a color reagent to measure uric acid in urine. This method is less subject to the interferences that make enzymatic methods unreliable and is inexpensive, simple, and fast.
Retention of antimony in skin biopsies of leishmaniasis patients after treatment with N-methylglucamine antimoniate
Authors: Dorea J G , Merchan-Hamann E , Ryan D E , Holzbecher J
Published: January 2020

Leishmaniasis patients were treated with N-methylglucamine antimoniate by intravenous injections of 10 or 20 mg of Sb per kilogram of body weight per day for 10 or 20 days. Digests of skin biopsies taken from the site of lesion before and after treatment were analyzed for antimony by instrumental neutron activation (INAA). The detection limit of the assay was 20 ng, and no Sb could be measured in digests of samples (less than 2.45 mg) taken before treatment. Biopsies taken after injections of Sb showed concentrations in the range of 8.32 to 70.68 ng/g skin. We discuss the usefulness of INAA in the study of Sb in small samples of tissues.
Measurement of pepsinogen group I in endoscopic gastroduodenal biopsies
Authors: Plebani M , Di Mario F , Dal Santo P L , Faggian D , Germana B , Vianello F , Naccarato R , Burlina A
Published: January 2020

A technique for determining pepsinogen group I (PGI) concentrations in endoscopic gastroduodenal biopsies is described and validated. This method has a good precision (intra-assay CVs 4.1% to 8.4%; between-assay CVs 4.6% to 9.3%), and analytical recovery is satisfactory (94% to 102%). Results were uninfluenced by the storage interval of the samples. At PGI-producing sites (i.e., fundus, corpus) peptic activity was greater than in the antrum and duodenum. PGI concentrations in serum and in gastroduodenal biopsies were not correlated. This simple, reliable method can quantify better than other assays peptic activity in humans, without being influenced by gastric secretory volumes. It will also facilitate prospective studies on the effect of various secretagogues in vivo and evaluations of the influence of anti-ulcer drugs on peptic secretion.
Effect of isoenzyme composition on Kodak Ektachem test results for creatine kinase, lactate dehydrogenase, alkaline phosphatase, and amylase
Authors: Gorus F K , De Pree L
Published: January 2020

Seventy-four patients' sera containing macroenzymes or other uncommon enzyme forms were analyzed for total creatine kinase, lactate dehydrogenase, alkaline phosphatase, or amylase activity on an Ektachem 700XR analyzer. The Ektachem test results correlated well with activities obtained by means of liquid reagents on RA-1000. For each enzyme tested, linear-regression analysis yielded data comparable with those for sera containing exclusively the more common isoenzymes. Increased concentrations of intestinal ALP, however, resulted in higher activities of total ALP being reported for the Ektachem.
Determination of urinary norepinephrine and epinephrine by liquid chromatography with fluorescence detection and pre-column derivatization
Authors: Moleman P , van Dijk J
Published: January 2020

We assayed norepinephrine and epinephrine by utilizing solvent extraction, fluorescence derivatization, and "high-performance" liquid chromatography. A 100-microL aliquot of urine was extracted twice according to Smedes et al. (J Chromatogr 1982;231:25-39) and subsequently incubated with 1,2-diphenylethylenediamine. A 100-microL aliquot of the resulting mixture was injected into a reversed-phase column, and norepinephrine and epinephrine were detected fluorometrically. The within-day CV was 3.0-4.0% and the between-day CV 3.4-7.1% for normal and high concentrations of both analytes. At low concentrations (15-40 nmol/L) these CVs were 4.2-6.8% and 6.8-9.2%, respectively. The detection limit of the assay was less than 0.4 nmol/L for each analyte. We discuss the critical steps for extraction, derivatization, and chromatography. The present method combines the qualities of high precision, specificity, and sensitivity.
Effects of methylamine and heparin on a rapid chromogenic assay of C1-esterase inhibitor in plasma
Authors: Munkvad S , Jespersen J , Gram J , Overgaard K , Rånby M
Published: January 2020

We describe a rapid assay of C1-esterase inhibitor (C1-inh) activity in plasma. After adding purified C1s serine protease (EC 3.4.21.42) in excess to plasma, we determine the residual C1s activity towards a new chromogenic tripeptide, CH3CO-Lys(CbO)-Gly-Arg-pNA. Optimal conditions include the addition of methylamine (final concentration 0.12 mol/L) to reduce the potential inhibitory capacity of alpha 2-macroglobulin towards C1s and the addition of heparin (final concentration 3000 int. units/L) to enhance the reaction of C1s with C1-inh. The correlation with C1-inh antigen concentrations in plasma was excellent. The estimated interassay CV was 4.3%, whereas the intra-assay CV was 2.0% for activity concentrations within the range of normal individuals (means +/- 2 SD: 70-124%), 1.3% at lower concentrations. The method is more convenient, rapid, and precise than previous methods, and C1-inh activity in plasma can be assessed within 30 min. We found that concentrations of C1-inh in plasma were low during open-heart bypass surgery.
Determination of reference intervals for serum magnesium
Authors: Duncanson G O , Worth H G
Published: January 2020

Using a discrete analyzer and a dye-binding method, we measured magnesium in 800 patients' samples received for routine analysis. By excluding data from samples for which the calcium and (or) alkaline phosphatase values were outside defined reference limits, we established a reference interval for magnesium. Because the data showed a gaussian distribution, we could use parametric analysis to establish age-related intervals for both males and females.
High predictivity of galactosyl-hydroxylysine in urine as an indicator of bone metastases from breast cancer
Authors: Moro L , Gazzarrini C , Modricky C , Rovis L , de Bernard B , Galligioni E , Crivellari D , Morassut S , Monfardini S
Published: January 2020

We measured the urinary excretion of galactosyl-hydroxylysine (GH) and hydroxyproline in two groups of women with breast cancer, with (M+, n = 24) and without (Mo, n = 30) clinical, scintigraphic, or radiological evidence of bone metastases. Both these compounds are excreted in larger amounts in the M+ group than in the Mo patients. However, GH, which is a specific marker for bone collagen, provides better predictivity for bone metastases than does hydroxyproline: 92% sensitivity and 90% specificity vs 74% and 79%, respectively, for hydroxyproline.
Screening with zinc protoporphyrin for iron deficiency in non-anemic female blood donors
Authors: Jensen B M , Sandø S H , Grandjean P , Wiggers P , Dalhøj J
Published: January 2020

Iron-depleted donors are at increased risk of developing anemia; if these donors could be identified by a screening test, iron supplementation or decreased donation frequency could be considered. Tests to determine serum ferritin, blood hemoglobin, and erythrocyte (Erc)-zinc protoporphyrin concentrations were examined in 679 consecutive female blood donors to identify donors with non-anemic iron deficiency. The test to determine serum ferritin is expensive and slow, whereas the two latter tests are rapid and less costly and could therefore be used for screening. Women in the fertile age groups had the lowest average serum ferritin values. In all, 93 women (13.7%) had depleted iron stores, as indicated by serum ferritin concentrations less than 14 micrograms/L. In these women, a much better correlation was found between Erc-zinc protoporphyrin and serum ferritin (rs = -0.49, P less than 0.001) than between blood hemoglobin and serum ferritin (rs = 0.31, P less than 0.01). These findings suggest that measurement of Erc-zinc protoporphyrin is superior to that of blood hemoglobin in identifying donors with non-anemic iron deficiency.
Radioligand binding assay of epidermal growth factor receptor: causes of variability and standardization of the assay
Authors: Dittadi R , Gion M , Brazzale A , Bruscagnin G
Published: January 2020

Although experimental evidence indicates a probable role of epidermal growth factor receptor (EGFr) in clinical oncology, no standardized method for its determination has been yet described, and discrepant results have been reported in clinical studies. In standardizing a radioligand binding assay for EGFr, we evaluated the causes of variability in each step of the assay. Entrapment of EGFr in the nuclear fraction and contamination of the crude membrane fraction by cytosol protein were eliminated through preliminary purification steps. Both Scatchard and Rosenthal analysis of the saturation reaction of the membrane fraction with a wide range of concentrations of 125I-labeled EGF revealed a double class of binding sites. Study of the saturation reaction showed a partial exchange of 125I-labeled EGF with endogenous EGF within 20 h. The present method--incubation of partly purified membrane fraction with 125I-labeled EGF, 0.5 nmol/L, with and without 100-fold excess of cold EGF, for 20 h at 26 degrees C, followed by centrifugation at 5000 x g for 30 min to separate membrane-bound 125I-labeled EGF--shows good sensitivity, precision, and accuracy; is reasonably simple; and may be suitable for routine clinical use.
Immunoradiometric assay of atrial natriuretic peptide in unextracted plasma
Authors: Tattersall J E , Dawnay A , McLean C , Cattell W R
Published: January 2020

We have developed and validated a two-site liquid-phase immunoradiometric assay (IRMA) of atrial natriuretic peptide (ANP) in unextracted human plasma. Both radiolabeled rabbit anti-ANP IgG and polyclonal mouse anti-ANP must bind to ANP for detection, and the assay is specific for peptides with both an intact C-terminus and a disulfide bridge. The assay sensitivity (detection limit) is 0.96 pmol/L, and the working range is 2.3-300 pmol/L, with the hook effect occurring above 500 pmol/L. Results for diluted plasma from normal subjects and from patients with renal failure paralleled the standard curve; analytical recovery of ANP added to such samples averaged 94%. The between- and within-assay CVs at 8 pmol/L were 10% and 5%, respectively. The assay is sufficiently sensitive and precise to detect the postural change in ANP concentrations in normal subjects.
Age and sex distribution of alkaline phosphatase isoenzymes by agarose electrophoresis
Authors: Van Hoof V O , Hoylaerts M F , Geryl H , Van Mullem M , Lepoutre L G , De Broe M E
Published: January 2020

We separated isoenzymes of alkaline phosphatase (ALP; EC 3.1.3.1) in 1383 sera of normal individuals (ages 4-65 years) by agarose electrophoresis with the Isopal system (Analis). As expected, the predominant isoenzyme in children was of bone origin, and almost all (99%) of the children had low activities of a second bone fraction, "bone variant" ALP. The "bone variant" disappeared after age 17 in girls and after age 20 in boys. The highest (median) bone ALP activity was reached at age 9 to 10 in girls and at age 13 to 14 in boys, followed by a gradual decline in girls and a steep decline in boys. During adulthood, activity of the bone fraction was constant and no significant differences were observed between sexes, neither for bone nor for liver ALP activity. The latter remained almost unchanged throughout life. We observed no high-Mr ALP activity in children, whereas sera from 60% of the adults contained low activities of high-Mr ALP. Intestinal ALP (soluble form) and "intestinal variant" ALP (hydrophobic form) were frequently present, in 21% and 37% of all samples, respectively. No significant differences were observed between age groups and sexes for the intestinal isoenzymes.
Unique structure of glycopeptide from alpha-fetoprotein produced in human hepatoma cell line, as determined by 1H-nuclear magnetic resonance spectroscopy
Authors: Terrana B , Tecce M F , Manetti R , Ceccarini C , Lamba D , Segre A L
Published: January 2020

Determination of alpha-fetoprotein is used in diagnosis of tumors and neural tube defects. A good reliable source of alpha-fetoprotein would be an obvious advantage to the preparation of diagnostic reagents and their standardization. We have recently developed a method for the production of alpha-fetoprotein from a human hepatoma cell line. This method, which is suitable for scaling up, allowed us to produce 40 g of alpha-fetoprotein from culture supernatant liquid through a simple purification procedure. We have previously shown this protein to be identical to alpha-fetoprotein produced from other sources. However, because the presence of different glycoforms has been reported in alpha-fetoprotein preparations, both from human sources and from other species, it was important to establish the type and extent of glycosylation of alpha-fetoprotein prepared by our method. By using 1H-NMR spectroscopy we were able to establish that our product contains a single N-linked biantennary, fully sialylated complex-type oligosaccharide, typical of human hepatomas.
Diagnosis of perioperative myocardial infarction by considering relationship of postoperative electrocardiogram changes and enzyme increases after coronary bypass operation
Authors: Griesmacher A , Grimm M , Schreiner W , Müller M M
Published: January 2020

We report the results of enzyme determinations in sera from 88 patients, 65 of whom showed inconspicuous reconvalescence, 14 who had myocardial infarction within 24 h (MI 1) after bypass surgery, and nine with myocardial infarction between 24 and 48 h postoperatively (MI 2). We wanted to determine whether the consequent measurement of activities of total creatine kinase (CK), CK isoenzyme MB (CK-MB), lactate dehydrogenase, alpha-hydroxybutyrate dehydrogenase, and aspartate aminotransferase, conducted as a part of routine laboratory diagnostics, provided meaningful information for diagnosing infarcts besides that obtained from the electrocardiogram. The postoperative mean values of the enzyme activities in blood were significantly different among the three groups; however, only a combined evaluation of CK and CK-MB by means of a discriminant analysis allowed the prediction of MI (sensitivity: MI 1 = 98.5%, MI 2 = 95.4%; specificity: MI 1 = 71.4%, MI 2 = 81.8%). CK greater than 600 U/L or CK-MB greater than 45 U/L supports the diagnosis of acute MI.
Use of polymerase chain reaction to detect heterozygous familial hypercholesterolemia
Authors: Keinänen M , Ojala J P , Helve E , Aalto-Setälä K , Kontula K , Kovanen P T
Published: January 2020

We used a modification of the polymerase chain reaction (PCR), involving two pairs of oligonucleotide primers, to detect a mutation in the low-density lipoprotein (LDL) receptor gene, commonly occurring among patients with familial hypercholesterolemia (FH) in Finland. This mutation, called FH-Helsinki, involves a large (about 9500 base pairs, bp) deletion in the LDL receptor gene extending from intron 15 to exon 18. For the PCR, one pair of primers was designed to cover both sides of the deletion in its immediate vicinity. In the presence of the deletion, the primers were brought close enough to each other to allow the amplification and electrophoretic detection of a 300-bp amplification product. In the absence of the deletion, no amplification occurred and this band accordingly was not visible in the gel. To render the interpretation of the results unequivocal, we designed a second pair of oligonucleotide primers. This pair of primers allowed another amplification product (158 bp) to appear in samples containing a normal exon 17, i.e., in DNA specimens from healthy subjects and FH heterozygotes with or without the FH-Helsinki deletion. The technique is easy to perform, avoids the use of radioactive reagents, and is applicable to the detection of any extensive DNA deletion.
Chemiluminescence immunoassay of cyclosporine in whole blood
Authors: Stabler T V , Siegel A L
Published: January 2020

We describe a chemiluminescent immunoassay (CLI) for measuring cyclosporine in whole blood. Its sensitivity and accuracy are comparable with those of an RIA method that makes use of the same specific monoclonal antibody. The comparison with the RIA method was excellent: y(RIA) = x(CLI) + 11.24 (r = 0.99). In our procedure the samples are incubated with cyclosporin C-hemisuccinate-aminobutyl-N-ethylisoluminol, antibody, and paramagnetic particles coated with second antibody. After magnetic separation and washing, the samples are incubated with 200 microL of NaOH (2 mol/L) at 60 degrees C for 30 min. The chemiluminescence generated by automated serial injections of solutions of microperoxidase and dilute (2 mL/L) H2O2 is measured for 5 s. The data are processed by using a spline fit of log B/Bo log conversion. This method is easy to perform and avoids the hazards and costs associated with isotopic waste disposal. The label is stable for at least three years.
Screening for drugs of abuse: effect of heat-treating urine for safe handling of samples
Authors: Wolff K , Shanab M A , Sanderson M J , Hay A W
Published: January 2020

Heating urine samples from high-risk patients for 1 h at 56 degrees C is no longer believed to completely inactivate human immunodeficiency virus (HIV; AIDS virus). To protect staff who are handling infectious samples such as those from drug-addiction units, heating at a higher temperature may be necessary. We report the stability to heat treatment (at 60, 70, and 100 degrees C) at pH 5.1 and 7.6 of some commonly abused drugs, namely, methadone, pethidine, amphetamine, the cocaine metabolite, benzoylecgonine, and the dextropropoxyphene metabolite nordextropropoxyphene. Heat-treating urine at 60 degrees C for 1.5 h or 70 degrees C for 1 h did not significantly affect the measured concentrations of these drugs. However, heat treatment at 100 degrees C for 1 h reduced the recovery of all the drugs. Benzoylecgonine and amphetamine were most susceptible to the different forms of heat treatment.
Cytokines in disease
Authors: Whicher J T , Evans S W
Published: January 2020

Cytokines are peptides used by immune and inflammatory cells to communicate with each other and to control the milieu interieur in which they operate. Recent evidence suggests that they are of immense importance in controlling the local and systemic events of the immune response, inflammation, hemopoiesis, healing, and the systemic response to injury. Many of them can now be measured by immunoassay, and the role of such measurements in the diagnosis and management of disease is actively under investigation. Similarly, the availability of recombinant DNA techniques to produce cytokines in almost unlimited quantities is leading to new and exciting therapeutic applications.
Magnetically tagged subsets of human lymphocytes for assays with laboratory robotics
Authors: Schröder U , Pero R W , Sjögren H O
Published: January 2020

We demonstrate the possibility of automation of whole-cell functionality assays, e.g., mitogen-activated DNA synthesis, DNA repair synthesis, and assessment of drug-metabolizing enzymes, by use of magnetic separation technology. We have attached antibody-coupled magnetic microspheres to the surface of human T-lymphocytes before performing various assays. Evaluating the biological functions of T-cells estimated by the DNA-synthesis assays showed that the presence of antibody-coupled magnetic microspheres did not affect the results (P greater than 0.05). The concentration of adenosine diphosphate ribosyltransferase (EC 2.4.2.30) was shown to be influenced by the magnetic microspheres. However, the amount of enzyme activity induced by oxidative stress was not significantly altered. The results from assays of the phase II drug-metabolizing enzymes glutathione transferase (EC 2.5.1.18) and epoxide hydrolase (EC 3.3.2.3) as well as evaluation of the proliferative response of polyclonal activators (phytohemagglutinin, staphylococcal enterotoxin A, and pokeweed mitogen) support our conclusion that assays can be performed on viable magnetized cells. The use of magnetized cells holds promise for further applications in automated genotoxic and immunological cell assays of mononuclear leukocyte subsets. Laboratory robotics will be essential in bringing these assays into routine use.
Development and performance of a highly sensitive and specific two-site immunometric assay of calcitonin gene-related peptide
Authors: Zaidi M , Seth R , Girgis S I , Self C H
Published: January 2020

Calcitonin gene-related peptide (CGRP), a potent endogenous circulating vasodilator, is produced by the alternative splicing of the calcitonin/CGRP gene and is expressed mainly in neural and cardiovascular tissues. We recently reported a highly sensitive radioimmunoassay of CGRP, based on an antiserum recognizing the C-terminus of the molecule. We have also found that circulating immunoreactive CGRP is heterogeneous; thus we are unable to measure selectively the intact molecule with our one-site competitive approach. We therefore attempted to construct a two-site immunometric assay involving two antibodies, one that detects the C-terminus and another that recognizes the midregion of the molecule. To enhance assay sensitivity, we applied a colorimetric amplification system to this assay. This rapid, robust, and reproducible assay provides more nearly accurate estimates of circulating CGRP and offers a sensitive and more specific alternative to the radioimmunoassay, with advantages in speed, simplicity, and convenience.
Methylmalonic acid concentrations in serum of normal subjects: biological variability and effect of oral L-isoleucine loads before and after intramuscular administration of cobalamin
Authors: Rasmussen K , Møller J , Ostergaard K , Kristensen M O , Jensen J
Published: January 2020

The clinical value of measuring concentrations of methylmalonic acid in serum (S-MMA) as an aid in the diagnosis of cobalamin deficiency has recently aroused interest. In 58 healthy subjects, ages 40-68 years, we found a 0.95 reference interval of 0.05-0.37 mumol/L (mean 0.21, SD 0.094). In 33 of the subjects, who were studied further, day-to-day variation (SD) was 0.031 mumol/L. Intake of food had no effect. Weekly and three-monthly intra-individual variations were both 0.038 mumol/L. In all seven subjects with S-MMA greater than 0.30 mumol/L, the concentrations declined significantly after intramuscular administration of cobalamin. No significant difference was found between mean serum cobalamin concentrations in these seven and in the remaining subjects. We have also established the normal response of S-MMA to standardized oral loading of L-isoleucine: 100 mmol caused a significant average S-MMA increase of 0.072 mumol/L before cobalamin administration vs 0.013 mumol/L after cobalamin, without significant relation to initial S-MMA values. Our results provide a necessary background for interpretation of S-MMA measurements in clinical studies.
Specific, sensitive, precise, and rapid functional chromogenic assay of activated first complement component (C1) in plasma
Authors: Munkvad S , Jespersen J , Sidelmann J , Gram J
Published: January 2020

We present a new functional assay for the first complement component (C1) in plasma, based on its activation by inhibition of the C1-esterase inhibitor (C1-inh) when monospecific antiserum to C1-inh is added to the plasma. After maximal activation, we can determine the concentration of activated C1 by using an amidolytic rate assay with a chromogenic substrate. We have optimized the assay conditions with respect to incubation time, concentration of antiserum to C1-inh, ionic strength, and pH. Our method determines specifically the concentration in plasma of free activated C1, not complexes of activated C1 with C1-inh, and is not influenced by the concentration of C1-inh in the test sample. Concentrations of C1 correlated significantly with activities determined by a hemolytic assay (r = 0.55, t = 4.09, P less than 0.001). The estimated interassay CV was 5% and the intra-assay CV was 1%. The sensitivity, imprecision, and practical test performance of our assay are superior to those of conventionally used hemolytic assays.
Rapid, simple, and reliable doctor's office test for antibodies to human immunodeficiency virus 1 in serum
Authors: Dafforn A , Irvine J D , Kurn N , Becker M , Bryning Z , Ullman E F
Published: January 2020

This "Unit Test Method" assay for detecting anti-human immunodeficiency virus 1 antibody is suitable for nonlaboratory testing and has a sensitivity comparable with that of present enzyme immunoassay methods. The method does not require instrumentation, gives a result in less than 15 min, and incorporates a procedural control. Little technical expertise and hands-on time are required of the user.
Interference by human anti-mouse antibodies in CA 125 assay after immunoscintigraphy: anti-idiotypic antibodies not neutralized by mouse IgG but removed by chromatography
Authors: Turpeinen U , Lehtovirta P , Alfthan H , Stenman U H
Published: January 2020

Falsely increased concentrations of the ovarian carcinoma-associated antigen, CA 125, were measured by a monoclonal antibody (MAb)-based double determinant immunoradiometric assay (IRMA) in patients who developed antibodies to mouse immunoglobulins (IgGs) after receiving injections of the same MAb as is used in the CA 125 IRMA. Addition of undiluted mouse serum or purified mouse IgG to the assay mixture failed to eliminate the falsely increased CA 125 concentrations in most of the samples, owing to the presence of anti-idiotype antibody. Because of their anti-idiotypic nature, the human anti-mouse antibodies (HAMAS) had only little effect on other immunometric assays, and this effect could be completely eliminated by addition of mouse IgG. To eliminate the effect of HAMA on the CA 125 assay, we studied the ability of various chromatographic methods to separate the interfering HAMA from CA 125. For measuring HAMA in serum and chromatographic fractions we developed a time-resolved fluoroimmunoassay. Adequate separation of CA 125 and HAMA was achieved by affinity chromatography of patients' sera with solid-phase Protein A, Protein G, cation-exchange chromatography on Mono S, and gel filtration on Superose 6. These results demonstrate that the interference can effectively be removed by rather simple chromatographic procedures.
Liquid-chromatographic measurement of biopterin and neopterin in serum and urine
Authors: Slazyk W E , Spierto F W
Published: January 2020

We report an improved "high-performance" liquid-chromatographic (HPLC) method for measuring biopterin and neopterin in serum and urine. Specimens are acidified, treated with iodine in 0.2 mol/L trichloroacetic acid, party purified on Bio-Rad MP-50 cation-exchange columns, and analyzed by reversed-phase HPLC with fluorometric detection. The minimal concentration of biopterin detectable is 0.3 micrograms/L in a 50-microL injection. The total CV is less than or equal to 10%. Improvements over other reported methods include the use of a single, simplified sample-preparation step with a Baker-10 SPE System, and a guard column to increase analytical column stability and analyte recovery. The assay is semiautomated to reduce technician time and improve precision. Mean observed values for biopterin and neopterin in sera of normal human adults were 1.64 and 5.52 micrograms/L, respectively. The mean ratio of neopterin to biopterin in acidified adult urine samples was lower than that found in matched nonacidified samples (n = 10). Serum specimens from diagnosed phenylketonuric (PKU) and hyperphenylalaninemic patients were also analyzed for biopterin and neopterin; the findings agreed with reported values for similar patients. One patient, previously identified as an atypical PKU patient, showed serum values of neopterin and biopterin suggestive of a defect in biopterin synthesis.
Determination of cocaine concentrations in plasma by high-performance liquid chromatography
Authors: Jatlow P , Nadim H
Published: January 2020

We describe a procedure for measuring concentrations of cocaine in plasma by reversed-phase high-performance liquid chromatography, with ion-pairing. The procedure involves solvent extraction followed by back-extraction with dilute acid. The n-propyl ester of benzoylecgonine is used as an internal standard. An evaporation step is not required, and concentrations as low as 5 micrograms/L can be quantified. Plasma concentrations of cocaine determined by high-performance liquid chromatography correlated well with those determined by a previously reported gas-chromatographic procedure with nitrogen detection.
Preanalytical increase of ammonia in blood specimens from healthy subjects
Authors: da Fonseca-Wollheim F
Published: January 2020

The course and magnitude of spontaneous increase in ammonia concentration in plasma on standing were investigated with EDTA-treated blood specimens from 36 healthy subjects with use of a sensitive and precise enzymic method. Over 90 min, the rates of increase were virtually constant at fixed temperature. The mean (and SE) rates at 0, 20, and 37 degrees C were 3.9 (0.23), 5.2 (0.23), and 25.2 (0.59) mumol/L per hour, respectively. At these temperatures, the plasma contributed at most 7%, 15%, and 10%, respectively, to the formation of ammonia in whole blood. In view of the medical needs and the measured rates of ammonia increase, an interval of 15 min between blood sampling and the start of centrifugation may be tolerated at a specimen temperature of 0 degree C. Rates of ammonia increase showed significant correlations with erythrocyte and platelet count as well as with the plasma activities of gamma-glutamyltransferase (EC 2.3.2.2) and alanine aminotransferase (EC 2.6.1.2).
Excretion of a Toxic Dose of Thallium
Authors: Chandler H A , Archbold G P R , Gibson J M , O'Callaghan P , Marks J N , Pethybridge R J
Published: January 2020

We report a successfully treated case of severe thallium intoxication that required 95 days of assisted ventilation and 224 days of hospitalization. Monitoring of the patient for 500 days by measuring thallium in whole blood, serum, and urine is documented, and the role of the laboratory and utility of the measurements are considered.
What should be monitored? The past, present, and future of physiological monitoring
Authors: Shoemaker W C
Published: January 2020

The status of conventional monitoring by vital signs and present concepts of invasive monitoring with the balloon-tipped pulmonary artery (Swan-Ganz) catheter are reviewed. Survivors of high-risk general surgery were observed to have cardiac index (CI) values averaging 4.5 L/min.m2, oxygen delivery (DO2) greater than 600 mL/min.m2, and oxygen consumption (VO2) greater than 170 mL/min.m2. By contrast, those who subsequently died during their hospitalization maintained relatively normal CI, DO2, and VO2 values. However, in the immediate postoperative period, values for other hemodynamic variables were not greatly different for survivors and nonsurvivors or different from the normal range. A predictive index based on these observations predicted outcome correctly in 94% of the subjects in a subsequent prospective study. The use of survivor values as appropriate therapeutic goals was tested in prospective randomized clinical trials and was found to reduce mortality and morbidity significantly. Simultaneous invasive and noninvasive hemodynamic and oxygen-transport monitoring systems were evaluated in high-risk postoperative patients to describe unanticipated adverse circulatory events. Before the monitored event, about three-fourths of the patients exhibited normal function. At the nadir, cardiac functions decreased in about two-thirds, perfusion decreased in more than one-half, and paO2 fell in only one-fourth. Two-thirds recovered with increased cardiac function, more than one-half had improved perfusion, and paO2 increased in fewer than one-fifth of monitored events. These data provide an information base for criteria needed to develop therapeutic decision rules for noninvasive monitoring systems. When noninvasive data are continuously displayed early in the course of critical illness and high-risk conditions, therapy may be instituted early, while physiological deficits are still minimal and easily reversible.
Metabolic integrity of specific organ systems
Authors: Marsh H M
Published: January 2020

This short review will address the potential uses for quantitative analyses of organ function in the critically ill patient. Multiple system failure is common in the critical-care unit, and the ability to measure reserves of organ function may enable earlier detection and treatment of this problem and provide a more accurate prognosis for such patients.
Critical-care medicine and the acute-care laboratory
Authors: Hall J R
Published: January 2020

Critical-care medicine today is practiced by anesthesiologists, internists, pediatricians, and surgeons. Outcome from today's management of critically ill patients is very good, yet associated costs are very high. Over one-half of the hospital costs of critically ill patients emanates from the intensive-care unit (ICU), although the ICU stay accounts for less than 20% of their time in the hospital. Outside of the operating room, the ICU is the most expensive location for patient care in the hospital, and laboratory tests are the most expensive single item. Plans for cost containment should incorporate the following: more effective data management, education of practitioners about appropriateness and costs of tests, conversion from laboratory measurements to appropriate in vivo and ex vivo measurements, and real-time utilization assessment. To provide high-quality, cost-effective critical care in the future, laboratorians and clinicians must work together today to meet the challenges of technology, data management, and staff education.
Intravascular monitoring: problems and promise
Authors: Halbert S A
Published: January 2020

Intravascular monitoring of arterial blood gases provides the means to detect precipitous change in cardiopulmonary function almost instantaneously, promising major improvement in the ability to manage critically ill patients. Attempts to miniaturize and repackage conventional electrodes for continuous intravascular use have met insurmountable problems, and only the application of new technology involving fiber-optic chemical sensors (FOCS) has proved that in vivo monitoring of blood gas chemistry is truly feasible. Although a wide range of analytes of clinical significance may potentially be monitored by using FOCS technology, the analytes of choice for the management of the critically ill patient are pHa, paCO2, and paO2, because of their dynamic nature and importance for critical-care diagnosis and monitoring. Although intravascular FOCS blood gas monitors currently under clinical investigation exhibit promising performance, clinical acceptance and commercial success will depend on the creative solution of several problems, the most challenging of which are related to physiological factors at the interface between the sensors and their intravascular sensing environment.
Automated blood-sample handling in the clinical laboratory
Authors: Godolphin W , Bodtker K , Uyeno D , Goh L O
Published: January 2020

The only significant advances in blood-taking in 25 years have been the disposable needle and evacuated blood-drawing tube. With the exception of a few isolated barcode experiments, most sample-tracking is performed through handwritten or computer-printed labels. Attempts to reduce the hazards of centrifugation have resulted in air-tight lids or chambers, the use of which is time-consuming and cumbersome. Most commonly used clinical analyzers require serum or plasma, distributed into specialized containers, unique to that analyzer. Aliquots for different tests are prepared by handpouring or pipetting. Moderate to large clinical laboratories perform so many different tests that even multi-analyzers performing multiple analyses on a single sample may account for only a portion of all tests ordered for a patient. Thus several aliquots of each specimen are usually required. We have developed a proprietary serial centrifuge and blood-collection tube suitable for incorporation into an automated or robotic sample-handling system. The system we propose is (a) safe--avoids or prevents biological danger to the many "handlers" of blood; (b) small--minimizes the amount of sample taken and space required to adapt to the needs of satellite and mobile testing, and direct interfacing with analyzers; (c) serial--permits each sample to be treated according to its own "merits," optimizes throughput, and facilitates flexible automation; and (d) smart--ensures quality results through monitoring and intelligent control of patient identification, sample characteristics, and separation process.
A noninstrumented quantitative test system and its application for determining cholesterol concentration in whole blood
Authors: Allen M P , DeLizza A , Ramel U , Jeong H , Singh P
Published: January 2020

A novel noninstrumented technology has been developed for quantifying analytes of clinical interest in biological fluids. Application of this technology is exemplified by the development of a quantitative cholesterol test with performance equivalent to state-of-the-art instrumented methods. The assay chemistry combines two separate processes located in different areas of a test strip: enzymatic action on serum cholesterol to produce hydrogen peroxide (5 x 10 mm enzyme reagent pad) and quantification of the hydrogen peroxide (5 x 70 mm measurement region). Color bands are formed in the measurement area through the use of a redox-coupled indicator system. The height of the color band on the strip is directly proportional to the sample cholesterol concentration. A one-step cassette contains all components necessary to run the test and includes blood filtration and automatic sample measurement, so that unmeasured finger-stick whole-blood specimens can be analyzed by the non-technically trained user. The test is complete in less than 15 min, is read visually like a thermometer, and gives results that are in excellent correlation with established instrumented methods.