bluuebunny/crossref_metadata_2025 huggingface dataset:
Factors Influencing the Reaction of α1-Fetoprotein with Concanavalin A and Lens Culinaris Agglutinin in Crossed Affinoimmunoelectrophoresis
Authors: Magne D , Seta N , Lebrun D , Durand G , Durand D
Published: January 2020

Concanavalin A (Con A) and lentil lectin (LCA) analysis of alpha-fetoprotein (AFP) glycosylation heterogeneity is used in a variety of clinical situations. We studied the influence of analytical conditions on the separation of AFP glycoforms by using lectin-crossed affinoimmunoelectrophoresis, regardless of the AFP concentration, which can vary over a wide range in biological fluids. We defined the optimal concentration of Con A (2 g/L) and LCA (0.35 g/L) in the first-dimension gel, together with the optimum antigen (AFP)/antibody ratio in the second-dimension gel. The presence of protein in the diluent used for AFP samples was found to change the shape of crossed affinoimmunoelectrophoresis patterns without changing the percentage composition of AFP fractions. The within-run CV was less than 4% for both lectins, and the between-run CV was less than 6.3%. The minimal quantity of AFP that provided a visible pattern with both lectins was 4 ng, corresponding to 50 microL of an 80 micrograms/L AFP sample. These technical conditions allow the cellular origin of AFP to be determined, regardless of the concentration in the sample. Typical AFP lectin patterns of secreting tumors are compared with fetal and cord serum AFP.
A Multicenter Evaluation of Lipid Profiling with a Compact Analyzer (Miles Clinistat)
Authors: Hardy R W , Ng R H , Hill R E , Walker S , Sparks K M , Kent S , Lakes C , Hiar C E , Valdes R , Statland B E
Published: January 2020

We evaluated the Clinistat Analyzer (Miles Inc., Diagnostics Division, Elkhart, IN) for measuring cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol at three medical centers. The system, based on multilayer film technology, uses precalibrated, dry film reagent disks. Ten microliters of serum is applied to the dry film reagent disk in the test procedure. For HDL-cholesterol measurement, serum is pretreated by precipitation with phosphotungstic acid and magnesium chloride. Total precision (CVs) of each of the three assays was less than or equal to 5%. The assay ranges were linear and satisfactory for clinical use. Patients' results compared well with established methods. No significant interferences were found with hemolysis, icterus, and lipemia.
Short- and Long-Term Variability of Selected Indices Related to Nutritional Status. II. Vitamins, Lipids, and Protein Indices
Authors: Gallagher S K , Johnson L K , Milne D B
Published: January 2020

Five free-living women (ages 23-38 years) who consumed a self-selected diet and five women (ages 23-44 years) residing in a metabolic unit who were fed constant diet were assessed for variation in vitamin and general chemistry indices. Blood was drawn from these women 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 of the indices. Analytical variability was determined by concurrently analyzing control samples prepared from plasma and serum pools. All samples were analyzed in duplicate. Of the measured indices, vitamins and lipids seemed to be the most variable. Diet had a significant effect only on ascorbic acid. We were unable to show any seasonal change for these analytes. Estimations of analytical variability, along with estimates of biological variability, and knowledge of dietary practices are essential when interpreting differences in analytes.
Enzyme-Linked Immunosorbent Assay of C-Erbb-2 Oncoprotein in Breast Cancer
Authors: Nugent A , McDermott E , Duffy K , O'Higgins N , Fennelly J J , Duffy M J
Published: January 2020

Amplification or increased expression of the c-erbB-2 gene has previously been reported to be a prognostic marker for breast cancer. Gene amplification is usually measured by Southern blotting, whereas increased protein expression is usually detected by immunocytochemistry. We measured c-erbB-2 protein with an enzyme-linked immunosorbent assay (ELISA). High concentrations of oncoprotein were found in 25 of 161 (16%) primary breast cancers and in 3 of 6 (50%) breast cancer metastases. High concentrations were not found in normal breast tissue or benign breast tumors. In the primary cancers, high concentrations of c-erbB-2 protein were found more frequently (a) in estrogen receptor-negative tumors than in estrogen receptor-positive tumors, (b) in progesterone receptor-negative tumors than in progesterone-positive tumors, and (c) in axillary node-positive cancers than in node-negative cancers. Patients with tumors containing high amounts of the c-erbB-2 protein had a significantly shorter (P less than 0.001) disease-free interval and overall survival rate than did patients with low amounts. We conclude that assay of c-erbB-2 protein by ELISA is simple, rapid, and quantitative and offers important prognostic information in breast cancer.
An In-House Opiate Enzymoimmunoassay Based on the Syva Emit® Principle
Authors: Colbert D L , Gooch J C
Published: January 2020

Many laboratories involved in drug-abuse screening use enzymoimmunoassays such as Syva's EMIT system. Although automation has been extensively used to reduce assay costs, further reductions can be achieved by the development of in-house reagents. We have developed our own reagents for the detection of opiates in urine by using the Syva EMIT principle and adapted it for use on an Eppendorf EPOS analyzer. The immunogen was produced from 3-carboxymethylmorphine, and morphine-3-glucuronide attached to glucose-6-phosphate dehydrogenase was used as the tracer. Excellent agreement with Syva's commercial assays was obtained.
Noninvasive Imaging and Spectroscopy--Broad Applications of Magnetic Resonance
Authors: Hornung P A , Schuff N
Published: January 2020

The present utility of nuclear magnetic resonance (NMR) spectroscopy in chemical analysis and magnetic resonance imaging (MRI) in the clinical environment has made this technology commonplace in the chemical industry, clinical medicine, and academic research. The attributes of nuclear magnetism that make the technique especially powerful in biology are discussed. This paper reviews the uses of NMR and MRI, with an emphasis on spatially resolved applications. These applications include imaging, localized spectroscopy, flow sensing, and diffusion mapping from using magnetic-field gradients. The limits of spatially resolved NMR and imaging will be examined in terms of both scientific principles and engineering practice. Block diagrams of both imaging and spectroscopy apparatus are presented and technical requirements of the critical components are discussed. Developing trends in sensing probes, magnets, and applications are highlighted.
Noninvasive Glucose Monitoring in Diabetic Patients: A Preliminary Evaluation
Authors: Robinson M R , Eaton R P , Haaland D M , Koepp G W , Thomas E V , Stallard B R , Robinson P L
Published: January 2020

Noninvasive monitoring of blood/tissue glucose concentrations has been successfully accomplished in individual diabetic subjects by using near-infrared (NIR) spectroscopy coupled with chemometric methods. Three different spectrometer configurations were tested: a) a Fourier-transform infrared spectrometer with an indium antimonide detector; b) a grating monochromator equipped with a silicon (Si) array detector, without fiber optics; and c) a grating monochromator equipped with an Si detector, with fiber-optic sampling. NIR spectra were obtained from diabetic subjects by transmission through the finger during a meal-tolerance test. The maximum range of observed plasma glucose concentrations obtained from the blood samples was 2.5-27 mmol/L. The NIR spectra were processed by using the chemometric multivariate calibration methods of partial least squares and principal component regression. The best calibration yielded a cross-validated average absolute error in glucose concentration of 1.1 mmol/L. This predictive ability suggests that noninvasive glucose determinations by NIR/chemometrics is a viable analytical method.
Near-Infrared Spectrophotometry: A New Dimension in Clinical Chemistry
Authors: Hall J W , Pollard A
Published: January 2020

The near-infrared (NIR) spectral region (700-2500 nm) is a fertile source of chemical information in the form of overtone and combination bands of the fundamental infrared absorptions and low-energy electronic transitions. This region was initially perceived as being too complex for interpretation and consequently was poorly utilized. Advances in chemometric techniques that can extract massive amounts of chemical information from the highly overlapped, complex spectra have led to extensive use of NIR spectrophotometry (NIRS) in the food, agriculture, pharmaceutical, chemical, and polymer industries. The application of NIRS in clinical laboratory measurements is still in its infancy. NIRS is a simple, quick, nondestructive technique capable of providing clinically relevant analyses of biological samples with precision and accuracy comparable with the method used to derive the NIRS models. Analyses can be performed with little or no sample preparation and no reagents. The success of NIRS in any particular case is determined by the complexity of the sample matrix, relative NIR absorptivities of the constituents, and the wavelengths and regression technique chosen. We describe the general approach to data acquisition, calibration, and analysis, using serum proteins, triglycerides, and glucose as examples.
Simultaneous Patient-Side Measurement of Hemoglobin, Glucose, and Cholesterol in Finger-Stick Blood
Authors: Sterling B , Kiang T , Subramanian K , Saltman M , Smart W , Tsay M , Sugarman J , Patel D , Monger D , Martin D
Published: January 2020

We describe a multianalyte assay system for patient-side use comprising single-use plastic cartridges and a small monitor. Hemoglobin, glucose, and cholesterol can be simultaneously measured in 3 min in an unmeasured volume of blood. The sample is drawn by capillary action into four channels for delivery to assay-specific stacks containing a set of closely apposed layers. The distal layer is a membrane that acts as the optical surface for reflectance optics. For glucose and cholesterol assays, erythrocytes are removed by a fibrous filter layer and oxidase-peroxidase chemical reactions contained in the optical membrane generate a colored product. For hemoglobin measurement, blood is lysed by detergent contained in a porous disk. The amount of color reaching the optical membrane is measured by fiber optics. To ensure fail-safe operation, sensors verify sample sufficiency and degree of hemolysis. The assays perform comparably with laboratory methods.
Simultaneous Detection of Seven Drugs of Abuse by the TriageTM Panel for Drugs of Abuse
Authors: Buechler K F , Moi S , Noar B , McGrath D , Villela J , Clancy M , Shenhav A , Colleymore A , Valkirs G , Lee T
Published: January 2020

This novel, competitive immunoassay simultaneously detects seven drugs of abuse in urine. A urine sample is placed in contact with lyophilized reagents, the reaction mixture is allowed to come to equilibrium (10 min), and then the whole mixture is applied to a solid phase that contains various immobilized antibodies in discrete drug-class-specific zones. After a washing step, the operator visually examines each zone for the presence of a red bar. The method incorporates present threshold concentrations that are independent for each drug. In the absence of drug or in the presence of drug in quantities less than the threshold concentration, no colored bar is visible. Samples containing drug(s) at or above the threshold concentration cause a red bar to appear for the appropriate drug(s). Positive and negative procedural control zones are incorporated into each determination. The performance of the assay methodology matches that of instrumented immunoassay systems.
Automated Total and Neonatal Bilirubin Values in Newborns: Is a Distinction Clinically Relevant?
Authors: Langbaum M E , Farber S J , Rosenthal P
Published: January 2020

Guidelines for managing hyperbilirubinemia in newborns were developed by using diazo methods that measure total and direct-reacting bilirubins and calculate an indirect fraction. The automated Kodak Ektachem system allows for measuring serum bilirubin by either of two dry-slide methods: TBIL, involving a modified diazo method, and NBIL, involving a dual-wavelength colorimetric method that fractionates and directly measures the unconjugated (Bu) and conjugated (Bc) bilirubins (Bu+Bc = neonatal bilirubin). The manufacturer recommends that NBIL be used in newborns less than 15 days old, which is impractical in a large, busy hospital laboratory. We compared NBIL and TBIL in 500 paired serum samples from infants less than 15 days old. We noted a statistically significant difference between TBIL and NBIL values (162.9, SD 70.4, vs 164.6, SD 69.2, mumol/L; P less than 0.0001), which was small and of no clinical significance. We conclude that TBIL values may be used with caution for newborn bilirubin screening. Furthermore, NBIL measurements are an acceptable alternative to diazo measurements for neonatal care, allowing the use of previously developed guidelines with NBIL values.
Measurement of Elastase and Cysteine Proteinases in Synovial Fluid of Patients with Rheumatoid Arthritis, Sero-Negative Spondylarthropathies, and Osteoarthritis
Authors: Huet G , Flipo R M , Richet C , Thiebaut C , Demeyer D , Balduyck M , Duquesnoy B , Degand P
Published: January 2020

Synovial fluid samples were collected from 45 patients with rheumatoid arthritis, spondylarthropathy, or osteoarthritis, to study their content of elastase (EC 3.4.21.37) and of cysteine proteinases (EC 3.4.22.1, 3.4.22.15). We measured both elastase complexed with alpha 1-proteinase inhibitor and elastase activity toward the substrate L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide. Cysteine proteinase activities were measured with the substrates N-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-AMC) and Z-Arg-Arg-AMC and the inhibitor E-64 [L-trans-epoxysuccinyl-leucyl-amido-(4-guanidino)-butane]. In all these enzyme assays, higher median values were obtained in inflammatory arthropathies than in osteoarthritis. The concentration of the elastase-alpha 1-proteinase inhibitor complex and of elastase and cysteine proteinase activities were statistically higher in patients with rheumatoid arthritis than in patients with osteoarthritis. The difference in results between patients with spondylarthropathy and patients with osteoarthritis was statistically significant only for the elastase-alpha 1-proteinase inhibitor complex. The median values of the complex and of both enzyme activities were higher in patients with rheumatoid arthritis than in patients with spondylarthropathy; however, the difference was statistically significant only for the cysteine proteinase activity measured with Z-Arg-Arg-AMC substrate. These results suggest that both elastase and cysteine proteinases, which are increased in patients with inflammatory arthritis, are involved in cartilage degradation in these arthropathies.
Macroamylases: Differences in Activity against Various-Sized Substrates
Authors: Rosenblum J L , Hortin G L , Smith C H , Pashos G E , Landt M
Published: January 2020

Hyperamylasemia caused by macroamylases can lead to the overdiagnosis of acute pancreatitis. We examined whether interference from macroamylase is less in assays that use high-molecular-mass (high-M(r)) substrates rather than oligosaccharide substrates. We hypothesized that high-M(r) substrates would be sterically excluded from macroamylasemic complexes and thus would be hydrolyzed less efficiently. Eighteen macroamylasemic samples were assayed by using red-dyed amylopectin or blue-dyed starch as polysaccharide substrates or by using maltoheptaose or maltotetraose as oligosaccharide substrates. The oligosaccharide substrates gave comparable results (y = 0.81x + 83), but we observed consistently lower activities for amylopectin than for maltotetraose (y = 0.32x + 38). We observed no bias among methods when nonmacroamylasemic specimens were analyzed. The mechanism of this difference was examined by adding antihuman pancreatic amylase antibodies to hyperamylasemic serum samples from patients without macroamylasemia and to purified human pancreatic or salivary isoamylases. In each case, polyclonal and monoclonal antibodies lowered amylase activity more in assays with complex polysaccharides than in those with oligosaccharides. The use of high-M(r) substrates diminishes interference, and detection of suspected macroamylasemia may be possible through comparing activities determined from automated methods that use different substrates.
Increased Concentrations of Total IgM At Clinical Onset of Type 1 (Insulin-Dependent) Diabetes: Correlation with IgM Binding to Cells. The Belgian Diabetes Registry
Authors: Decraene T , Vandewalle C , Pipeleers D , Gorus F K
Published: January 2020

Eighty patients with type 1 (insulin-dependent) diabetes (ages 0-39 years) were consecutively recruited by the Belgian Diabetes Registry. Sera obtained at clinically diagnosed onset (i.e., before start of insulin therapy or within 7 days of initial treatment) were analyzed for total IgM concentrations and for IgM binding to fixed rat splenocytes (IgM-LyAb) and permeabilized rat islet cells (IgM-ICAb). Comparison of results with those in age- and sex-matched control subjects, by fluorescence-activated cell-sorter analysis, indicated greater concentrations of IgM-LyAb and IgM-ICAb in sera from the patients. IgM antibodies reacted indiscriminately with islet beta and islet endocrine non-beta cells. The prevalence of IgM-ICAb, but not of IgM-LyAB, was significantly (P less than 0.05) higher in patients than in the control subjects. Of the ICAb-positive patients, 54% were also LyAb-positive, whereas none of the control subjects were doubly positive. IgM-ICAb and IgM-LyAb binding signals were positively correlated. Serum IgM concentrations were significantly (P less than 0.001) greater in patients than in control subjects and were significantly correlated with IgM-LyAb (P less than 0.001) and IgM-ICAb (P less than 0.01). The positivity for IgM binding was not, however, merely a reflection of total IgM, because no such correlation was found in sera from seven patients with Waldenström macroglobulinemia. Clinical onset of type 1 diabetes is apparently accompanied by increased production of IgM. The correlation between IgM concentrations and IgM binding to islet cells might reflect polyclonal activation or natural autoantibodies.
Minimum Distinguishable Difference in Concentration: A Clinically Oriented Translation of Assay Precision Summaries
Authors: Sadler W A , Murray L M , Turner J G
Published: January 2020

A trend to market-driven health care in many parts of the world is focusing increasing attention on getting maximum value from available resources. Laboratories are not exempted. Well-informed clinical input has a potentially valuable role in any laboratory rationalization process. However, a communication difficulty exists in the sense that, although laboratory workers, commercial developers, regulatory bodies, etc., are thoroughly conditioned to using assay coefficient of variation as a general performance measure (for excellent reasons), this is not necessarily the most intuitive or informative scale from a clinician's perspective. Here we use routine clinical data from an immunoradiometric assay of thyrotropin to illustrate, first, a general approach to estimation and prediction of reproducibility, and second, an alternative summary that expresses the discriminatory power of an assay. This latter measure, our experience suggests, is more suited to the way clinicians perceive assays and assay results. The overall aim is improved clinician/laboratory communication.
Detection and Confirmation of Cocaine and Cocaethylene in Serum Emergency Toxicology Specimens
Authors: Puopolo P R , Chamberlin P , Flood J G
Published: January 2020

We report the performance of a liquid chromatography (LC) assay for detection and confirmation of cocaine and cocaethylene in serum. A photodiode array (PDA) detector monitors the cyano column effluent at 230 nm for analyte detection. Confirmation of a peak's identity is performed by use of only the 226-254-nm segment of the spectra rather than a broader range such as 210-340 nm. Use of the narrower range improves the assay's sensitivity by almost 10-fold. Analyte values of 10 micrograms/L are confirmed routinely. Intra-assay imprecision at 100, 20, and 10 micrograms/L for cocaine is 5.1%, 5.7%, and 6.6% (CV), respectively. Absolute recovery of the analytes exceeds 80%. In a split-sample study (33 positives and 74 negatives), each of this method's cocaine findings were confirmed. Cocaethylene was found in 15 of these samples. A complete chromatogram can be available 15 min after the start of the procedure. The method's focus on the active drug (rather than urinary metabolites) and its ability to detect and confirm many other drugs make it well suited for emergency toxicology testing.
Increased Concentrations of Type IV (7 S) Collagen in Sera of Hyperthyroid Patients with Graves Disease
Authors: Shima T , Yano Y , Goto H , Misaki M
Published: January 2020

Serum concentrations of type IV collagen (7 S) were determined in 29 patients with untreated hyperthyroidism and 30 healthy subjects. Serum 7 S collagen was significantly higher (P less than 0.0001) in the hyperthyroid patients (6.3, SD 1.3, micrograms/L) than in the healthy control subjects (3.9, SD 0.6, micrograms/L). No difference in serum concentrations of 7 S collagen were observed between patients with normal liver function and those with abnormal liver function. Serum concentrations of 7 S collagen correlated positively with serum concentrations of free triiodothyronine (r = 0.41, P less than 0.05). In the hyperthyroid patients, 7 S collagen concentrations in serum gradually fell into the normal range as thyroid function became normalized. Thus, hyperthyroidism is one of the diseases in which serum concentrations of 7 S collagen are increased.
Automated Capillary Electrophoresis Applied to Therapeutic Drug Monitoring
Authors: Evenson M A , Wiktorowicz J E
Published: January 2020

Chromatographic separations in conjunction with physical chemical detection methods can provide extremely accurate and highly precise measurements of drugs in biological matrices. Immunochemical methods for drug analyses, on the other hand, although usually fully automated, often lack accuracy, may be subject to matrix interference effects, and frequently exhibit nonlinearities that contribute to suboptimal analytical results. Automated capillary electrophoresis (CE) promises the high accuracy and precision of physical methods with no operator intervention once the sample is loaded into the sample cup. A clean-up step is necessary for the analysis of drugs in biological matrices, but that could also be fully automated. Reagent costs per test with CE approach zero, because of the use of extremely small volumes of buffers, standards, and controls. We present data supporting CE as an alternative separation strategy for 28 commonly used therapeutic drugs. These can be resolved in less than 16 min with an optimized micelle-based buffer system.
An Oral-Diffusion-Sink Device for Extended Sampling of Multiple Steroid Hormones from Saliva
Authors: Wade S E
Published: January 2020

Performance of a new oral-diffusion-sink (ODS) device for sampling the steroid hormones cortisol, progesterone, estradiol, and testosterone from human saliva was tested in vitro. The tested device differed from an earlier ODS sampler by using a polymeric composition of beta-cyclodextrin rather than an antiserum to bind analytes diffusing into the device. The capacity of the cyclodextrin-driven device to accumulate the steroid hormones from a simple buffer, human saliva, and saliva fortified with high concentrations of glucocorticosteroids was evaluated. Further, the ability of this device to accurately register the average analyte concentration resulting from treatments simulating physiological episodic secretion events was tested. Each steroid showed a characteristic rate of uptake that was unaffected by saliva or high physiological concentrations of competing steroids, and ODS uptake resulting from temporally varied concentrations of the hormone in the medium accurately reflected the time-integrated average concentration. This ODS provides a potential noninvasive and unobtrusive means of sampling the tissue-available concentrations of multiple steroid hormones, with averaging over physiological secretion events.
Analysis of Cholesterol in All Lipoprotein Classes by Single Vertical Ultracentrifugation of Fingerstick Blood and Controlled-Dispersion Flow Analysis
Authors: Kulkarni K R , Garber D W , Schmidt C F , Marcovina S M , Ho M H , Wilhite B J , Beaudrie K R , Segrest J P
Published: January 2020

This new, highly sensitive analytical system, based on controlled dispersion of the flowing sample, gives a rapid, continuous, and direct analysis for cholesterol in all lipoprotein classes, separated by single vertical-spin density-gradient ultracentrifugation. In this Vertical Auto Profile-II fingerstick system, designated VAP-IIfs, a narrow-bore Teflon coil serves as the reactor with no segmentation of the analytical stream by air bubbles, in contrast to the Technicon AutoAnalyzer used in the VAP-I method. Concentrations of high-, low-, intermediate-, and very-low-density lipoprotein cholesterol and lipoprotein(a) cholesterol are determined by decomposing the spectrophotometric absorbance curve for the continuous analysis of the centrifuged sample, with use of software developed in this laboratory. Total cholesterol is determined from the total area under the absorbance curve. For assaying total cholesterol, the CV between aliquots within a rotor ranged from 1.35% to 3.15%; the CV between rotors was 2.45%. Because only 18 microL of sample is required, VAP-IIfs can be readily adapted to analysis for lipoprotein cholesterol profiles in capillary blood samples. Total cholesterol values by VAP-IIfs for fingerstick and venous samples from 23 subjects agreed well: slope = 1.01 (SD 0.03), intercept = -21 (SD 51) mg/L, Sy/x = 50 mg/L, and r = 0.992. Results by VAP-IIfs also correlated highly with results for duplicate samples analyzed at the Northwest Lipid Research Laboratories.
Analytical performance evaluation of EMIT II monoclonal amphetamine/methamphetamine assay: more specificity than EMIT d.a.u. monoclonal amphetamine/methamphetamine assay
Authors: Dasgupta A , Saldana S , Kinnaman G , Smith M , Johansen K
Published: January 2020

We evaluated a new EMIT II monoclonal amphetamine/methamphetamine assay for screening human urine by comparing it with the EMIT d.a.u. monoclonal amphetamine/methamphetamine assay and a fluorescence polarization assay. The EMIT II assay has a cutoff of 1 mg/L d-methamphetamine. The EMIT II and EMIT d.a.u. assays were run on a BM/Hitachi 704 analyzer; for the fluorescence polarization assay we used a TDx analyzer. All EMIT II positive samples were also analyzed by the fluorescence polarization assay. We used gas chromatography/mass spectrometry (GC/MS) for confirmation of the presence of amphetamine or methamphetamine. Within-run CVs for the Level 1 (1 mg/L) and Level 2 (3 mg/L) calibrators for the EMIT II assay were 0.47% and 0.53%, respectively. Corresponding between-run CVs were 1.48% and 1.60%, respectively. Of the 1007 samples screened for amphetamines, 50 were positive by the EMIT d.a.u. assay; 21 samples (not a subset of the 50 samples) were positive by the EMIT II assay. However, 19 samples that tested positive by EMIT II also tested positive by the EMIT d.a.u. assay. Subsequent testing of the EMIT II positive samples by the fluorescence polarization assay detected in six positive samples. By means of chiral derivatization wer identified two specimens containing primarily l-isomers of amphetamine and methamphetamine. Sympathomimetic amines were identified in several of the samples not containing amphetamine or methamphetamine.
Discordant results for determinations of triglycerides in pig sera
Authors: Tuten T , Robinson K A , Sgoutas D S
Published: January 2020

We recently determined triglyceride concentrations in pig sera by three fully enzymatic methods (Kodak Ektachem 700, Hitachi 707, and Abbott EPx) and obtained significantly lower values than those obtained with chemical or enzymatic methods based on chemical hydrolysis. All methods used involve microbial lipases for liberating glycerol from glycerides and glycerol phosphate dehydrogenases or oxidases for subsequent oxidation. The methods were validated against reference methods by using fresh human sera and survey materials. The discordant results were not from matrix sample-method interaction but from incomplete hydrolysis of pig serum triglycerides by the lipolytic enzymes. When serum triglycerides from 10 pigs showing the highest biases were hydrolyzed by microbial lipases and the reaction mixture was subjected to thin-layer and gas-liquid chromatography, the predominant end products were palmitoyl monoglyceride and a mixture of free fatty acids with the following composition (fatty acid as percent of total +/- SD): 16:0, 7.8 +/- 2; 18:0, 5.4 +/- 2.2; 18:1, 53 +/- 12; 18:2, 31 +/- 4.6; and 18:3, 2.5 +/- 1. Assuming that the lipases exhibit the usual specificity toward the 1 and 3 positions of the triglyceride, the data suggest that, in pig, triglycerides 18:1 and 18:2 occupy the 1 and 3 positions and 16:0 (palmitic acid) predominantly occupies the 2 position. Triglycerides of this structure may not be well hydrolyzed by the typical lipolytic enzymes in clinical assays.
Phenylalanine determined in plasma with use of phenylalanine dehydrogenase and a centrifugal analyzer
Authors: Vilaseca M A , Farré C , Ramón F
Published: January 2020

Quantitative determination of plasma phenylalanine (Phe) is essential for the diagnosis of phenylketonuria (PKU) and the control of dietary therapy of PKU patients. We have adapted a spectrophotometric method (Clin Chim Acta 1991;201:95-8) based on phenylalanine dehydrogenase (EC 1.4.1.-) for rapid and accurate determinations of Phe with the Cobas Fara II centrifugal analyzer. The method is based on the NAD(H)-dependent reductive deamination of Phe with large amounts of phenylalanine dehydrogenase, which catalyzes the formation of phenylpyruvate at pH 10.8. Combining the specificity of the phenylalanine dehydrogenase with the precision, accuracy, and considerable time-saving of an automated system is useful for monitoring PKU patients in a children's hospital.
Robotic chromatography: development and evaluation of automated instrumentation for assay of glycohemoglobin
Authors: Herold C D , Andree K , Herold D A , Felder R A
Published: January 2020

The measurement of glycohemoglobin (GHb) by boronate affinity chromatography is useful in monitoring long-term glucose control in diabetic subjects. The inherent disadvantage of this method is the hands-on time required because the hemoglobin fractions are separated on individual disposable columns. To overcome this disadvantage, we have programmed a Hamilton Microlab 2200 automated pipetting cartesian robot to complete the procedure, from the aspiration of blood from the sample-collection tube to the transfer of the separated hemoglobin fractions to a microtiter plate for absorbance measurement. This automated robotic system can analyze 96 specimens, including patients' samples and control material, in approximately 3 h. The precision (CV) of the method ranged from 1.6% to 3.5% within-run and from 2.7% to 3.5% day-to-day. The results correlated with those obtained with the Accuflex semiautomated robot, which used the identical disposable column, and those obtained with a Primus high-performance liquid chromatograph, which used a regenerated microparticle column. Automation of the GHb procedure allowed improved throughput, reduced labor cost, improved precision, and offered greater laboratory safety.
C-reactive protein and its cytokine mediators in intensive-care patients
Authors: Sheldon J , Riches P , Gooding R , Soni N , Hobbs J R
Published: January 2020

C-reactive protein (CRP) is an acute-phase protein produced by the liver during bacterial infections and inflammation. The cytokines interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF) are widely reported to induce synthesis of CRP by hepatocytes both in vitro and in vivo. We investigated the relation between CRP and its cytokine mediators in 64 critically ill patients during their treatment in the intensive-care unit. Plasma CRP and IL-6 concentrations were significantly lower in patients without any evidence of infection than in those with clinical infection; plasma IL-1 beta concentrations showed no significant difference between any of the groups, but plasma TNF concentrations were lower in patients with evidence of infection. Significant correlation was seen between plasma concentrations of CRP and IL-6 when the latter was measured by bioassay; however, IL-6 showed, at best, only a 50% predictive value for a change in CRP concentration.
Early diagnosis of ectopic arginine vasopressin secretion
Authors: Coyle S , Penney M D , Masters P W , Walker B E
Published: January 2020

We describe a patient who presented with the syndrome of inappropriate antidiuretic hormone secretion (SIADH) 2 months before clinical evidence of bronchogenic malignancy. Because of the potential for the ectopic production of atrial natriuretic peptide (ANP) to mimic SIADH, both hormones were measured in this hyponatremic patient to seek a possible marker of tumor activity. A hypertonic saline infusion at presentation revealed excessive osmotically decoupled secretion of arginine vasopressin but a normal ANP response.
Increase of plasma proapolipoprotein A-I in patients with liver cirrhosis and its relationship to circulating high-density lipoproteins 2 and 3
Authors: Suehiro T , Yamamoto M , Yoshida K , Ohno F
Published: January 2020

We determine the concentration of proapolipoprotein (proapo) A-I and its ratio with total apolipoprotein (apo) A-I (proapo A-I/total apo A-I) in plasma of patients with liver disease; we used a noncompetitive sandwich method, an enzyme-linked immunosorbent assay. The mean (SD) proapo A-I concentrations in patients with decompensated or compensated liver cirrhosis were higher than in normal subjects: 88 (25), 105 (36), and 69 (25) mg/L, respectively. The mean (SD) ratio (expressed as %) for each of these types of liver cirrhosis was also higher than in normal subjects: 10.0 (3.5), 10.2 (3.9), and 4.6 (1.6), respectively. In the patients, the proapo A-I concentration was positively correlated with the concentration of high-density lipoprotein subtype 2 cholesterol (HDL2-C) (r = 0.736), and the proapo A-I/total apo A-I ratio was correlated inversely with the HDL3-C concentration (r = -0.609). The activity of proapo A-I converting enzyme in patients with liver cirrhosis (62 +/- 30 nmol/h per liter) was significantly (P < 0.01) lower than that in normal subjects (172 +/- 55 nmol/h per liter). The increases of the plasma proapo A-I concentration and ratio in patients with liver cirrhosis may be caused by a decreased production of the converting enzyme in the liver. The increase of plasma proapo A-I may thus also affect the circulating HDL subtypes.
Limitations of quality control in physicians' offices and other decentralized testing situations: the challenge to develop new methods of test validation
Authors: Baer D M , Belsey R E
Published: January 2020

Traditional quality-control methods are effective for detecting systematic error caused by deterioration of reagents or instruments, but ineffective for detecting sporadic error, which is more likely to occur in low-volume testing environments. Decentralized testing performed by individuals without formal laboratory training has a high potential for sporadic errors. New methods for validating test results, used each time a test result is produced, should replace current quality-control procedures. Under the rules of CLIA '88, manufacturers and the US Food and Drug Administration have an opportunity to develop new approaches to test validation.
Assay instrument-dependent matrix effects in standardization of cholesterol measurements
Authors: Waymack P P , Miller W G , Myers G L
Published: January 2020

Human serum-based frozen reference materials have been used by the Centers for Disease Control and Prevention (CDC)-National Heart, Lung and Blood Institute Lipid Standardization Program to improve the precision and accuracy of blood cholesterol measurements. Occasionally, laboratories in the program have had problems obtaining results for patients' fresh serum samples equivalent to those obtained with frozen CDC standardization pools. This incompatibility of sample, reagent, instrument, and assay characteristics has been labeled broadly as a "matrix effect," which usually is attributed to unknown characteristics of the processed pool material. In this study we showed that a large negative bias obtained with CDC pools was attributable to use of the sample blank mode on the Cobas-Bio analyzer. However, under the same conditions, fresh patients' serum samples were analyzed accurately. The use of a blank absorbance immediately after mixing sample and reagents (the "autoblank" mode) allowed the instrument to accurately analyze both fresh serum samples and CDC standardization pools and thus allowed the documentation of traceability of the cholesterol measurements to the National Reference System for Cholesterol.
Passive hemagglutination inhibition test for diagnosis of brown recluse spider bite envenomation
Authors: Barrett S M , Romine-Jenkins M , Blick K E
Published: January 2020

Our goal was to recreate a passive hemagglutination inhibition (PHAI) test to diagnose brown recluse spider (BRS; Loxosceles reclusa) bite envenomation for treatment trials. Guinea pigs received intradermal injections of concentrated spider venom from the following species: Loxosceles reclusa, Argiope aurantia, Argiope trifasciata, Phidippus audax, and Lycosa frondicola. Skin lesion exudate was collected and tested with the BRS venom PHAI assay. From 51 separate collections of exudate, test sensitivity was 90% as long as 3 days after venom injection. Specificity was 100% with venom from the other spider species listed above in vivo (7 test samples) and in vitro (5 test samples), as well as with random bacterial exudate with and without added serial dilutions of BRS venom (10 test samples). The test was reproducible over repetitive assays to within one 10-fold dilution. A positive PHAI test result could function as an entry criterion for BRS bite victims in human treatment trials.
Enzymatic rate assay of creatinine in serum and urine
Authors: Fujita T , Takata S , Sunahara Y
Published: January 2020

A two-step method for assaying creatinine in serum and urine samples, suitable with automated analyzers, is reported. Reagent 1, for the first step, contains a blanking system [creatine amidinohydrolase (CRTase), urease, glutamate dehydrogenase, NADPH, and 2-oxoglutarate] and a NADPH-regenerating system [Mg(2+)-dependent isocitrate dehydrogenase (ICD), MgCl2, and excess isocitrate]. Reagent 2, for the second step, contains the metal-chelating reagent trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CyDTA) and a trigger system [creatinine amidohydrolase (CRNase)]. When a specimen is mixed with reagent 1, all the creatine, urea, and NH3 present are removed by the blanking and NADPH systems. On adding reagent 2, CyDTA inactivates ICD to inhibit the NADPH system. Simultaneously, the creatinine (1 mol) in the specimen is hydrolyzed into creatine by CRNase, and then releases NADP+ (2 mol) through the blanking system. Our optimized method can determine creatinine linearly up to 500 mg/L, with within-day CVs < 1.2% and day-to-day CVs < 2.7%.
Quantification of the cross-link pentosidine in serum from normal and uremic subjects
Authors: Takahashi M , Kushida K , Kawana K , Ishihara C , Denda M , Inoue T , Horiuchi K
Published: January 2020

Pentosidine is a fluorescent cross-link compound that accumulates in human tissues from uremic and diabetic patients. Using SP-Sephadex C-25 pretreatment before reversed-phase HPLC, we developed a method for quantifying pentosidine in the acid hydrolysate of serum. We examined concentrations of pentosidine in serum from 98 patients with end-stage renal disease requiring hemodialysis and from 33 normal control subjects. The mean (+/- SD) concentration of pentosidine was significantly greater in serum from uremic patients than that from control subjects (1267 +/- 695 nmol/L vs 77 +/- 40 nmol/L, P = 0.0001). There was a significant correlation between serum pentosidine concentrations and age in control subjects (r = 0.453, P < 0.01), but not in uremic patients. Serum pentosidine significantly increased with the duration of hemodialysis treatment (r = 0.272, P < 0.05). The greater pentosidine concentrations in uremic patients may be caused by the increased synthesis of pentosidine (perhaps because of the retention of pentosidine precursors), by the retention of pentosidine molecule itself in end-stage renal failure, or both.
Homogeneous immunofluorometric assays of alpha-fetoprotein with macroporous, monosized particles and flow cytometry
Authors: Frengen J , Schmid R , Kierulf B , Nustad K , Paus E , Berge A , Lindmo T
Published: January 2020

We evaluated two homogeneous immunofluorometric assays (IFMAs) of alpha-fetoprotein (AFP) based on new macroporous acrylate particles combined with flow cytometry. The standard IFMA, requiring 1 h of incubation, provided a working range from 1.8 to > 900 kIU/L (CV < 10%) and a detection limit of 0.6 kIU/L. Use of overnight incubation and a lower particle concentration extended the working range by 1 decade in the lower end. Analytical recoveries for the standard IFMA varied between 97% and 108%. The slope and y-intercept of the regression line correlating measurements by the standard IFMA and a routine immunoradiometric assay were not significantly different from 1 and 0, respectively (P > 0.5), and the correlation coefficient was 0.996. High precision and warning of spuriously high measurements were obtained by including in each sample separate particle types for detecting instrument instability and measuring nonspecific binding only.
Relation between serum and whole-blood ethanol concentrations
Authors: Rainey P M
Published: January 2020

An algorithm is suggested for interpretation of serum ethanol concentrations under legal statutes that specify whole-blood alcohol concentrations. The algorithm uses the distribution of individual serum:whole-blood alcohol concentration ratios to allow calculations at various levels of confidence that can be related to legal standards of evidence. Serum:whole-blood alcohol concentration ratios were determined for 211 patients. The ratios ranged from 0.88 to 1.59 (median 1.15). The distribution of ratios was positively skewed, but the logarithms of the ratios were normally distributed. This allowed the parametric calculation of a range of ratios of 0.90-1.49 for the central 99% of the population and a range of 0.95-1.40 for the central 95%. The serum:whole-blood alcohol concentration ratio was independent of both the serum alcohol concentration (R2 = 0.005) and the hematocrit (R2 = 0.03).
Solid-phase extraction protocol for isolating retinol-d4 and retinol from plasma for parallel processing for epidemiological studies
Authors: Dueker S R , Lunetta J M , Jones A D , Clifford A J
Published: January 2020

Solid-phase extraction permits the parallel processing of samples in large numbers. We have applied this technique to the isolation of retinol isotopomers from plasma of humans participating in a study of vitamin A stable isotope dilution. The isotopomers were analyzed by gas chromatography/mass spectrometry. The extraction involves the separation of retinol from its aqueous matrix with a C18 silica-based sorbent followed by removal of lipid contaminants with an aminopropyl silica-based sorbent. Overall recovery of retinol from plasma was 47.2% +/- 1.8%. Purity of the retinol isolated from plasma is comparable with that obtained with a single HPLC method. This method permits the preparation of 32 samples per day by one analyst. Elimination of the need for HPLC permits sample preparation in the field with a minimum of equipment and technical skill.
Improved quantitative Apt test for detecting fetal hemoglobin in bloody stools of newborns
Authors: Liu N , Wu A H , Wong S S
Published: January 2020

We devised an improved spectrophotometric method, based on the differential susceptibility of various hemoglobins to alkaline denaturation, to measure the percentage of fetal hemoglobin (HbF) in bloody meconium so as to determine the source of the blood. The oxyhemoglobin of a sample was spectrophotometrically scanned from 450 to 650 nm before and after the addition of sodium hydroxide. With Allen baseline correction, the ratio of absorbance at 576 nm was linearly proportional to the percentage of HbF in the specimen as described by the equation: ratio = 0.01 (%HbF) + 0.0045 (r = 0.9971, Sy/x = 0.047). Preliminary studies on samples containing 10% or 80% HbF revealed that the method was fairly precise. The accuracy of this test was verified by comparing the results (y) with those obtained by radial immunodiffusion (x): y = 0.997x + 2.93 (r = 0.91). This preliminary study demonstrates that the modified test is simple, fast, and free from bilirubin interference. It should be useful for the diagnosis of gastrointestinal bleeding of the newborn on a stat basis.
Automated enzymatic analysis of inulin
Authors: Summerfield A L , Hortin G L , Smith C H , Wilhite T R , Landt M
Published: January 2020

We have developed an automated enzymatic assay for quantitation of inulin in plasma and urine that can be performed on the Cobas FARA II. In the assay, inulinase hydrolyzes inulin to fructose, and sorbitol dehydrogenase converts fructose to sorbitol with consumption of NADH, which is detected by spectrophotometry. The method incorporates a sample blank (inactivated inulinase) for each specimen to subtract contributions of endogenous fructose. Recovery of fructose or inulin was near 100%, with linearity to 300 mg/L. The enzymatic assay (y) agreed well with an anthrone comparison method (x) for analysis of inulin in both urine specimens (y = 1.00x - 138; Sy/x = 714) and plasma specimens (y = 1.00x - 3.5; Sy/x = 5.5). Glucose at 300 mg/L yielded an apparent inulin value of 1.3 mg/L in the enzymatic assay, but reacted at nearly 10% equivalency in the anthrone assay. Interferences from sorbitol, mannitol, and xylitol were negligible. CVs for day-to-day precision studies were 1-4%. The automated enzymatic assay of inulin is faster and avoids the use of caustic reagents required by the classical anthrone method.