bluuebunny/crossref_metadata_2025 huggingface dataset:
Automated Homogeneous Liposome Immunoassay Systems for Anticonvulsant Drugs
Authors: Kubotsu K , Goto S , Fujita M , Tuchiya H , Kida M , Takano S , Matsuura S , Sakurabayashi I
Published: January 2020

We developed automated homogeneous immunoassays, based on immunolysis of liposomes, for measuring phenytoin, phenobarbital, and carbamazepine from serum. Liposome lysis was detected spectrophotometrically from entrapped glucose-6-phosphate dehydrogenase activity. The procedure was fully automated on a routine automated clinical analyzer. Within-run, between-run, dilution, and recovery tests showed good accuracies and reproducibilities. Bilirubin, hemoglobin, triglycerides, and Intrafat did not affect assay results. The results obtained by liposome immunoassays for phenytoin, phenobarbital, and carbamazepine correlated well with those obtained by enzyme-multiplied immunoassay (Syva EMIT) kits (r = 0.995, 0.986, and 0.988, respectively) and fluorescence polarization immunoassay (Abbott TDx) kits (r = 0.990, 0.991, and 0.975, respectively). The proposed method should be useful for monitoring anticonvulsant drug concentrations in blood.
Radioimmunoassay of Beta-Endorphin in Ventricular and Lumbar Cerebrospinal Fluid
Authors: Bach F W , Schmidt J F , Faber T
Published: January 2020

We describe a sensitive beta-endorphin (beta-EP) radioimmunoassay specific for beta-EP 1-31 applied to human ventricular and lumbar cerebrospinal fluid (CSF). Specificity was documented by reversed-phase HPLC of CSF pools. Simultaneous ventricular and lumbar CSF samples from 13 patients suspected of having normal-pressure hydrocephalus showed median beta-EP values of 2.2 (range 1.7-4.0) and 4.8 (2.8-14.6) pmol/L, respectively. Ventricular and lumbar beta-EP concentrations were positively correlated (Spearman r = 0.72, P = 0.013). The beta-EP rostral-caudal gradient was closely related to the CSF protein gradient. HPLC profiles of beta-EP immunoreactivity were similar in ventricular and lumbar CSF with both C- and N-terminal antisera. beta-EP concentrations did not vary in the first 12 mL of lumbar CSF, tapped in 3-mL portions [F(3,32) = 0.42, P = 0.74]. The beta-EP concentration in lumbar CSF from 15 children in remission from acute leukemia [23.4 (15.0-27.1) pmol/L] was higher than in 54 healthy adults [11.7 (10.9-13.3) pmol/L; P less than 0.01]. There was no effect of sex or age on CSF beta-EP in adults. beta-EP in lumbar human CSF may indicate di- and mesencephalic beta-EP neuronal activity.
Dissociation-Enhanced Lanthanide Fluorescence Immunoassay of Lipoprotein(a) in Serum
Authors: Jürgens G , Hermann A , Aktuna D , Petek W
Published: January 2020

Lipoprotein(a), a human serum lipoprotein structurally related to low-density lipoprotein (LDL), contains in addition to apolipoprotein B (apo B) apolipoprotein(a) [apo(a)], a glycoprotein with a strong homology to plasminogen. Lp(a) is a risk factor for coronary heart disease and ischemic cerebrovascular disease. Several immunological techniques are used to quantify Lp(a) in human serum, including radioimmunoassays, rocket immunoelectrophoresis, and enzyme-linked immunosorbent assays. Only the last method is suitable for large-scale clinical studies. We describe another solid-phase immunoassay, based on the dissociation-enhanced lanthanide fluorescence system Delfia (Wallac Oy), and outline the technical details. A polyclonal antiserum directed against Lp(a) was used as the capture antibody. Two kinds of detection antibodies were applied, a polyclonal antiserum against apo B and the polyclonal antiserum against Lp(a). The results agree excellently with the values estimated by rocket immunoelectrophoresis. This assay is easily established, measures Lp(a) in a wide concentration range, and is suitable for screening large populations.
Improved Electrochemiluminescent Label for DNA Probe Assays: Rapid Quantitative Assays of HIV-1 Polymerase Chain Reaction Products
Authors: Kenten J H , Gudibande S , Link J , Willey J J , Curfman B , Major E O , Massey R J
Published: January 2020

We describe the characterization and utility of a new electrochemiluminescent (ECL) label for oligonucleotides, utilizing phosphoramidite chemistry. This phosphoramidite of the tris(2,2-bipyridine)ruthenium(II) complex, bis(2,2-bipyridine)(4-[4-(2-cyanoethoxy-N,N-diisopropyl-amino) phosphinoxybutyl]4'-methyl)2,2-bipyridine ruthenium(II) dihexafluorophosphate or Origen phosphoramidite, enables the direct incorporation of the label during automated DNA synthesis. Efficiency of this automated synthesis allows the direct utilization of probes without further purification. Introduction of this labeling group is reproducible, and the ECL signal recovered is not influenced by hybridization. Furthermore, neither hybridization kinetics nor hybrid stability was affected by our label. We also demonstrate the utility of these labels for the development of rapid assays with oligonucleotides direct from automated synthesis. The clinical utility of these labeled oligonucleotides is shown with assays of total nucleic acid, extracted from peripheral blood lymphocytes of patients with acquired immunodeficiency syndrome (AIDS), to detect the human immunodeficiency virus (HIV-1). The results demonstrate the ability of the assay to quantify 30-2000 copies of HIV1 gag genes and to rapidly detect (less than 45 min) HIV-1 gag genes in a nonseparation assay. The application of this assay to clinical samples demonstrates the utility of these assays for rapid and quantitative analysis.
Charts of operational process specifications ("OPSpecs charts") for assessing the precision, accuracy, and quality control needed to satisfy proficiency testing performance criteria
Authors: Westgard J O
Published: January 2020

“Operational process specifications” have been derived from an analytical quality-planning model to assess the precision, accuracy, and quality control (QC) needed to satisfy Proficiency Testing (PT) criteria. These routine operating specifications are presented in the form of an “OPSpecs chart,” which describes the operational limits for imprecision and inaccuracy when a desired level of quality assurance is provided by a specific QC procedure. OPSpecs charts can be used to compare the operational limits for different QC procedures and to select a QC procedure that is appropriate for the precision and accuracy of a specific measurement procedure. To select a QC procedure, one plots the inaccuracy and imprecision observed for a measurement procedure on the OPSpecs chart to define the current operating point, which is then compared with the operational limits of candidate QC procedures. Any QC procedure whose operational limits are greater than the measurement procedure's operating point will provide a known assurance, with the percent chance specified by the OPSpecs chart, that critical analytical errors will be detected. OPSpecs charts for a 10% PT criterion are presented to illustrate the selection of QC procedures for measurement procedures with different amounts of imprecision and inaccuracy. Normalized OPSpecs charts are presented to permit a more general assessment of the analytical performance required with commonly used QC procedures.
Proficiency testing as a regulatory device: a CAP perspective
Authors: Hamlin W
Published: January 2020

The history and development of the proficiency testing programs of the College of American Pathologists are reviewed. Important considerations of external surveys include target value assignment and determination of acceptable ranges. "Blind" proficiency testing and on-site evaluation provide alternative methods of laboratory evaluation but are limited by practical logistics.
The Impact of New Regulations on Laboratory Testing in Physicians' Offices
Authors: Loschen D J
Published: January 2020

The reaction of the clinician to the specter of regulation of any part of his or her practice mirrors the reaction of the laboratorian to the implementation of Medicare and Clinical Laboratory Improvement Amendments legislation in 1965 and 1967, respectively. Whether the regulatory burdens that will be visited upon these laboratories are justified or necessary is arguable; the fact of the upcoming regulation is not. The volume and breadth of testing in physicians' office laboratories (POLs) has increased exponentially since passage of the Diagnosis Related Group legislation by Congress in 1983, an increase made possible by remarkable developments in technology. State regulatory initiatives and private accrediting agencies have been perceived as being inadequate to prevent the proliferation of poorly controlled testing in the nontraditional laboratory environment. The testing menu of a given POL varies according to the scope of clinical services offered; the size of the practice group; the funding available for equipment and personnel acquisition; and the general availability of hospital, reference, and consultative laboratory services. Physicians who offer laboratory services as part of their practices must now prepare their laboratories to meet whatever requirements are mandated by regulation. This will include acquisition of trained personnel, improvement of instrumentation and methodologies, participation in proficiency testing, establishment of comprehensive quality-assurance programs, and adequate documentation of laboratory services. Organized medicine should devote its energies to assisting with needed educational processes to assure the survival of POLs.
Dependence of Free Thyroxine Estimates Obtained with Equilibrium Tracer Dialysis on the Concentration of Thyroxine-Binding Globulin
Authors: Nelson J C , Wilcox R B , Pandian M R
Published: January 2020

Some equilibrium dialysis determinations of free thyroxine (T4) vary directly with thyroxine-binding globulin (TBG) concentration. This apparent TBG dependence has been limited to methods involving radiolabeled T4 added to the dialysis system (tracer dialysis). In this study we compared tracer dialysis with direct dialysis for determining free T4 and obtained the following results (mean +/- SD) for patients with hypothyroxinemia of nonthyroidal illness (23.8 +/- 10.7 vs 24.2 +/- 10.9 pmol/L, P greater than 0.8), patients with congenital TBG deficiency (11.4 +/- 2.2 vs 16.2 +/- 7.1 pmol/L, P greater than 0.05), normal control subjects (32.7 +/- 6.5 vs 18.5 +/- 5.8 pmol/L, P less than 0.001), and pregnant women (31.2 +/- 8.7 vs 12.1 +/- 2.6 pmol/L, P less than 0.001). Direct dialysis determinations were independent of TBG and total T4. Tracer determinations were greater than direct determinations in normals, a discrepancy that increased in pregnancy. Tracer determinations correlated significantly with total T4 and TBG concentrations (P less than 0.001). TBG and total T4 dependence in the tracer method was attributable to small overestimations of the free fraction of T4. Similar overestimations multiplied by increasing total T4 concentrations resulted in greater errors. Relative to results for normal sera, the tracer method overestimated free T4 when total T4 was increased and underestimated free T4 when total T4 was decreased.
Design and Preliminary Performance Characteristics of a Newly Proposed Reference Cell for Ionized Calcium in Serum
Authors: D'Orazio P , Bowers G N
Published: January 2020

A novel flow-through design for a calcium ion-selective liquid membrane and half-cell is proposed. The membrane is based on a neutral carrier for calcium and has the same quantitative chemical composition as that used in most commercial analyzers for ionized calcium (iCa2+). The membrane and half-cell follow the specifications in the Stage 1, Draft 4 document for a Reference Method for iCa2+ of the IFCC Expert Panel on pH, Blood Gases, and Electrolytes. Measurements of iCa2+ in serum with this cell confirm prior observations that protein adsorption on the membrane surface causes a rapid but reversible shift in the cell calibration signal and possible systematic errors in the measurement of iCa2+ in serum. When the protein adsorption phenomenon is maintained at equilibrium during calibrations and sample measurements, the new cell can be used to reproducibly assign iCa2+ concentrations to serum pools. These calibrated pools may then be used as reference materials and to resolve the biases known to exist among commercial iCa2+ analyzers.
Effect of Lyophilization on Determinations of Lipoprotein(a) in Serum
Authors: Sgoutas D S , Tuten T
Published: January 2020

We studied the effect of lyophilization of serum on the determination of serum lipoprotein(a) [Lp(a)], using two enzyme-linked immunosorbent assays (ELISAs) and one immunoturbidimetric assay (ITA). After adjusting for the effect of dilution due to the reconstitution of lyophilized serum, Lp(a) values obtained for lyophilized sera were consistently lower than the original values for the corresponding fresh samples, the difference ranging from 17% to 83% for the monoclonal-polyclonal antibody-based ELISAs and from 12% to 49% for the exclusively polyclonal ITA. Statistical analysis showed that differences between either ELISA and the ITA were significant but not the differences between the ELISAs. Moreover, the effect was not qualitatively or quantitatively uniform, which suggested that the effect was serum-source dependent. Addition of sucrose (600 mmol/L) to sera before lyophilization decreased but did not eliminate the effect. Apparently, serum pools in lyophilized form are not a suitable reference material for Lp(a) determination. Because different methods produce different results for Lp(a) in the same common reference material intended for standardization of Lp(a) methodology, any Lp(a) method should be tested for the effect of lyophilization if lyophilized samples are to be analyzed.
Effects of Temperature on Optical Absorbance Spectra of Oxy-, Carboxy-, and Deoxyhemoglobin
Authors: Steinke J M , Shepherd A P
Published: January 2020

The optical absorbance spectra of oxy-, carboxy-, and deoxyhemoglobin were recorded at wavelengths from 479 to 651 nm and at temperatures of 20, 30, and 40 degrees C. As noted in earlier reports, a major effect of lowering the temperature was an increase in the absorptivities at or near the absorbance maxima. However, at other wavelengths, reducing the temperature increased, decreased, or caused no change in absorbance. At wavelengths where temperature-induced shifts did occur, the absorbance change appeared to be a linear function of temperature. Unlike previous reports, the data presented here are quantitative and thus can be used to predict temperature-induced errors in spectrophotometric measurements of the relative concentrations of these hemoglobin species. Examples are given of the error that would occur in a widely used CO-Oximeter, the IL482, if it were not temperature controlled. Thus, the data presented here should be particularly useful to the operators and designers of spectrophotometric instruments such as oximeters, CO-Oximeters, and hemoglobinometers.
Sensitive, Direct Procedures for Simultaneous Determinations of Iron and Copper in Serum, with Use of 2-(5-Nitro-2-Pyridylazo)-5-(N-Propyl-N-Sulfopropylamino)Phenol (Nitro-PAPS) as Ligand
Authors: Yamashita S , Abe A , Noma A
Published: January 2020

We developed a direct, simple, and sensitive procedure for the simultaneous colorimetric assay of iron and copper in serum, using sodium dodecyl sulfate-ascorbic acid to dissociate iron and copper from transferrin and ceruloplasmin, respectively. We also use a new water-soluble reagent, 2-(5-nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol disodium salt (nitro-PAPS) and thioglycolic acid to eliminate interference from copper in the measurement of iron. Within- and between-run precisions of the present method were 2.5-2.8% for iron and 1.8-4.6% for copper. The proposed method is susceptible to interference by hemoglobin and lipemia, especially for the iron assay. Linear-regression analyses of results of the proposed method with those of the bathophenanthroline method for iron and of the atomic absorption spectroscopic method for copper correlated well (r = 0.996, Sy/x = 0.73 and r = 0.959, Sy/x = 1.11, respectively).
Diagnostic Significance of Methemoglobin Determination in Colorless Cerebrospinal Fluid
Authors: Trbojević-Cepe M , Vogrinc Z , Brinar V
Published: January 2020

The presence of various heme derivatives can be demonstrated spectrophotometrically in colorless cerebrospinal fluid (CSF). Because of the high sensitivity of the method, it may detect compounds that reflect a "traumatic tap" rather than a disease process. However, the presence of methemoglobin excludes the possibility of a hemorrhagic CSF being caused by traumatic lumbar puncture. Here we describe a highly sensitive spectrophotometric method involving measurement at the Soret band (400-420 nm) to detect methemoglobin (greater than or equal to 15%) in trace amounts of hemoglobin mixture (less than 0.3 mumol/L). We demonstrated methemoglobin in colorless CSF samples in 9% of 454 patients with cerebrovascular pathology and in 4% of 449 patients with other neurological diseases (n = 449). In a group of 21 patients with verified acute cerebral hematomas, methemoglobin was confirmed in 66% of colorless CSF samples after ultrafiltration. We conclude that routine spectrophotometric analysis of all CSF samples is very useful, allowing detection of xanthochromic compounds in patients with small cerebral and subdural hematomas as well as in those with minimal subarachnoid hemorrhages, hemorrhagic infarctions, or bleedings from aneurysms and neoplasms.
Direct Enzyme Immunoassay of Estradiol in Serum of Women Enrolled in an in Vitro Fertilization and Embryo Transfer Program
Authors: Bouve J , De Boever J , Leyseele D , Bosmans E , Dubois P , Kohen F , Vandekerckhove D
Published: January 2020

We present a fast and simple direct enzyme immunoassay for the sexual steroid hormone estradiol-17 beta in serum of women enrolled in an in vitro fertilization and embryo transfer program. We added 25 microL of standards or samples and 125 microL of a mixture of monoclonal antibody and displacing agents to the wells of a microtiter plate previously coated with a second antibody. After 30 min 50 microL estradiol-horseradish peroxidase conjugate is added. Total incubation time is 75 min. The immunoassay is stopped by washing the microtiter plate. The amount of bound conjugate is then measured at 450 nm with hydrogen peroxide as a substrate and 3,3',5,5'-tetramethylbenzidine as chromogen. The detection limit is 0.24 nmol/L. The assay is thus limited in its application. Analytical recovery is 90.8% (range 80.4-102.9%); within- and between-assay CVs range between 1.3% and 15.4%. Total assay time, including preparation of reagents and data reduction, is only 2.5 h. Results of this enzyme immunoassay compare well with those obtained with four different commercial radioimmunoassays (r = 0.88-0.93).
ROC Curve Analysis: An Example Showing the Relationships Among Serum Lipid and Apolipoprotein Concentrations in Identifying Patients with Coronary Artery Disease
Authors: Zweig M H , Broste S K , Reinhart R A
Published: January 2020

Clinical accuracy, defined as the ability to discriminate between states of health, is the fundamental property of any diagnostic test or system. It is readily expressed as clinical sensitivity and specificity, and elegantly represented by the receiver operating characteristic (ROC) curve. To demonstrate the use of ROC curves, we reexamine a study of the ability of serum lipid and apolipoprotein measures to discriminate among degrees of coronary artery disease in patients undergoing coronary angiography. ROC curve analysis reveals that none of these indexes is highly accurate, but demonstrates a modest increase in the accuracy of apolipoprotein over lipid indexes.
Assessment of Urea and Other Uremic Markers for Quantification of Dialysis Efficacy
Authors: Vanholder R C , De Smet R V , Ringoir S M
Published: January 2020

To validate azotemic markers as an index for intradialytic changes in solute concentration, we compared eight solutes (pseudouridine, xanthine, hypoxanthine, peak 4, peak 5, p-hydroxyhippuric acid, indoxyl sulfate, and hippuric acid) with five classical azotemic markers (urea, creatinine, uric acid, phosphate, and potassium). We determined concentrations by reversed-phase HPLC coupled to ultraviolet absorption or photometrically. Seven compounds showed significant intercorrelation (P less than 10(-5)): urea, pseudouridine, uric acid, peaks 4 and 5, p-hydroxyhippuric acid, and creatinine. The hippuric acid concentration change after dialysis correlated with the change for these seven compounds and also with indoxyl sulfate, hypoxanthine, potassium, and the group of unidentified ultraviolet-absorbing HPLC peaks accumulating in uremia. We conclude that urea only partially represents the concentration changes of other retention compounds after dialysis; alternative markers, e.g., hippurate, should be considered.
Macroamylases: Differences in Activity Against Various-Size 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-weight rather than oligosaccharide substrates. We hypothesized that high-molecular-weight 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); 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 anti-human pancreatic amylase antibodies to hyperamylasemic serum samples from patients without macroamylasemia and 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-molecular-weight substrates diminishes interference, and detection of suspected macroamylasemia may be possible through comparison of activities determined from automated methods that use different substrates.
Frozen Human Serum Reference Material for Standardization of Sodium and Potassium Measurements in Serum or Plasma by Ion-Selective Electrode Analyzers
Authors: Gunaratna P C , Koch W F , Paule R C , Cormier A D , D'Orazio P , Greenberg N , O'Connell K M , Malenfant A , Okorodudu A O , Miller R
Published: January 2020

Three interlaboratory round-robin studies (RR1, RR2, and RR3) were conducted to identify a serum-based reference material that would aid in the standardization of direct ion-selective electrode (ISE) measurements of sodium and potassium. Ultrafiltered frozen serum reference materials requiring no reconstitution reduced between-laboratory variability (the largest source of imprecision) more than did other reference materials. ISE values for RR3 were normalized by the use of two points at the extremes of the clinical range for sodium (i.e., 120 and 160 mmol/L), with values assigned by the flame atomic emission spectrometry (FAES) Reference Method. This FAES normalization of ISE raw values remarkably improved all sources of variability and unified the results from seven different direct ISE analyzers to the FAES Reference Method value. Subsequently, a three-tiered, fresh-frozen human serum reference material was produced to the specifications developed in RR1-RR3, was assigned certified values for sodium and potassium by Definitive Methods at the National Institute of Standards and Technology (NIST), and was made available in 1990 to the clinical laboratory community as a Standard Reference Material (SRM); it is now identified as SRM 956. Albeit retrospectively, we show how applying an FAES normalization step identical to that used in RR4/5 to the ISE data for SRM 956 after the NIST Definitive Method values were known, consistently moved the ISE results for RR3 closer to the true value for Na+ and K+.
HemoCue: Evaluation of a Portable Photometric System for Determining Glucose in Whole Blood
Authors: Ashworth L , Gibb I , Alberti K G
Published: January 2020

We assessed the HemoCue system for measuring glucose in 5 microL of whole blood. A glucose dehydrogenase-based reaction is used with dried reagents contained in disposable microcuvettes, which are filled with blood by capillary action. Automated hexokinase and YSI 23AM glucose analyzer methods were used for comparison. Overall imprecision (CV) was better than 4.5%, with no significant differences in results between three different HemoCue photometers and four batches of microcuvettes. Regression slopes (+/- SE) were 0.947 (0.011) with the YSI and 0.966 (0.015) with the hexokinase method. Analytical recovery of added glucose was 101-106%, and the system functioned with hematocrits up to 0.65. Bilirubin up to 453 mumol/L did not interfere, but high concentrations of endogenous (greater than 3 mmol/L) and exogenous triglycerides gave positive interference. The system proved stable and robust under a wide range of storage and handling conditions; performance was impaired only at high ambient temperature (37 degrees C). We conclude that the HemoCue system should prove useful for glucose measurement; further testing outside the laboratory is warranted.
Intact Parathyroid Hormone: Performance and Clinical Utility of an Automated Assay Based on High-Performance Immunoaffinity Chromatography and Chemiluminescence Detection
Authors: Hage D S , Taylor B , Kao P C
Published: January 2020

The performance and clinical utility of an automated assay of intact parathyroid hormone (parathyrin, PTH) are evaluated. The method is based on the extraction of PTH from plasma by an HPLC column containing immobilized anti-(44-68 PTH) antibodies. The PTH retained is detected with a postcolumn reactor and use of anti-(1-34 PTH) chemiluminescent-labeled antibodies. The total cycle time of the assay is 6.5 min per injection after a 1-h incubation. The lower limit of detection for PTH in a 66-microL plasma sample was 0.5 pmol/L based on peak heights and 0.2 pmol/L based on peak areas. Mean analytical recovery for PTH added to plasma was 97%. The within-day precisions (CVs) for 4.2 and 30 pmol/L PTH plasma samples were 9.2% and 5.6% and the day-to-day precisions were 10.3% and 5.7%, respectively. No significant interferences from 1-34, 44-68, or 53-84 PTH fragments were noted, even at highly increased concentrations of fragments. The correlation of results with those of a manual assay of intact PTH was 0.97, and the results showed good agreement with disease state for patients with hypo- or hyperparathyroidism. The specificity of the assay for primary hyperparathyroidism was greater than 95%. We discuss the advantages (speed and quality control) of this approach over current immunoassays and the potential use of this method for detecting other analytes.
Ciliated Enteric Duplication Cyst Presenting as A Pancreatic Cystic Neoplasm: Report of A Case with Cyst Fluid Analysis
Authors: Pins M R , Compton C C , Southern J F , Rattner D W , Lewandrowski K B
Published: January 2020

Pancreatic cysts include inflammatory pseudocysts, cystic tumors (serous and mucinous), and various rare cystic lesions. We report a case of a ciliated enteric duplication cyst that presented on computed tomographic scan as a pancreatic cystic neoplasm. Cyst fluid analysis revealed markedly increased concentrations of carcinoembryonic antigen and CA 125 and increased fluid viscosity. These features are typical for a mucinous cystic neoplasm of the pancreas and demonstrate a potential pitfall associated with the diagnosis of pancreatic cysts by chemical analyses. Enteric duplications involving the pancreas are rare, usually of gastric origin, and usually communicate with their enteric source. Morphological and histochemical analysis suggest that this cyst was of caudal foregut origin. This is the first reported case of a ciliated foregut cyst involving the pancreas. The chemical characteristics of the cyst fluid of these lesions have not been described previously.
Progress Toward the Development of an Implantable Sensor for Glucose
Authors: Wilson G S , Zhang Y , Reach G , Moatti-Sirat D , Poitout V , Thévenot D R , Lemonnier F , Klein J C
Published: January 2020

The development of an electrochemically based implantable sensor for glucose is described. The sensor is needle-shaped, about the size of a 28-gauge needle. It is flexible and must be implanted subcutaneously by using a 21-gauge catheter, which is then removed. When combined with a monitoring unit, this device, based on the glucose oxidase-catalyzed oxidation of glucose, reliably monitors glucose concentrations for as long as 10 days in rats. Various design considerations, including the decision to monitor the hydrogen peroxide produced in the enzymatic reaction, are discussed. Glucose constitutes the most important future target analyte for continuous monitoring, but the basic methodology developed for glucose could be applied to several other analytes such as lactate or ascorbate. The success in implementation of such a device depends on a reaction of the tissue surrounding the implant so as not to interfere with the proper functioning of the sensor. Histochemical evidence indicates that the tissue response leads to enhanced sensor performance.
Portable Simultaneous Multiple Analyte Whole-Blood Analyzer for Point-of-Care Testing
Authors: Schembri C T , Ostoich V , Lingane P J , Burd T L , Buhl S N
Published: January 2020

We describe a portable clinical chemistry analyzer for point-of-care measurements of multiple analytes in less than 10 min from approximately 40 microL of whole blood (fingerstick or venous). Whole blood is applied directly to a 7.9-cm-diameter, single-use plastic rotor containing liquid diluent and greater than or equal to 4-12 tests in the form of 1- to 2-mm-diameter dry reagent beads. The reagent/rotor is immediately placed in a portable instrument along with a ticket/label results card. As the instrument spins the rotor, capillary and rotational forces process the blood into diluted plasma, distribute the patient's diluted sample to cuvettes containing the reagent beads, and mix the diluted sample with the reagents. The instrument monitors the chemical reactions optically at nine wavelengths; sample volume and temperature are also measured optically. The calibration data for each reagent are read from a bar code on the periphery of each rotor. The instrument processes all the measurements to calculate, store, print, and communicate the results. Each reagent/rotor contains an enzymatic control that must be within a defined range before the results from that analysis are reported.
Eliminating Interference from Heterophilic Antibodies in a Two-Site Immunoassay for Creatine Kinase Mb by Using F(ab')2 Conjugate and Polyclonal Mouse IgG
Authors: Vaidya H C , Beatty B G
Published: January 2020

Heterophilic antibodies interfere in two-site immunoassays. Our purpose was to screen for the samples containing heterophilic antibodies that react with mouse immunoglobulins and to use them to determine how to eliminate interference in various two-site immunoassays being developed in our laboratory. Of approximately 2600 samples screened, 81 had heterophilic antibodies. When creatine kinase MB (CKMB) concentration was measured with intact antibody conjugate in these 81 samples, 18 (22%) samples had apparent CKMB values significantly greater than values measured with Hybritech's "Tandem -E CKMB immunoenzymetric" assay and Corning agarose-gel electrophoresis. Adding up to 133 mg/L of polymerized IgG or up to 1666 mg/L polyclonal mouse IgG in the assay did not eliminate the interference in all the samples. However, adding F(ab')2 conjugate plus 40 mg/L of polymerized IgG or 83 mg/L of polyclonal mouse IgG eliminated the interference in all the samples. This approach was also effective in eliminating the interference in 15 samples containing 4.7-165.2 mg/L of human antimouse antibody (HAMA). Combined use of F(ab')2 conjugate and polyclonal mouse IgG is recommended to eliminate interference from heterophilic antibodies that react with murine immunoglobulins or HAMA in two-site murine-antibody-based assays.
Assay of Endopeptidase-24.11 Activity in Plasma Applied to In Vivo Studies of Endopeptidase Inhibitors
Authors: Yandle T , Richards M , Smith M , Charles C , Livesey J , Espiner E
Published: January 2020

We developed a fluorometric assay for endopeptidase-24.11 (EC 3.4.24.11) in human plasma. Substrate [glutaryl-Ala-Ala-Phe-amidomethylcoumarin(AMC)] was incubated with plasma (20 microL, 30 min, pH 7.6) with (control) or without the endopeptidase-24.11 inhibitor phosphoramidon. Further incubation with aminopeptidase M released free AMC. Within-assay CVs were 4.5% and 8.6%, respectively, at 3.31 and 0.27 nmol of AMC released per milliliter per minute. The between-assay CV was 10.4% at 0.31 nmol/mL per minute and the detection limit was 0.05 nmol/mL per minute. A highly skewed distribution of endopeptidase-24.11 in 41 normal samples was found, ranging from 0.12 to 6.84 nmol/mL per minute (median = 0.44). Mean endopeptidase-24.11 concentrations were significantly higher in hypertensive subjects (0.68 nmol/mL per minute) than in normotensive subjects (0.34 nmol/mL per minute; P less than 0.05). Compared with placebo administration, the oral endopeptidase-24.11 inhibitor UK 79300 significantly inhibited the plasma enzyme at doses of 100 mg (twice daily). Although in normotensive subjects the enzyme was unaffected with doses of 25 mg, the same dose (25 mg) inhibited the plasma enzyme in hypertensive subjects. No activity was detected in sheep plasma, but addition of exogenous endopeptidase-24.11 to sheep plasma in vitro allowed in vivo assessment of the effect of infused endopeptidase-24.11 inhibitor SCH 39370.
Identification of the Steroids in Neonatal Plasma that Interfere with 17 Alpha-Hydroxyprogesterone Radioimmunoassays
Authors: Wong T , Shackleton C H , Covey T R , Ellis G
Published: January 2020

Neonatal plasma contains interferents that increase the apparent 17 alpha-hydroxyprogesterone (17-OHP) content measured by direct (no-extraction) radioimmunoassay. We fractionated extracts from neonatal plasma pools by liquid chromatography with a Sephadex LH-20 column and measured 17-OHP immunoreactivity by a direct test kit. We found immunoreactivity in the free steroid and glucuronide fraction and also in the monosulfate fraction. We analyzed these two fractions by reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry. We collected fractions and assayed for 17-OHP immunoreactivity. The HPLC fractions containing the interfering steroid monosulfates were analyzed by ion-spray mass spectrometry and, after solvolysis, by gas chromatography-mass spectrometry. Several monosulfates were identified, including those of 17 alpha-hydroxy-pregnenolone, 16 alpha-hydroxypregnenolone, pregnenolone, and several pregnenetriols. 17 alpha-Hydroxypregnenolone sulfate was the most significant interferent. Other commercially available 17-OHP assays showed similar interference when used without an extraction step. Kit manufacturers should select antibodies and protocols to minimize cross-reaction with sulfates, especially 17 alpha-hydroxypregnenolone sulfate.
Comparison of Assay Kits for Unconjugated Estriol Shows that Expressing Results as Multiples of the Median Causes Unacceptable Variation in Calculated Risk Factors for Down Syndrome
Authors: Reynolds T , John R
Published: January 2020

We compared the performance of two methods for assaying unconjugated estriol in serum: the modified Amerlex third-trimester RIA kit, as used in seminal papers on unconjugated estriol in Down syndrome screening, and the new optimized Amerlex-M second-trimester kit. The significant difference between the results of each assay could cause unacceptable changes in the detection rate and false-positive rate of Down syndrome screening programs, especially if previously published values for estriol are used in the risk calculation. It is not possible to define new calculation parameters for every assay kit because new parameters will need to be defined every time kit changes occur, which would require a large collection of samples from Down syndrome pregnancies for standardization. Possible solutions to this problem are discussed.
Nonradioactive Quantification of Low Concentrations of Hemoglobin a by HPLC for Midtrimester Prenatal Diagnosis of β-Thalassemia
Authors: Maiavacca R , Tedeschi S , Mosca A , Calmi S , De Leonardis P , Todaro C , Zuliani G , Buscaglia M , Cantù-Rajnoldi A
Published: January 2020

The usual methods for prenatal diagnosis of beta-thalassemia and other hemoglobinopathies by assay of fetal blood erythrocytes are either complex (analysis of globin chains synthesis by carboxymethylcellulose chromatography) or only semiquantitative [isoelectric focusing of hemoglobin (Hb)]. To further simplify the diagnostic procedure and to obtain quantitative data, we measured the small concentrations of Hb A in fetal erythrocytes by using a high-pressure liquid chromatography (HPLC) instrument (DIAMAT-TM; Bio-Rad) equipped with the new column proposed for measuring Hb A2. We analyzed 212 uncontaminated fetal blood samples obtained by cordocentesis between the 18th and 22nd weeks of pregnancy, using the HPLC procedure, and compared the results with those obtained by the above-named methods. The Hb A values obtained ranged between 0% and 8.5%; they were less than or equal to 1.8% in 44 fetuses affected by homozygous beta-thalassemia and greater than 2.5% in 168 unaffected fetuses. The method was simple, rapid, and reproducible (CV 3.2%) and there was good correlation between Hb A concentrations determined by HPLC and the beta/gamma ratio determined by carboxymethylcellulose chromatography (r = 0.7687; P less than 0.0001). No false-negative or false-positive results were observed, and there was no overlap of values between affected and unaffected fetuses.
Medical-care systems for long-duration space missions
Authors: Houtchens B A
Published: January 2020

As in the opening of frontiers on Earth, human physiological maladaptation, illness, and injury--rather than defective transportation systems--are likely to be the pace-limiting variables in efforts to expand the presence of humans into the solar system. Because of the inability of individuals to return to Earth rapidly and conveniently, the capability of delivering medical care on site will be key to the success of a manned space station, lunar base, and Mars mission. Spaceflight medical care equipment must meet stringent constraints of size, weight, and power requirements, and then must function accurately in remote, self-contained, microgravity settings after extended intervals of storage, with neither expert operators nor repair technicians on site. Satisfying these unusually rigorous requirements will require sustained direct involvement of clinically up-to-date health-care providers, medical scientists, and biomedical engineers, as well as astronauts and aerospace engineers and managers. Solutions will require validation in clinical settings with real patients, as well as in simulated operational settings.
The clinical chemistry and immunology of long-duration space missions
Authors: Wu A H , Taylor G R , Graham G A , McKinley B A
Published: January 2020

Clinical laboratory diagnostic capabilities are needed to guide health and medical care of astronauts during long-duration space missions. Clinical laboratory diagnostics, as defined for medical care on Earth, offers a model for space capabilities. Interpretation of laboratory results for health and medical care of humans in space requires knowledge of specific physiological adaptations that occur, primarily because of the absence of gravity, and how these adaptations affect reference values. Limited data from American and Russian missions have indicated shifts of intra- and extracellular fluids and electrolytes, changes in hormone concentrations related to fluid shifts and stresses of the missions, reductions in bone and muscle mass, and a blunting of the cellular immune response. These changes could increase susceptibility to space-related illness or injury during a mission and after return to Earth. We review physiological adaptations and the risk of medical problems that occur during space missions. We describe the need for laboratory diagnostics as a part of health and medical care in space, and how this capability might be delivered.
Preliminary evaluation of an experimental clinical chemistry analyzer developed for space medicine
Authors: Wu A H , Gornet T G , Schenkel O , Smith-Cronin L , Graham G A , Tonnesen A S , McKinley B A
Published: January 2020

An experimental clinical chemistry analyzer system was designed and built to demonstrate the feasibility of clinical chemistry as part of a medical-care system at NASA's planned space station Freedom. We report the performance of the experimental analyzer, called a medical development unit (MDU), for selected analytes in a laboratory setting in preparation for a preliminary clinical trial at patients' bedsides in an intensive-care unit. Within-run CVs ranged from 0.7% for sodium to 7.1% for phosphorus; day-to-day CVs ranged from 1.0% for chloride to 23.4% for calcium. Correlation of patients' blood sample analyses compared well with those by Ektachem E700 and other high-volume central laboratory analyzers (r ranged from 0.933 for creatine kinase MB isoenzyme to 0.997 for potassium), except for hemoglobin (r = 0.901) and calcium (r = 0.823). Although several CVs obtained in this study exceeded theoretical desired precision limits based on biological variations, performance was adequate for clinical laboratory diagnosis. We examined the effect of potentially interfering concentrations of hemoglobin, bilirubin, and lipids: the only effect was negative interference with calcium analyses by high concentrations of bilirubin. We also examined the effects of preanalytical variables and the performance of experimental sample-transfer cups designed to retain sample and reference liquid in microgravity. Continued development of the MDU system is recommended, especially automation of sample processing.
Reliability and feasibility of pregnancy home-use tests: laboratory validation and diagnostic evaluation by 638 volunteers
Authors: Daviaud J , Fournet D , Ballongue C , Guillem G P , Leblanc A , Casellas C , Pau B
Published: January 2020

All 27 home-use tests sold in France in 1989 for the self-diagnosis of pregnancy were evaluated. The kits were first tested by qualified clinical chemistry technologists. Eleven kits with 100% specificity and 100% sensitivity were retained for the diagnostic study. Each of 638 laywomen was given a kit and asked to perform the assay with a coded urine specimen containing either no human chorionic gonadotropin (hCG) or an hCG concentration adjusted to the claimed detection limit (1 DL) or twice the detection limit (2 DL). After testing, each participant filled out a detailed questionnaire. The results showed a diagnostic specificity of 86-100% for 10 kits but a diagnostic sensitivity of 85-100% for only 5 kits at 2 DL and for only 2 at 1 DL. Among the 478 positive urine samples distributed, 230 were falsely interpreted as negative. The main explanation for such a high percentage of false-negative results was difficulty in understanding the explanatory leaflets accompanying the kits and hence in reading the results, regardless of the socioeconomic situation of the participant. We conclude that pregnancy home-use tests should be subjected to rigorous analytical controls and evaluated by a panel of potential users before being released on the market.
Differences in serum potassium concentrations in normal men in different geographic locations
Authors: Reidenberg M M , Gu Z P , Lorenzo B , Coutinho E , Athayde C , Frick J , Alvarez F , Brache V , Emuveyan E E
Published: January 2020

Hypokalemia has been associated with the taking of gossypol, a potential oral antifertility drug for men. Because the frequency of this response differed in different parts of the world, this study was done to learn if "normal" serum [K+] also differed. [K+] was measured by flame photometry in serum from apparently normal men from Austria (n = 30), China (53), Brazil (100), the Dominican Republic (38), and the US (103), and in plasma from Nigerian men (82). The mean (SD) for [K+] in Chinese men, 3.82 (0.27) mmol/L, was lower than that in Brazilians [4.06 (0.29) mmol/L], Austrians [4.14 (0.44) mmol/L], Dominicans [4.37 (0.33) mmol/L], or Americans [4.38 (0.37) mmol/L]. Apparently there are regional differences in average serum [K+], with men in China having lower serum [K+] than men elsewhere. This may predispose them to hypokalemia.
HPLC of folinic acid diastereoisomers and 5-methyltetrahydrofolate in plasma
Authors: Etienne M C , Speziale N , Milano G
Published: January 2020

We present a rapid, sensitive, and automated HPLC method with direct resolution of l-folinic acid (l-FA), d-folinic acid (d-FA), and 5-methyltetrahydrofolate (5MTHF) from plasma samples. Plasma (500 microL) is first extracted on solid phase (RP-18 cartridge). The dI-FA peak is collected on-line from a reversed-phase column (RP-8, 119 x 2 mm, 4 microns: HPLC 1) and then automatically loaded onto a chiral stationary phase (human serum albumin, 150 x 4.6 mm, 7 microns: HPLC 2). The same mobile phase flows in both systems (0.2 mol/L Na2HPO4:1-propanol, 98:2, pH 6.2). HPLC 1 allows quantification of 5MTHF by absorption at 313 nm; HPLC 2, the quantification of l-FA and d-FA by electrochemical detection in the oxidation mode (total run time 18 min). Recoveries are > 80%. CVs for intra- and interassay reproducibilities are < 5% and 15%, respectively. Linearity of the response (0.1-1 mumol/L and 1-50 mumol/L, r = 0.99, P < 0.01) is satisfactory. The sensitivity limit is 50 nmol/L for 5MTHF and 20 nmol/L for l-FA and d-FA. This assay is substantially improved over existing methods regarding feasibility and is being used in pharmacokinetic investigations in cancer patients.
Detection and characterization of blocking-type anti-acetylcholine receptor antibodies in sera from patients with myasthenia gravis
Authors: Hara H , Hayashi K , Ohta K , Itoh N , Nishitani H , Ohta M
Published: January 2020

We developed a highly sensitive, convenient assay for measuring blocking-type anti-acetylcholine receptor antibodies, which inhibit the binding of 125I-labeled alpha-bungarotoxin (alpha-BuTx) to the acetylcholine receptor (AChR). This procedure detected inhibitory activities in sera from 76% of patients with myasthenia gravis. Results of an experiment done with synthetic peptide corresponding to the alpha-BuTx binding region in the alpha-subunit of Torpedo AChR suggested that this inhibition is due to nonspecific steric hindrance caused by the binding of antibodies to a region other than the alpha-BuTx site, rather than by direct binding to the latter site. The inhibitory activities of the blocking-type antibodies and the titers of non-blocking-type antibodies were correlated. Moreover, the blocking-type antibodies could dissociate 125I-labeled alpha-BuTx from 125I-labeled alpha-BuTx-human AChR complex, and their dissociation activities showed good correlation with the inhibitory activities.