bluuebunny/crossref_metadata_2025 huggingface dataset:
Multicomponent analysis for alkaline phosphatase isoenzyme determination by multiple linear regression
Authors: Tillyer C R , Rakhorst S , Colley C M
Published: January 2020

Alkaline phosphatase (EC 3.1.3.1) isoenzymes in serum may be determined by multicomponent analysis of the enzyme activities in the presence of multiple inhibitors. To determine inhibition coefficients of the isoenzymes, we used multiple linear regression analysis to compare alkaline phosphatase activities in the presence of known inhibitors with electrophoretically determined isoenzyme activities in plasma and serum samples. All possible combinations of exactly determined and overdetermined linear systems of inhibitors were ranked according to their prediction error to select an optimum set. The best multicomponent system for prediction included the use of levamisole, phenylalanine, and heat inhibition at 56 degrees C and 65 degrees C to determine bone, hepatic, intestinal, and placental isoenzymes. Consideration of the hepatic isoenzyme as liver and macromolecular fractions resulted in significantly worse predictions. Error analysis involving repeat determinations and a simplex optimization of the inhibition coefficients indicated that the inaccuracy of the comparison electrophoretic method may have been a major factor affecting poor isoenzyme prediction in some samples.
ELISA of IgG antibody to oxidized low-density lipoprotein: effects of blocking buffer and method of data expression
Authors: Craig W Y , Poulin S E , Nelson C P , Ritchie R F
Published: January 2020

We describe an ELISA for serum IgG antibodies against malondialdehyde-modified low-density lipoprotein (mLDL). Optimal antigen concentration, serum dilution, and dilution of enzyme-conjugated second antibody were 25 mg/L, 1:250, and 1:5000, respectively, when 5 g/L human serum albumin was used for blocking. When data were expressed as mLDL/LDL (the ratio of IgG binding to mLDL vs LDL), within-run and between-run CVs were 7.0% and 8.9%, respectively. Antibody concentrations expressed as mLDL/LDL or as mLDL-LDL (the difference between IgG binding to mLDL and to LDL) were higher in women with systemic lupus erythematosus (n = 20) than in controls (n = 20) (P < 0.001). With bovine serum albumin or Superblock blocking buffers, only the mLDL-LDL data were significant. Thus, the choice of blocking agent and the method of data expression should be carefully considered when assaying IgG antibodies against mLDL.
Realistic modeling of clinical laboratory operation by computer simulation
Authors: Vogt W , Braun S L , Hanssmann F , Liebl F , Berchtold G , Blaschke H , Eckert M , Hoffmann G E , Klose S
Published: January 2020

An important objective of laboratory management is to adjust the laboratory's capability to the needs of patients' care as well as economy. The consequences of management may be changes in laboratory organization, equipment, or personnel planning. At present only one's individual experience can be used for making such decisions. We have investigated whether the techniques of operations research could be transferred to a clinical laboratory and whether an adequate simulation model of the laboratory could be realized. First we listed and documented the system design and the process flow for each single laboratory request. These input data were linked by the simulation model (programming language SIMSCRIPT II.5). The output data (turnaround times, utilization rates, and analysis of queue length) were validated by comparison with the current performance data obtained by tracking specimen flow. Congruence of the data was excellent (within +/- 4%). In planning experiments we could study the consequences of changes in order entry, staffing, and equipment on turnaround times, utilization, and queue lengths. We conclude that simulation can be a valuable tool for better management decisions.
Simultaneous macroamylasemia and macrolipasemia
Authors: Zaman Z , Van Orshoven A , Mariën G , Fevery J , Blanckaert N
Published: January 2020

The first case of the simultaneous presence of macroamylasemia and macrolipasemia in a patient with gluten enteropathy (celiac disease) is described. Both macroenzymes were formed by association of polyclonal IgA with amylase and lipase. Both macroenzymes had molecular masses > 300 kDa.
Application of the clearance concept to hyponatremic and hypernatremic disorders: a phenomenological analysis
Authors: Shoker A S
Published: January 2020

The kidney and its response to the antidiuretic hormone (ADH) are the principal protective mechanisms necessary to maintain a normal plasma tonicity (osmolality). Hence, determination of the response of the ADH-renal axis to an abnormal plasma tonicity is an important step to understanding water homeostasis. Determination of free water clearance is the most direct clinical method to measure the ability of the kidney to reabsorb or excrete water; it can be used as a sensitive method to study water metabolism, describing the abnormal water homeostasis in simple quantitative terms. A positive electrolyte-free water clearance denotes the excretion of excess free water. A negative electrolyte-free water clearance indicates reabsorption of excess free water. During hypertonicity, an increased concentration of ADH enhances renal reabsorption of free water. With diminished ADH secretion and normal renal function, a substantial volume of free water is cleared in response to hypotonic stimuli. A positive free water clearance > 0.4 L/day in hypertonic conditions or a negative free water clearance during hypotonicity confirms an abnormal ADH-renal axis response.
Comparable detection of acute myocardial infarction by creatine kinase MB isoenzyme and cardiac troponin I
Authors: Adams J E , Schechtman K B , Landt Y , Ladenson J H , Jaffe A S
Published: January 2020

Although measurement of cardiac troponin I (cTnI) is, in some situations, more specific for detection of cardiac injury than is measurement of the MB isoenzyme of creatine kinase (MBCK), its sensitivity and specificity relative to MBCK for detection of myocardial infarction has not been established. Accordingly, we studied prospectively 199 consecutive patients admitted to the coronary care unit. Values of MBCK and cTnI mass were determined in all samples. Of the 188 patients admitted with a suspicion of acute myocardial ischemia, 89 were diagnosed as having an acute myocardial infarction on the basis of the patterns of MBCK values. Eighty-six of these patients also had increased cTnI (concordance, 96.6%); three did not. Of the patients diagnosed as without infarction, five with unstable angina and symptoms in the day(s) prior to admission had increased cTnI, for a cTnI specificity of 94.9%. Receiver operating characteristic curve analysis indicated that cTnI and MBCK had statistically indistinguishable diagnostic accuracies for the detection of acute myocardial infarction.
Improved HPLC-radioimmunoassay for quantifying angiotensin II in plasma
Authors: Voelker J R , Cobb S L , Bowsher R R
Published: January 2020

To measure the octapeptide angiotensin II (Ang II) in plasma, we developed a sensitive, specific assay that interfaces solid-phase extraction, HPLC, and RIA. A reversed-phase HPLC system involving isocratic elution at 38 degrees C with a volatile mobile phase of acetonitrile and the ion-pairing reagent heptafluorobutyric acid produced baseline separation of angiotensin peptides. Ang II was collected as a single fraction, concentrated by evaporation to dryness, and measured by RIA after resuspension in RIA buffer. Even including column washing between sample injections to prevent carryover of plasma constituents, two plasma extracts could be processed per hour by HPLC. Assay validation experiments demonstrated < 2% cross-reactivity with Ang II-related peptides; a 75% recovery from plasma at physiological concentrations of Ang II; intra- and interassay precision (CVs) of 6.2% and 10.3%, respectively; and a lower limit of quantification of 1.3 ng/L. Two clinical protocols designed to measure plasma Ang II concentration under basal and stimulated conditions confirmed the utility of the assay.
Indoxyl-beta-D-glucuronide, the primary emitter of low-level chemiluminescence in plasma of hemodialysis patients
Authors: Agatsuma S , Sekino H , Nagoshi T , Watanabe H
Published: January 2020

Characteristic light emission induced by the oxidation of hydroxyl radicals has been found in plasma of hemodialysis patients (Agatsuma et al., Clin Chem 1992;38:48-55). We purified a primary emitter, a chemiluminescent component peaking at 430 nm, by anion-exchange chromatography and reversed-phase HPLC. By using proton nuclear magnetic resonance and authentic indoxyl compounds, we determined the primary emitter to be indoxyl-beta-D-glucuronide. Absorption and fluorescence spectra of the purified sample coincided well with those of authentic indoxyl-beta-D-glucuronide, as did the peak in the chemiluminescence emission spectrum. Retention time of the purified sample on reversed-phase HPLC, measured by fluorescence, was also in accordance with that of indoxyl-beta-D-glucuronide. To our knowledge, this is the first identification of a primary emitter of low-level chemiluminescence from a biological source.
Benefits and risks of emerging genetic technologies: the need for regulation
Authors: Holtzman N A
Published: January 2020

Efforts to localize human genes to specific chromosomes and then to identify these genes and their role in specific diseases are increasing. These developments will expand the repertoire of genetic tests that are claimed to be predictive of future disease in the person being tested (pre-symptomatic tests) or in future offspring of those being tested (tests for reproductive options). In single-gene (mendelian) disorders, such tests may fail to detect all of the alleles capable of causing disease. In multifactorial disorders, positive results of tests for specific alleles will not always predict future disease because the other necessary factors may be absent. Policies are already in place to provide safe and effective clinical laboratory tests and interventions for subjects with positive test results, and to assure the quality of the laboratories performing such tests, but are not widely known or enforced for genetic tests.
Use of small-sample-based reference limits on a group basis
Authors: van der Meulen E A , Boogaard P J , van Sittert N J
Published: January 2020

The evaluation of a biochemical or hematological quantity measured in a study group of employees during occupational health assessments involves a comparison with a reference sample group. Part of this evaluation consists of checking whether the percentage of values larger than a predetermined upper reference limit is significantly larger than the percentage normally expected (2.5%, if the 97.5 percentile is used as the upper reference limit). The reference limit, however, is estimated from a random reference sample, the size of which, for many reasons, may be relatively small; as a consequence, the reference limit estimate will be imprecise. In situations in which the reference sample size is smaller than or not much larger than the study sample size, this imprecision results in the usual binomial test of significance being highly inappropriate. We provide an exact nonparametric test valid for all reference sample sizes.
Molecular diagnosis of acute intermittent porphyria by analysis of DNA extracted from hair roots
Authors: Schreiber W E , Fong F , Jamani A
Published: January 2020

Analysis for mutations in the porphobilinogen deaminase gene offers a more definitive diagnosis of acute intermittent porphyria (AIP) than do conventional biochemical tests. We used single-strand conformation polymorphism analysis followed by direct sequencing to identify a new G-->A mutation at the last position of intron 7 in a patient with AIP. The mutation disrupts the invariant AG dinucleotide at the 3' splice acceptor site and therefore interferes with mRNA processing. To identify other individuals who inherited this mutation, we analyzed five hairs with intact roots collected by each participating family member and sent to us by mail. DNA was extracted from the hair roots and amplified by the polymerase chain reaction. The amplified products were digested with the restriction enzyme BsaJI to confirm the presence or absence of the mutation. All six family members who were known to have AIP tested positive, as did three members who had not been previously diagnosed. Hair roots provide a convenient, accessible, and economical alternative to blood as a source of DNA for molecular diagnostic testing.
Micromachined analyzers on a silicon chip
Authors: Arquint P , Koudelka-Hep M , van der Schoot B H , van der Wal P , de Rooij N F
Published: January 2020

For an example of a silicon-based micromachined analyzer, we describe a combined PO2, PCO2, and pH sensor designed for extracorporeal blood gas monitoring. The clinically well-accepted amperometric (PO2) and potentiometric (PCO2, pH) sensing principles are used, realized in a planar and miniaturized form on a single silicon chip (6 x 22 mm). The transducer part of the chip is fabricated by standard silicon technology. Polyacrylamide and polysiloxane polymeric layers, which are used as internal electrolyte and gas-permeable membrane, respectively, are deposited and patterned by photopolymerization. The entire sensor is fabricated on the wafer level by using integrated-circuit-compatible processes, thus allowing mass production. By integrating a flow-through channel directly on the chip, the sample size and the reagent consumption are substantially reduced. The device was characterized in aqueous solutions and in blood intended for transfusion. The sensor has a typical sensitivity of 0.36 nA/mmHg (PO2), -39 mV/decade (PCO2), and 51 mV/pH (pH); low drift; and a functional lifetime of > 2 months. The analytical precision in the physiologically expected range is better than 2 mmHg for the PO2 and PCO2 sensor, and 0.02 pH unit for the pH sensor.
Improvements to immunoassays by use of covalent binding assay plates
Authors: Douglas A S , Monteith C A
Published: January 2020

The uniformity of Xenobind covalent binding microwell plates was compared with that of passive binding plates by binding streptavidin to the plate surface, reacting the streptavidin with biotinylated alkaline phosphatase, and developing color with phenolphthalein monophosphate. Absorbance of the covalent binding plates at 570 nm was 0.95 A (CV 1.9%) compared with 0.35-0.5 A (CV 6.7-16.9%), for passive plates. No edge effect was observed with Xenobind covalent plates. Use of both plates in a model immunoassay gave a CV of 4% for the covalent binding plate and 12-15% for the passive binding plates. Moreover, the background absorbance (color development when no antigen was present) for the covalent binding plates was < 0.05 A in all cases, whereas that for the passive binding plates ranged up to 0.45 A. The high background on passive plates results from displacement of capture antibody and (or) blocking agent from the plate, thereby providing a place for detector antibody binding.
Natural history and risk factors for early human atherogenesis. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group
Authors: Strong J P
Published: January 2020

A multi-institutional study, Pathobiological Determinants of Atherosclerosis in Youth (PDAY), was initiated to document the natural history of atherosclerosis, its relationship to risk factors, and the pathobiology of lesion development in young subjects. Pathology laboratories in nine centers collected arteries and tissues from > 2000 persons, ages 15-34 years, whose deaths were attributed to homicides, accidents, or suicides. Arteries were evaluated for lesions, and risk factors were analyzed in a central laboratory. Postmortem risk factors include serum lipoproteins, serum thiocyanate (smoking), glycohemoglobin (diabetes), thickness of panniculus adiposus (obesity), changes in small renal arteries (hypertension), and apoprotein isoforms. This PDAY study documents the development of atherosclerosis at an early age and shows that the recognized risk factors for coronary heart disease are associated with lesion development in the arteries of these young subjects. The findings provide a strong justification for reducing risk factors in young persons.
Rapid, automated quantification of total human chorionic gonadotropin in serum by a chemiluminescent enzyme immunometric assay
Authors: Vankrieken L , De Hertogh R
Published: January 2020

Total human chorionic gonadotropin (hCG-hCG beta) in serum was assayed with the immulite system, a fully automated random-access immunoassay analyzer that has a unique centrifugal procedure for solid-phase washing and a chemiluminescent detection system. The broad range of the hCG calibration curve (up to 5000 IU/L) is achieved by using a small serum sample size (5 microL), which provides sufficient volume for low-end sensitivity and prevents the possible high-dose hook effect in the working range of the assay. Within-run imprecision (CV) ranged from 3.9% to 5.9% for hCG between 10.5 and 2908 IU/L. Between-run imprecision ranged from 8.8% to 12.7% for hCG mean concentrations from 11.4 to 88.4 IU/L. The antibodies used in the immulite hCG assay system gave little or no interferences when high amounts of follitropin, lutropin, and thyrotropin were added. A complete recognition of the free beta-subunit of hCG was obtained (+/- 180%). In sera from women with molar pregnancies, we observed no high-dose hook effect at hCG concentrations up to 3000 kIU/L. The broad range of hCG concentrations encountered throughout normal pregnancy (up to 200 kIU/L) requires an extended working range to avoid high dilutions. In early pregnancy, accuracy in the range of 1000-5000 IU/L is enhanced by avoiding dilutions.
Can molecular markers now be used for early diagnosis of malignancy?
Authors: Duffy M J
Published: January 2020

Most of the presently available cancer markers are neither specific for malignancy nor allow early diagnosis. However, the recent elucidation of the molecular events occurring during tumorigenesis may provide new markers that are likely to be both specific for cancer and sensitive for early disease. The key molecules undergoing alterations during carcinogenesis are the cellular oncogenes and suppressor genes. Alterations in these genes can be detected in cells shed from malignant and premalignant lesions. Thus, mutant p53 genes have been found in urine from patients with bladder cancer, mutant ras genes in stools from patients with colorectal and pancreatic cancers, and both mutant p53 and ras genes in sputum from patients with lung cancer. These findings show that the genetic alterations in cancer can be detected in fluids or secretions that had contact with the malignant tissue. The preliminary studies, however, had small numbers of both patients and controls and used time-consuming, labor-intensive, and expensive assays. For routine applications, these assays must be simplified, automated, and tested for sensitivity, specificity, and predictive value.
Free and complexed prostate-specific antigen (PSA): in vitro stability, epitope map, and development of immunofluorometric assays for specific and sensitive detection of free PSA and PSA-alpha 1-antichymotrypsin complex
Authors: Pettersson K , Piironen T , Seppälä M , Liukkonen L , Christensson A , Matikainen M T , Suonpää M , Lövgren T , Lilja H
Published: January 2020

Generation of 15 monoclonal antibodies (MAbs) allowed construction of epitope maps and specific two-site immunofluorometric assays for free prostate-specific antigen (PSA) and PSA complexed with alpha 1-antichymotrypsin (ACT). Close correlation of PSA concentrations obtained with the use of different assays of free PSA suggested extensive similarity in immunodetection of free PSA in serum. Assays of the PSA-ACT complex overestimated the concentration of PSA-ACT in serum because of nonspecific adsorbance of ACT or cathepsin G-complexed ACT to the solid phase. This interference was substantially decreased in the presence of heparin. In studying the stability of purified PSA and PSA-ACT complexes formed in vitro, we found that the free PSA was stable during storage for 4 weeks at 35 degrees C, whereas PSA-ACT complexes largely dissociated in these conditions. The instability of PSA-ACT complexes was counteracted by storage at low temperatures, by adjusting the pH of the storage buffer between 6.8 and 7.4, and through addition of 100-1000-fold molar excess of native ACT. The ease of calibration and the accuracy of free PSA assays in comparison with assays of the PSA-ACT complex suggest that measurements of free to total PSA most accurately reflect the inverse of the proportion of PSA complexed to ACT in serum.
AquaLite bioluminescence assay of thyrotropin in serum evaluated
Authors: Sgoutas D S , Tuten T E , Verras A A , Love A , Barton E G
Published: January 2020

We studied a new commercially available thyrotropin (TSH) assay, the AquaLite Bioluminescence TSH-Immunoassay (SeaLite Inc.). This assay has a detection limit of 0.005 mIU/L and a functional sensitivity of 0.017 mIU/L and meets the requirements of a third-generation TSH assay. Using this assay, we measured serum TSH in 153 euthyroid individuals and in the following patients: 32 primary hypothyroids; 38 primary hyperthyroids; 35 with thyroid cancer receiving suppressive therapy with levothyroxine (L-T4); 33 receiving replacement L-T4 aimed at reaching and maintaining a euthyroid status; 23 with subclinical hyperthyroidism; and 52 hospitalized for nonthyroidal illnesses (NTI). The AquaLite TSH assay perfectly discriminated hypothyroid and untreated hyperthyroid patients from euthyroid subjects and clearly discriminated between overtly and mildly hyperthyroid patients. Intermethod comparisons showed that the AquaLite and the Nichols assays were more effective than the ACS-180 and the TOSOH assays in discriminating among hyperthyroid patients, including patients over-treated with L-T4.
Detection of point mutations in T lymphocytes
Authors: van Zeeland A A , Jansen J G , Mohn G R , Vrieling H , Tates A D , Lohman P H
Published: January 2020

We exposed experimental animals to a series of alkylating agents that induced mutations at the X-linked hprt gene of T lymphocytes. We then isolated the mutant cells and analyzed the molecular nature of the mutations by amplification of hprt cDNA sequences with the use of reverse transcriptase PCR followed by DNA sequence analysis, and then correlated the mutational spectra obtained to the spectra of DNA adducts caused by the alkylating agents used. The nature of the base-pair changes causing the mutations was characteristic for the reaction pattern of the genotoxic agent with DNA. However, we also found a clear influence of DNA repair processes; i.e., in those cells that were able to remove certain types of DNA damage, the class of mutations expected from that type of damage was reduced.
Prostatic acid phosphatase assay with self-indicating substrate 2,6-dichloro-4-acetylphenyl phosphate
Authors: Osawa S , Iida S , Yonemitsu H , Kuroiwa K , Katayama K , Nagasawa T
Published: January 2020

We characterized six self-indicating substrates, synthesized as the derivative compounds of acetylphenyl phosphate, for serum prostatic acid phosphatase (PAP) activity. One of the substrates, 2,6-dichloro-4-acetylphenyl phosphate (DCAPP), is superior to others in terms of stability, affinity, and low Km for PAP. The hydrolyzed product, 2,6-dichloro-4-acetylphenol (DCAP), has a maximum absorption at 334.2 nm, a pKa of 4.15, and a molar absorptivity at 340 nm of 21,490 L.mol-1.cm-1 in citrate-HCl buffer, pH 5.4. PAP activity was assessed by subtracting tartaric acid-inhibited acid phosphatase activity from total acid phosphatase activity. Our assay system involving DCAPP is a unique kinetic method that shows good reproducibility, wide analytical dynamic range, and high specificity for PAP. Moreover, it is easily adaptable to automated analyzers because the product, DCAP, can be monitored at 340 nm.
Ultrasensitive assay of prostate-specific antigen used for early detection of prostate cancer relapse and estimation of tumor-doubling time after radical prostatectomy
Authors: Yu H , Diamandis E P , Prestigiacomo A F , Stamey T A
Published: January 2020

We used an ultrasensitive prostate-specific antigen (PSA) assay with a detection limit of 0.02 microgram/L for long-term monitoring of PSA changes in 5 patients who were cured by radical prostatectomy and in 10 patients who had failed prostatectomies; 5 patients who underwent cystoprostatectomy were also evaluated with one sample after surgery. Relapse-free periods, determined on the basis of criteria designed specifically for the ultrasensitive assay or proposed for other currently available PSA assays, were calculated for the patients with failed prostatectomies. Tumor-doubling times were also calculated, postsurgery, according to a model that assumes exponential tumor growth over time. We found that prostate cancer relapse, on average, could be diagnosed 420 or 883 days earlier with the ultrasensitive assay than with assays having detection limits of 0.1 or 0.3 microgram/L, respectively. Tumor-doubling times, calculated after radical prostatectomy, ranged from 67 to 568 days among the 10 patients. We also present evidence that even more-sensitive PSA assays might be able to further reduce the relapse-free periods in approximately 50% of the prostate cancer patients who ultimately relapse.
Two alpha-chain hemoglobin variants, Hb Broussais and Hb Cemenelum, characterized by cation-exchange HPLC, isoelectric focusing, and peptide sequencing
Authors: Turpeinen U , Sipilä I , Anttila P , Karjalainen U , Kuronen B , Kalkkinen N , Ahola T , Stenman U H
Published: January 2020

We here report the characteristics of two rare alpha-chain hemoglobin (Hb) variants. The variants were found during quantification of HbA1c by cation-exchange HPLC with the Diamat glycohemoglobin analyzer. They were further characterized by isoelectric focusing and PolyCAT A cation-exchange chromatography. The structure of the abnormal Hbs was established by amino acid analysis after separation of the globin chains by reversed-phase chromatography, digestion with trypsin, separation of the peptides by reversed-phase chromatography, and amino acid sequencing. These studies showed that the two variants were Hb Broussais [alpha 90 (FG2)Lys-->Asn] and Hb Cemenelum [alpha 92 (FG4)Arg-->Trp].
Chromatographic evaluation of minor hemoglobins: clinical significance of hemoglobin A1d, comparison with hemoglobin A1c, and possible interferences
Authors: Bissé E , Huaman-Guillen P , Wieland H
Published: January 2020

Using an HPLC procedure, we evaluated > 10 minor hemoglobins (Hbs) in healthy adults, individuals on long-term aspirin therapy, diabetic subjects, and uremic patients. Hb A1c and Hb A1d3 were the most abundant and important minor Hb components, respectively, accounting for 4.10% +/- 0.50% and 3.46% +/- 0.43% of total Hb in 361 healthy subjects. Acetylated Hb A was a potential interferent in the measurement of Hb A1c. The amounts of both Hb A1d3 and Hb A1carb were significantly increased in uremic patients, indicating that these Hb adducts may be carbamylated. There was a significant correlation (r = 0.69, P < 0.0001) between the amounts of Hb A1d3 and plasma urea in uremic patients. Nonuremic subjects were clearly separated from uremic patients with regard to the Hb A1d3 content of their total Hb. Our data suggest that Hb A1d3 is useful for assessment of the uremic state and that the combination of Hb A1c and Hb A1d3 could be well-suited for simultaneous control of carbohydrate and urea metabolism.
Rapid gas-chromatographic assay of lactulose and mannitol for estimating intestinal permeability
Authors: Celli M , D'Eufemia P , Dommarco R , Finocchiaro R , Aprigliano D , Martino F , Cardi E , Giardini O
Published: January 2020

We developed a gas-chromatographic method to determine urinary mannitol and lactulose. The procedure for purification of urine by a resin was optimized for purification of analytes and high recovery; the aliquot of resin chosen (500 mg) was kept in contact with the urine for 1 min. The recoveries of mannitol and lactulose were > 85% at concentrations that include both normal and pathological values. Sugars were converted to oximes before the silylation step to avoid multiple peaks for the anomeric forms. The calibration was linear over the range 0.1-1 microgram of sugar injected. Analytical recovery of the sugars ranged from 90% to 95.3% for mannitol and from 90.4% to 95.8% for lactulose. The mean within-day imprecision (CV) was 6.2% for mannitol and 4.7% for lactulose; the between-day CV was 6.7% for mannitol and 5.1% for lactulose. A lactulose/mannitol ratio of 0.035 completely differentiated 28 normal children and 28 children with active gluten-sensitive enteropathy, whose mean ratios were 0.022 (SD 0.007) and 0.084 (SD 0.054), respectively.
Diagnosis of human heritable diseases--laboratory approaches and outcomes
Authors: Dowton S B , Slaugh R A
Published: January 2020

Detection of mutant human genes is rapidly becoming an integral part of clinical practice. Human disease may arise by genetic deletion, insertion, fusion, point mutation, or amplification of unstable sequences. Such changes in structure may occur in germ cells or somatically. Rapid advances in understanding the complex nuclear and mitochondrial genomes necessitates deployment of a variety of methods to identify aberrant genes. These techniques include polymerase chain reaction, Southern transfer, and allele-specific hybridization studies, as well as methods to unmask mismatches between mutant and normal sequences. Development of protein truncation tests has added a vehicle for assessing larger DNA segments for mutations that cause premature translational termination. Linkage analysis remains an important tool where direct assay of disease-causing mutations is not possible. Considerations of confidentiality, informed consent, and insurability are important whenever genetic testing is used. These issues will assume increasing importance as presymptomatic testing for heritable predispositions emerges for common conditions.
For goodness' sake: expected therapeutic benefit as a basis for healthcare delivery
Authors: Denton T A , Diamond G A
Published: January 2020

The current debate on our healthcare system has focused primarily on the cost of care. Because of drastically rising costs and their burden on our economy, government and the private sector have developed many approaches to reduce these costs. Managed care, special contracting arrangements, and government fiat have all been used to stem the tide of rising costs--with variable success. We propose that the primary goal of healthcare is the provision of patient benefit, and we describe a model that calculates an expected benefit in terms of survival and quality of life. We have applied this model to a cohort of patients undergoing coronary angiography to determine the distributions of benefit. Furthermore, we describe a reimbursement strategy that relates the expected therapeutic benefit to the reimbursement received for that therapy--the greater the benefit, the greater the reimbursement. The future of our healthcare system lies in keeping the patient at the center of the debate on the delicate balance between optimal care and societal cost.
Immunoradiometric method and electrophoretic system compared for quantifying bone alkaline phosphatase in serum
Authors: Van Hoof V O , Martin M , Blockx P , Prove A , Van Oosterom A , Couttenye M M , De Broe M E , Lepoutre L G
Published: January 2020

Agarose electrophoresis (Isopal, Beckman) and an immunoradiometric assay (IRMA) involving specific monoclonal antibodies (Ostase, Hybritech), two methods for the quantification of serum bone alkaline phosphatase (ALP, EC 3.1.3.1), a marker of osteoblastic activity, were compared in 293 patients: 79 with end-stage renal failure treated with hemodialysis and 214 with malignant disease. Overall correlation between the two methods was good (r = 0.92), except (a) for low values of bone ALP and (b) in some samples with high total liver ALP activity--both due to considerable cross-reactivity of the anti-bone ALP antibodies of the Ostase kit with liver ALP. This interference was not constant and was not evenly distributed across all concentrations of bone ALP. Low bone ALP determined with the IRMA (< or = 5 micrograms/L) was confirmed by electrophoresis (< or = 21 U/L), but bone ALP activity determined by electrophoresis to be low (< or = 21 U/L) was not correlated with the IRMA results. After standardizing our results by computing z-values for bone ALP, delta z (= zOstase - zIsopal) was significantly correlated with liver ALP activity (r = 0.73, P < 0.0001). We conclude that the IRMA for quantifying bone ALP is acceptable as a screening method. However, when high values for bone ALP are found with the Ostase method, confirmation by electrophoresis remains mandatory to rule out cross-reactivity with high amounts of liver ALP. For detecting low bone ALP activities, electrophoresis remains the method of choice.
Multienzyme control serum (Seraclear-HE) containing human enzymes from established cell lines and other sources. 2: Evaluation as candidate working enzyme Reference Material for alanine and aspartate aminotransferases
Authors: Nakano N I , Eto A , Chikaura Y , Oishi T
Published: January 2020

Seraclear-HE, containing seven enzyme analytes from human sources, was evaluated as an intermethod calibrator for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) to transfer Reference Method values to seven routine methods, including one based on hydrogen peroxide detection for possible unification of values (interlaboratory comparability of data). The commutabilities of AST from erythrocytes and ALT from a hepatoma cell line were studied between the consensus methods of Japan Society of Clinical Chemistry (chosen as the Reference Methods) and each of the automated routine methods at reaction temperatures of 30 degrees C and 37 degrees C. For AST, calibration of patients' sera with Seraclear-HE decreased average intermethod variation (CV) from 12% to 2%; for ALT, the decrease was from 20% to 3%. For both enzymes, Seraclear-HE was judged to be commutable between the Reference Methods and each of the methods investigated. The limitations for such use are discussed.
Flexible protocols improve parallel experimentation throughput
Authors: Aarts R J , Lindsey J S , Corkan L A , Smith S F
Published: January 2020

Advanced chemical workstations offer the potential to substantially improve the productivity of experimental research. To fully exploit such technologies, effective scheduling of experiments is crucial. Chemists tend to define experimental protocols with rigid time constraints, although often the scientific objectives can be achieved without adhering to such constraints. Investigation of a scheduling algorithm that allows flexible time constraints shows that improvements in workstation throughput as great as 50% can be reached by modest flexibility in the timing of operations in the experiments. Several heuristics that might be used with the scheduling algorithm were tested; a heuristic that schedules long experiments while first keeping the workstation busy was shown to be a good general choice.
Mycophenolic acid binding to human serum albumin: characterization and relation to pharmacodynamics
Authors: Nowak I , Shaw L M
Published: January 2020

Mycophenolate mofetil, the prodrug form of the immunosuppressive agent mycophenolic acid (MPA), is currently in clinical trials evaluating its effectiveness in transplant recipients. In this study, we validated an ultrafiltration system for the reliable measurement of free MPA. Using this technique, we evaluated factors that might be important in modulating the free fraction of this drug. Human serum albumin (HSA), high concentrations of the primary glucuronide metabolite of MPA, and sodium salicylate significantly affected MPA binding. For HSA the mean +/- SE binding capacity (Bmax) and the dissociation constant (Kd) were 1095 +/- 34 mumol/L and 12.98 +/- 0.93 mumol/L, respectively. The dose for 50% inhibition (IC50) of inosine monophosphate dehydrogenase isoform II by MPA increased 5.4-fold as the concentration of HSA added to the enzyme reaction mixture increased from 0 to 50 g/L (0-724 mumol/L). Furthermore, the IC50 MPA concentration for phytohemagglutinin A-stimulated human peripheral blood mononuclear cells increased 4.8-fold when incubations were performed in the presence of 10 g/L (145 mumol/L) HSA vs no added HSA. These data support the hypothesis that the pharmacological activity of MPA is a function of unbound drug concentration.
Nonradioactive quantification of mdr1 mRNA by polymerase chain reaction amplification coupled with HPLC
Authors: van Hille B , Lohri A , Reuter J , Herrmann R
Published: January 2020

We describe a new strategy for quantifying mRNA by using polymerase chain reaction (PCR) coupled with HPLC. PCR-amplified products are directly analyzed with a specific HPLC column and are quantified by standardization against a housekeeping gene, beta-actin. To evaluate the experimental conditions, we examined the multidrug-resistance-associated mdr1 gene expression in two drug-sensitive cell lines (RPMI-8226 and KB-3-1) and in their drug-resistant sublines. This revealed a significant overexpression of the mdr1 gene in chemoresistant sublines that paralleled the degree of in vitro drug resistance and the amounts of mRNA determined by other methods. Results were consistently reproducible, with imprecision (CV) < 25%. The reverse transcription option PCR/HPLC protocol described here is a sensitive, nonradioactive method for quantifying mdr1 mRNA in cell lines or clinical tumor samples, and can be applied to the simultaneous quantification of several mRNA species.
Characterization of a monoclonal antibody (HB-22) and development of an ELISA for human apolipoprotein A-I
Authors: Rothwell T C , Kamanna V S , Jin F Y , Koren E , Foley T , Kashyap M L
Published: January 2020

We describe the production and characterization of a high-affinity monoclonal antibody, HB-22, for apolipoprotein (apo) A-I, a major protein of human high-density lipoproteins (HDL). Including Tween 20 in the reaction mixture increased the binding capacity of HB-22 to apo A-I. HB-22 showed monospecific reactivity with HDL or apo A-I, displaying no cross-reactivity with apo A-II, intermediate-, low-, or very-low-density lipoproteins. Immunoaffinity columns with HB-22 (in the absence of Tween 20) showed an immunosorbent capacity of 80 micrograms of apo A-I per milligram of antibody. The immunosorbent capacity of HB-22 for apo A-I was similar in plasma samples from normolipidemic, hypercholesterolemic, or hypertriglyceridemic patients. Comparative binding studies demonstrated that compared with other available monoclonal apo A-I antibodies, HB-22 had the greatest apparent affinity for binding to HDL. A competitive ELISA developed by utilizing HB-22 could detect as little as 20 ng of apo A-I in the reaction mixture. The intra- and interassay CVs of the ELISA were 5.4% and 9.5%, respectively.
A model for medical decision making and problem solving
Authors: Werner M
Published: January 2020

Clinicians confront the classical problem of decision making under uncertainty, but a universal procedure by which they deal with this situation, both in diagnosis and therapy, can be defined. This consists in the choice of a specific course of action from available alternatives so as to reduce uncertainty. Formal analysis evidences that the expected value of this process depends on the a priori probabilities confronted, the discriminatory power of the action chosen, and the values and costs associated with possible outcomes. Clinical problem-solving represents the construction of a systematic strategy from multiple decisional building blocks. Depending on the level of uncertainty the physicians attach to their working hypothesis, they can choose among at least four prototype strategies: pattern recognition, the hypothetico-deductive process, arborization, and exhaustion. However, the resolution of real-life problems can involve a combination of these game plans. Formal analysis of each strategy permits definition of its appropriate a priori probabilities, action characteristics, and cost implications.
Probable impact of the 1994 elections on laboratory medicine
Authors: Foster H S
Published: January 2020

During the past quarter century, federal health policy makers concerned themselves with: (a) improving the quality of healthcare delivered to the American public; (b) increasing access to needed healthcare services; and (c) curtailing the escalating cost of such services. These goals led Congress to expand the role of the federal government in regulating the delivery of healthcare. The enactment of the Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) was a significant and widely discussed example of how Congress, when controlled by the Democrats, sought to correct healthcare problems and achieve federal objectives. In November 1994, the Republicans won majorities in both the Senate and the House, promising to reduce the federal government's power. Many now believe that CLIA '88, or significant parts of it, could be substantially modified as part of this effort. This paper addresses the developments that led the Democrats to seek enactment of CLIA '88 and the likely arguments that may be offered by the Republicans to lessen the rigor and scope of the law.
Value-added laboratory medicine in an era of managed care
Authors: McDonald J M , Smith J A
Published: January 2020

Clinical laboratory consultants (M.D.s and Ph.D.s) must add value and medical relevance to the healthcare system to earn and maintain their roles in an era of managed care. Service opportunities include new and expanded roles in system-wide (a) managing of point-of-care testing, (b) informatics and information systems, (c) clinical consultation, (d) resource management, (e) management of utilization, (f) improving healthcare quality assurance, and (g) technology assessment and implementation. Hypothesis-driven research focused on the linkage between the laboratory and outcome analysis, clinical practice guidelines, total quality improvement, technology assessment, and healthcare policy provides major opportunities to complete for expanding research support. Changes in clinical laboratory consultant training are necessary to provide the knowledge, skills, and experience required to provide value-added and medically relevant services. Clinical laboratory consultants with Ph.D. degrees should initially be trained broadly in all areas of the laboratory, with emphasis on system-wide issues, and then trained as specialized scientists.
Two sensitive time-resolved fluoroimmunoassays for cellular fibronectin
Authors: Kropf J , Gressner A M
Published: January 2020

Two sensitive sandwich-type immunoassays for determination of cellular fibronectin (cFN) in cell culture supernatants and in human plasma were developed. Both assays used a monoclonal antibody with specificity against the EDA sequence, which is characteristic for the cellular form of human and rat FN. Assay 1 involves binding of FN on gelatin-coated microwells followed by reaction with the anti-cFN antibody, whereas in assay 2 the anti-cFN antibody was immobilized first and detection was with an anti-FN antiserum. Time-resolved fluorescence spectrophotometry with measurement of an Eu3+ chelator after dissociation of the solid-phase complexes with urea/sodium dodecyl sulfate in the presence of excess Eu3+ was the detection method. The detection limit of the new assays was between 2.6 and 4.0 micrograms/L cFN. In serial dilution of human plasma samples, parallelism with the calibration curve was obtained over the whole measuring range (12-1000 micrograms/L) with assay 2, whereas assay 1 had deviations of the dilution curves at concentrations > or = 100 micrograms/L. The between-run CVs for assays 1 and 2 were 11.4% and 7.2%, respectively, at a concentration of 200 micrograms/L (median value of 18 experiments). Respective within-series CVs of 4.3% and 4.7% were obtained at the same concentration. The recovery of added cFN from human plasma was between 90% and 96%.
Assay of magnesium in serum and urine with use of only one enzyme, isocitrate dehydrogenase (NADP+)
Authors: Fujita T , Kawakami Y , Kohda S , Takata S , Sunahara Y , Arisue K
Published: January 2020

We report a method for assaying magnesium in serum and urine involving only one enzyme, isocitrate dehydrogenase (NADP+)(EC 1.1.1.42), which requires magnesium ion for activity. The enzymatic reduction of NADP+ by isocitrate increases in rate linearly up to at least 20 mmol/L magnesium in the presence of appropriate concentrations of the two metal-chelating reagents, EDTA and glycol ether diamine-N,N,N',N'-tetraacetate. Within-run (n = 20) CVs and day-to-day (n = 10) CVs for sera are < or = 1.5% and < or = 2.6%, respectively. Analytical recovery of magnesium in sera averages 96-100%. This method is not affected by bilirubin, hemoglobin, or lipemia. The method (y) gives the following results correlating with atomic absorption spectrophotometry (x): y = 1.03x + 0.06 mmol/L (n = 62, r = 0.995, Sylx = 0.03) for sera, and y = 1.03x - 0.10 mmol/L (n = 62, r = 0.989, Sylx = 0.19) for urines; with the calmagite method (x): y = 0.99x + 0.04 mmol/L (n = 62, r = 0.991, Sylx = 0.03) for sera, and y = 0.98x + 0.03 mmol/L (n = 62, r = 0.999, Sylx = 0.02) for urines.
Decoupling spatial and temporal processes for clinical analyzers
Authors: Mazza J , Huber M , Frye S
Published: January 2020

The separation of time and space in processing a sample greatly simplifies the design of automation for clinical testing. The efficient spatial arrangement of analytical units and sample manipulators has become a more complex task because of the degree of automation required on today's state-of-the-art analyzer. Minimization of sample volume and the reduction of overall analyzer size further complicate the design problem. We report the development of a proprietary method of decoupling the temporal and spatial elements required for analysis of samples. This process is based on number theory and can be used to optimize the distance between the physical processing stations while allowing these same stations to operate on samples over a substantial range of times. The technique is versatile and can also be used when it is desirable to sequentially move groups of items from location to location.
The scope and impact of thyroid disease
Authors: Wartofsky L
Published: January 2020

Aspects of the incidence and demographics of common thyroid disorders in the US (and elsewhere, to a lesser extent) are reviewed. The impact of healthcare reform and the efforts of managed care organizations to impose cost-effective management for the diagnosis and treatment of thyroid disorders are bringing unusual pressures to bear on both clinical laboratories and practicing endocrinologists. I discuss the potential dangers of utilization of suboptimally focused diagnostic approaches and of the inefficiencies in clinical management by primary-care providers, who often lack sufficient expertise, as opposed to endocrinologists. More than dollars are at stake, and the suboptimal management of common thyroid disorders presents several significant risks. Finally, I propose a general blueprint for the ongoing development of a structure for continuing quality improvement of the laboratory and clinical diagnosis, treatment, and long-term follow-up of patients with thyroid disease.