bluuebunny/crossref_metadata_2025 huggingface dataset:
Robotics and the changing face of the clinical laboratory
Authors: Boyd J C , Felder R A , Savory J
Published: January 2020

Rapid changes in healthcare coupled with parallel advances in technology have stimulated the evolution of new approaches for laboratory automation. In particular, the emergence of commercially available laboratory robotic systems offers promise for streamlining the clinical laboratory. Increasing cost-containment pressures make the application of this technology extremely attractive, and several organizations have begun to systematically integrate robotic devices into their laboratory automation schemes. Integration of these technologies, however, presents many challenges for software developers, instrument manufacturers, and laboratory workers. Differing needs across laboratories require flexibility and intelligence in robots, instruments, and control systems. Standardization of mechanical and electronic interfaces will be key to making these systems easy to integrate. Systems engineering, aided by simulation modeling and artificial intelligence schemes, will be important to assist in the design of optimal configurations. Software for the overall control of integrated automation will be needed that can be tailored by the laboratorian to fit the requirements of the individual laboratory. Thus, laboratory workers will need to be actively involved in implementing this new wave of laboratory automation, becoming well-versed in computers, electronics, and systems engineering.
COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine diagnostic PCR
Authors: DiDomenico N , Link H , Knobel R , Caratsch T , Weschler W , Loewy Z G , Rosenstraus M
Published: January 2020

The COBAS AMPLICOR system automates amplification and detection of target nucleic acids, making diagnostic PCR routine for a variety of infectious diseases. The system contains a single thermal cycler with two independently regulated heating/cooling blocks, an incubator, a magnetic particle washer, a pipettor, and a photometer. Amplified products are captured on oligonucleotide-coated paramagnetic microparticles and detected with use of an avidin-horseradish peroxidase (HRP) conjugate. Concentrated solutions of amplicon or HRP were pipetted without detectable carryover. Amplified DNA was detected with an intraassay CV of < 4.5%; the combined intraassay CV for amplification and detection was < 15%. No cross-reactivity was observed when three different target nucleic acids were amplified in a single reaction and detected with three target-specific capture probes. The initial COBAS AMPLICOR menu includes qualitative tests for diagnosing infections with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, and hepatitis C virus. All tests include an optional Internal Control to provide assurance that specimens are successfully amplified and detected.
Reticulocyte quantification by Coulter MAXM VCS (volume, conductivity, light scatter) technology
Authors: Buttarello M , Bulian P , Prà M D , Barbera P , Rizzotti P
Published: January 2020

In this study the ability of the Coulter MAXM analyzer to quantify reticulocytes was evaluated. The results were compared with those obtained by a microscopic method according to NCCLS H44-P recommendations and with the results from the automated analyzer Sysmex R-1000. Duplicate samples from 330 patients were analyzed. The reference intervals obtained with the three methods were: MAXM 0.37-1.80%, median 0.83%; manual 0.40-2.30%, median 1.00%; R-1000 0.60-1.95%, median 1.06%. The imprecision (CV) at all concentrations is lower than the microscopic method (low 16.1% vs 67%; normal 16.9% vs 28.9%; high 9.5% vs 13.0%). The MAXM shows a good overall correlation with the microscopic method (intercept 0.01, slope 0.89, r2 = 0.87) despite evidence of a significant overestimation at low concentrations [difference (D) 0.30] and a moderate underestimation at normal (D -0.15) and high (D -1.04) concentrations; the same behavior is shown in comparison with results from the R-1000, with an overall underestimation by MAXM (D -0.26). When compared with the microscopic method, MAXM shows a modest sensitivity at low reticulocyte counts (54.8%) and satisfactory sensitivity for high counts (87.3%), with an overall agreement of 81.3%.
Metal excretion and magnesium retention in patients with intermittent claudication treated with intravenous disodium EDTA
Authors: Guldager B , Jørgensen P J , Grandjean P
Published: January 2020

Sixty patients with intermittent claudication participated in a double-blind placebo-controlled trial of 20 courses of intravenous chelation therapy with 3 g of disodium EDTA vs placebo during 5-9 weeks. After the first infusion, the 24-h urinary excretion of lead and zinc was approximately 25-fold higher in the EDTA-treated group; relative differences for copper and calcium were smaller. Urinary magnesium excretion in the EDTA-treated group was one-third less than in the control group. After the treatment period, the blood lead concentration had decreased by approximately 73% and the serum zinc concentration by approximately 34%; other changes in blood concentrations were negligible. The loss of essential minerals and the possible redistribution of lead in the body may constitute a disadvantage that should be taken into account in repeated intravenous EDTA treatment.
Routine clinical monitoring of sirolimus (rapamycin) whole-blood concentrations by HPLC with ultraviolet detection
Authors: Napoli K L , Kahan B D
Published: January 2020

During phase I/II clinical trials of sirolimus (rapamycin; SRL), therapeutic drug monitoring was performed with a multistep liquid-liquid extraction of 1-mL aliquots of whole blood followed by reversed-phase HPLC with ultraviolet detection. Blood was sampled according to a standardized protocol and clinical status. SRL concentrations were interpolated from calibration curves with a linear range of 0-50 micrograms/L and 1 microgram/L lower limit of quantification. Quality control was monitored over 68 consecutive analytical runs by using frozen aliquots of SRL-supplemented pooled whole blood at 4, 12, and 32 micrograms/L. These samples showed mean concentrations of 3.7 +/- 0.6, 10.9 +/- 1.1, and 29.6 +/- 2.6 micrograms/L, respectively. This method for therapeutic drug monitoring of SRL permits one full-time technician to analyze 100 clinical specimens per week with a 24-h turnaround time. With this method, a strong linear relation (r2 = 0.946, Sy/x = 0.41, n = 115) between the average SRL concentration over a 24-h period and the SRL concentration at the 24th h was revealed.
High correlation but lack of agreement between direct high-performance gel chromatography analysis and conventional indirect methods for determining lipoprotein cholesterol
Authors: Krause B R , Schork N J , Kieft K A , Smith M P , Maciejko J J
Published: January 2020

Low-density lipoprotein-cholesterol (LDL-C) is currently estimated clinically by using the Friedewald formula, when plasma triglycerides are < 4000 mg/L, or as the difference between infranatant and high-density lipoprotein-cholesterol (HDL-C) values after ultracentrifugation of plasma at native density, when plasma triglycerides are > or = 4000 mg/L (beta quantification). HDL-C is measured by selective precipitation of apolipoprotein B-containing lipoproteins from whole plasma or from the density > 1.006 kg/L infranatant. We compared these conventional methods for LDL-C and HDL-C with "high-performance" gel chromatography (HPGC), a method that directly and simultaneously measures both LDL-C and HDL-C in a single, microliter volume of plasma. Not surprisingly, we found that the results by all these methods were highly correlated. However, LDL-C values were significantly higher and HDL-C values significantly lower by the direct HPGC method than by the conventional methods (paired t-test). In addition, both Bland-Altman plots and concordance correlation analyses indicated lack of agreement between the methods' results in the majority of patients' subgroups.
Method for determination of bone alkaline phosphatase activity: analytical performance and clinical usefulness in patients with metabolic and malignant bone diseases
Authors: Withold W , Schulte U , Reinauer H
Published: January 2020

We report the performance characteristics of an assay for determination of bone alkaline phosphatase (ALP) activity after immunoadsorption in microplate wells. Between-run imprecision was between 7.1% and 11.2%. The detection limit was 1.0 U/L. Comparisons with an immunoradiometric test for determination of bone ALP mass concentrations yielded the following regression equation: y = 3.11 + 1.33x with y, the bone ALP activity concentration (U/L) (and x, the bone ALP mass concentration microgram/L) (r +=0.974, n = 103). Using sera from patients with liver diseases and sera from patients with secondary hyperparathyroidism yielded a cross-reactivity of 20% for circulating liver ALP (and its membrane-bound isoform). In patients receiving renal transplants, Z-score analysis revealed that after transplantation the increase in bone ALP activity is more pronounced than total ALP activity. In tumor patients, receiver-operating characteristic analysis revealed that bone ALP activity shows the same diagnostic efficacy as total ALP activity in the detection of bone metastases (as assessed by bone scintigraphy). In multiple myeloma patients, suppressed osteoblast activity was well detectable by bone ALP activity determination.
Rapid automated determination of adenosine deaminase and lysozyme for differentiating tuberculous and nontuberculous pleural effusions
Authors: Villena V , Navarro-Gonzálvez J A , García-Benayas C , Manzanos J A , Echave J , López-Encuentra A , Arenas Barbero J
Published: January 2020

The catalytic concentration of pleural adenosine deaminase (ADA) and the ratio of pleural lysozyme (PL) to serum lysozyme (SL) were measured in consecutive patients (49 tuberculous and 179 nontuberculous) with two automated procedures in a Hitachi 717 analyzer. Using sensitivity and specificity curves, we established cutoff values at 33 U/L for ADA and 1.7 for the PL/SL ratio. The sensitivity of ADA activities for tuberculous effusion was 90%, specificity 85%. Combining ADA with the PL/SL ratio enhanced specificity to 99%. However, high values for ADA and lysozyme ratios are not, alone or in combination, sensitive or specific enough to replace pleural biopsy or culture of pleural fluid for the diagnosis of tuberculous empyema.
Short- and long-term reproducibility of the 1-h 50-g glucose challenge test
Authors: Phillipov G
Published: January 2020

A study was undertaken to assess the short-term (intrapregnancy) and long-term (interpregnancy) variability of the 1-h 50-g oral glucose challenge test (GCT). Two groups of pregnant women had GCTs in consecutive pregnancies, 1 (n = 77) and 2 (n = 43) years apart. Their results were compared with published results for a group (n = 53) who had GCTs on consecutive days. Robust estimates of the mean error variance (sigma2 error = sigma2 within-individual + sigma2 analytical) were calculated on log10-transformed data and were for the three groups 0.003995, 0.002603, and 0.0026249 (mg/dL)2, respectively. There was no significant difference between the group variances, establishing that the short- and long-term reliability of the GCT is comparable. Sigma2 between-individuals was estimated from the GCT values for 2695 pregnant women tested during the same period and was the main component (67.1%) of the total sample variation (sigma2 between-individuals / sigma2 population). Estimates of the population mean, sigma2 between-individuals, and sigma2 error were used to compute the probability that an observed GCT value had a true value equal to or greater than the consensus threshold of 7.8 mmol/L (140 mg/dL).
Sensitive, specific quantitative analysis of tacrolimus (FK506) in blood by liquid chromatography-electrospray tandem mass spectrometry
Authors: Taylor P J , Jones A , Balderson G A , Lynch S V , Norris R L , Pond S M
Published: January 2020

The capacity of liquid chromatography-tandem mass spectrometry (LC-MS2) to detect and define individual components in a complex mixture has been utilized to develop a quantitative assay of the potent immunosuppressant drug, tacrolimus. Trough blood concentrations were measured in 175 samples obtained over several weeks after liver transplantation from seven patients. The assay was linear over the range of 0.2 to 100 micrograms/L. Imprecision was <8%, and accuracy was 99-101%. The turnaround time for a batch of 20 samples was 2.5 h. No interference from any of the other drugs being administered to the patients was evident. An ELISA also performed on the same samples overestimated the concentrations substantially, as indicated by a plot of the difference between the results for the two methods vs their mean. The favorable characteristics of the LC-MS2 assay, especially its sensitivity and specificity, will facilitate detailed pharmacokinetic studies of tacrolimus, particularly under circumstances in which metabolism is perturbed by either hepatic dysfunction or drug interactions.
Simultaneous determination of ethylene glycol and its major toxic metabolite, glycolic acid, in serum by gas chromatography
Authors: Yao H H , Porter W H
Published: January 2020

We developed a gas-chromatographic procedure for the simultaneous determination of ethylene glycol (EG) and its major toxic metabolite, glycolic acid (GA), suitable for clinical use in instances of EG intoxication. After serum protein precipitation with acetonitrile (containing internal standard), the supernate is treated with 2,2-dimethoxypropane (containing dimethylformamide) to remove water, and the volume is then reduced by evaporation to <100 microL of dimethylformamide (but not to dryness). After trimethylsilyl derivatization, the resulting derivatives are analyzed by capillary column gas chromatography. Only 100 microL of serum is required and the entire determination, including calibrators and controls, takes <2 h. The method gives a linear response to at least 10 g/L EG and 5 g/L GA and has a limit of detection <10 mg/L. Intraassay CV is < or = 2.8% for EG (100 and 1000 mg/L) and GA (100 and 500 mg/L); between-day CV is < or = 6.5%. The absolute recovery from serum was 91% for EG and 77-82% for GA (200 and 2000 mg/L each). Relative to calibrators prepared bovine serum albumin (70 g/L), the recovery was 99-104% for EG (100 - 5000 mg/L) and 95-105% for GA (50 - 2500 mg/L). No clinically important interference was detected for >60 exogenous or endogenous compounds and drugs.
Effect of cigarette smoking on pentane excretion in alveolar breath
Authors: Euler D E , Davé S J , Guo H
Published: January 2020

The concentrations of acetone, isoprene, and pentane in alveolar breath were examined in 50 smokers and 50 nonsmokers by gas chromatography. The baseline pentane in smokers was 0.17 +/- 0.03 nmol/L (mean +/- SE), which was not different from pentane in nonsmokers (0.23 +/- 0.03 nmol/L). There were also no differences between smokers and nonsmokers in the concentrations of acetone and isoprene. Serial breath samples were obtained from 15 smokers before smoking and at 5, 15, and 60 min after smoking. Although acetone was not altered by smoking, isoprene increased by 86% +/- 26% 5 min after smoking (P <0.001) and returned to baseline 10 min later. Pentane increased by 456% +/- 156% 5 min after smoking (P <0.001) and remained increased 10 min later (204% +/- 73% of baseline, P <0.05). Isoprene concentrations in mainstream cigarette smoke were >5000 times greater than breath concentrations, whereas pentane could not be detected in mainstream smoke. Because pentane is produced from the peroxidation of n-6 polyunsaturated fatty acids, the results provide evidence that cigarette smoking causes an immediate increase in lipid peroxidation.
Quantification of fecal carbohydrates by near-infrared reflectance analysis
Authors: Stein J , Purschian B , Zeuzem S , Lembcke B , Caspary W F
Published: January 2020

Established methods for quantitative analysis of fecal carbohydrates (CHO) are time consuming, require unpopular sample handling, and are therefore rarely performed. The aim of this study was to evaluate the efficiency, validity, and practicability of near-infrared reflectance analysis (NIRA), compared with standard methods for measurement of fecal CHO. Excretion of fecal CHO was cross- validated spectrophotometrically with the anthrone method. Fecal CHO concentrations ranged form 2.7 to 24.5 g/kg wet weight. Methods comparison showed linear regression over the entire range of diagnostic relevance with a correlation coefficient of 0.869 (S(y/x) +/- 0.31). Repeated measurements from the same stool collections obviated the need for homogenization. These results indicate that NIRA may be a new, reliable, and accurate test in the diagnosis of CHO malabsorption.
Liquid-chromatographic study of purine metabolism abnormalities in purine nucleoside phosphorylase deficiency
Authors: Chantin C , Bonin B , Boulieu R , Bory C
Published: January 2020

Using HPLC methods, we measured the concentrations of nucleosides and nucleotides for a patient with no purine nucleoside phosphorylase (PNP; EC 2.4.2.1) enzymatic activity. Concentrations of inosine and guanosine were abnormally high in urine and plasma, whereas guanosine diphosphate (GDP) and guanosine triphosphate (GTP) concentrations in erythrocytes were depleted. The unusual presence of deoxyribonucleosides (deoxyinosine and deoxyguanosine) and deoxyribonucleotides (dGDP and dGTP) was also notable. Thus, HPLC represents an accurate and useful tool for the study of purine metabolic disorders.
Simultaneous measurement of ethanol and ethyl-d5 alcohol by stable isotope gas chromatography-mass spectrometry
Authors: Dean R A , Thomasson H R , Dumaual N , Amann D , Li T K
Published: January 2020

Efforts to define gender- and ethnic-dependent differences in ethanol first-pass metabolism by gastric mucosa and liver have been limited by a lack of analytical tools that distinguish ethanol concurrently administered by oral and intravenous routes. A stable isotope gas chromatography-mass spectrometry method for simultaneous measurement of ethanol and ethyl-d5 alcohol in serum was developed to meet this need. The assay was linear from 1 to 30 mmol/L. The limit of quantification was 1 mmol/L. Analytical imprecision (CV) was <10%. Analytical recovery was >90%. Specificity was based on retention time and reproducibility of ion ratios. The assay was free from interference by other volatile alcohols. Simultaneous oral administration of ethanol and ethyl-d5 alcohol produced nearly identical pharmacokinetic profiles. Simultaneous oral ingestion of ethanol and intravenous infusion of ethyl-d5 alcohol, adjusted for gastric emptying time, revealed decreased bioavailability of ethanol by the oral route. The method described is sufficient to study the first-pass metabolism of ethanol.
Effect of antibody specificity on results of selected digoxin immunoassays among various clinical groups
Authors: Datta P , Xu L , Malik S , Landicho D , Ferreri L , Halverson K , Roby P V , Zebelman A M , Kenny M A
Published: January 2020

We examined the specificity of three automated digoxin immunoassays (Abbott TDxFLx Digoxin II assay, Baxter-Dade Stratus II Digoxin assay, and Ciba Corning ACS Digoxin assay) applied without modification to (a) sera from 229 digoxin-free patients in 12 cohorts associated with nonspecific or endogenous digoxin-like immunoreactive factor (DLIF) interference, and (b) drug-free serum supplemented with the major metabolites and analogs of digoxin. We observed three patterns of apparent digoxin results among the DLIF samples: one common to kidney and liver failure patients, where TDx and Stratus assays showed significant positive results; one common to newborns and cord blood, where only the TDx assay had significant interference; and one from cardiac surgery patients, where the Stratus assay alone showed interference. Of the three assays, the ACS had the least interference from DLIF. The assays also behaved differently with respect to cross-reactivity with digoxin metabolites, digitoxin, and digitoxin metabolites. The ACS assay again had the least analog or metabolite cross-reactivity. The three methods agreed well on digoxin-positive specimens, with a mean bias of <0.15 microgram/L digoxin for each and discrepancies (defined as >3 SD between the assay pairs compared) of only 3-5%.
Improved fluorometric quantification of urinary xanthurenic acid
Authors: Liu M , Wang G R , Liu T Z , Tsai K J
Published: January 2020

Measurement of urinary xanthurenic acid (XA) has been used clinically to study a variety of disorders caused by vitamin B6 deficiency. To obviate some cumbersome steps of current methods for measuring XA in human urine, we have developed a simple fluorometric method. We apply the urine sample to a solid-phase extraction column containing trimethylaminopropyl group bound to silica, which enables us to purify and concentrate the XA from the urine without contamination from various tryptophan metabolites. The XA in the acidic eluate can then be quantified fluorometrically. The linearity of the proposed method extends from 0.2 to 10.0 mg/L. The method is precise, yielding day-to-day CVs for two pooled control specimens (1.08 and 1.90 mg/L) of 1.2% and 2.6%, respectively. Correlation studies with an established HPLC method and with a spectrophotometric procedure showed correlation coefficients of 0.99 and 0.98, respectively. Interference from vitamin C, uric acid, salicylate, acetaminophen, vanillylmandelic acid, and homovanillic acid was insignificant. The proposed method for urinary XA is rapid, simple, and suitable for routine use in the clinical laboratory.
Rapid quantification of alpha-tocopherol in plasma and low- and high-density lipoproteins
Authors: Teissier E , Walters-Laporte E , Duhem C , Luc G , Fruchart J C , Duriez P
Published: January 2020

We have developed two methods for measuring the alpha-tocopherol content in plasma and lipoproteins (LDL and HDL). In procedure 1, plasma or lipoproteins are deproteinized with ethanol containing delta-tocopherol as internal standard and then extracted with hexane or ethyl acetate. The organic layer is removed and evaporated, and the residue is redissolved in methanol and injected into a reversed-phase HPLC. In procedure 2, plasma or lipoproteins are diluted in a methanol and ethanol mixture containing the same internal standard. The solution is vortex-mixed, centrifuged, and directly injected into the column. The tocopherols are eluted with an isocratic methanol mobile phase at a flow rate of 1 mL/min and detected by fluorescence (lambda(exc)= 295 nm, lambda(em)= 330nm). Recoveries are approximately 100% in both cases. Between-run CVs were 8.39% for procedure 1 and 6.55% for procedure 2. Small sample requirement, simplicity of sample preparation, short assay time, and good reproducibility make procedure 2 ideal for clinical or research use. This method was applied to determination of alpha-tocopherol in plasma of patients whose diet was supplemented with alpha-tocopherol and in LDL and HDL.
Chronic increased serum lipase without evidence of pancreatitis: tumor-derived lipase?
Authors: Donnelly J G , Ooi D S , Burns B F , Goel R
Published: January 2020

A 51-year-old man developed a large retroperitoneal tumor with liver and lymph node metastases; there was no radiological evidence of pancreatic involvement. Despite the progression of disease, results of laboratory tests, notably serum amylase, were normal except for minor increases in aspartate aminotransferase and gamma-glutamyltransferase and a marked increase in lipase. The increased lipase was not attributable to formation of macroenzyme. To determine the source of the lipase, we fractionated serum and a tumor biopsy homogenate, using electrophoresis. The lipase pattern obtained from the patient's serum differed from that seen in serum from a patient with acute pancreatitis. Additionally, the lipase pattern obtained from a homogenate of biopsy sample from the retroperitoneal tumor did not match the pattern observed for normal pancreas. Apparently, the source of this increased serum lipase activity was the nonpancreatic tumor.
Tumor markers cancer antigen 15.3, carcinoembryonic antigen, and tissue polypeptide antigen for monitoring metastatic breast cancer during first-line chemotherapy and follow-up
Authors: Sölétormos G , Nielsen D , Schiøler V , Skovsgaard T , Dombernowsky P
Published: January 2020

We investigated whether model systems integrating stochastic variation into criteria for marker assessment could be used for monitoring metastatic breast cancer. A total of 3989 serum samples was obtained from 204 patients receiving first-line chemotherapy and from 112 of these patients during follow-up. Each sample was analyzed for cancer antigen 15.3, carcinoembryonic antigen, and tissue polypeptide antigen. The efficiency for identifying progression and nonprogression was 94% during therapy and 85% during follow-up, with no false-positive marker results for progressive disease. At clinical progressive disease, the median positive lead time was 35 days during therapy and 76 days during follow-up. Tumor marker assessment may document that a therapy is effective and ought to be continued in spite of adverse toxic effects, and that a treatment is ineffective and should be stopped to prevent unnecessary toxicity. Marker information may also be useful in studies investigating whether early treatment during follow-up will alter the prognosis of metastatic breast cancer.
Markers of activated coagulation and their usefulness in the clinical laboratory
Authors: Tripodi A , Mannucci P M
Published: January 2020

Currently, information on hypercoagulability can be achieved directly--through measuring the enzymatic forms of coagulation zymogens generated during coagulation activation--or indirectly--through measuring the activation peptides generated when zymogens are activated or the enzyme-inhibitor complexes formed by inhibition of the enzymes by their plasmatic inhibitors. On the basis of published results, markers of activated coagulation are considered useful for investigating mechanisms that regulate hemostasis. They can also be used to better characterize patients at increased thrombotic risk. However, they should be considered indices of hypercoagulability, not of the risk of thrombosis, until prospective studies can demonstrate that alterations of these markers can predict the occurrence of thrombosis. For diagnosing acute thrombosis, their usefulness is questionable; they are less effective than markers of fibrinolysis activation such as the D-dimer. Finally, their use to monitor anticoagulant treatment is still premature and needs investigation in well-designed clinical studies.
Determination of shelf life and activation energy for tumor necrosis factor-alpha in cerebrospinal fluid samples
Authors: Cruz Ruiz M , Recio Quijano F , López Cortés L F , Hebles Duvison M , Vázquez Rubio R
Published: January 2020

The cytokine rumor necrosis factor-alpha (TNF-alpha) plays a prominent role in the inflammatory response and has been quantified in several kinds of body fluids. A safe method for keeping samples under the best storage conditions for later studies is clearly needed. We estimated TNF-alpha shelf life by using an accelerated stability testing protocol based on the Arrhenius equation. We investigated two kinds of samples: cerebrospinal fluid (CSF) normal pool (from patients without meningitis) and CSF pathological pool (from patients with pyogenic meningitis). Results of the stress protocol indicated that storing both normal and pathological CSF samples at -70 degrees C would maintain sample stability-i.e., retain at least 90% of the original TNF-alpha content-for approximately 5 years. At -20 degrees C and 4 degrees C, the projected stability time at which the same recovery would be obtained was 190 and 90 days, respectively.
Ultrasensitive detection of prostate-specific antigen by a time-resolved immunofluorometric assay and the Immulite immunochemiluminescent third-generation assay: potential applications in prostate and breast cancers
Authors: Ferguson R A , Yu H , Kalyvas M , Zammit S , Diamandis E P
Published: January 2020

We report an ultrasensitive time-resolved immunofluorometric assay (TRIFA) for prostate-specific antigen (PSA). The assay is an improvement of our previous report (Clin Chem 1993;39:2108-14) and includes the utilization of two monoclonal antibodies and a one-step incubation period, which greatly reduces analysis time. The new method demonstrates a superior lower analytical limit of detection (< or = 1 ng/L), a wide dynamic range, absence of a hook effect at 10(6) ng/L PSA, and equimolarity for free PSA and PSA-antichymotrypsin complex. Also, we have compared several aspects of our TRIFA with a commercially available third-generation assay (Immulite). An evaluation of breast tumor cytosol extracts from 315 patients shows PSA immunoreactivity > 15 ng/g of total protein in 28% and 23% by TRIFA and Immulite analysis, respectively. Both methods demonstrate a significant association between breast tumor PSA immunoreactivity and progesterone and estrogen receptor positivity (P <0.001). Analysis of serum samples obtained for monitoring of postradical prostatectomy patients reveals significant PSA changes at concentrations undetectable by conventional methods. The significance of these results as well as the potential applications of ultrasensitive PSA assays in breast and prostate cancers are discussed.
Fluorometric detection of HIV-1 genome through use of an internal control, inosine-substituted primers, and microtiter plate format
Authors: Gérard B , Peponnet C , Brunie G , Cavé H , Denamur E , d'Auriol L , Monplaisir N , Simon F , Elion J , Grandchamp B
Published: January 2020

We describe a PCR-based fluorometric assay for the detection of the HIV-1 genome. This technique consists of a reverse hybridization with oligonucleotide probes covalently coated onto a microtiter plate as a solid support. Several improvements to the PCR amplification and detection steps gave greater sensitivity and specificity for HIV-1 screening and resulted in a convenient and rapid technique. False-positive results were avoided by using uracyl DNA glycosylase. False-negative results from the presence of PCR inhibitors were detected by coamplifying an internal control with the viral sequence. False-negative results from viral genome variability were limited by using two pairs of primers and by incorporating inosine at the primer positions corresponding to viral polymorphic nucleotides. Furthermore, the hybridization buffer and enzymatic reaction were optimized to increase the assay's sensitivity. The sensitivity and specificity of the fluorometric detection were similar to those of radioisotopic oligonucleotide solution hybridization; however, hands-on time was reduced, and the use of radioactivity was eliminated. We have used this technique routinely on 115 samples and obtained 100% specificity and high sensitivity (only one false-negative result) according to viral culture and (or) serological status of the patients.
Variability in cholesterol measurements: comparison of calculated and direct LDL cholesterol determinations
Authors: Schectman G , Patsches M , Sasse E A
Published: January 2020

Calculated low-density lipoprotein cholesterol (LDL-C) concentrations determined from the Friedewald equation have a large intraindividual CV, in part because the calculation incorporates the variability of cholesterol, high-density lipoprotein cholesterol (HDL-C), and triglyceride measurements. We studied whether a new assay that measures LDL-C directly will reduce this variability and reduce the need for averaging serial specimens. Four blood samples were obtained 1 week apart from 35 mildly hypercholesterolemic subjects and analyzed for total cholesterol, triglycerides, and HDL-C. LDL-C was calculated by the Friedewald equation, and was also measured directly with a commercially available direct LDL-C assay. The intraindividual CV for the direct and calculated LDL-C assays were similar [CV of direct LDL-C assay (mean +/- SE): 6.8 +/- 0.5% vs calculated LDL-C: 7.3 +/- 0.6%; difference 0.44%, 95% confidence interval: -0.7-1.5%]. For both assays, at least two blood tests were required from each subject to reduce total variability of LDL-C to less than or equal to 5%. We conclude that the direct LDL-C assay did not reduce the variability in LDL-C compared with the conventional LDL-C calculation. However, it may have a specific role in lipid disorder evaluation and (or) monitoring when triglycerides are increased or the LDL-C value alone is needed.
Determination of ionized magnesium in platelets and correlation with selected variables
Authors: Niemela J E , Snader B M , Elin R J
Published: January 2020

We describe a method for determining the intracellular ionized magnesium concentration ([Mg2+]i) in platelets by using the fluorescent probe FURAPTRA. We determined the dissociation constant (KD) of FURAPTRA for Mg2+ (2.26 +/- 0.29 mmol/L), within-day assay variability (CV = 6.8%), among-day intraindividual variability (CV = 11.0%), variability after a 4-h delay in processing the blood specimen (t = 1.2, P >0.2; F = 6.2, P <0.02), and the reference interval (0.23-0.59 mmol/L) for this assay. We also evaluated the correlation between platelet [Mg2+]i and concentrations of selected serum electrolytes, proteins, and total cholesterol; age; body mass index; and gender. Only the inverse correlation between platelet [Mg2+]i and serum total cholesterol concentration in men was significant (r=-0.66, P <0.005).
Food and Drug Administration (FDA)'s impact on laboratory performance: FDA's perspective
Authors: Gutman S
Published: January 2020

The Division of Clinical Laboratory Devices is responsible for the premarket review of in vitro diagnostic devices (laboratory tests). We currently process >1000 diverse applications per year. New versions of old devices are handled as premarket notifications, so-called 510(k) submissions. The review objective is to establish that the new product is "substantially equivalent" to its predicate. Fundamentally new devices are handled as premarket applications. The review objective is to establish de novo that the product is ¿safe and effective.¿ A central regulatory issue over the past several years has been the development of a standardized model for scientific review. The Food and Drug Administration contributes to the quality of in vitro diagnostic devices by providing oversight and objective review, by setting thresholds for product safety and effectiveness, and by ensuring that organized data and appropriate labeling is present in support of a device's intended use.
Federal reimbursement to laboratories
Authors: Logue J
Published: January 2020

The change from a predominately fee-for-service payment environment to managed care has significantly reduced revenues for many clinical laboratories. Medicare is rapidly becoming the most favorable payor in areas with high managed-care penetration. This trend has not gone unnoticed by Congress, and congressional and regulatory initiatives are rapidly moving to reduce federal laboratory reimbursement. The efforts by both the private and public payors to further restrict payments could have a profound effect on the scope of testing offered by hospital-based laboratories. On-site nonemergent testing capability will be dependent on the cost to provide the service and the level of reimbursement. Laboratory providers have an opportunity to influence the extent of laboratory cost-reducing initiatives. To effect congressional or regulatory change requires an understanding of the federal Medicare payment system and a well-organized effort.
Impact of cost cutting on laboratories: new business strategies for laboratories
Authors: Ash K O
Published: January 2020

Cost reduction is the primary force driving healthcare reform. To survive and thrive in these tumultuous times, laboratories must adapt and implement new business strategies. Business paradigm shifts create opportunities for organizations with a plan; a wait-and-see attitude forecasts failure. Drawing upon an 11-year experience with the "ARUP business model," this work will highlight business strategies that have contributed to the success of this university-based reference laboratory. In the future, successful laboratories will implement new business strategies to become more effective members of the emerging integrated healthcare delivery teams. Within the laboratory, traditional organizational disciplinary boundaries, i.e., chemistry, microbiology, and hematology, are melding together to increase efficiency. Laboratorians must become influential members of institutional healthcare delivery teams formed to control utilization. Laboratory services are being adjusted to optimize patient care. Incremental pricing is only one of the strategies to be implemented to expand outpatient business to those in the region. Expanded computer capabilities, client services, specimen handling, marketing, and sales are also required. On a regional basis, service laboratories are increasingly joining forces to increase efficiency while at the same time improving the quality of patient care.
A computer program that periodically monitors the ability to interpret the antinuclear antibody test
Authors: Astion M L , Wener M H , Hutchinson K , Olsen G B , Orkand A R , Pagliaro L J
Published: January 2020

Our laboratory has been developing computer programs that help medical technologists improve their performance of the microscope-based immunofluorescence assay for antinuclear antibodies (ANA). This image-based laboratory test has been associated with poor reproducibility. We have previously described our first program, ANA-Tutor, which systematically teaches the ANA test by using approximately 150 processed digital images of ANA test results. The program we describe here, Pattern Plus Auditor, is a logical extension to ANA-Tutor. Pattern Plus Auditor tests the ability of laboratory personnel to interpret the ANA test, and tracks individual and laboratory performance over time. The program consists of image-based questions that test a variety of ANA staining patterns, including homogeneous, speckled, centromere, nucleolar, mixed patterns, and rare patterns. For each question, the program provides correct answers with explanations and color overlays that highlight key image features. By entering the proper password, users gain access to exam results for individuals and for the laboratory as a whole. Results are available for the current exam, any previous exam, or cumulatively on all exams to date. Intralaboratory testing with computer programs such as Pattern Plus Auditor might be a useful part of quality-assurance procedures for many image-based laboratory tests.
Novel homogeneous liposomal immunoassay for colorimetric estimation of serum IgG anticardiolipin antibodies
Authors: Frost S J , Chakraborty J , Firth G B
Published: January 2020

Liposomes entrapping the dye sulforhodamine B were used to develop an assay for anticardiolipin antibodies (ACAs). In the presence of magnesium ions, IgG ACAs induced liposomal lysis and the resulting absorbance changes were dependent on the amount of ACAs present. The liposomal assay (y) showed similar intraassay imprecision and detection limit to an ELISA for IgG ACAs (x), with a correlation coefficient of 0.90 (y = 1.05x - 1.61). No correlation with ELISA IgM ACA measurements was observed. Although ELISA remains the method of choice, particularly in examining different ACA classes, the liposomal assay offers advantages of speed and potential for processing large numbers of samples for IgG ACAs. This may facilitate study of the significance of increased IgG ACAs in the groups of conditions with which they are associated, and perhaps enable more laboratories to perform the test.
Linearity and stability of the AVL and Nova magnesium and calcium ion-selective electrodes
Authors: Rehak N N , Cecco S A , Niemela J E , Hristova E N , Elin R J
Published: January 2020

We studied the stability and linearity of the AVL and Nova Mg and Ca ion-selective electrodes and the relation between the ionized Ca and ionized Mg results reported by each analyzer. The response of the electrodes to different concentrations of Mg and Ca was determined for saline solutions, aqueous solutions, and serum samples. The electrodes from both manufacturers demonstrated acceptable stability for the time of the study. The response of the electrodes was linear within the range specified by each manufacturer, but relative nonlinearity and the values for the linear limits differed between the AVL and Nova analyzers. The ionized Mg results varied with the concentration of Ca. The relation between ionized Ca and ionized Mg results was nonlinear and differed between the AVL and Nova electrodes. Intermethod comparison between the electrodes showed poor agreement for ionized Mg results, especially at low and high concentrations of total Ca and total Mg.
Metabolism of anabolic androgenic steroids
Authors: Schänzer W
Published: January 2020

Anabolic androgenic steroids (AAS) are misused to a high extent in sports by athletes to improve their physical performance. Sports federations consider the use of these drugs in sports as doping. The misuse of AAS is controlled by detection of the parent AAS (when excreted into urine) and (or) their metabolites in urine of athletes. I present a review of the metabolism of AAS. Testosterone is the principal androgenic steroid and its metabolism is compared with that of AAS. The review is divided into two parts: the general metabolism of AAS, which is separated into phase I and phase II metabolism and includes a systematic discussion of metabolic changes in the steroid molecule according to the regions (A-D rings), and the specific metabolism of AAS, which presents the metabolism of 26 AAS in humans.
Use of precision profiles to evaluate precision of the automated leukocyte differential
Authors: Hübl W , Tlustos L , Bayer P M
Published: January 2020

The commonly used methods of assessing the precision of the automated leukocyte differential have certain drawbacks that affect the validity and comparability of results. In the present report, we introduce a procedure based on building precision profiles from a large number of within-run imprecision experiments. The profiles are fitted to the function for the CV of proportions, which yields the number of theoretically differentiated leukocytes. Differences between fitted curves are evaluated for statistical significance by the F-test. As an example, we compared the precision of two hematology analyzers, a flow-cytometric technique involving fluorescence-labeled monoclonal antibodies, and the manual differential. We were able to establish definite differences in precision between different analyzers and different leukocyte classes. Our data also indicated that conventional within-run imprecision studies may completely misjudge analyzer precision. Furthermore, we could demonstrate that the precision of analyzers that analyze a fixed amount of blood rather than a fixed number of leukocytes is strongly influenced by the leukocyte count of the sample, leading to high imprecision for leukopenic samples. We believe the proposed procedure is a useful addition to currently used protocols; it yields clear results and creates a statistical basis of comparison between various instruments and techniques of differentiation.
Robust, reliable biosensor for continuous monitoring of urea during dialysis
Authors: Della Ciana L , Caputo G
Published: January 2020

We developed a new urea sensor for the on-line monitoring of hemodialysis adequacy. The biosensor consisted of an immobilized urease cartridge placed between magnetoinductive conductivity cells. The biosensor output was taken as the conductivity difference between these cells. The device was placed on the ultrafiltrate line of a paired filtration dialysis system. The amount of urease present in the cartridge was sufficient for the complete conversion to ammonium carbonate of urea up to 35 mmol/L. Agreement was good between the urea concentration by the biosensor method and an automated analyzer for seven patients: range 8.07-30.3 mmol/L (22.6-84.8 mg/dL blood urea nitrogen, BUN); intercept 0.20 +/- 0.1 mmol/L (0.55 +/- 0.4 mg/dL BUN); slope 1.01 +/- 0.01; r 0.997; S(y/x) 0.40 mmol/L (1.11 mg/dL BUN). The device proposed meets the requirements of accuracy, cost, ruggedness, and ease of use (no calibration required) for a biosensor to be used for continuous monitoring of hemodialysis.
Validation of HPLC-amperometric detection to measure serotonin in plasma, platelets, whole blood, and urine
Authors: Pussard E , Guigueno N , Adam O , Giudicelli J F
Published: January 2020

We describe an isocratic liquid-chromatographic method with amperometric detection for determination of serotonin by rapid sample preparation. Platelet-poor plasma and platelets were injected after a single deproteinization step with perchloric acid. Addition of sodium borohydride to whole blood avoids oxidation of serotonin during the deproteinization step without any chromatographic interferences. We purified urinary serotonin by two successive cationic and anionic extraction steps. After urine dilution, urinary 5-hydroxyindoleacetic acid (5-HIAA) was measured under the same chromatographic conditions. Platelet serotonin concentrations correlated more closely with whole-blood serotonin concentrations after correction for platelet number than with concentrations expressed in nmol/L. This suggests that whole-blood serotonin measurements should be corrected for platelet count to eliminate the variability of circulating platelets. Combined determination of serotonin in whole blood and urine and of 5-HIAA in urine provides a useful tool for detecting and monitoring carcinoid tumors.
Development of a two-site solid-phase immunochemiluminescent assay for measurement of dimeric inhibin-A in human serum and other biological fluids
Authors: McConnell D S , Padmanabhan V , Pollak T B , Groome N P , Ireland J J , Midgley A R
Published: January 2020

Inhibin is a heterodimeric glycoprotein that inhibits the secretion of follitropin from the pituitary and has been isolated in two distinct forms composed of a common alpha subunit and either a beta A or beta B subunit. Utilizing paired monoclonal antibodies specific to the alpha and beta A subunit, we have developed an immunochemiluminescent assay for dimeric inhibin-A. The assay is capable of quantifying free and bound inhibin-A in human serum and follicular fluid. The limit of detection is 10 ng/L. Related proteins exhibit little cross-reactivity or interference. Recovery is excellent. Whereas samples from men and postmenopausal women are near the detection limit of the assay, inhibin-A is higher in the luteal than the follicular phase of normally cycling women, 20-fold higher during in vitro fertilization treatment, and approximately 200-fold greater in pregnancy. The assay measures inhibin-A in follicular fluid from a variety of other species.
Rapid method for visual identification of specific DNA sequences based on DNA-tagged liposomes
Authors: Rule G S , Montagna R A , Durst R A
Published: January 2020

We describe a rapid method for visually determining specific DNA sequences at femtomole concentrations. Liposomes, encapsulating a red dye and labeled with oligonucleotide, were used in a capillary migration-sandwich hybridization assay. Capture probe was immobilized on nitrocellulose strips, and liposomes, migrating along each strip, formed a visually discernible band in the presence of target DNA. One femtomole of synthetic target sequence could be detected in < 10 min. Sufficiently stringent hybridization conditions can be used to allow the discrimination of a 10% mismatch sequence from perfectly complementary DNA. A 366-base PCR product was detected at 200 fmol.