bluuebunny/crossref_metadata_2025 huggingface dataset:
Two-dimensional gel electrophoresis detects prostate-specific antigen–α1-antichymotrypsin complex in serum but not in prostatic fluid
Authors: Qian Yi , Sensibar Julia A , Zelner David J , Schaeffer Anthony J , Finlay Judith A , Rittenhouse Harry G , Lee Chung
Published: January 2020

We investigated the interaction between prostate-specific antigen (PSA) and 1-antichymotrypsin (ACT) in prostatic secretions, identifying PSA and ACT in human serum, prostatic fluid, and seminal plasma by two-dimensional gel electrophoresis (2-D PAGE). Both PSA and ACT were detected in all three body fluids, but PSA-ACT complex was detected only in serum. Moreover, the 2-D PAGE Western blot staining profile for ACT from serum differed from that for prostatic fluid or seminal plasma. Incubation of prostatic fluid with purified ACT led to formation of PSA-ACT complex. Incubation of prostatic fluid with purified PSA, however, failed to form the complex, suggesting that the ACT in prostatic fluid was inactive or inhibited. Given that physiological concentrations of zinc inhibited the formation of PSA-ACT complex, we consider zinc a possible physiological inhibitor of the formation of the PSA-ACT complex. These results indicate that the failure to detect the PSA-ACT complex in prostatic fluid could be related to the inactivation of ACT, the presence of inhibitors (e.g., zinc), or simply the PSA:ACT ratio in the fluid.
Fluid elements—a concept for automation of diagnostic tests
Authors: Van Damme Henk , Van Velthoven Thea , Kaelen Erik , Pelssers Eduard
Published: January 2020

Constructs consisting of a channel, a membrane, and an absorber are designed for autonomously carrying out various liquid-handling functions of analytical tests. These so-called fluid elements can be used to set up various circuits for conducting several kinds of analytical tests. To demonstrate the feasibility of this concept, we constructed such a circuit and used it to perform, with two handling steps, an ELISA of hepatitis B surface antigen. The detection limit of the assay was comparable with those of state-of-the-art ELISAs for screening blood, and results could be obtained within a total test time of 20 min. We anticipate that this concept of automation may also serve as a basis for new, highly simplified immunoanalyzers.
Whole-blood test for total cholesterol by a self-metering, self-timing disposable device with built-in quality control
Authors: Law Wai Tak , Doshi Sonal , McGeehan John , McGeehan Susan , Gibboni David , Nikolioukine Yuri , Keane Robert , Zheng Jennifer , Rao Jagan , Ertingshausen Gerhard
Published: January 2020

A whole-blood test for total cholesterol has been developed that is performed in a low-cost disposable flow device without user intervention (after sample addition). The device meters the sample, separates plasma from erythrocytes, and precisely times plasma flow into various reagent compartments, including a quality-assurance chamber. Test results are displayed as a well-defined and easily readable color bar. A quality-control window attests to the integrity of the test components. Here, we describe the assembly and individual functions of the device and report its performance characteristics. Precision and accuracy studies in four clinical studies at independent locations yielded total imprecision of <5% and an average bias of 1.35% vs the Abell–Kendall method.
Serum ionized magnesium in chronic alcoholism: is it really decreased?
Authors: Hristova Elena N , Rehak Nadja N , Cecco Stacey , Ruddel Mark , Herion David , Eckardt Michael , Linnoila Markku , Elin Ronald J
Published: January 2020

Chronic alcoholism is associated with a marked deficit in total magnesium (tMg). However, little is known about the status of the physiologically active form, ionized magnesium (iMg). We assessed serum iMg (measured with two ion-selective electrodes, AVL 988-4 and NOVA CRT) and tMg concentrations in chronic alcoholics at admission (n = 31) and after abstinence (n = 13) and compared these results with those for a control group (n = 40). At admission, the tMg and NOVA iMg concentrations in alcoholics (0.78 ± 0.020 and 0.38 ± 0.016 mmol/L, respectively) were significantly less (P <0.001) than in the controls (0.85 ± 0.008 and 0.50 ± 0.006 mmol/L). The AVL iMg results, however, did not differ significantly between the two groups: 0.53 ± 0.013 vs 0.56 ± 0.006 mmol/L, respectively (P >0.05). The mean iMg between the two analyzers differed significantly in both groups (P <0.001). After 3 weeks of abstinence, the alcoholics showed a significant increase in tMg (P <0.001) and in both NOVA and AVL iMg values (P <0.01 for each). tMg concentrations were positively correlated with the AVL iMg values in both alcoholics and controls but correlated positively with the NOVA iMg results only in the controls. Thus, the altered status of iMg is instrument-dependent, and the usefulness of the measurement in alcoholics is yet to be determined.
Automated liquid-chromatographic analyzer used for toxicology screening in a general hospital: 12 months’ experience
Authors: Sadeg Nouredine , François Gilles , Petit Brigitte , Dutertre-Catella Hélène , Dumontet Michel
Published: January 2020

We evaluated the clinical utility of an automated HPLC system (Remedi, Bio-Rad) for identification of drugs and metabolites in biological fluids. Serum or urine or both from 354 consecutive cases of poisoning were analyzed by the system and by a set of fluorescence polarization immunoassay (FPIA, Abbott) and thin-layer chromatographic (TLC) procedures. Antidepressants and most phenothiazines were recognized by the new system. Comparison of Remedi results with final clinical diagnoses yielded diagnostic specificity and sensitivity of 80% and 90%, respectively. Remedi detected 26 additional compounds that were neither reactive in the immunoassay screening tests nor detected by TLC procedures. Because the Remedi expands the range of drugs covered by the immunoassays and provides a rapid, preliminary report in emergency situations, we conclude that this system can be a useful complementary technique in the clinical toxicology laboratory. Although urine toxicological screening seemed adequate for a good toxicological report, blood analysis allows extra toxicokinetic data such as blood concentrations and half-life estimations.
Immunoassay of insulin-like growth factor binding protein-1
Authors: Khosravi M Javad , Diamandi Anastasia , Mistry Jehangir
Published: January 2020

Accurate measurement of insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is important for precise definition of its physiological roles and potential diagnostic values. Because altered phosphorylation results in altered IGFBP-1 immunoreactivity, current assays may significantly underestimate or fail to detect physiological changes in the IGFBP-1 concentrations. We developed three ELISAs (ELISA 1–3) using a common capture but three different detection antibodies. IGFBP-1 in serum, synovial fluid (SF), cerebrospinal fluid (CSF), and amniotic fluid (AF) were measured before and after treatment with alkaline phosphatase (ALP). Among the methods, only ELISA-1 was unaffected by IGFBP-1 phosphorylation and generated identical results before and after ALP treatment. The serum and SF values by ELISA-2 and -3 were lower by ∼4- to 10-fold, but increased after ALP treatment to within 66–98% of those by ELISA-1. The medians in AF, and to a lesser extent in CSF, by all methods were similar and did not change significantly after dephosphorylation. ELISA-1 showed excellent correlation with ELISA-2, ELISA-3, and a commercial IGFBP-1 IRMA only after ALP-treated samples were analyzed by the comparative methods. ELISA-1 is highly specific for IGFBP-1 and demonstrated acceptable analytical performance characteristics.
Prostate-specific antigen: characterization of epitopes by synthetic peptide mapping and inhibition studies
Authors: Corey Eva , Wegner Sandra K , Corey Michael J , Vessella Robert L
Published: January 2020

To improve our understanding of the prostate-specific antigen (PSA) antigenic regions, we studied the association targets of one anti-PSA polyclonal antibody and 10 anti-PSA monoclonal antibodies (mAbs). We also examined the ability of the mAbs to inhibit PSA enzymatic activity and block the association of PSA with α1-antichymotrypsin (ACT). Linear epitope mapping with a polyclonal antibody indicated the presence of six major antigenic regions in PSA. Examination of the panel of mAbs established that three of them bind to linear epitopes. Five of the mAbs inhibited >90% of PSA enzymatic activity. However, inhibition of PSA enzymatic activity and hindrance of PSA-ACT association by mAbs cannot be used to predict whether the mAbs bind to free PSA, the PSA-ACT complex, or both. Some of the mAbs may block PSA-ACT association through peripheral occlusion of the binding site, or through induction of conformational changes in PSA.
Computer-aided identifications of thin-layer chromatographic patterns in broad-spectrum drug screening
Authors: Siek Theodore J , Stradling Corey W , McCain Marc W , Mehary Timothy C
Published: January 2020

We have developed a systematic thin-layer chromatographic (TLC) technique for detecting and identifying drugs and drug metabolites on 10-cm-long silica-gel plates with organic binder (fluorescent indicator); a computer program (SPOT CHEK) assists in matching the data from a particular chromatogram with those obtained for known drugs recovered from serum, urine, or other specimens. The plates are developed in a single mobile phase. Visualization and detection reagents used to characterize unknowns include fluorescamine, ferric chloride/perchloric acid/nitric acid, Dragendorff, Marquis, Mandelin, and iodinated Dragendorff solutions, 254 nm ultraviolet light, and vapor from chlorine or hydrochloric acid. Detection limits of 5–200 ng per sample spot were obtained for drugs in the database. The computer program database is based on nine reaction responses plus the plate zone locations for 243 drug substances but requires entry of only one TLC property to generate a matching list. We ran the program with an IBM-compatible 386/486 PC using an MS-DOS operating system (version 6.2).
Effects of reducing reagents and temperature on conversion of nitrite and nitrate to nitric oxide and detection of NO by chemiluminescence
Authors: Yang Fan , Troncy Eric , Francœur Martin , Vinet Bernard , Vinay Patrick , Czaika Guy , Blaise Gilbert
Published: January 2020

To measure the concentration of nitrites and nitrates by chemiluminescence, we examined the efficiency of five reducing agents [V(III), Mo(VI) + Fe(II), NaI, Ti(III), and Cr(III)] to reduce nitrite (NO2−) and (or) nitrate (NO3−) to nitric oxide (NO). The effect of each reducing agent on the conversion of different amounts of NO2− and (or) NO3−(100–500 pmol, representing concentrations of 0.4 to 2 μmolar) to NO was determined at 20 °C for NO2− and at 80 °C for NO3−. The effect of temperature from 20 to 90 °C on the conversion of a fixed amount of NO2− or NO3− (400 pmol or 1.6 μmolar) to NO was also determined. These five reducing agents are similarly efficient for the conversion of NO2− to NO at 20 °C. V(III) and Mo(VI) + Fe(II) can completely reduce NO3− to NO at 80 °C. NaI and Cr(III) were unable to convert NO3− to NO. Increased temperature facilitated the conversion of NO3− to NO, rather than that of NO2− to NO. We evaluated the recovery of NO2− and NO3− from plasmas of pig and of dog. Recovery from plasma of both animals was reproducible and near quantitative.
Vitamine—vitamin. The early years of discovery
Authors: Rosenfeld Louis
Published: January 2020

In 1905, Cornelius Adrianus Pekelharing found that animals fed purified proteins, carbohydrates, fats, inorganic salts, and water would thrive only if small amounts of milk were added to the diet. He concluded that the milk contained some unrecognized substance that in very small quantities was necessary for normal growth and maintenance. In 1911, Casimir Funk isolated a concentrate from rice polishings that cured polyneuritis in pigeons. He named the concentrate “vitamine” because it appeared to be vital to life and because it was probably an amine. Although the concentrate and other “accessory food substances” were not amines, the name stuck, but the final “e” was dropped. In 1913 two groups discovered a “fat-soluble” accessory food substance. Initially believed to be a single vitamin, two separate factors were involved. One, effective against xerophthalmia, was named vitamin A; the other, effective against rickets, was named vitamin D. The factor that prevented scurvy was isolated in 1928. Known as “water-soluble C,” it was renamed ascorbic acid.
Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification
Authors: Gelmini Stefania , Orlando Claudio , Sestini Roberta , Vona Giovanna , Pinzani Pamela , Ruocco Luca , Pazzagli Mario
Published: January 2020

We describe a PCR-based assay for determining c-erbB-2 oncogene amplification in breast cancer in which we use the TaqManTM system. Two fluorogenic probes anneal to the target between primers for c-erbB-2 and β-globin genes and contain both a reporter dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethyl-rhodamine). During the extension phase of the PCR cycle, the 5′→3′ exonuclease activity of Taq polymerase cleaves the hybridized fluorogenic probe, resulting in an increase of fluorescence emission of the reporter dye that is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. Assay performance showed adequate precision and a lower detection limit and good correlation with the results obtained in the same samples by a competitive PCR assay (n = 25, r = 0.94, P <0.01). This homogeneous assay is time-saving, avoids usually cumbersome postamplification procedures (that can be additional sources of inaccuracy and contamination), and seems suitable for determination of c-erbB-2 oncogene amplification in tumor specimens.
Construction of cDNA bank from biopsy specimens for multiple gene analysis of cancer
Authors: Ishikawa Takashi , Ichikawa Yasushi , Miura Yasuhiko , Momiyama Mobuyoshi , Keller Cylia , Koo Kenneth , Akitaya Tatsuo , Shimada Hiroshi , Mitsuhashi Masato
Published: January 2020

We constructed a “cDNA bank” of human colorectal cancer and surrounding normal tissues with our unique mRNA assay system. Total nucleic acids extracted from patients’ tissues were applied to 96-well plates, where poly(dT) sequences of oligonucleotides were immobilized. After hybridization, the cDNA was reverse-transcribed on the plate with the captured mRNA as a template, followed by synthesis of double-stranded (ds) cDNA. The resulting sense cDNA was removed from the plate, then used in PCR for analysis of various genes. The sense strand of the cDNA was repeatedly synthesized by using the immobilized antisense cDNA as a template even from plates used once and stored at 4 °C for as long as 6 months. Furthermore, the results of PCR could be easily compared among different specimens if the same amount of total mRNA were applied to the plate for the ds cDNA synthesis. This demonstrated that the cDNA bank constructed from clinical materials provides almost unlimited supplies of cDNA for multiple gene analysis of cancer.
Automation of human sperm cell analysis by flow cytometry
Authors: Ferrara Fulvio , Daverio Rita , Mazzini Giuliano , Bonini Pierangelo , Banfi Giuseppe
Published: January 2020

Semen sample analysis is routinely performed by microscopical evaluation and manual techniques by laboratory operators; the analysis is affected by a wide imprecision related to variability among observers, influencing its clinical validity. Our aim was to automate sperm analysis with the use of flow cytometry for evaluation of cell counts and typing and with the use of a new membrane-permeant nucleic acid stain for evaluation of sperm viability. Statistical analysis of the comparison between manual and automated methods for sperm counts was performed by the Bland and Altman method; the mean difference was 0.243 × 106 sperms/mL. The precision of the flow cytometric analysis was evaluated with whole sperm; the between-run CV was 7.5% and the within-run CV was 2.5%. Data observed suggest that flow cytometric sperm analysis, with high precision and accuracy and low costs, can be proposed for routine use in clinical laboratories.
Three generations of high-density lipoprotein cholesterol assays compared with ultracentrifugation/dextran sulfate–Mg2+ method
Authors: Harris Neil , Galpchian Vartouhi , Thomas Jennifer , Iannotti Elizabeth , Law Terence , Rifai Nader
Published: January 2020

We report on the analytical performance of three generations of HDL-cholesterol assays: phosphotungstic acid/Mg2+, Spinpro®, and a homogeneous method, N-geneous™. The run-to-run imprecision (CV) of all assays was ≤4.9%, and all results correlated highly with those of a modified reference procedure (r ≥0.96). At triglycerides concentrations <4000 mg/L, these field methods showed an acceptable systematic error (y = 1.12x − 47, 1.05x − 23, and 0.96x + 8 for the phosphotungstate, Spinpro, and N-geneous assays, respectively), and the total error of the field methods met the current National Cholesterol Education Program (NCEP) performance goal of ≤22%. Regression analyses of results for samples with triglycerides >4000 mg/L produced the following results for the above respective assays: y = 1.08x − 4.2, 1.02x + 3.6, and 0.85x + 108. In this hypertriglyceridemic group, only the N-geneous assay (at an HDL-cholesterol content of 240 mg/L) had a total error (35%) that exceeded the NCEP limit. Bilirubin and ascorbate produced a negative interference with the phosphotungstate and Spinpro assays but had little effect on the N-geneous assay.
Gastric tubes, meals, acid, and analysis: rise and decline
Authors: Rosenfeld Louis
Published: January 2020

The stomach tube was first used to administer food and medication or to remove poisonous substances. Later, it served to aspirate the stomachs of patients with gastric retention. Chemical analysis of stomach contents after a meal was first suggested in 1871 and quickly became an important laboratory procedure as various test-meal stimuli and more flexible tubes were developed. Quantitative estimations of free and total acidity were made by titration with 0.01 mol/L sodium hydroxide and specific indicators. Pentagastrin has supplanted secretagogues such as histamine and betazole; meal stimulation, tubeless tests, and other tests of gastric secretion are no longer used clinically. Tests of gastric acid secretion have been used in the diagnosis of upper gastrointestinal lesions and to help select the type of surgical procedure for gastric and duodenal ulcers, but the tests have decreased in importance because of their limited diagnostic sensitivity and specificity. Today, fiberoptic endoscopy is replacing gastric analysis as well as radiologic examination.
Quality: the next six months
Authors: Laessig Ronald H , Ehrmeyer Sharon S
Published: January 2020

The eclectic mix of participants in the forum had a surprisingly singular focus when it came to the topic of quality in clinical laboratories. All sensed that the time is right for a transition from laws, rules, and inspections to a true quality-based system. Such a system can achieve the goals, implicit and explicit, that are the rationale for the multiplicity of regulations affecting today’s laboratories. A true quality-based system has great potential benefits to laboratories, regulators, and manufacturers, and ultimately to our true customers, the patients. The benefits include lower costs, superior products, and better test results; in short, better patient care. This transition will be possible only through formation of a “Quality Alliance,” composed of those skilled in the “theory” of quality—laboratory personnel, manufacturers, and regulators, acting as one to implement the quality system. The Quality Alliance requires a team of individuals with different skills, aligned as one, for the purpose of achieving a common goal. On the basis of views expressed in this Forum, our collective future will be defined by the evolving Quality Alliance, an alliance focused on true quality systems in clinical laboratories.
Initial evaluation of cystatin C measurement by particle-enhanced immunonephelometry on the Behring nephelometer systems (BNA, BN II)
Authors: Finney Hazel , Newman David J , Gruber Walter , Merle Peter , Price Christopher P
Published: January 2020

Serum cystatin C has been suggested as a new marker of glomerular filtration rate (GFR). We describe a fully automated and rapid particle-enhanced nephelometric immunoassay (PENIA) for measuring serum cystatin C on the Behring nephelometer systems (BNA, BN II). Each sample is analyzed in 6 min with as many as 75 samples per batch. The assay covers the range 0.23–7.25 mg/L, up to seven times the upper limit of normal. The intra- and interassay imprecision are <3.3% and <4.5%, respectively. There is absolute linearity across the assay range (r2 = 0.997), with analytical recovery by cystatin C addition between 95% and 109% (mean 102%). Hemoglobin (≤8.0 g/L), bilirubin (≤488 μL), triglycerides (≤23 mmol/L), rheumatoid factor (≤2000 kIU/L), and myeloma paraprotein (≤41 g/L) do not interfere with the assay. This assay agreed well with an in-house particle-enhanced turbidimetric immunoassay (PETIA) (mean difference = 1.73 ± 2.10) and a commercial PETIA (mean difference = 1.13 ± 0.86). This is a new assay by which cystatin C may be effectively used as a marker of GFR estimation.
Gastrointestinal absorption, tissue retention, and urinary excretion of dietary aluminum in rats determined by using 26Al
Authors: Jouhanneau Philippe , Raisbeck Grant M , Yiou Françoise , Lacour Bernard , Banide Hélène , Drüeke Tilman B
Published: January 2020

We used accelerator mass spectrometry (AMS) and 26Al to study the plasma concentration, urinary excretion, and retention in bone, brain, and liver of a single dose of a dietary concentration of aluminum ingested either with or without citrate by 2-month-old Wistar rats. In the absence of citrate, cumulative urinary excretion and skeleton retention were each ∼0.05% of the total 26Al dose ingested. 26Al retention in brain and liver were ∼4 × 10−8 and 2 × 10−6, respectively. Concomitant citrate intake increased these median values by about two- to fivefold, although this factor was highly variable in individual rats. Independent of citrate administration, 90% of the26Al excreted in urine (measured cumulatively over 30 days) was excreted within the first 48 h. Uptake by bone was rapid (∼1 h) and permanent over the 30-day duration of the experiment.
Evaluation of two homogeneous methods for measuring high-density lipoprotein cholesterol
Authors: Huang Yi-Chang , Kao Jau-Tsuen , Tsai Keh-Sung
Published: January 2020

We evaluated the performance of two homogeneous assays for quantifying HDL cholesterol (HDL-C) and compared them with the phosphotungstic acid (PTA)/MgCl2 assay. Both homogeneous HDL-C assays were precise, having a within-run CV of <1.20% and a between-run CV of <4.07%. The HDL-C values (y) measured by the two homogeneous methods correlated well with those by the PTA/MgCl2 method (x): y = 1.00x + 64.98 mg/L, r = 0.987, Sy|x = 27.99 mg/L (n = 152) for the polyethylene glycol-modified enzymes/α-cyclodextrin sulfate (PEGME) assay (Kyowa), and y = 0.84x + 106.51 mg/L, r = 0.984, Sy|x = 26.10 mg/L (n = 152) for the polyanion–polymer/detergent (PPD) assay (Daiichi). The specificity of the PEGME method seemed better than that of the PPD method, as the PPD method was markedly interfered with by supplemental LDL-C. Addition of 20 g/L triglycerides produced a negative error of ∼18% in both homogeneous assays. Bilirubin and hemoglobin had little influence on the PEGME method; hemoglobin had little effect on the PPD method. Bilirubin, however, markedly decreased the readings by the PPD method. We found the PEGME assay superior to the PPD assay for routine HDL-C testing, because the PPD assay is relatively inaccurate and not specific.
Reference values for retinol, tocopherol, and main carotenoids in serum of control and insulin-dependent diabetic Spanish subjects
Authors: Olmedilla Begoña , Granado Fernando , Gil-Martinez Enrique , Blanco Inmaculada , Rojas-Hidalgo Enrique
Published: January 2020

To establish reference ranges for use in clinical and epidemiological studies, we determined concentrations of retinol, α-tocopherol, β-carotene, α-carotene, β-cryptoxanthin, lutein, zeaxanthin, and lycopene in 450 Spanish control subjects and 123 Spanish patients with insulin-dependent diabetes mellitus (IDDM). Results were grouped according to sex, and samples were collected throughout the year. Concentrations of retinol were significantly lower and β-carotene and α-carotene were higher in women than in men, both in controls and IDDM subjects, whereas β-cryptoxanthin concentrations were higher only in control women. Conditional logistic regression analysis showed that retinol, β-carotene, and lycopene were the variables associated with diabetes. In comparison with other populations, our controls showed, in general, ordinary concentrations of retinol, comparatively low β-carotene and high β-cryptoxanthin concentrations, and a relatively high α-tocopherol/cholesterol ratio.
Platelet distribution width for differential diagnosis of thrombocytosis
Authors: Osselaer Jean-Claude , Jamart Jacques , Scheiff Jean-Marie
Published: January 2020

Differential diagnosis of thrombocytosis is not always obvious. The routine clinical chemistry laboratory classically provides only limited help in distinguishing between reactive thrombocytosis (RT) and autonomous thrombocytosis, where platelet production escapes normal regulatory processes, and which is seen in myeloproliferative diseases (MPD) such as essential thrombocythemia and polycythemia vera. We explored the clinical use of platelet distribution width (PDW) in the differential diagnosis of thrombocytosis. During a 3-month period, 250 patients presenting with a platelet count >500 × 109/L were studied; 174 were classified as having RT, 42 had a diagnosis of MPD, and 34 patients were excluded because they had a hemopathy different from MPD, and either did or did not present a known etiologic factor for RT. First, we determined that in the RT group the value of PDW was closely linked to both mean platelet volume (MPV) and platelet count (PLT) (PDW = 79.5 − 0.005 PLT − 3.5 MPV; r = 0.848, R2 = 0.720). Therefore a new parameter, PDWresidual, was defined (PDWresidual = PDWobserved − PDWexpected). Second, the discrimination between reactive and autonomous thrombocytosis obtained with PDWresidual was compared with that obtained with either PDW, MPV, or PLT. PDWresidual proved much more powerful than each of the other parameters used separately: 76% of MPD patients had a PDWresidual above the 95th percentile value of the RT population and none of the MPD patients had a PDWresidualbelow the 50th percentile. Thus, the combined interpretation of PLT, MPV, and PDW through the use of a PDWresidualappears highly useful in the differential diagnosis of thrombocytosis. Also, through simple modeling, more information can be drawn from parameters such as PDW that hitherto were mostly discarded as being without clinical interest.
Enzyme immunoassay for measuring 25-hydroxyvitamin D3 in serum
Authors: Lind Charlotte , Chen Jiewei , Byrjalsen Inger
Published: January 2020

We developed a rapid, competitive enzyme immunoassay (EIA) for measuring 25-hydroxyvitamin D3 [25(OH)D3] in serum. The EIA was based upon 25(OH)D3-3-hemisuccinate covalently coupled to secondary amino groups grafted onto the polystyrene surface of microtiter wells. Optimal coupling conditions were established, and we found that inclusion of 40 μmol/L chloramine T, an agent not previously described for use in coupling to these plates, resulted in both more reproducible coupling as well as more than a twofold increase in the coupling efficiency. Before EIA, 25(OH)D3 was extracted from the serum samples by acetonitrile, and the redissolved extract was incubated with polyclonal rabbit antibody raised against 1,25-dihydroxyvitamin D3-3-hemisuccinate conjugated to bovine serum albumin. Peroxidase-labeled antibody raised in goat against rabbit immunoglobulins was used for detection. The detection limit of the EIA was 4.4 μg/L; recovery 102%; on-plate CV 11%; within-run CV including extraction 12%, and between-run CV 15%. There was no clinically important cross-reactivity with other vitamin D metabolites, and results obtained by the EIA were compared with results obtained by a previously described RIA.
Growth hormone (GH) assays: influence of standard preparations, GH isoforms, assay characteristics, and GH-binding protein
Authors: Jansson Chatarina , Boguszewski Cesar , Rosberg Sten , Carlsson Lena , Albertsson-Wikland Kerstin
Published: January 2020

The impact of the adoption of the new biosynthetic growth hormone (GH) WHO International Reference Preparation (IRP 88/624), and the recommendation to report results in μg/L instead of mU/L, is described. Conversion factors were determined by comparing both the linear and nonlinear relations of the GH values. The Pharmacia polyclonal IRMA (p-IRMA) and the DELFIA® monoclonal time-resolved immunofluorometric assay (trIFMA) with kit calibrators calibrated either against the pituitary-derived WHO IRP 80/505 or the new 88/624 were evaluated. Conversion factors of 4.17 mU/L = 1 μg/L for the p-IRMA and 4.31 mU/L = 1 μg/L for the trIFMA were necessary. Different cross-reactivity patterns for the deaminated and dimer 22-kDa, 20-kDa, and 17-kDa GH isoforms were found. Expected GH recovery was similar when the measured values were adjusted according to the results of the cross-reactivity study.
Cardiac troponin I, cardiac troponin T, and creatine kinase MB in dialysis patients without ischemic heart disease: evidence of cardiac troponin T expression in skeletal muscle
Authors: McLaurin Mary D , Apple Fred S , Voss Ellen M , Herzog Charles A , Sharkey Scott W
Published: January 2020

Serum cardiac troponin T (cTnT) concentrations are frequently increased in chronic dialysis patients as measured by the first-generation ELISA immunoassay, as is creatine kinase (CK) MB mass in the absence of acute ischemic heart disease. We designed this study to compare four serum markers of myocardial injury [CK-MB mass, first-generation ELISA cTnT, second-generation Enzymun cTnT, and cardiac troponin I (cTnI)] in dialysis patients without acute ischemic heart disease. We also evaluated skeletal muscle from dialysis patients as a potential source of serum cTnT. No patients in the clinical evaluation group (n = 24) studied by history and by physical examination, electrocardiography, and two-dimensional echocardiography had evidence of ischemic heart disease. Biochemical markers were measured in serial predialysis blood samples with specific monoclonal antibody-based immunoassays. For several patients at least one sample measured above the upper reference limit: CK-MB, 7 of 24 (30%); ELISA cTnT, 17 of 24 (71%); Enzymun cTnT, 3 of 18 (17%); and cTnI, 1 of 24 (4%). In a separate group of dialysis patients (n = 5), expression of cTnT, but not cTnI, was demonstrated by Western blot analysis in 4 of 5 skeletal muscle biopsies. Chronic dialysis patients without acute ischemic heart disease frequently had increased serum CK-MB and cTnT. The specificity of the second-generation cTnT (Enzymun) assay was improved over that of the first-generation (ELISA) assay; cTnI was the most specific of the currently available biochemical markers. cTnT, but not cTnI, was expressed in the skeletal muscle of dialysis patients.
Cardiac troponin I and T alterations in hearts with severe left ventricular remodeling
Authors: Ricchiuti Vincent , Zhang Jianyi , Apple Fred S
Published: January 2020

Cardiac troponin T (cTnT) and troponin I (cTnI) have been suggested as new, more specific markers of myocardial cellular damage. The objective of this study was to examine how the distributions of cTnI and cTnT were affected in postinfarction left ventricular remodeled (LVR) myocardium. At 2 months postinfarct in a porcine heart failure model, both Western blot and biochemical assay analyses were performed on left ventricular myocardium remote from the infarct zone in ligation animals (n = 8). Results were compared with data from the left ventricular myocardium from similar sized healthy (control) pigs (n = 7). Autoradiograms from Western blot analysis showed that the protein mass for cTnI and cTnT in LVR hearts decreased 80% (P <0.001) and 40% (P <0.02), respectively, when compared with nondiseased tissue. Similarly, the concentrations for cTnI and cTnT in LVR hearts decreased 42% (P <0.05) and 70% (P <0.001), respectively, compared with nondiseased normal tissue. The clinical assumption is that the appearance of cTnI and cTnT in the blood is proportional to chronic loss of cTnI and cTnT from injured myocardium associated with left ventricular remodeling.
Microalbuminuria and borderline-increased albumin excretion determined with a centrifugal analyzer and the Albumin Blue 580 fluorescence assay
Authors: Kessler Manfred A , Meinitzer Andreas , Petek Walter , Wolfbeis Otto S
Published: January 2020

We report a new automated fluorescence assay for determination of albumin in urine. The dye Albumin Blue 580 specifically binds to albumin with exhibition of strong red fluorescence. The albumin concentration is calculated from emission intensity at 616 nm (excitation at 590 nm) and a calibration curve. Two Cobas Fara programs cover working ranges of 2–200 and 1–50 mg/L with detection limits of 1.4 and 0.4 mg/L, respectively. Within-run CVs (n = 10) ranged from 1.7% (189 mg/L) to 8.9% (7.2 mg/L) for 2–200 mg/L and from 2.9% (43.3 mg/L) to 5.7% (2.3 mg/L) for the 1–50 mg/L range. A test of urine samples (n = 100) submitted to routine analysis gave results that agreed well with those by the Behring nephelometric assay: AB 580 = 0.922 (± 0.010) BNA + 4.16 (± 0.78). No interference was detected from other urine components, including several proteins and 46 drugs. The high specificity and sensitivity make the method ideal for determination of microalbuminuria. In addition, the method is fast, inexpensive, and well-suited for clinical laboratory application and thus may be used instead of immunoassays.
Methods for detection of point mutations: performance and quality assessment
Authors: Nollau Peter , Wagener Christoph , Division on behalf of the IFCC Scientific , Techniques Committee on Molecular Biology
Published: January 2020

We give an overview of current methods for the detection of point mutations as well as small insertions and deletions in clinical diagnostics. For each method, the following characteristics are specified: (a) principle, (b) major modifications, (c) maximum fragment size that can be analyzed, (d) ratio and type of mutations that can be detected, (e) minimum ratio of mutant to wild-type alleles at which mutations can be detected, and (f) detection methods. Special attention is paid to the possibilities of quality assessment and the potential for standardization and automation.
Simple triple-label detection of seven cystic fibrosis mutations by time-resolved fluorometry
Authors: Heinonen Pia , Iitiä Antti , Torresani Toni , Lövgren Timo
Published: January 2020

We describe a simple hybridization assay performed in microtitration wells with use of DNA probes labeled with three different lanthanide chelates for detection of seven mutations that cause cystic fibrosis. The assay is based on DNA amplification of four fragments containing the mutations (ΔF508, G1717→A, G542X, R553X, 3905 insertion T, W1282X, and N1303K) by PCR, followed by hybridization with short, allele-specific oligonucleotide probes labeled with europium, terbium, or samarium chelates. Because the technology makes it possible to hybridize three DNA probes simultaneously in one reaction, all 14 mutation-related alleles were detected in a total of five reaction wells. Blood spot specimens, obtained from children with cystic fibrosis, their parents, and their siblings, have been assayed, and for all the probes the positive signal-to-noise ratios are >10. Solution hybridization utilizing triple-label time-resolved fluorometry combined with PCR is a suitable procedure for large-scale screening and automation.
Analytical and clinical evaluation of the Immulite® estradiol assay in serum from patients undergoing in vitro fertilization: estradiol increase in mature follicles
Authors: Dancoine François , Couplet Gisèle , Buvat Jacques , Guittard Catherine , Marcolin Gérard , Fourlinnie Jean-Claude
Published: January 2020

We examined an immunoassay for estradiol (E2) on the Immulite®—an automated, random access chemiluminescent immunoassay system—to determine its accuracy and precision required for in vitro fertilization (IVF) studies. The assay, which has a reportable range from 73 to 7300 pmol/L, demonstrated good linearity under dilution, a detection limit of 44 pmol/L, and interassay CVs of 12.6% and 7.6% at 466 and 6164 pmol/L, respectively. In a retrospective analysis of 545 serum samples, the assay showed adequate agreement with an antibody-coated-tube RIA. The two E2 assays showed good agreement, even on samples from patients receiving a variety of different estrogen replacement therapies. Longitudinal studies of individual IVF cycles showed good parallelism between the automated system and the RIA, and results by the automated assay correlated well with the total number of follicles.
Mass spectrometric characterization of nicked fragments of the β-subunit of human chorionic gonadotropin
Authors: Liu Chuanliang , Bowers Larry D
Published: January 2020

We describe the use of an HPLC/MS technique for the characterization of nicked fragments of hCG β-subunit. After reductive alkylation of the nicked hCG β-subunit with vinylpyridine, endoproteinase Glu-C or trypsin was used to digest the protein to produce peptides that could be analyzed by HPLC/electrospray ionization MS. Human leukocyte elastase digestion was used to produce an experimentally nicked hCG. Two nicking sites were observed, between amino acids 42Thr and 43Arg and between 44Val and45Leu. The former site has not been previously reported for elastase digestion. The structures of the fragments were confirmed by HPLC/MS after removal of the oligosaccharide by direct mass measurement and by mass determination of their proteolytic digests. Without the glycopeptidase treatment, the microheterogeneity of the two N-linked oligosaccharides could be deduced from the spectra of the proteolytic fragments. Nicking with elastase was found to alter the oligosaccharide structures. Nicked β-subunit samples isolated from the urine of choriocarcinoma patients were also analyzed and the location of the nicking site(s) agreed with that determined by classical techniques. Important differences in the oligosaccharide structures were also observed in these samples, including the presence of triantennary oligosaccharides not found in hCG from healthy subjects. These findings demonstrate the potential of HPLC/MS for characterization of glycoprotein standard preparations.
Correlation between plasma 5-aminolevulinic acid concentrations and indicators of oxidative stress in lead-exposed workers
Authors: Costa Cristine A , Trivelato Gilmar C , Pinto Adriana M P , Bechara Etelvino J H
Published: January 2020

5-Aminolevulinic acid (ALA), a heme precursor accumulated in acute intermittent porphyria and lead poisoning, undergoes metal-catalyzed aerobic oxidation at physiological pH to yield reactive free radical species (O2−·>, HO·, and ALA·). We analyzed the relationships between plasma ALA concentrations, blood concentrations of lead, protoporphyrin IX (PP-IX), superoxide dismutase (SOD), and methemoglobin (metHb), and urine chemiluminescence (CL) in samples collected from lead-exposed workers. All variables measured were substantially (P <0.01) higher (2–8-fold) in the lead-exposed workers (n = 60). Plasma ALA concentrations were, on average, 6-fold higher in lead-exposed workers. We observed positive linear relationships between ALA and lead (r = 0.992), ALA and PP-IX (r = 0.891), ALA and metHb (r = 0.984), lead and SOD (r = 0.948), ALA and urine CL (r = 0.987), and lead and PP-IX (r = 0.993). These data are consistent with our free radical hypothesis for lead poisoning, where ALA distribution to and accumulation in several organs may trigger oxidative stress responses.
Endocrine aspects of anabolic steroids
Authors: Wu F C W
Published: January 2020

Understanding of the mechanism of androgen action has been enhanced by advances in knowledge on the molecular basis of activation of the androgen receptor and the importance of tissue conversion of circulating testosterone to dihydrotestosterone and estradiol. New evidence supports the view that supraphysiological doses of anabolic steroids do have a definite, positive effect on muscle size and muscle strength. However, the nature of the anabolic action of androgens on muscle is currently unclear and may involve mechanisms independent of the androgen receptor. The dose–response relationships of anabolic actions vs the potentially serious risks to health of androgenic-anabolic steroids (AAS) use are still unresolved. Most of the adverse effects of AAS are reversible but some are permanent, particularly in women and children. The reported incidence of acute life-threatening events associated with AAS abuse is low, but the actual risk may be underrecognized or underreported; the exact incidence is unknown. The long-term consequences and disease risks of AAS to the sports competitor remain to be properly evaluated.
Immunoprocedures for detecting human chorionic gonadotropin: clinical aspects and doping control
Authors: Stenman Ulf-Håkan , Unkila-Kallio Leila , Korhonen Juha , Alfthan Henrik
Published: January 2020

The pregnancy hormone human chorionic gonadotropin (hCG) is also present at low concentrations in plasma and urine of men and nonpregnant women. hCG immunoreactivity occurs in various molecular forms: Besides the intact hCG heterodimer, considerable amounts of proteolytically cleaved forms, free subunits, and fragments are found in plasma and urine. Especially in urine, proteolytic fragments constitute a major part of the hCG immunoreactivity. The different forms of hCG cross-react to various degrees in immunoassays and constitute a problem for standardization of specific hCG determinations. After injection of hCG (10 000 IU of Pregnyl®; Organon), above-normal concentrations of hCG can be detected in serum and urine for 7–11 days. Most immunoassays for hCG also measure hCGβ. Quantitative hCG determinations are mainly performed on serum samples, and very few commercial hCG determinations have been validated for determination of urine samples. Considerable care must therefore be exercised when utilizing such assays to analyze urines for doping control.
Counterpoint To (measure apo)B or not to (measure apo)B: a critique of modern medical decision-making
Authors: Sniderman Allan D
Published: January 2020

The measurement of apo B provides critical information that is complementary to that provided by the plasma and lipoprotein lipids for the assessment of coronary risk and the choice of appropriate pharmacological therapy. Why then is this measurement not in more widespread clinical use? I suggest two explanations. First, against the evidence, there is a lingering perception that problems persist in its measurement in routine clinical practice. Far from this being the case, however, the measurement of apo B has met every reasonable standard of laboratory precision and reliability to allow its widespread introduction in clinical laboratories. The second impediment is that the introduction of new tests has become subject to the authority of consensus conferences, a new approach to medical decision-making. The number of such conferences is increasing astronomically, and their reports are major determinants of clinical practice and allocation of resources. Notwithstanding the benefits they have brought, here I argue that, just as with any other scientific method, the merits of this new method of decision-making need to be examined critically; for if we do not, a process that was established to introduce change may, in fact, retard it or destroy it altogether.
Extraction of glyceric and glycolic acids from urine with tetrahydrofuran: utility in detection of primary hyperoxaluria
Authors: Dietzen Dennis J , Wilhite Timothy R , Kenagy David N , Milliner Dawn S , Smith Carl H , Landt Michael
Published: January 2020

Primary hyperoxaluria (PH) is an autosomal recessive metabolic abnormality characterized by excessive oxalate excretion leading to nephrocalcinosis and progressive renal dysfunction. Type I primary hyperoxaluria (PH I) results from a deficiency of alanine:glyoxylate aminotransferase, whereas type II disease has been traced to a deficiency of d-glycerate dehydrogenase. The two syndromes are often distinguished on the basis of organic acids that are coexcreted with oxalate: glycolate and l-glycerate in type I and type II disease, respectively. Routine organic acid analysis with diethyl ether extraction followed by gas chromatographic analysis failed to detect normal and increased concentrations of these diagnostic metabolites. Subsequent extraction of urine with tetrahydrofuran (THF), however, extracted 75% of added glycerate, 42% of added glycolate, and 75% of added ethylphosphonic acid (internal calibrator). THF extraction was analytically sensitive enough to allow determination of normal excretion of glycolate (14–72 μg/mg creatinine) and glycerate (0–5 years, 12–177 μg/mg creatinine and >5 years, 19–115 μg/mg creatinine). Four of five patients with PH I and both patients with type II disease were correctly identified. Thus, THF extraction is a convenient adjunct to routine organic acid analysis and facilitates the detection of PH.
Creatinine measurement proficiency testing: assignment of matrix-adjusted ID GC-MS target values
Authors: Carobene Anna , Ferrero Carlo , Ceriotti Ferruccio , Modenese Annalisa , Besozzi Marco , De Giorgi Elena , Franzin Michela , Franzini Carlo , Kienle Marzia Galli , Magni Fulvio
Published: January 2020

The results of an external quality-assessment experiment for serum creatinine measurement are described. Fifty-one laboratories performed quintuplicate analyses during three different analytical runs on six lyophilized sera and two frozen human serum pools. Isotope dilution gas chromatography–mass spectrometry (ID GC-MS) target values were assigned to all the materials. Intralaboratory within- and between-run imprecision results were very similar for all the materials tested (CV ≤2.20% and ≤4.70%, respectively). The overall imprecision obtained was high (CV 6.5–20.0%) because of increased interlaboratory–intermethod variability. A significant positive bias (+9.2–+43.7%) was found for all the materials at lower creatinine concentration. By using two human sera at different concentrations, we could calculate the constant and the proportional calibration bias displayed by each peer group. The majority of the lyophilized materials showed a behavior divergent from the frozen pools, indicating matrix-related problems. We propose a new algorithm for calculating matrix bias correction factor instrument–reagent specific for each material.