bluuebunny/crossref_metadata_2025 huggingface dataset:
Competitive particle concentration fluorescence immunoassay for measuring 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (lometrexol) in serum
Authors: Taber Larry D , O'Brien Peter , Bowsher Ronald R , Sportsman J Richard
Published: January 2020

A competitive particle concentration fluorescence immunoassay (PCFIA) is described for measuring 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (lometrexol; Lilly) in human serum. b-Phycoerythrin-labeled lometrexol competes with free lometrexol for binding to a limiting concentration of lometrexol-specific antibodies immobilized by a second antibody to submicrometer-diameter polystyrene particles in specially designed 96-well plates. Reaction particles are washed and concentrated onto filter membranes in the wells of the plates and the fluorescence is measured at 575 nm. The method, including sample preparation and data reduction, is automated and can be completed in less than 2 h. The assay has a standard curve maximum measurable concentration of 1000 micrograms/L and a minimum detectable concentration of 0.1 microgram/L. Analytical recovery of lometrexol in serum is quantitative at concentrations greater than 1 micrograms/L. Intra- and interassay coefficients of variation at 50 micrograms/L in serum are 7.1% (n = 9) and 7.5% (n = 33), respectively. The cross-reactivity of naturally occurring folates, folic acid analogs, and the anti-cancer agent methotrexate is minimal. We report the use of the PCFIA during Phase I clinical studies designed to evaluate the pharmacokinetics of lomextrexol after intravenous administration to cancer patients.
Comparison of nucleic acid hybridization and cytologic examination for detection of human papillomavirus infection, with evaluation of two commercially available hybridization kits
Authors: Chimera J A , Anderson S M , Noell H , Rizk V
Published: January 2020

Human papillomavirus (HPV) infections were detected by analyzing exfoliated cervical cells for HPV DNA by use of nucleic acid hybridization; the results were correlated with cytologic findings on Papanicolaou smears. HPV infection was diagnosed in 154 women (20%) by either morphologic evidence on cervical smears or nucleic acid hybridization. Many of these women (38%; 58/154) exhibited Papanicolaou smears with no morphologic evidence of HPV infection. In those patients with cytologic evidence of HPV infection, only 28% were positive for HPV DNA. HPV 16 and (or) 18 were the most common types (27%) detected in women with cervical intraepithelial neoplasia, whereas all HPV groups tested were equally represented in patients with normal cervical smears. We also present an assessment of 17,000 clinical specimens submitted to this laboratory for analysis of HPV DNA.
Computerized method for validating laboratory reference ranges for triiodothyronine and thyroxin immunoassays
Authors: Luttrell Brian , Watters Sall
Published: January 2020

We used a computer-based method to help validate the reference ranges of assays for triiodothyronine (T3) and thyroxin (T4). A retrospective search of a database of laboratory results for the previous six months identified all patients with apparent euthyroid status, as defined by methods independent of the immunoassay under review. A computer-generated reference group (CGR Group) of 2001 records had a gaussian distribution of T4 values and a reference range (mean +/- 2 SD) of 56-161 nmol/L, compared with the supplier's suggested range for euthyroid subjects (58-148 nmol/L) and an in-house range of 60-144 nmol/L for a group of 97 normal subjects. A similar CGR Group of 1902 records gave a reference range for T3 of 0.7-2.1 nmol/L (manufacturer's range 0.8-2.8; normal subjects 0.8-2.2). An attempt to devise a reference range for thyrotropin failed when we found that its concentration in the population of patients with normal values for thyroid hormones was distributed differently from that in the normal population. The method is intended to be used in addition to conventionally derived ranges based on results for healthy subjects. It allows the laboratory to conveniently verify the reference ranges for T3 and T4 assays at regular intervals by using very large samples with appropriate age, sex, and weight distribution, drawn from the population of patients' samples submitted for analysis.
Lipase activity in serum measured with Ektachem is often increased in nonpancreatic disorders
Authors: Tetrault Gregory A
Published: January 2020

For patients with symptoms of pancreatitis, measurement of amylase in serum reportedly is more sensitive than that of lipase in acute pancreatitis, whereas lipase reportedly is more specific. However, serum lipase activities exceeding the upper reference limit (URL) have been reported for many patients who did not have pancreatitis. I reviewed the serum lipase and amylase concentrations of 493 consecutive inpatients and emergency department patients for whom both tests were ordered. Serum lipase and amylase activities, determined with an Ektachem 700 analyzer, were less than or equal to URL for 390 patients (83%) and greater than URL for 103. Medical records of 101 of these 103 were reviewed; 18 had acute or chronic relapsing pancreatitis. In this latter group, serum lipase values greater than URL had 100% sensitivity and 84% specificity; those of serum amylase greater than URL had 72% sensitivity and 88% specificity. However, the test combination of serum lipase greater than URL and serum amylase less than or equal to URL also occurred in 84% of the patients in which review of the medical records revealed nonpancreatic gastrointestinal or hepatobiliary disorders as the primary problem (n = 55). Therefore, serum lipase activity measured with the Ektachem assay is also often increased in patients with intra-abdominal disorders that appear to be nonpancreatic.
Health care in the developing world: need for appropriate laboratory technology
Authors: Heuck Claus C , Deom Andrȳ
Published: January 2020

Health care technology is rapidly advancing, and the development of equipment based on new principles has had a strong impact on laboratory services in Western countries. The use of such technologies frequently creates problems in countries of the Third World, because the effects of several interacting elements are not always taken into account. Not only technical factors but also socioeconomic and political factors, as well as differences in diseases in developing countries, must be considered. Standardization of equipment and methodology, and adaptation to local health systems, will facilitate improvement of laboratory services. Training and incentives for local initiatives are fundamental elements for the provision of cost-effective health care.
Development, validation, and certification by isotope dilution gas chromatography-mass spectrometry of lyophilized human serum reference materials for cortisol (CRM 192 and 193) and progesterone (CRM 347 and 348)
Authors: Thienpont Linda , Siekmann Lothar , Lawson Alexander , Colinet Elisabeth , De Leenheer Andrȳ
Published: January 2020

The Community Bureau of Reference of the European Communities has produced four batches of lyophilized serum Certified Reference Materials, two for cortisol (CRM 192 and 193) and two for progesterone (CRM 347 and 348). For cortisol, one of the pools consisted of serum from healthy blood donors, whereas the second batch was supplemented with pure cortisol. The progesterone Reference Materials contained only endogenous hormone concentrations. Assessment of vial-to-vial variability in the cortisol and progesterone concentrations showed no between-sample inhomogeneity, and the materials were stable. The quality of the materials was therefore considered sufficient for certification of the values for the cortisol and progesterone concentrations by a collaborative study involving several laboratories from the European Communities, using isotope dilution gas chromatography-mass spectrometry. Inaccuracy in reconstitution of the lyophilized materials was less than 0.3%; imprecision of sampling was less than 0.2%. For determinations of cortisol and progesterone concentrations, the mean within-laboratory coefficients of variation (CVs) were 1.76% (CRM 192), 1.19% (CRM 193), 1.64% (CRM 347), and 1.75% (CRM 348). The between-laboratory CVs were greater: CRM 192, 1.79%; CRM 193, 1.48%; CRM 347, 2.08%; and CRM 348, 2.16%. The concentrations in the reconstituted Reference Materials were certified to be 273 nmol/L in CRM 192 and 763 nmol/L in CRM 193 for cortisol and 10.13 nmol/L in CRM 347 and 40.3 nmol/L in CRM 348 for progesterone. Uncertainties at the 0.95 confidence level--6 (CRM 192), 14 (CRM 193), 0.21 (CRM 347), and 1.0 nmol/L (CRM 348)--were considered compatible with the intended use of the materials.
Comparison of four methods for free thyroxin
Authors: Deam D , Goodwin M , Ratnaike S
Published: January 2020

We performed a limited evaluation of four free thyroxin (FT4) reagent kits: the Amerlex-M (AFT4), the Amerlite (LFT4), the MagicLite (MFT4), and the GammaCoat two-step RIA (GFT4). FT4 was measured in specimens from 201 subjects: 19 healthy controls, 14 patients who were thyrotoxic, 13 who were hypothyroid, 59 who had a past history of thyroid disease, seven with thyroxin autoantibodies, seven who were taking amiodarone, and 82 who had no clinical indication of thyroid hormone abnormality. Of these 201 subjects, 78 had a low serum albumin (less than 35 g/L) and 27 had severe nonthyroidal illness. We also investigated 60 pregnant subjects. We found no correlation between thyroxin-binding globulin (TBG) and FT4 in any of the assays, and only the AFT4 method showed a significant correlation with albumin concentrations. The presence of autoantibodies to thyroxin affected the results of all methods except the GFT4 method. All methods showed a decrease in mean FT4 values in late pregnancy. Correlation of patients' clinical state and FT4 results suggested that the reference ranges published by the manufacturers need to be modified for our laboratory.
IgA anti-gliadin antibodies in adult celiac disease
Authors: Hill P G , Thompson S P , Holmes G K
Published: January 2020

We have improved the enzyme-linked immunosorbent assay for IgA-class antibodies to gliadin in serum by evaluating earlier publications. We also assess the value of measuring these antibodies when screening for adult celiac disease and monitoring dietary compliance. Of 61 adults with untreated celiac disease, 57 had abnormal results, giving a sensitivity for the test of 0.93. Patients (n = 283) attending a gastroenterology clinic formed the control group. The predictive values of positive results and negative results were 50% and 99.7%, respectively, indicating that the test has a role in helping select those subjects in whom small bowel biopsy is indicated. Adults with celiac disease, after two years on a strict gluten-free diet, and normal subjects showed no significant difference in serum IgA-class anti-gliadin antibody concentrations. The test thus provides objective evidence of dietary compliance in addition to its role as a screening test.
Simple enzyme immunoassay for the simultaneous measurement of whole choriogonadotropin molecules and free beta-subunits in sera of women with abnormal pregnancies or tumors of the reproductive system
Authors: Choi M J , Choe I S , Kang H K , Lee J S , Chung T W
Published: January 2020

A multiple enzyme immunoassay (multi-EIA) was developed to quantify whole-molecule human choriogonadotropin (w-hCG) and free hCG beta-subunits (hCG-beta) simultaneously. A clone of a specific monoclonal antibody was coupled to solid phase; two other clones of different monoclonal antibodies were conjugated to horseradish peroxidase (HRP; EC 1.11.1.7) and alkaline phosphatase (AP; EC 3.1.3.1), respectively. These two enzyme conjugates were mixed together to measure w-hCG or hCG-beta, depending on the selection of the enzyme substrate. To measure w-hCG and hCG-beta simultaneously, both enzyme substrates were used with the blended enzyme conjugates. The assay is simple and reproducible, and can be completed within 2 h with high specificity and sensitivity. We measured w-hCG and hCG-beta in the sera of women with abnormal pregnancies and in patients with tumors of the reproductive system, and observed different hCG-beta/w-hCG ratios in patients with various types of trophoblastic tumors.
Interferences in current methods for measurements of creatinine
Authors: Weber J A , van Zanten A P
Published: January 2020

We measured the interference of carbonyl compounds, drugs, and other substances in human serum on the determination of creatinine by the two-point, fixed-time kinetic modification of the Jaffé reaction as well as by four enzymatic methods. We added known concentrations of the interfering substances to a solution of creatinine in water. For bilirubin, we used both pooled normal sera with added bilirubin and icteric patient sera. The magnitude of interferences varies widely from method to method. Carbonyl compounds, dopamine, cephalosporines, and bilirubin interfere with the Jaffé reaction. Bilirubin, creatine, dopamine, ascorbic acid, and sarcosine interfere with the enzymatic methods. We conclude that the elimination of interferences in the determination of creatinine has still not been achieved.
Evaluation of an assay of the free beta-subunit of choriogonadotropin and its potential value in screening for Down's syndrome
Authors: Spencer Kevin
Published: January 2020

In this study I investigated the analytical and clinical performance of the measurement of the free beta-subunit of choriogonadotropin (hCG) in normal pregnancies and in pregnancies affected by Down's syndrome. Free beta-hCG in maternal serum has been shown to be increased in Down's syndrome-affected pregnancies and is proportionally increased in more cases than is total hCG. This study confirms previous findings of low concentrations of unconjugated estriol and alpha-fetoprotein in maternal serum in Down's syndrome-affected pregnancies. Using a multivariate risk analysis of maternal age and concentrations of alpha-fetoprotein, unconjugated estriol, and hCG in maternal serum, I determined that, at a risk cutoff value of 1 in 300, 52% of Down's cases could be detected with total hCG in the calculation, compared with 66% with the free beta-hCG concentration. The false-positive rate was 5.9% in both cases. Therefore, free beta-hCG can be used effectively in a screening program for Down's syndrome; however, further studies are required to ascertain whether the measurement of free beta-hCG has any advantages over the use of total hCG for detecting Down's syndrome.
Multiple forms of alkaline phosphatase in plasma of hemodialysis patients
Authors: Tibi L , Chhabra S C , Sweeting V M , Winney R J , Smith A F
Published: January 2020

We used quantitative assays to measure the activity of the bone, liver, and intestinal forms of alkaline phosphatase in plasma in 75 patients with endstage chronic renal failure undergoing hemodialysis. The results were correlated with radiological and other biochemical indices of bone disease and with biochemical indices of liver disease. The total activity of alkaline phosphatase in plasma increased in 28 patients. In 10 of these patients, nine of whom had increased activity of gamma-glutamyltransferase in plasma, the increase in total activity of alkaline phosphatase was from the liver isoenzyme alone (nine patients) or from the liver and bone isoenzymes together (one patient). Intestinal alkaline phosphatase in plasma, although greater than 23 U/L in eight patients, was solely responsible for the increase in total alkaline phosphatase in one patient (who had normal gamma-glutamyltransferase). Bone alkaline phosphatase in plasma was increased in 25 patients, seven of whom had normal total alkaline phosphatase, and was closely correlated (r = 0.78) with osteocalcin concentration in plasma, which was increased in a much greater proportion of patients (99%). Both total and bone alkaline phosphatase were correlated with parathyrin in plasma (r = 0.46 and 0.50, respectively) and with osteocalcin (r = 0.60 and 0.78, respectively). Osteocalcin and bone alkaline phosphatase, but not parathyrin, decreased with age, implying that the skeletal response to parathyrin may be age dependent. In patients with increased total alkaline phosphatase undergoing hemodialysis, the concurrent measurement of gamma-glutamyltransferase may help identify whether the enzyme increase originates from the liver or bone, but this approach wrongly identified the source of the increase in three of 28 patients. Therefore, we recommend a separate measurement of the bone isoenzyme of alkaline phosphatase.
Assessing acute parathyroid responsiveness in hemodialysis patients by measuring intact parathyrin in pre- and post-dialysis serum
Authors: Dilena Beverly A , White Graham H
Published: January 2020

We measured pre- and post-dialysis concentrations of ionized calcium (iCa) in whole blood, total calcium (tCa) in plasma, and intact parathyrin (PTH) in serum of 19 patients undergoing maintenance hemodialysis. Plasma tCa was inappropriately increased relative to iCa in 63% of the specimens; the iCa correlated with the PTH concentration in 12 of 19 pre-dialysis specimens, whereas tCa correlated with PTH in only five patients. During dialysis, 16 patients had analytically significant changes in iCa (i.e., exceeded the analytical imprecision of 0.04 mmol/L). Pre- and post-dialysis concentrations of PTH were normal in six patients, four of whom showed a detectable response to changes in iCa. Ten patients had increased PTH in at least one specimen; of these, eight had responsive parathyroid glands. Five of the 16 patients had an increased set point for calcium. The minimal PTH responses of two patients suggested refractory hyperparathyroidism. We conclude that routine estimation of iCa, rather than tCa, in dialysis patients markedly improves the identification of patients at risk for secondary hyperparathyroidism, and that measurement of intact PTH in pre- and post-dialysis serum offers a simple means of assessing parathyroid responsiveness in dialysis patients.
One-step chromogenic equivalent of activated partial thromboplastin time evaluated for clinical application
Authors: Ponjee Gabriëlle E , Vader Huib L , de Wild Net J , Janssen Ger W T , van der Graaf Fedde
Published: January 2020

We evaluated the clinical usefulness of a recently developed semi-automated one-step chromogenic equivalent of activated partial thromboplastin time (APTT; Behring). This simple test is easily adaptable for automation. Generally, the results with this chromogenic one-step APTT were at least as precise as those obtained with comparative coagulometric methods. The chromogenic one-step APTT showed, both in vitro and in vivo, adequate sensitivity to congenital intrinsic factor deficiency but no sensitivity to Factor VII deficiency. Unlike a two-step coagulometric APTT (Dade), the one-step chromogenic APTT seemed sensitive to activation products of the contact system, which are present in immunoadsorbed factor-deficient plasma. The in vitro sensitivity of the chromogenic APTT to heparin was comparable with that of a coagulometric APTT, but the sensitivity to heparin in patients' samples differed slightly. The chromogenic APTT is relatively insensitive to anomalies in the fibrinogen-fibrin conversion. Finally, we observed discrepancies between the chromogenic and coagulometric APTT results for plasma of patients with disseminated intravascular coagulation. We conclude that this one-step chromogenic APTT warrants further evaluation for possible use as a routine test for the clinical laboratory.
Clinical validation of a second-generation fructosamine assay
Authors: Cefalu William T , Bell-Farrow Audrey D , Petty Marie , Izlar Camille , Smith Jeffrey A
Published: January 2020

The serum fructosamine assay, used to monitor short-term clinical glycemic control, reportedly has several technical drawbacks. However, technical improvements have resulted in a new second-generation assay of fructosamine. We evaluated this second-generation assay (from Roche Diagnostics) in 529 nondiabetic and diabetic patients and found a highly significant correlation with results of the first-generation assay (r = 0.91, P less than 0.001). Use of the second-generation assay with samples from patients classified on the basis of glycemic control according to their glycohemoglobin (GHb) values, enabled us to discriminate between the nondiabetics, diabetics with "good/moderate" control (i.e., GHb less than 10%), and diabetics with "poor" control (GHb greater than or equal to 10%). We evaluated the validity of the second-generation assay to assess short-term glycemic control in 23 non-insulin-dependent diabetic patients who participated for 10 weeks in an intensive intervention program designed to rapidly normalize the clinical glycemic profile. Results correlated significantly with the one-week average capillary blood glucose concentration (CBG) and with the three-week average CBG in all 23 patients. In addition, the second-generation fructosamine assay results demonstrated a significant decrease at each week of study, as did the average CBG. Results of the first- and second-generation assays correlated significantly at each week of study. GHb correlated significantly with both the second- (r = 0.78, P less than 0.001) and first-generation fructosamine assay results (r = 0.77, P less than 0.001) for the baseline blood samples of the intervention study, but this correlation decreased (to r = 0.35, P = 0.09 and r = 0.34, P = 0.09, respectively) by the conclusion of the study.
Multicenter evaluation of new enzyme-linked immunoassays of follitropin and lutropin in serum or plasma
Authors: Thijssen J H , Wood W G , Kessler A C , Griesser H W , Bauer O , Bieglmayer C , Eugui J , Mirabel J L , Gaessler H , Klinga K
Published: January 2020

Results from a multicenter evaluation of two new enzyme-linked immunosorbent assays [Enzymun-Test for follitropin (FSH) and lutropin (LH)] are presented and compared with results from 11 other commercial immunoassays, radioactive as well as nonradioactive. Enzymun-Test FSH and LH assays are suitable for automated systems and manual applications. The tests were reproducible (CV less than 5%), highly specific, and sensitive enough (less than 0.5 int. unit/L) to measure the hormones directly in almost all patients' samples, except for LH measurements in prepubertal children. We did not find interference by heterophilic antibodies or other factors. A comparison of assays for FSH found very good agreement among all modern two-site assays; competitive immunoassays almost invariably yielded systematically lower results for FSH, probably because of the heterogeneity of the International Reference Preparation (2nd IRP FSH, 78/549). For LH also we found good agreement, with no systematic differences among the various reagents. Guidelines for reference values with the new reagents are given.
Automated measurement of amylase isoenzymes by a double kinetic assay with "blocked" beta-2-chloro-4-nitrophenyl maltopentaoside as substrate and with wheat germ inhibitor
Authors: Abe A , Nishimura T , Noma A , Hamano K
Published: January 2020

We evaluated the enzymic mechanism by which 3-keto butylidene-beta-2-chloro-4-nitrophenyl maltopentaoside (3KB-G5-CNP) serves as a substrate for serum pancreatic (p-) and salivary (s-) amylases. In aliquots of the reaction mixture, three kinds of beta-2-chloro-4-nitrophenyl oligosaccharides (glucose, maltoside, and maltotrioside) were separated from the substrate by high-performance liquid chromatography. Both isoenzymes behaved nearly identically and produced almost the same products. We automated a double kinetic procedure for determining total (t-) and p-amylase with use of a selective inhibitor from wheat germ in a single channel on the Hitachi 7050 analyzer. Within- and between-run CVs were, respectively, 0.5% and 1.7% for t-amylase (240 U/L), and 0.7% and 2.3% for p-amylase (230 U/L). The test results varied linearly with concentrations up to approximately 2000 U/L for t- and p-amylase activities. p/s ratios varied from 0.2 to 5.0. Results correlated well with those obtained by the monoclonal inhibition method (r = 0.992).
Interference in thyroid-function tests in postpartum thyroiditis
Authors: John R , Othman S , Parkes A B , Lazarus J H , Hall R
Published: January 2020

Three women are described from a study of patients with postpartum thyroiditis whose sera gave spuriously increased concentrations of free thyroid hormone because of antibody binding of radiolabeled thyroxin (T4) and triiodothyronine (T3) analogs. All of the women showed increased serum concentrations of thyroid autoantibodies. The antibody binding of radiolabeled analogs and its effect on free T4 and free T3 assays disappeared by 48 weeks postpartum. Postpartum women who develop thyroid autoantibodies have approximately 2% prevalence of increased binding of radiolabeled analogs, which can result in an interference in thyroid hormone assays involving T4 and T3 analogs.
S-troponin T in suspected ischemic myocardial injury compared with mass and catalytic concentrations of S-creatine kinase isoenzyme MB
Authors: Gerhardt W , Katus H , Ravkilde J , Hamm C , Jørgensen P J , Peheim E , Ljungdahl L , Löfdahl P
Published: January 2020

In a multicenter study we compared three tests for ischemic myocardial injury (IMI): a new, automated enzyme immunoassay for S-troponin T (S-TNT; Boehringer Mannheim) and two S-creatine kinase (CK) isoenzyme MB assays (mass and catalytic concentrations). For critical evaluation of clinical sensitivity, we studied 243 cases with an IMI prevalence of 43% and an 18% prevalence of cases with unstable angina. Relative peak values of S-TNT and S-CK-MB (mass) after onset of pain were four- to fivefold higher than S-CK-MB (catalytic) results. Increases of S-TNT and S-CK-MB (mass), even though still within their reference ranges, indicated minor myocardial damage in about one-third of the cases primarily classified as unstable angina. The diagnostic window for S-TNT ranged from hours to weeks after the acute episode. The time courses were frequently biphasic, with the initial S-TNT peak closely paralleling that of the mass concentrations of S-CK-MB. With a biological half-life for S-TNT of 2 h, the prolonged increases in S-TNT indicate a continuous release of S-TNT from necrotizing cells. Clinical specificities of S-TNT and S-CK-MB (mass) were greater than that of S-CK-MB (catalytic), even in the presence of 30% to 40% severe skeletal muscle injuries. The combination of S-TNT and S-CK-MB (mass) is excellent for detection of acute IMI, including minor myocardial damage.
Prevalence of familial dysalbuminemic hyperthyroxinemia in serum samples received for thyroid testing
Authors: Arevalo G
Published: January 2020

The prevalence of familial dysalbuminemic hyperthyroxinemia (FDH), a condition sometimes mistaken for hyperthyroidism, has not been clearly established. I present a study of the prevalence of FDH in serum samples received for thyroid-function tests in a reference laboratory. A prospective study of 15,674 serum samples was carried out over 24 months, of which 13,232 cases were from women (84.42%) and 2442 were from men (15.58%). FDH was diagnosed in 26 cases, 22 in women and four in men. Therefore, the prevalence of FDH in the total number of samples from both sexes was 0.17%, 0.17% in women, and 0.16% in men, which is consistent with a dominant autosomal type of familial transmission. These findings demonstrate that cases of FDH occur frequently; therefore, every laboratory must be prepared to recognize them and thus avoid an incorrect diagnosis of the patient's thyroid function.
What can be achieved in an external quality-assessment scheme with a small number of participants: four years of experience with thyroid-related tests in Israel
Authors: Kahana L , Yechieli H
Published: January 2020

The Israel External Quality-Assessment scheme (EQAS) provided service from 1985 to 1989, primarily to laboratories in Israel. Participation was voluntary and confidential, and involved 30-35 laboratories performing thyroid-related function tests. Scheme design included 36 human serum specimens distributed to the participants at the beginning of each year. For 12 months, three specimens were analyzed each month and the results were reported to EQAS on pre-agreed dates. Monthly reports sent by EQAS to participants after analysis of the results included the overall consensus mean used as target value, between-laboratory agreement, individual laboratory bias, and recovery data. The overall consensus mean calculated as all-laboratory trimmed mean was validated by reproducibility and recovery studies. Samples with undetectable concentrations of thyrotropin (TSH), obtained from patients proven thyrotoxic, were used to validate measurement of very low TSH concentrations. During the scheme, liquid serum was found superior to lyophilized specimens for distribution to the participants. The scheme helped stimulate major improvements in between-laboratory agreement, especially for low TSH concentrations, with CVs decreasing from 180% in the first year to 20% in the fourth year. Each laboratory's performance also improved as judged by the median bias and variability of bias and by the considerable decrease in the number of laboratories with unacceptable performance for all tests. Method-related differences in performance were observed despite the small number of participants. Better methods, e.g., a radioimmunometric method for TSH, were detected and adopted by the participants, with concomitant improvement in performance. Transfer to analog procedures for free thyroxin, in addition to, but mostly instead of, thyroxin, was also documented, with no gain in analytical performance. Despite this being a small scheme, most of the goals achievable with a large scheme were realized.
Polymerase chain reaction and Q beta replicase amplification
Authors: Cahill P , Foster K , Mahan D E
Published: January 2020

The polymerase chain reaction (PCR) and Q beta replicase are two methods in which nucleic acid polymerases are used for amplification. Although these approaches share many similar problems concerning target contamination and probe specificity, they differ dramatically in their mechanisms of action and modes of application. The PCR method amplifies target sequences between two priming oligonucleotides and in essence amplifies a portion of the analyte. Q beta replicase, on the other hand, amplifies a specific template molecule hybridized to target sequences and therefore amplifies a signal component of the system. For this reason, Q beta replicase amplification has applications in areas other than for the detection of nucleic acid sequences. The requirements for application and the advantages of both PCR and Q beta replicase amplification are reviewed.
Multiple labeling and time-resolvable fluorophores
Authors: Diamandis E P
Published: January 2020

A new time-resolved fluorescence immunoassay system involving use of the europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid as label is reviewed. This stable chelate by itself is not very fluorescent but, used in multiple labeling strategies, improves the achievable detection limits. By using multiple labeling, streptavidin tailing, and Eu3+ activation, one can create a very stable, easy-to-use reagent that is suitable for devising highly sensitive immunoassays and other biotechnological assays. This reagent, a streptavidin-based macromolecular complex, is able to detect approximately 300,000 molecules (approximately 0.5 amol) of alpha-fetoprotein in a model noncompetitive immunoassay.
Optical biosensor assay (OBA)
Authors: Tsay Y G , Lin C I , Lee J , Gustafson E K , Appelqvist R , Magginetti P , Norton R , Teng N , Charlton D
Published: January 2020

We describe a new biosensor immunoassay involving optical diffraction to detect clinically important analytes in human body fluids. A silicon wafer is used as a support for immobilization of antigen or antibody. The protein-coated surface is illuminated through a photo mask to create distinct periodic areas of active and inactive protein. When the surface is incubated with a positive sample, antigen-antibody binding occurs only on the active areas. Upon illumination with a light source such as a laser, the resulting biological diffraction grating diffracts the light. A negative sample does not result in diffraction because no antigen-antibody binding occurs to create the diffraction grating. The presence or absence of a diffraction signal differentiates between positive and negative samples, and the intensity of the signal provides a quantitative measure of the analyte concentration. The technique is demonstrated with a quantitative assay of choriogonadotropin in serum.
Elimination of matrix and spectral interferences in the measurement of lead and cadmium in urine and blood by electrothermal atomic absorption spectrometry with deuterium background correction
Authors: D'Haese P C , Lamberts L V , Liang L , Van de Vyver F L , De Broe M E
Published: January 2020

Direct measurement of lead and cadmium in blood and urine by electrothermal atomic absorption spectrometry with deuterium background correction (D2-AAS) is prone to severe matrix and spectral interferences. We overcame these effects by coating the L'vov platform with ammonium molybdate, reducing the atomization time, introducing a post-atomization cooling step, carefully selecting ashing and atomization temperatures, and using an appropriate procedure for matrix modification. To determine Pb and Cd in blood and urine, we used matrix-matched calibration curves. With the proposed procedure for sample preparation, both Pb and Cd in whole blood can be determined in the same diluted sample. Results obtained by D2-AAS correlate closely with those by Zeeman-corrected AAS. Detection limits (mean blank + 3 SDblank) for Pb in urine and blood were 4 micrograms/L. For cadmium, the detection limits were 0.4 and 0.1 micrograms/L for urine and blood analysis, respectively. Between-run CVs were less than 5.0%.
Application of Neural Networks to the Interpretation of Laboratory Data in Cancer Diagnosis
Authors: Astion Michael L , Wilding Peter
Published: January 2020

Neural networks are a relatively new method of multivariate analysis. The purpose of this study was to investigate the ability of neural networks to differentiate benign from malignant breast conditions on the basis of the pattern of nine variables: patient age, total cholesterol, high-density lipoprotein cholesterol, triglycerides, apolipoprotein A-I, apolipoprotein B, albumin, the tumor marker CA15-3, and the Fossel index (measurement of methylene and methyl line-widths in proton NMR spectra). The laboratory analyses were made with blood plasma or serum specimens. The neural network was "trained" with 57 patients: 23 patients with breast malignancies and 34 patients with benign breast conditions. A neural network with nine input neurons, 15 hidden neurons, and two output neurons correctly classified all 57 patients. The ability of the network to predict the diagnoses of patients that it had no encountered in training was tested with a separate group (cross-validation group) of 20 patients. The network correctly predicted the diagnoses for 80% of these patients. For comparison we analyzed the same sets of 57 training patients and 20 cross-validation patients by quadratic discriminant function analysis. The quadratic discriminant function, calculated from the same 57 patients used to train the neural network, correctly classified 84% of the 57 patients, and correctly diagnosed 75% of the 20 cross-validation patients. The results suggest that neural networks are a potentially useful multivariate method for optimizing the diagnostic utility of laboratory data.
Hydroxyl Radical-Induced Characteristic Chemiluminescent Spectra from Plasma of Hemodialysis Patients
Authors: Agatsuma Shinichi , Nagoshi Toshiyuki , Kobayashi Masaki , Usa Masashi , Watanabe Haruo , Sekino Hiroshi , Inaba Humio
Published: January 2020

Plasma from hemodialysis patients evoked weak photon emissions (chemiluminescence) in a characteristic emission spectrum with a peak at 430 nm, attributed to attack by hydroxyl radicals generated from the iron-catalyzed breakdown of hydrogen peroxide (Fenton reaction), whereas plasma from normal healthy subjects showed a rather weak red chemiluminescence peak at around 680 nm, similar to that resulting from attack by hydroxyl radicals. However, the addition of hydrogen peroxide in the absence of divalent irons induced almost the same red chemiluminescent emission spectrum in both plasmas. The HPLC-gel-filtration chromatography carried out with both plasmas revealed that a primary emitter evoking a peak emission at 430 nm was located in the fraction of lower-molecular-mass substances in fractionated plasma from hemodialysis patients. In contrast, the elution peaks evoking red chemiluminescence with the addition of hydrogen peroxide were mainly observed for the higher-molecular-mass fraction, as determined by gel chromatography of both plasmas. Therefore, the observation of a chemiluminescence peak at 430 nm, induced by the generation of hydroxyl radicals, correlated well with chemiluminescent emissions in plasma samples from patients with chronic renal failure. Spectral analyses of clinical samples that show weak chemiluminescence by forced oxidation by such an active oxygen may provide a new and more sensitive method for diagnosing metabolic disorders.
Development of Time-Resolved Immunofluorometric Assay of Vascular Permeability Factor
Authors: Yeo Kiang-Teck , Sioussat Tracy M , Faix James D , Senger Donald R , Yeo Tet-Kin
Published: January 2020

We describe a two-site time-resolved immunofluorometric assay for guinea pig vascular permeability factor (VPF) for quantifying VPF in different biological fluids. Antibody against the carboxy terminus (C-IgG) is immobilized on microtiter wells, and antibody against the amino terminus (N-IgG) is labeled with Eu(3+)-chelate. Line 10 tumor culture medium, known to be rich in VPF, is assayed in a two-step incubation. Bound Eu3+ is then quantified by dissociation into a fluorescent enhancement solution, with measurement of the time-resolved fluorescence. The analytical sensitivity is 0.35 VPF unit, and the intra-assay CV is about 20%. The assay is specific for VPF, because pre-treatment with the appropriate C- or N-peptide, or pre-extraction of VPF, greatly decreases fluorescence. The VPF immunoassay is highly correlated (r2 = 0.94) with the Miles permeability assay, the classical bioassay of VPF. In addition, the immunofluorometric assay is about 30-fold more sensitive than the Miles assay.
Direct, Simplified, and Sensitive Assay of Angiotensin II in Plasma Extracts Performed with a High-Affinity Monoclonal Antibody
Authors: Simon D , Romestand B , Huang H , Badouaille G , Fehrentz J A , Pau B , Marchand J , Corvol P
Published: January 2020

A very simple, fast, and sensitive RIA of angiotensin (Ang) II has been developed, based on a monoclonal antibody with high affinity and specificity, making possible the direct measurement of circulating Ang II in human plasma after solid-phase extraction. The purified monoclonal antibody 4D8 has an association constant of 1.3 x 10(11) L/mol with Ang II and a cross-reactivity of < 1% for Ang I. The assay can detect as little as 0.8 fmol of Ang II in 2 mL of plasma and is not influenced by the presence of Ang I. Analytical recoveries between 112% and 116% were obtained for Ang II added to human plasma at physiological concentrations. Comparison of the RIA with a reversed-phase, high-performance liquid chromatographic method followed by RIA to measure Ang II in human plasma samples from normal and hypertensive subjects--and from normotensive subjects before and after an acute inhibition of angiotensin-converting enzyme with captopril (50 mg)--showed a high degree of correlation (r2 = 0.93) between the two methods.
Sensitive, Specific Radioimmunoassay for Quantifying Pergolide in Plasma
Authors: Bowsher R R , Apathy J M , Compton J A , Wolen R L , Carlson K H , DeSante K A
Published: January 2020

Pergolide, a synthetic ergoline with potent dopaminergic activity, is used to treat Parkinson disease. The low plasma concentrations of pergolide achieved during therapy complicate the development of a method for its analysis. Because radioimmunoassay successfully measures other structurally related ergolines in physiological fluids, we undertook the development of a radioimmunoassay of pergolide. The detection limit of the radioimmunoassay is 21 ng/L with an optimal working range from 100 to 1000 ng/L. We maximized assay specificity by using a monoclonal antibody that displayed low cross-reactivity with pergolide sulfoxide, a major metabolite found in animals. The radioimmunoassay has performed acceptably for > 2 years during toxicology studies with rats and rhesus monkeys and in clinical studies involving patients with Parkinson disease. We consider the radioimmunoassay a valid method for quantifying therapeutic concentrations of pergolide in plasma.
Determination of Fetal Hemoglobin by Time-Resolved Immunofluorometric Assay
Authors: Turpeinen U , Stenman U H
Published: January 2020

We have developed a "sandwich"-type time-resolved immunofluorometric assay (IFMA) for fetal hemoglobin (HbF) in hemolysates from adults and newborns, amniotic fluid, and plasma, based on a polyclonal and a monoclonal antibody against human fetal hemoglobin. Microtiter wells are coated with polyclonal capture antibody, and the gamma-chain-specific monoclonal tracer antibody is labeled with a europium chelate. In a simple and fast assay procedure, prediluted hemolysates are incubated in the microtiter wells first with capture antibody for 1 h and, after washing, for 1 h with tracer antibody. The wells are washed and the fluorescence of europium is measured. The mean analytical recovery is 102% and results by IFMA agreed well with values obtained by high-performance liquid chromatography. The analytical range of IFMA is large and well suited for clinical purposes. The detection limit of the assay is 0.2 microgram/L and the measuring range extends to 500 micrograms/L.
Precipitation and Characterization of an Aluminosilicate from ALCL3-Na2SiO3-HCL in Serum, of Interest for Alzheimer Disease
Authors: Bilinski H , Horvath L , Trbojevic-Cepe M
Published: January 2020

A precipitation experiment was performed with human serum to model aluminosilicate formation in brains of patients with Alzheimer disease. Aluminum and (or) silicate ions were added to serum in a 1:2 molar ratio at pH 7.4. Precipitates formed immediately and were left for 24 h at 37 degrees C before filtration. Silicate and aluminosilicate formed precipitates with human serum proteins albumin, transferrin, and IgG. In untreated samples, the IgG/albumin ratio increased slightly compared with the ratio in dried serum. Diethylbarbiturate-washed precipitates had a significantly lower protein content than did untreated ones. The IgG/albumin ratio increased considerably in the sample containing aluminosilicate. We conclude that IgG is the sodium dodecyl sulfate-soluble human protein most firmly bound to the aluminosilicate matrix. From 27Al magic-angle-spinning nuclear magnetic resonance (MAS NMR), a pronounced peak was found at 52.79 ppm and a minor peak at 0.53 ppm, suggesting that 4-coordinated aluminum predominates and that 6-coordinated aluminum is present in a smaller proportion. The 29Si MAS NMR spectrum shows a poorly ordered structure. The aluminosilicate formed also contains the cations Na+ > K+ > Ca2+ > Mg2+ and anions Cl- > PO4(3-). Rather than looking for aluminum toxicity to explain the effects of Alzheimer disease, one should consider that by precipitating such a composite phase, the balance of cations, anions, and proteins in human serum is changing.
Specific Assay of Serum Lactate Dehydrogenase Isoenzyme 1 by Proteolysis with Alpha-Chymotrypsin and Protein Denaturation
Authors: Shirahase Y , Watazu Y , Kaneda N , Uji Y , Okabe H , Karmen A
Published: January 2020

We devised a method for assaying serum lactate dehydrogenase isoenzyme 1 (LD-1) activity specifically by preincubation with alpha-chymotrypsin and guanidine. Cleavage of phenylalanine bonds in the loop of A and B subunits of LD-3, LD-4, and LD-5 isoenzymes (residues 117-119) by incubation with alpha-chymotrypsin for a short time completely inactivated these isoenzymes and partially inactivated LD-2. Addition of guanidine (0.50 mol/L, pH 7.8) to the incubation mixture containing the chymotrypsin completed the inactivation of LD-2. As much as 4000 U/L of LD-2, LD-3, LD-4, and LD-5 were inactivated, whereas LD-1 was affected only slightly. Results by this method (y) correlated well with those by the Roche Isomune immunochemical LD-1 method (x): y = 0.98 x -0.11, r = 0.99 (n = 60). Within-run CVs were 0.5-2.5%. Several common interferents had no effect. In 500 healthy people, serum LD-1 ranged between 66 and 130 U/L, with a mean +/- SD of 88 +/- 15 U/L.
Cross-Reactivity of Anti-Digoxin Antibodies with Digitoxin Depends on Tracer Structure
Authors: Stenzel R , Reckmann B
Published: January 2020

The most common methods for measuring digoxin concentrations in serum are immunoassays. The prerequisite for exact determination of the digoxin value is an antibody that specifically binds digoxin. Because digitoxin differs from digoxin only in the C-12 hydroxy group, it is difficult to obtain anti-digoxin antibodies that do not cross-react with this compound. During the development of a fluorescence polarization immunoassay (FPIA) for digoxin, we investigated digoxin tracers with different structures. We found that in FPIA the digitoxin cross-reactivity of an antibody could be reduced by varying the structure of the tracer molecule.
Sandwich Immunoassay for Intact Human Osteocalcin
Authors: Hosoda K , Eguchi H , Nakamoto T , Kubota T , Honda H , Jindai S , Hasegawa R , Kiyoki M , Yamaji T , Shiraki M
Published: January 2020

To overcome the problems of limited-region specificity associated with conventional radioimmunoassay (RIA), we developed a sandwich enzyme immunoassay (EIA) for intact human osteocalcin (hOC). For this EIA we used antibodies to the N- and C-terminal regions of hOC that were raised against an N-terminal 20-residue peptide and a C-terminal 7-residue peptide, both synthetic. Immunoassay profiles of tryptic digests of hOC and serum from patients with renal failure, fractionated by reversed-phase HPLC, facilitated direct demonstration of the region specificity of this method. A preliminary study of serum osteocalcin concentrations in patients with renal failure further confirmed this specificity, showing lower positive rates obtained by this method than by conventional RIA. The cross-reactivity data of hOC with bovine and rat osteocalcins by the sandwich method indicated its species specificity. These studies demonstrate the superior specificity of this sandwich EIA compared with conventional RIA and thus confirm its potential diagnostic superiority.
Plasma Lipoprotein(a) Values and Severity of Coronary Artery Disease in a Large Population of Patients Undergoing Coronary Angiography
Authors: Labeur C , De Bacquer D , De Backer G , Vincke J , Muyldermans L , Vandekerckhove Y , Van der Stichele E , Rosseneu M
Published: January 2020

To determine possible associations between lipoprotein(a) [Lp(a)] and the severity of coronary artery lesions, we measured lipid, apolipoprotein, and Lp(a) in a large population of Belgian patients (n = 1054) undergoing coronary angiography. In both women and men, univariate analysis demonstrated significant differences in the Lp(a) concentrations according to the severity of the coronary stenosis. However, after adjustment for possible confounding factors, many of these differences were attenuated, indicating that other variables that differentiate patients from control subjects also influence Lp(a) distribution. Differences in lipid, apolipoprotein, and Lp(a) concentrations between male and female patients are discussed.