bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative analyses of the interaction between calcium ions and human serum albumin
Authors: Kragh-Hansen U , Vorum H
Published: January 2020

We examined the suitability of nine organic buffers for studying calcium binding to albumin by equilibrium dialysis. Results obtained with defatted human serum albumin showed that 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 2-([tris(hydroxymethyl)methyl] amino)ethanesulfonic acid were superior. Scatchard analysis of the experimental data from an extensive study performed in HEPES at pH 7.4 and 20 degrees C revealed (putting n(i) = 1, i = 1-4) k1 = 367 L/mol, k2 = 314 L/mol, k3 = 291 L/mol, and k4 = 179 L/mol. The very weak binding was characterized as n5 = 10 and k5 = 40 L/mol. The results were also analyzed in terms of stoichiometric association constants. The constants K1 and K2 were calculated to be 1513 and 647 L/mol, respectively, whereas the other constants were considered undeterminable. pH studies showed that in the interval 6.8-7.4, binding was not influenced by changes in acidity. Increasing pH to above the physiological value resulted in increased binding. At pH 8.0, k1 was increased almost fourfold, whereas k2 and k3 were approximately doubled. These findings indicate that the neutral to basic transition is important for the calcium-binding properties of albumin. The transition is a reversible, gradual conformational change of the protein at pH 6-9.
Distribution of cyclosporin G (NVa2 cyclosporin) in blood and plasma
Authors: Yatscoff R W , Honcharik N , Lukowski M , Thliveris J , Chackowsky P , Faraci C
Published: January 2020

We report here on the distribution of cyclosporin G (CsG), an analog of cyclosporin A, in whole blood. CsG has a temperature-dependent distribution between erythrocytes (RBCs) and plasma. After 30 min at 37 degrees C, the plasma/whole blood and plasma/RBC ratios were relatively constant (0.7-0.8) up to CsG at 1000 micrograms/L. At 5000 micrograms/L, this ratio increased to 1.0-1.1 for plasma/whole blood and 1.7 for plasma/RBC. The primary CsG metabolites GM1 and GM9 were sequestered within RBCs to a greater extent than was the parent drug. In whole blood, approximately 2% of CsG was bound to granulocytes, 6% to lymphocytes, and 50-55% to RBC, and 35-40% was found in the plasma fraction. The free fraction of the drug as determined by ultracentrifugation was 5-6% and 13-17% of total drug at 37 and 4 degrees C, respectively. In plasma the drug was primarily associated with high-density lipoprotein (50-60%) and to a lesser degree with low-density (20-30%) and very-low-density (10%) lipoproteins.
Laboratory diagnosis of medium-chain acyl-coenzyme A dehydrogenase deficiency by the amplification refractory mutation system
Authors: Tsai M Y , Schwichtenberg K , Tuchman M
Published: January 2020

We used the amplification refractory mutation system (ARMS)--a polymerase-chain-reaction-based method--to detect the 985 A-to-G mutation of the gene coding for the enzyme medium-chain acyl-CoA dehydrogenase (MCAD). The 985 A-to-G allele is thought to account for approximately 89% of the mutant alleles in this disorder and at least one copy was reported to be present in 98% of affected individuals. ARMS provided a simple and robust method that reliably identified the 985 A-to-G mutant allele in patients either homozygous or heterozygous for this allele. Combined with organic acid analysis, ARMS can provide definitive diagnosis for the great majority of the patients with MCAD deficiency. The method, therefore, should be useful in clinical laboratories involved with the diagnosis of inborn errors of metabolism.
Assessment of the role of G proteins and inositol phosphate production in the action of gonadotropin-releasing hormone
Authors: Hawes B E , Conn P M
Published: January 2020

The first step in gonadotropin-releasing hormone (GnRH) action involves the binding of GnRH to a plasma membrane receptor. Calcium has been implicated as a second messenger molecule. More recently, it has been suggested that the products of phosphoinositide metabolism may act as a second messenger for GnRH-stimulated release of luteinizing hormone (LH). To be considered a second messenger, however, a candidate molecule must meet three requirements: in the second messenger's presence, (a) GnRH should stimulate increased production of inositol phosphate; (b) inositol phosphate production, stimulated by any means, should provoke LH release; and (c) inhibition of inositol phosphate production should block GnRH-stimulated release of LH.
Simplified enzymatic assay of angiotensin-converting enzyme in serum
Authors: Groff J L , Harp J B , DiGirolamo M
Published: January 2020

We report a simple, enzymatic method for determining angiotensin-converting enzyme (ACE; EC 3.4.15.1) in serum. The proposed method features coupling an established reaction catalyzed by gamma-glutamyltransferase (GGT; EC 2.3.2.2) to the ACE reaction, which releases glycylglycine from the artificial substrate hippuryl-glycyl-glycine. The glycyl-glycine released by the ACE reaction becomes rate-limiting in the GGT reaction, in which it participates as a receptor substrate for a gamma-glutamyl group transferred from a donor substrate, L-gamma-glutamyl-3-carboxy-4-nitroanilide. The reaction releases 3-carboxy-4-nitroaniline, which is monitored spectrophotometrically at 410 nm. The resulting rate of change in absorbance is linearly related to the glycyl-glycine concentration and therefore to the activity of ACE. The linear range of the method extends from ACE values < 50 to at least 1300 U/L of serum. Good precision is indicated by a low CV for replicate analyses (3.6% and 4.6% for within-run and day-to-day assays, respectively, for normal ACE activity, and 3.1% within-run for high ACE activity). Results also correlate well with those of an established colorimetric method (r = 0.978). The major advantages of the method are its procedural simplicity, limited cost, use of readily available reagents, applicability to isoenzyme studies, and adaptability to automation.
Behavior of tumor markers CA19.9, CA195, CAM43, CA242, and TPS in the diagnosis and follow-up of pancreatic cancer
Authors: Banfi G , Zerbi A , Pastori S , Parolini D , Di Carlo V , Bonini P
Published: January 2020

We compared the recently proposed tumor markers CA195, CA242, and CAM43 with a widely used antigen, CA19.9, and a circulating marker of cellular proliferation, TPS, to define their specificity, sensitivity, and cost-benefit ratio. The tumor markers were measured in 41 pancreatic carcinoma patients and in two control groups, the first comprising 19 patients with benign pancreatic diseases, the second comprising 41 healthy blood donors. Sensitivities were 79% for CA19.9, 57% for CA242, 60% for CAM43, 76% for CA195, and 98% for TPS. Specificities calculated for the group with pancreatic diseases were 60% for CA19.9, 84% for CA242, 95% for CAM43, 53% for CA195, and 22% for TPS. Specificities for the blood donor group were 100% for CA19.9, 93% for CA242, 98% for CAM43, 85% for CA195, and 88% for TPS. Positive values for the tumor markers appeared from second stage (Hermreck classification). Metastases, invasion of lymph nodes, and coupling of cancer-associated antigens did not significantly modify marker sensitivity. In pancreatic carcinoma, CA19.9 showed good sensitivity (79%) and high specificity (60-100%). In view of their own advantages (e.g., high specificity of CAM43, high sensitivity of TPS in recurrences) and limits (e.g., low sensitivity of CAM43, very low sensitivity of TPS), the other markers could be used alone or with CA19.9. Two pairs of tumor markers showed high similarity in our study: CA19.9 and CA195, and CAM43 and CA242.
Aberrant PO2 values in proficiency testing
Authors: Fonzi C E , Clausen J L , Mahoney J
Published: January 2020

We prospectively determined the frequency of aberrant vials of fluorocarbon/buffer used for proficiency testing of measurements of pH, PCO2, and PO2, using 20 duplicate vials from 12 lots of fluorocarbon/buffer and two arterial blood gas analyzers in eight reference laboratories. We defined aberrant vials as vials for which both duplicate measurements differed from the mean value of repeated measurements for the specific instrument (for each lot of testing materials) by > 0.04 for pH, > 10% of the mean or 3.0 mm Hg, whichever was greater, for PCO2; or > 10% of the mean or 6 mm Hg, whichever was greater, for PO2. Four of 1620 vials (0.25%) were aberrant, all based on PO2 measurements (< 70 mm Hg); all would have failed in both American/California Thoracic Societies and College of American Pathologists proficiency programs. The average intra-instrument SDs of repeated measures (range of mean values: pH, 7.181-7.631; PCO2, 12.7-65.9; PO2, 32.5-150.1) were 0.0055 for pH, 0.67 mm Hg for PCO2, and 1.65 mm Hg for PO2. Deliberate contamination of the fluorocarbon emulsion with room air, as might occur during sampling from the vial, indicated that only minor increases in PO2 (e.g., 1.0 mm Hg at PO2 of 56 mm Hg) occur when samples are aspirated. Larger increases in PO2 (mean 7.1 mm Hg at a PO2 of 66 mm Hg) occurred when the syringe samples were contaminated with room air. We conclude that isolated aberrant measurements of PO2 in blood gas proficiency testing attributable to vial contents can occur, but the frequency is very low.
Monoclonal antibody-based immunoenzymometric assays of retinol-binding protein
Authors: Pereira A B , Nishida S K , Vieira J G , Lombardi M T , Silva M S , Ajzen H , Ramos O L
Published: January 2020

Retinol-binding protein (RBP) is a low-molecular-mass protein (21 kDa), easily filtered in renal glomeruli and very efficiently reabsorbed by the proximal convoluted tubules (PCTs). In PCT dysfunction, high concentrations of RBP are found in urine. Several methods have been used to determine RBP in serum or urine. We describe the production, selection, labeling, and utilization of anti-RBP monoclonal antibodies in two- or one-step immunoenzymometric assays for the determination of RBP. The one-step assay has good precision, with within-run and between-run CVs < 6.6% and 5.9%, respectively. Comparison with radial immunodiffusion (x) showed good agreement: y = 0.068 mg/L + 0.899x (n = 24). Comparison between the one-step (y) and two-step (x) versions of the assay also showed a very good correlation: y = 212 micrograms/L + 0.910x. The one-step assay has been adopted for routine work; it detects transthyretin-bound as well as free RBP and may have clinical usefulness in evaluating the functional status of PCTs.
Rate nephelometric assay of serum lipoprotein(a)
Authors: Gillery P , Arthuis P , Cuperlier C , Circaud R
Published: January 2020

This nephelometric assay of serum lipoprotein(a) [Lp(a)] is characterized by the use of a specific antibody to generate a high rate of light-scatter formation and the elimination of nonspecific reactions from serum samples by diluting samples in phosphate-buffered saline containing polymer enhancer polyethylene glycol (PEG), 40 g/L, and detergent before the assay. We reacted 100 microL of sixfold-diluted serum in 500 microL of buffer containing PEG with 42 microL of pure polyclonal rabbit antiserum (Dakopatts) directed against human Lp(a) and monitored the reaction by rate nephelometry with the Array Protein System nephelometer (Beckman). The standard curve for the reaction was linear in the Lp(a) range 10-1280 mg/L; antigen excess occurred between 1300 and 1400 mg/L. Calibration was performed with serial dilutions of a standard serum. Precision studies showed within-run and between-run CVs of < 2.1% and 6.9%, respectively. The nephelometric results (y) for 100 serum samples were highly correlated with those obtained by radial immunodiffusion (x) calibrated with the same materials: y = 1.07 (+/- 0.03) x - 16.2 (+/- 8.1) mg/L (r = 0.974, P < 0.001). Storing serum for 3 weeks at 4 degrees C or 3 months at -80 degrees C did not affect the results.
Low-molecular-mass proteinuria as a marker of proximal renal tubular dysfunction in normo- and microalbuminuric non-insulin-dependent diabetic subjects
Authors: Holm J , Hemmingsen L , Nielsen N V
Published: January 2020

We determined the urinary excretion, expressed as the protein/creatinine ratio (morning urines), of albumin (a marker of glomerular dysfunction) and retinol-binding protein (RBP; a low-molecular-mass protein marker of tubular proteinuria) in 102 non-insulin-dependent diabetic patients. There was a statistically significant (P < 0.0001) correlation (rho = 0.38) between the urinary excretion values of the two proteins. The population could be divided into four subgroups: 32 with normal excretion values, 15 with above-normal urinary excretion of RBP, 24 with above-normal urinary excretion of albumin, and 31 patients with above-normal urinary excretion of both proteins. No patients had above-normal serum creatinine concentrations or above-normal serum RBP concentrations. This seems to exclude "tubular overflow proteinuria" as the cause of the increased urinary excretion of RBP seen in some patients with non-insulin-dependent diabetes. Our data suggest the presence of a state of proximal tubular dysfunction in these patients.
Rapid determination of serum myoglobin with a routine chemistry analyzer
Authors: Bakker A J , Boymans D A , Dijkstra D , Gorgels J P , Lerk R
Published: January 2020

A turbidimetric immunoassay system (Turbitime system, Behringwerke AG) allows rapid determination of myoglobin in serum. We adapted the reagents for this myoglobin assay (Turbiquant myoglobin) for use with a Hitachi 717 analyzer. No high-dose hook effect was observed up to 15,000 micrograms/L. Interassay CVs were 4.6% (mean = 72.0 micrograms/L; n = 9) and 2.5% (mean = 365.6 micrograms/L; n = 11). The calibration curve was stable for at least 1 month. Hemolysis did not interfere, and turbidity from lipemia interfered only when absorbance exceeded 2.0 A. Results of this method (y) correlated well with those by the Turbitime method (y = 1.256x - 44.1 micrograms/L; n = 91; r = 0.991) and by a commercially available radioimmunoassay (Byk-Sangtec; y = 0.739x - 42.2 micrograms/L; n = 94; r = 0.991). The upper limit (95th percentile) of the reference interval for myoglobin was estimated at 57.9 micrograms/L. The positive predictive value for results of myoglobin at admission was 89% with this upper reference limit and 99% with 100 micrograms/L, whereas the negative predictive value was about 60% for both limits.
International Federation of Clinical Chemistry standardization project for measurements of apolipoproteins A-I and B. III. Comparability of apolipoprotein A-I values by use of international reference material
Authors: Marcovina S M , Albers J J , Henderson L O , Hannon W H
Published: January 2020

In the third phase of the International Federation of Clinical Chemistry (IFCC) study for the standardization of apolipoprotein (apo) measurements, the preparation SP1-01, selected as the candidate international reference material for apo A-I, was investigated for its ability to transfer an accuracy-based value to the immunoassay calibrators and to produce comparability of the values for patients' samples. An apo A-I value of 1.50 g/L (SD 0.08 g/L) was assigned to SP1-01 by a highly standardized RIA calibrated with purified apo A-I for which the mass value had been determined by amino acid analysis. According to a common detailed protocol, the participants transferred the mass value from SP1-01 to the calibrator of each method. To confirm that uniformity of calibration ensures comparability of the values over a wide range of apo A-I values, each laboratory analyzed 50 fresh-frozen samples from individual donors, using an approach similar to that adopted by the Cholesterol Reference Laboratory Network. The consensus mean value for each sample was in excellent agreement with the value assigned by the Northwest Lipid Research Laboratories, with the average absolute bias between assigned and consensus value being 0.01 g/L. The among-laboratory CV on each of the 50 samples ranged from 2.1% to 5.6% (mean 3.6%), demonstrating that comparable apo A-I results can be obtained by a variety of immunochemical methods through the use of certified reference material. Based on the results obtained in these studies, SP1-01 has been approved as Apolipoprotein A-I International Reference Material by the World Health Organization.
Assessment of copper status: effect of age and gender on reference ranges in healthy adults
Authors: Milne D B , Johnson P E
Published: January 2020

We measured major indices related to copper nutritional status in 55 men and 86 women between ages 20 and 83 years who were in apparent good health. Plasma copper concentrations and both immunoreactive and enzymatically measured ceruloplasmin were significantly higher in women than in men and were higher in women taking oral contraceptives. Plasma copper, immunoreactive ceruloplasmin, and cytochrome-c oxidase in platelets and mononucleated leukocytes tended to increase with age. The ratio of enzymatic to immunoreactive ceruloplasmin, erythrocyte superoxide dismutase, and 67Cu uptake by erythrocytes were not significantly affected by either age or gender. Thus, factors other than copper nutriture--such as age, gender, and hormone use--need to be considered when using many of these indicators to evaluate copper nutritional status.
Childhood porphyrias: implications and treatments
Authors: Badcock N R , O'Reilly D A , Zoanetti G D , Robertson E F , Parker C J
Published: January 2020

In describing the clinical, biochemical, and family findings in five children with porphyria, we examine initial treatments and, where appropriate, the effectiveness of long-term therapy. We note that porphyria diagnosis, particularly in childhood, relies heavily on specialist laboratory investigations. Because disease expression in some porphyrias requires exposure to precipitating factors, it may be prevented or delayed by their avoidance.
Chemistry with confidence: should Clinical Chemistry require confidence intervals for analytical and other data?
Authors: Henderson A R
Published: January 2020

Confidence intervals are not commonly provided with analytical or other data reported in Clinical Chemistry although P values are. However, confidence intervals provide an explicit demonstration of the direction and magnitude of uncertainty and are intuitively easy to grasp, unlike P values. It is therefore argued that the Journal should adopt a policy requiring the provision of confidence intervals. Such a policy would improve the statistical rigor of Journal reports.
Determination of one thousandth of an attomole (1 zeptomole) of alkaline phosphatase: application in an immunoassay of proinsulin
Authors: Cook D B , Self C H
Published: January 2020

Enzyme amplification has proved to be a highly sensitive quantification technique for immunoassays. We have shown that by using a fluorescent end-point, even more sensitive enzyme amplification assays can be generated than hitherto reported. We describe some general properties of this system and demonstrate its application in an assay for human proinsulin in plasma. The detection system can be used to measure less than one thousandth of an attomole (1 zeptomole) of alkaline phosphatase, equivalent to about 350 molecules of alkaline phosphatase per well of a microtiter plate. We have used this system to construct a proinsulin assay with a sensitivity of 0.017 pmol/L.
Cardiac-specific immunoenzymometric assay of troponin I in the early phase of acute myocardial infarction
Authors: Larue C , Calzolari C , Bertinchant J P , Leclercq F , Grolleau R , Pau B
Published: January 2020

The screening by immunoenzymometric assay (IEMA) of 784 monoclonal antibody (MAb) combinations resulted in the selection of an optimal pair of MAbs for measuring human cardiac troponin I (TnI). Using a one-step IEMA described here, we were able to detect TnI within the range of 0.2-20 micrograms/L in 30 min at room temperature. No cross-reactivity was observed with the skeletal isoforms of troponin up to a concentration of 500 micrograms/L. This assay was used to measure cardiac TnI in the plasma of 43 patients with acute myocardial infarction (AMI). TnI was detected relatively early after the onset of chest pain (4.3 +/- 2.1 h, mean +/- SD); the peak occurred after 12.2 +/- 4.6 h in a population that had undergone fibrinolysis. TnI disappearance was generally observed between 5 and 9 days after the onset of chest pain. No cardiac TnI could be detected in 145 healthy donors or in a control group of 6 patients (with skeletal damage or rhabdomyolysis). This assay allows a specific diagnosis of AMI in its early acute phase, with a high diagnostic specificity and sensitivity.
In vitro effect of elastase and cathepsin G from human neutrophils on creatine kinase and lactate dehydrogenase isoenzymes
Authors: Stark J A , Henderson A R
Published: January 2020

The polymorphonuclear granulocyte, or neutrophil, has been implicated as a mediator of tissue-destructive events because it releases the preformed proteolytic enzymes elastase and cathepsin G, and, as a result of myeloperoxidase action, hypochlorous acid. We show that elastase inactivates and fragments creatine kinase isoenzymes CK-2 and CK-3, and, to a lesser extent, lactate dehydrogenase (LD) isoenzyme LD-1, whereas cathepsin G acts only on CK-2. Both neutrophil enzymes act on LD-3. The course of inactivation was followed by measuring the loss of catalytic activity at 37 degrees C. The evidence for fragmentation was obtained by gel filtration; electrophoresis after sample treatment with sodium dodecyl sulfate and 2-mercaptoethanol was less satisfactory for this purpose. Hypochlorous acid inactivates CK activity by about 75% at concentrations as low as 8 mumol/L and totally at concentrations > 140 mumol/L, whereas LD activity is not affected until concentrations exceed 200 mumol/L. After a myocardial infarction, the number of neutrophils increases; they are triggered and concentrate around damaged myocardial tissue. Our data suggest that neutrophils may inactivate and fragment "cardiac" enzymes released from such damaged tissue.
Can medical decisions be standardized? Should they be?
Authors: Werner M
Published: January 2020

After a variety of regulatory and payment schemes have failed to stem the rising tide of health care cost, the Omnibus Budget Reconciliation Act of 1989 mandates the creation of a system of decision rules called practice parameters for appropriate medical action in many circumstances. A large body of practice guidelines already exists, but lacks the internal coherence required of a policy tool. Professional organizations therefore have developed attributes to achieve uniform style. However, little has been said or published about the constraints that might be imposed on the structure and content of an efficient and coherent system. The arguments presented here lead to the following conclusions: (a) process control is an inefficient tool to manage outcome--standards should reflect product control; (b) guidelines that proscribe are more likely to be supported by scientific data and consensus than those that prescribe; (c) the decision thresholds contained in such directives are policy choices rather than scientific imperatives; (d) neither decision analysis nor artificial intelligence is likely to readily influence medical decisions; and (e) as suggested by operations research, the development of practice parameters should concentrate on issues of therapeutic management in preference to issues of diagnostic activity.
Polymorphism of apolipoprotein E, lipoprotein(a), and other lipoproteins in children with type I diabetes
Authors: Salzer B , Stavljenić A , Jürgens G , Dumić M , Radica A
Published: January 2020

We assessed the effect of particular apolipoprotein (apo) E phenotypes, lipoprotein(a) [Lp(a)], and other lipoproteins on the development of dyslipoproteinemia in 450 patients with type I diabetes, ages 13-14 years. The control group consisted of 450 healthy school children of both sexes, ages 13-14 years. Both groups were found to be normolipidemic, but the concentration of Lp(a) was significantly (P < 0.05) higher in the diabetic children than in the control group. Apo E 3/2 and apo E 4/4 phenotypes were more frequent in the group of diabetics. Diabetics with the apo E 3/3 phenotype had higher concentrations of very-low-density lipoprotein (VLDL) and Lp(a), and lower concentrations of low-density lipoprotein (LDL) than the apo E 3/3 nondiabetics. For apo E 3/2 phenotypes, total cholesterol, LDL cholesterol, LDL, apo A-I, and Lp(a) concentrations were higher in the diabetic children than in the control group; for apo E 4/3 phenotypes, this was true for triglycerides and VLDL cholesterol. The distribution of Lp(a) lipoprotein concentrations between 0.01 and > 0.5 g/L indicated a more frequent occurrence of higher Lp(a) values in diabetic children than in the control group. Results of this study indicate that an increased concentration of Lp(a) lipoprotein and apo E 3/2 and apo E 4/3 phenotypes contribute to the expression of dyslipoproteinemia in type I diabetes in childhood.
Recombinant creatine kinase proteins and proposed standards for creatine kinase isoenzyme and subform assays
Authors: Friedman D L , Kesterson R , Puleo P , Wu A H , Perryman M B
Published: January 2020

We developed standards for creatine kinase (CK; EC 2.7.3.2) assays by expressing human CK cDNAs in COS cells. Cells were transiently transfected with full-length cDNAs for CK subunits M and B, individually and in combination; and subsequently, high concentrations of CK activity were present in the cell lysate (1.2 U/mg protein). These proteins exhibited the characteristic isoenzyme-specific electrophoretic mobilities for CK MM and BB isoenzymes. We also produced subforms of CK MM and MB, identical to the modified CK variants produced in plasma after muscle or myocardial injury, by mutating the cDNA for the CK M subunit to delete the carboxy-terminal lysine residue. When this construct was cotransfected with the normal cDNAs for CK M and B, five electrophoretically distinct CK isoenzymes were detected by nondenaturing electrophoresis: MM3, MM2, MM1, MB2, and MB1. These proteins retained 100% of their activity after storage of the cell lysates -20 or 4 degrees C for 3 months.
Adsorption losses from urine-based cannabinoid calibrators during routine use
Authors: Blanc J A , Manneh V A , Ernst R , Berger D E , de Keczer S A , Chase C , Centofanti J M , DeLizza A J
Published: January 2020

The major metabolite of cannabis found in urine, 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (delta 9-THC), is the compound most often used to calibrate cannabinoid immunoassays. The hydrophobic delta 9-THC molecule is known to adsorb to solid surfaces. This loss of analyte from calibrator solutions can lead to inaccuracy in the analytical system. Because the calibrators remain stable when not used, analyte loss is most probably caused by handling techniques. In an effort to develop an effective means of overcoming adsorption losses, we quantified cannabinoid loss from calibrators during the testing process. In studying handling of these solutions, we found noticeable, significant losses attributable to both the kind of pipette used for transfer and the contact surface-to-volume ratio of calibrator solution in the analyzer cup. Losses were quantified by immunoassay and by radioactive tracer. We suggest handling techniques that can minimize adsorption of delta 9-THC to surfaces. Using the appropriate pipette and maintaining a minimum surface-to-volume ratio in the analyzer cup effectively reduces analyte loss.
Influence of hemoglobin variants and derivatives on glycohemoglobin determinations, as investigated by 102 laboratories using 16 methods
Authors: Weykamp C W , Penders T J , Muskiet F A , van der Slik W
Published: January 2020

Influences of hemoglobin (Hb) variants (HbSS, HbCC, beta-thalassemia, HbAE, HbAS, HbAC, hereditary persistent HbF) and Hb derivatives (carbamylated- and acetylated-Hbs, Schiff base, and those formed in stored blood) on results of glyco-Hb assays by 102 laboratories using 16 different methods were investigated. Affinity chromatography shows deviating results only with homozygous Hb S and C. Correct interpretation of results from patients with decreased erythrocyte half-lives requires previous knowledge on this condition. Measurements of HbA1c by HPLC and electrophoresis are obviously unsuitable for homozygous hemoglobinopathies; for heterozygous hemoglobinopathies and Hb synthesis variants, HbA1c should be expressed as percentage of HbA0 + HbA1c; abnormal Hbs are usually recognized; both carbamylated- and acetylated-Hbs interfere and Schiff base must be eliminated. Except for stored blood, all Hb variants and derivatives gave erroneous results with disposable ion-exchange columns. Dako's immunoassay is not affected by Hb derivatives; glycated Hb variants are not recognized as glyco-Hb and percentages are consequently too low. Glyco-Hb by the immunoassay of Bayer (performed by one laboratory) is not affected by Hb variants and derivatives.
A 10-year analysis of "revenues," costs, staffing, and workload in an academic medical center clinical chemistry laboratory
Authors: Benge H , Csako G , Parl F F
Published: January 2020

From 1980 to 1990 we found progressive increases in workload (number of billable tests; 12.1% per year), staffing [number of full-time equivalents (FTEs); 5.6% per year], "revenues" (gross billings; 25.8% per year), and direct cost (12.9% per year) in the clinical chemistry laboratory of a large tertiary-care university medical center. The increase in direct cost was mainly attributable to an increase in salary cost (23.7% per year), whereas the impact of increasing "consumable" cost was relatively small (5.3% per year). In fact, after adjustment for inflation, the consumable cost was virtually unchanged or decreased during the 10-year study period. Initially, consumables represented about 60% of the direct cost, and the remaining 40% was for salaries. After 1982/83, however, the relative contribution of consumables and salaries to direct cost gradually reversed. Because the workload grew at a higher rate than staffing, the workload per FTE increased from 1980 to 1990. This was paralleled by gradual increases in both "revenue" per FTE and salary per FTE in actual dollars, but by lesser increases to no increases in inflation-corrected dollars. After adjusting for inflation with different indices, the direct cost per test, the consumable cost per test, and the salary cost per test either remained unchanged or decreased in the 1980s. The findings are discussed in the context of technical advancements in laboratory testing, nationwide shortages of medical technologists, and implementation of prospective fixed-fee reimbursement practices during the study period.
Pharmacokinetics of ethanol in saliva: comparison with blood and breath alcohol profiles, subjective feelings of intoxication, and diminished performance
Authors: Jones A W
Published: January 2020

The concentration-time profiles of ethanol were determined for capillary blood, end-expired breath, and saliva after 21 healthy men ingested ethanol at 0.68 g/kg body weight. Near the time of obtaining body fluids, the volunteers estimated their feelings of intoxication, and body sway (with open and closed eyes), hand tremor, positional alcohol nystagmus (PAN), and roving ocular movements (ROM) were quantitatively recorded. The concentration-time profiles of ethanol in blood, breath, and saliva agreed well within individuals but there were large variations between subjects. The mean saliva-ethanol profiles ran slightly above those for blood and breath. Subjective ratings of intoxication and impairment of body function (standing steadiness and hand steadiness) were highest at the time of reaching the peak concentrations of ethanol in body fluids. PAN was evident in most subjects between 60 and 120 min after the start of drinking, whereas ROM appeared mainly during the postabsorptive phase of ethanol kinetics (120-420 min). The blood ethanol concentration thresholds were between 500 and 700 mg/L (50-70 mg/dL) when the diminished performance had recovered to baseline values.
Rapid Zeeman atomic absorption determination of copper in serum and urine
Authors: Wang S T , Demshar H P
Published: January 2020

In this simple, quick procedure for determining copper in human serum and urine, the serum and urine specimens were analyzed directly after dilution with a solution of HNO3 and Triton X-100, 1 mL/L each. We calibrated with aqueous standards for quantitation in Zeeman background atomic absorption spectrometry. By modifying the drying and pyrolysis stages of the graphite furnace atomic absorption spectrometer, we reduced the analytical time to 30 s per determination. The within-run imprecision (CV) is 2.6% and 3.4% and the between-run imprecision is 0.9% and 2.5% for serum and urine copper at concentrations of 30.4 and 2.70 mumol/L, respectively. The accuracy of this fast method was verified by analyzing the National Institute of Standards and Technology Standard Reference Materials SRM 1598 bovine serum and SRM 2670 urine (agreement with certified values within 0.1 mumol/L for serum and within 0.02 mumol/L for urine), by analytical recovery studies (98% recovered in serum, 100% recovered in urine), and by comparison with our normal routine method. We also used the Quebec Interlaboratory Comparison Program to validate the analytical performance. From the precision and accuracy studies, we conclude that this fast-furnace program is a rapid, simple, and reliable method for determining copper in serum and urine.
AccuMeter noninstrumented quantitative assay of high-density lipoprotein in whole blood
Authors: Liu V Y , Lin T Y , Schrier W , Allen M , Singh P
Published: January 2020

A novel noninstrumented technology (ChemTrak AccuMeter) for the quantitative measurement of analytes in biological fluids was reported at the 1990 Oak Ridge Conference (Clin Chem 1990;36:1591-7). This instrument-free technology has been adapted for the quantitative measurement of high-density lipoprotein (HDL). An in situ lipoprotein separation method has been developed and incorporated into the test for HDL determination. The sensitivity of the assay system has been adjusted so that HDL is measured over a clinically significant range of 250 to 1000 mg/L. This compares to a range of 1000 to 4000 mg/L for the total cholesterol in the earlier reported assay. Like the AccuMeter total cholesterol test, the Accumeter HDL test system is self-contained and consolidates blood separation, lipoprotein separation, and specimen measurement into a single step. The test procedure is simple, and the results are accurate.
Ability of two new thyrotropin (TSH) assays to separate hyperthyroid patients from euthyroid patients with low TSH
Authors: Taimela E , Tähtelä R , Koskinen P , Nuutila P , Forsström J , Taimela S , Karonen S L , Välimäki M , Irjala K
Published: January 2020

We evaluated the ability of new thyrotropin (TSH) assays to separate hyperthyroid (n = 50) patients from clinically euthyroid subjects with low TSH values (nodular goiter, n = 20, and nonthyroidal illness, n = 22). Only patients whose serum TSH was < 0.1 mIU/L by immunoradiometric assay were included. We used a new immunofluorometric method based on time-resolved fluorescence (TR-IFMA) and a new immunochemiluminometric assay (ICMA) to measure TSH in serum. Although the differences between the hyperthyroid patients and the euthyroid patients differed from each other by both methods (P = 0.0012 for TR-IFMA and P < 0.0001 for ICMA), there was no cutoff point that could definitely separate the groups. Thus, it is not possible to draw any definite conclusions on whether a patient is hyperthyroid or not, solely on the basis of TSH concentration measured with these new TSH assays.
Specificity of antigen assays of plasminogen activator inhibitor in plasma: Innotest PAI-1 immunoassay evaluated
Authors: Meijer P , Pollet D E , Wauters J , Kluft C
Published: January 2020

We evaluate a new commercial enzyme immunoassay (EIA) of plasminogen activator inhibitor-1 (PAI-1) in plasma, the Innotest PAI-1. Because we wanted to measure PAI-1 in blood samples, we developed a procedure for evaluating the specificity of the assay for different PAI-1 forms in their natural environment. All molecular forms were prepared from a plasma that contained only active PAI-1. The recovery of the different molecular forms of PAI-1, relative to active PAI-1 (100%), was 99% +/- 7% for PAI-1 complexed with recombinant tissue-type plasminogen activator (t-PA), 104% +/- 4% for PAI-1 complexed with melanoma t-PA, 94% +/- 11% for PAI-1 complexed with high-M(r) urokinase, and 113% +/- 3% for latent PAI-1. The parallelism between the calibration curve of the EIA and the serial dilutions of the different PAI-1 forms was considered acceptable for clinical purposes. In selected clinical plasma samples, the PAI-1 values obtained with the Innotest PAI-1 EIA correlated well with those of the TintElize PAI-1 EIA (r = 0.913, n = 106); the observed correlation of the Innotest measurements with PAI activity was r = 0.795 (n = 79). The Innotest PAI-1 antigen assay appears to detect all molecular forms of PAI-1 to a similar degree, and comes close to being the so-called grand total assay for detecting the total molecular concentration of PAI-1 in plasma.
Two-site enzyme immunoassay of CD4 and CD8 molecules on the surface of T lymphocytes from healthy subjects and HIV-1-infected patients
Authors: Carriere D , Fontaine C , Berthier A M , Rouquette A M , Carayon P , Laprode M , Juillard R , Jansen A , Paoli P , Paolucci F
Published: January 2020

A highly sensitive two-site enzyme immunoassay (Capcellia) was developed to determine the concentration of CD4 and CD8 molecules expressed on the surface of human T lymphocytes. This assay, performed in one step (20 min), involves the specific immunocapture of T lymphocytes and reaction of the CD4 or CD8 molecules with an enzyme-labeled monoclonal antibody (mAb). The results were expressed as molar concentrations of the T-cell markers on the basis of results obtained with calibrated CD4 and CD8 standards. The assay was sensitive enough to detect 0.4 pmol/L CD4 or 0.8 pmol/L CD8, which corresponded to approximately 20 x 10(6) CD4+ or CD8+ T cells per liter of blood. Mean concentrations in healthy adults were 17.2 pmol/L for CD4 and 22.1 pmol/L for CD8. The CD4 concentration was < 8 pmol/L in 50% of HIV-1-infected patients and in 95% of AIDS patients. Given the epitopic specificity of the mAb to CD4 we used, these values correspond to the concentration of CD4 molecules free of envelope glycoprotein (gp)120.
Radioimmunoassay of 2-hydroxyestrone in urine
Authors: McGuinness B J , Power M J , Fottrell P F
Published: January 2020

Catechol estrogens such as 2-OH estrone are interesting estrogen metabolites formed in several human tissues and excreted in urine. We developed and thoroughly validated a radioimmunoassay for urinary 2-OH estrone that has several advantages over published RIAs. Because we developed a relatively specific antiserum, we did not include a preliminary chromatographic step to eliminate cross-reacting urinary steroids. We hydrolyzed urinary steroid conjugates with beta-glucuronidase from Helix pomatia because recoveries after acid hydrolysis were only 49.6% compared with 73.8% after enzyme hydrolysis. Published RIAs for urinary 2-OH estrone use acid hydrolysis. Our RIA measured 2-OH estrone independently of the volume of sample, and the detection limit was between 100 and 240 ng/L (10-24 pg per tube). The ED50 was 370 ng/L, and inter- and intraassay CVs for low, medium, and high concentrations were 22.5%, 22.8%, and 19.9%, and 17.4%, 14.3%, and 10.8%, respectively. Median concentrations measured in 14 controls and 33 postmenopausal patients with breast cancer were 0.96 and 1.55 micrograms/g creatinine, respectively, but there was considerable overlap between values from controls and patients.
Four rapid serum-urine combination assays of choriogonadotropin (hCG) compared and assessed for their utility in quantitative determinations of hCG
Authors: Mishalani S H , Seliktar J , Braunstein G D
Published: January 2020

We evaluated the performance of four visually read pregnancy tests (TestPack Plus hCG Combo, ICON II hCG, SureCell hCG-Serum/Urine and PregnaGen 1-Step) designed to detect increased concentrations of choriogonadotropin (hCG) in either serum or urine samples. The biochemical sensitivities and specificity in both serum and urine samples were similar for each kit. All kits correctly identified pregnancy serum samples: The TestPack Plus hCG Combo and SureCell hCG-Serum/Urine were 100% specific; the other two kits exhibited a few false-positive results. For urine samples the ICON II hCG test was 100% sensitive, and the other three were 99.5% sensitive, with false-positive urine results occasionally reported by the PregnaGen 1-Step and ICON II hCG tests. Quantitative hCG concentrations could be estimated in pregnancy serum samples, but not urine samples, through determination of the time elapsed from the sample application or addition of the final reagent to the first appearance of a positive result.
High-performance assays of small molecules: enhanced sensitivity, rapidity, and convenience demonstrated with a noncompetitive immunometric anti-immune complex assay system for digoxin
Authors: Self C H , Dessi J L , Winger L A
Published: January 2020

We report the first clinical application of a noncompetitive immunometric assay system that provides advantages for the rapid and robust assay of small molecules similar to those realized for larger molecules with two-site immunometric assays. This anti-immune complex assay is based on the interaction of a receptor such as a primary antibody with its ligand, such that new binding sites, recognizable by a secondary antibody, are formed. In this report the system is applied to the measurement of digoxin in serum. Utilizing an anti-complex antibody that recognizes a digoxin-bound primary antibody with affinity > 2000-fold over its binding to the primary antibody alone, we show that this anti-complex assay system provides a high-performance assay for serum samples, being conveniently simple (immobilized primary antibody binds digoxin and then labeled secondary antibody so that when excess unbound label is washed away the immunometric readout reflects the digoxin concentration), rapid (incubation time 1-10 min), sensitive (detection limit 30 ng/L), precise (3-4% within-run CV, 1-8% total CV), and free from interference from digoxin-like immunoreactive factors.
Six osteocalcin assays compared
Authors: Díaz Diego E M , Guerrero R , de la Piedra C
Published: January 2020

We evaluated comparatively six commercially available osteocalcin kits: ELSA-OST-NAT IRMA (CIS), ELSA-Osteo IRMA (CIS), Osteocalcin IRMA (Nichols), OSTK-PR RIA (CIS), OSCA Test Osteocalcin RIA (Henning), and Osteocalcin RIA (Nichols). All of them presented acceptable values of variance, sensitivity, and serial dilutions. However, only the ELSA-OST-NAT, ELSA-Osteo, and OSCA Test kits had analytical recoveries near 100% for added purified human osteocalcin; the other assays showed important differences in standardization. After treating samples with antibody against amino acids 43-49 of human osteocalcin, only the ELSA-OST-NAT IRMA detected no residual osteocalcin. Stability of serum values at room temperature and at 4 degrees C is good in the ELSA-Osteo and Osteocalcin IRMAs and in the Henning RIA if serum samples are collected in special tubes available with the kit. Differences in stability and after antibody treatment can be due to the degradation of intact osteocalcin molecules in serum and to osteocalcin fragments detected by assays. Results by different assays were well correlated for samples from controls and postmenopausal osteoporotic women but not for patients with impaired renal function or Paget disease of bone, probably because of osteocalcin fragments accumulated in serum of these patients and differences in specificity of the assays.
Allelic D variants of transferrin in evaluation of alcohol abuse: differential diagnosis by isoelectric focusing-immunoblotting-laser densitometry
Authors: Bean P , Peter J B
Published: January 2020

In the diagnosis of alcohol abuse transferrin (Tf) allelic D variants generate false-positive test results for carbohydrate-deficient transferrin (CDT) as assessed by their electrophoretic migration patterns. The predominant Tf C1 allele encodes a protein for which the most prevalent isoform has a pI of 5.4, i.e., four sialic acids and two bound ion molecules. Carriers of allele D encode Tfs with different amino acid sequences, for which the pI is > 5.7, despite their identical iron and carbohydrate composition. We used isoelectric focusing, immunoblotting, and laser densitometry (IEF-IB-LD) to distinguish Tf D variants from CDT. Alcohol abusers carrying the D chi allele tested CDT+; D chi nondrinkers were CDT-. Although normal controls (< 15 g of alcohol per day for 7-10 consecutive days) carrying variants D1, D2, or D chi exhibited abnormal IEF banding patterns, they did not generate false-positive results for CDT. D3 variants expressed isoforms that migrate at the same pI as CDT bands. Thus, IEF-IB-LD yields a highly resolved banding pattern to distinguish most Tf D variants from CDT.
In vitro analysis of cell-mediated immunity: clinical relevance
Authors: Shearer G M , Clerici M
Published: January 2020

Cellular immunity, a major component of the immune system, contributes to protection against infections and plays a role in the rejection of foreign allografts. T helper cells and the antigen-presenting cells with which they interact provide important functions for cellular immune responses by generating helper signals via soluble molecules known as lymphokines or cytokines. As described here, in vitro analysis of markers of cellular immune function can be used for clinical diagnosis and assessment of therapeutic efficacy and to elucidate the mechanisms of immune dysregulation. Not all helper signals necessarily have the same augmenting effect, because immunoregulatory cytokines that up-regulate the cellular arm of the immune system can conversely down-regulate the humoral arm, and vice versa. The interplay between cross-regulatory cytokines determines which component of the immune system is dominant and can influence the clinical outcome of immunologically controlled diseases. Given the apparent dramatic effects of these cytokines, assays performed in future clinical laboratories should include profiles of immunoregulatory cytokines.
Factors influencing changes in the clinical immunology laboratory
Authors: Nakamura R M , Bylund D J
Published: January 2020

The clinical immunology laboratory is primarily concerned with tests for diagnosing disease and monitoring the therapy and course of diseases mediated by immune mechanisms. In the future, the immunology laboratory will provide test services on a disease-oriented basis and will utilize assays of humoral and cellular immunity, as well as other assays based on molecular techniques. The laboratory scientist will work closely with clinicians and provide interactive consultation. The major factors that will influence future laboratory tests are: (a) governmental regulations, (b) healthcare economics, (c) advances in technology, and (d) advances in development and use of therapeutic agents. Here I discuss the interplay of these factors and their likely effect on the future role of the immunology laboratory.
Verification of reference ranges by using a Monte Carlo sampling technique
Authors: Holmes E W , Kahn S E , Molnar P A , Bermes E W
Published: January 2020

We have investigated the application of Monte Carlo significance tests to the verification of reference ranges in the context of the transfer of an established range from one laboratory to another. Here we present an introduction to the Monte Carlo technique, outline a procedure for performing these tests using a commercially available software program, and demonstrate some of the operating characteristics of the tests when they are used to compare samples of different sizes and variances.