Run Coefficients Of Variation Research Articles

Simple kinetic method for assessing catalase activity in biological samples

A novel kinetic method for measuring catalase activity in biological samples was evaluated. The principle of the current method is based on the oxidation effect of unreacted hydrogen peroxide (H2O2) on pyrogallol red (PGR) using the catalytic effects of molybdenum. The decrease in the absorbance of PGR in the presence of H2O2 with time from 0.5 to 4.5 min was directly proportional to the concentration of H2O2, and, in turn, directly proportional to catalase activity. Erythrocyte lysate homogenates were used to measure catalase activity and the results of the current method were significantly correlated to those of the ammonium peroxovanadate method. The 3.1% within run and 4.7% between run coefficients of variation indicated the high precision of the present novel method. The validation process confirmed that the diagnostic method is appropriate for different types of biological samples. Here, we describe a rapid, relatively easy, and reliable method for measuring catalase activity. The assay could be applied as a diagnostic tool and is suitable in research contexts.•A novel kinetic method for measuring catalase activity in biological samples was evaluated.•The validation process confirmed that the diagnostic method is appropriate for different types of biological samples.•The assay could be applied as a diagnostic tool and is suitable in research contexts.

Abstract 5384: Analytic validation of an assay to detect androgen receptor splice variant ARv7 protein expression on circulating tumor cells from prostate cancer patients

Abstract Circulating tumor cells (CTCs) can provide information on drug target expression, response to therapy, and disease prognosis from a non-invasive blood draw. Currently, investigating biomarkers on CTCs is difficult due to challenges of developing multiplexed assays that also identify rare cells. Presence of the androgen receptor splice variant ARv7 in prostate cancer cells is associated with resistance to second generation anti-androgen therapies. We report here the analytical validation of an immunofluorescence assay for characterization of ARv7 protein expression on CTCs using the RareCyte platform - an end-to-end platform that combines CTC sample preparation, multiparameter fluorescence staining, digital imaging, and single cell retrieval. Blood samples spiked with positive and negative cell lines for ARv7 expression were processed using the AccuCyte Sample Preparation System. Slides were auto-stained by immunofluorescence with the RarePlex ARv7 CTC Panel Kit comprised of a three-channel CTC detection base plus an ARv7 biomarker channel. The detection base consists of a nuclear dye, anti-CD45 antibody to exclude white blood cells, and cocktailed antibodies to cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM). Stained slides were imaged with the CyteFinder Instrument. CTCs were identified using machine learning-based algorithms and confirmed by user review. Mean fluorescence intensity (MFI) measurements were used as a metric for ARv7 expression on confirmed CTCs. Analytic validation studies of the AR-V7 CTC assay were performed using 22RV1 (high), LNCaP (low), and BT-474 (negative) cell lines. Performance characteristics tested for ARv7 included accuracy, sensitivity, specificity, repeatability, and inter-stainer run coefficient of variation. Performance metrics for CTC recovery were calculated on spike-in and clinical samples. For recovery calculations, the number of CTCs found with the ARv7 assay was compared to the number of CTCs found with the CTC detection base assay. An ARv7 MFI threshold that segregated negative and positive cell lines was statistically defined. This threshold identified 80% of 22RV1 cells as positive for ARv7, 97% of BT-474 cells as negative, with an overall accuracy of 90%. When the assay was applied to clinical prostate cancer samples, staining with proper nuclear localization was observed. CTC recovery was at least as high with the ARv7 assay as with the base CTC detection assay. Citation Format: Daniel Campton, Edward Lo, Lillian Costandy, Brady Gardner, Ryan Houston, Jeffery L. Werbin, Kyla Teplitz, Daniel E. Sabath, Alisa C. Clein, Celestia S. Higano, Tanisha M. Mojica, Kristin Province, Eric P. Kaldjian, Arturo B. Ramirez, Tad George. Analytic validation of an assay to detect androgen receptor splice variant ARv7 protein expression on circulating tumor cells from prostate cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5384.

An amperometric biosensor for specific detection of glycated hemoglobin based on recombinant engineered fructosyl peptide oxidase

Here, we present a specific biosensor based on the detection of glycated hemoglobin (HbA1c) proteolytic digestion product, fructosyl valyl histidine (Fru-ValHis). A recombinant engineered fructosyl peptide oxidase (FPOX) enzyme with improved specificity was immobilized on the electrode surface modified by chitosan (CHIT), graphene oxide (GO) and gold nanoparticles (AuNPs). The biosensor exhibited a linear response toward different concentrations of Fru-ValHis ranging from 0.1 to 2 mM with a sensitivity of 8.45 µA mM-1 cm−2. Detection limit of the current biosensor for Fru-ValHis was 0.3 µM as the lowest quantity required giving a signal to a background. Analytical recovery of added Fru-ValHis in whole blood was 95.1–98.35% for FPOX/AuNPs/GO/CHIT/FTO electrode. For Fru-ValHis determination by FPOX-AuNPs-GO-CHIT/FTO electrode, within-run coefficient of variation (CV) was between 1.3% and 2.4% and between run CV was between 2.1% and 3.5%. A significant change in electron transfer resistance after the incubation of FPOX-modified electrode with Fru-ValHis was observed, while no response was achieved with control, indicating specific measurement of Fru-ValHis. Moreover, designed biosensor measured HbA1c in human blood samples and the results were well agreed with that obtained with NORUDIA™ N HbA1c diagnostic kit. Overall, suitable specificity of the engineered FPOX made the bioelectrode responded well to the Fru-ValHis level, which demonstrates a promising application for specific detection of HbA1c biomarker.

Setting up of 15 POC blood gas analyzers at Montpellier Hosptital (France)

The purpose of this article is to describe the setting up of 15 blood gas analyzers GEM(®) Premier™ 4000 (IL) at Montpellier hospital. This experience includes analytical characterization (within and between run coefficient of variation) using GSE and GHE IL controls, correlation of 35 samples with a routinely used laboratory blood gas analyzer (Cobas b221, Roche(®)). We shall also develop the training, the habilitation and its follow-up for the user staff (450 people) of the different hospital's units in the aim of the accreditation.

Improved Mass Spectrometry Assay For Plasma Hepcidin: Detection and Characterization of a Novel Hepcidin Isoform

Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25+40 isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF) platforms. As expected, implementation of hepcidin-25+40 as internal standard in our weak cation exchange (WCX) TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9) with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and standardization of plasma hepcidin assays.

New Enzymatic Colorimetric Method for the Quantitative Determination of Phenylalanine in Dry-Blood Spots

Phenylketonuria (PKU) is an autosomal recessive disorder, which is characterized by severe mental retardation, microcephaly and seizures. The symptoms of this disease can be prevented if detected soon after birth. Therefore, blood Phenylalanine (Phe) measurement is essential for the early diagnosis, treatment and dietary monitoring of PKU patients. The goal of this research was to introduce a rapid, precise and effective enzymatic colorimetric method using the recombinant Bacillus badius phenylalanine dehydrogenase (PheDH; EC 1.4.1.20) for the quantitative determination of Phe in dry-blood spots. This test was based on the enzymatic reaction of PheDH coupled with an artificial electron acceptor system composed of phenazine methosulfate (PMS) and iodonitro tetrazolium chloride (INT). This assay system contained PMS to transfer the electrons of NADH to INT, enabling the formation of formazan with an absorbance at 490 nm. Calibration curve was plotted and the experimental data were fitted by linear regression analyze. The regression equation and correlation coefficient (R 2 ) were Y= 0.0109x + 0.032 and R 2 =0.996, respectively. This method showed a recovery in the range of 95.1%-102.6% and had the limit detection of 0.5 mg/dl for Phe. The between run coefficients of variation (CVs) mean was between 3.8% and 9.1%. The within-run CVs was between 8.5% and 18.6%. Furthermore, no interferences from other amino acids and Phe derivatives were observed. Altogether, we here presented a quick and reliable enzymatic colorimetric assay for application in newborn screening and monitoring of PKU patients.

Performance Evaluation of an Enhanced-Sensitivity Interferon-gamma ELISpot Assay Kit (132.5)

Abstract Human Interferon-gamma (IFN-y) ELISpot assays are designed for the detection of IFN-y secreting cells at the single cell level. We have developed and validated an ELISpot assay with enhanced sensitivity (SC) and compared its performance relative to a common commercial kit (CK). Three manufactured lots of ELISpot kits (SC1-3) and one CK were analyzed in cellular titration experiments using PHA stimulation of cryopreserved PBMC from four subjects. The minimum quantity of cells required to elicit a single spot-forming cell (SFC) per well was 25,000 for CK compared to 7,000 for SC. In many cases, > 50,000 cells were required to measure a response using the CK. Slopes of log/log plots were 2.3 for CK versus 1.7 for SC, with no overlap at the 95% CI level. At the upper detection limit (200,000 cells/well), the CK averaged 108±46 SFC/well, while SC 1-3 measured 264±79, 377±120 and 231 ±95 SFC/well, respectively, demonstrating a broader linear range. Differences in sensitivity and range are possibly due to spot size, SC produced spots 9 x 10-3 mm2 (95% CI 7-11) while CK averaged 19 x 10-3 mm2 (95% CI 17-21), P < 0.001. The same PBMC were also analyzed using CEF (CMV, EBV and Influenza A – specific peptide pools) and CMV peptide stimulants and media controls. For CEF, the mean values for SC 1-3 were 100.0, 90.0, and 86.6 SFC/well, vs. 77.7 for CK. The CMV responses were 104.3, 80.2 and 74.4 SFC/well for SC 1-3 vs. 77.1 for CK. PBMC added with no stimulus, gave backgrounds of <2 SFC/well in all cases. The average within run coefficients of variation (CV) were similar- 10.2, 14.1 and 18.9 for SC 1-3 and 13.8 for CK when stimulated using CEF and 20.8 to 26.4 with CMV. No significant differences were observed for means or variability between CK and SC 1-3. In summary, SeraCare has developed and validated an IFN-y ELISpot kit having an enhanced dynamic range and sensitivity.

Comparison of tunable bandpass reaction cell inductively coupled plasma mass spectrometry with conventional inductively coupled plasma mass spectrometry for the determination of heavy metals in whole blood and urine

A Dynamic Reaction Cell™ inductively coupled plasma mass spectrometer (DRC-ICP-MS) was evaluated for the determination of arsenic, lead, cadmium, mercury, and thallium in urine and whole blood. Reaction cell conditions were evaluated for suppression of ArCl + and CaCl + polyatomic interferences. The reaction gas was 5% hydrogen in argon. Lead, cadmium, mercury, and thallium were determined with the reaction cell vented. Mixture of 2.5% t-butanol, 0.5% HCl, and 2 mg Au/l plus Ga, Rh, and Bi internal standards was used to dilute whole blood and urine. Calibration was achieved using aqueous acidic standards spiked into urine matrix. Urine and whole blood addition calibration curves were nearly identical for all five elements. DRC-ICP-MS detection limits were equivalent or better than conventional ICP-MS. Within run coefficients of variation (CV's) were nearly the same for DRC-ICP-MS and conventional ICP-MS for National Institute of Standards and Technology (NIST) SRM 2670 and BioRad Lyphochek Urine Metals Control. DRC-ICP-MS within run CV's for As, Pb, Cd, and Hg were 1.9%, 4%, 1.7%, and 1.7%, respectively, for NIST 2670 and 2.9%, 1.8%, 3.4%, 1.7%, and 1.0% for BioRad urine. BioRad Lyphochek Whole Blood control concentrations and CV's were: 78 μg/l (3.8%), 284 μg/l (0.52%), and 544 μg/l (0.9%). With the exception of mercury day-to-day CV's for certified whole blood and urine controls were less than 4% on both the DRC-ICP-MS and conventional ICP-MS.

Microassay of Phosphate Provides a General Method for Measuring the Activity of Phosphatases Using Physiological, Nonchromogenic Substrates Such as Lysophosphatidic Acid

Since measurement of lysophosphatidate phosphatase activity is important in studies of tumorigenesis, we attempted to develop a simpler alternative to the more complex methods currently available. Measuring the phosphate released would permit use of the same method for a variety of phosphatases with physiological substrates, many of which are nonchromogenic. The Malachite green method of K. Itaya and M. Ui (1966, Clin. Chim. Acta 14, 361) has adequate sensitivity for quantitating phosphatase activity in biological samples. In samples with high endogenous phosphate concentrations pretreatment with 50 mg Dowex 1 × 10 (100–200 mesh, OH− form) usually permitted reliable determination of phosphatase activity. For 34 consecutive runs the mean relative difference [(phosphorus activity − vitamer activity)/phosphorus activity] obtained from the simultaneous measurement of both the phosphate released and the corresponding organic product (pyridoxal and pyridoxine) was −0.03 ± 0.09. The within run and between run coefficients of variation (three runs of four to five replicates) were 0.05 and 0.04, respectively. Pyridoxine 5′-phosphate hydrolase activity (pH 10) in cultured skin cells (normal and cancerous) ranged from 2 to 12 nmol phosphorus/min · mg protein. Lysophosphatidate phosphatase activity (pH 7.4) ranged from 3 to 14 nmol phosphorus/min · mg protein. The current approach permits the measurement of phosphatase activity with a single method using a variety of substrates and incubation conditions.

Evaluation of an EIA method for measuring serum levels of the estrogen metabolite 2-hydroxyestrone in adults

Two-hydroxyestrone (2OHE-1) and 16α-hydroxyestrone (16OHE-1) are two estrogen metabolites that may play important roles in the development or promotion of breast cancer. Our study assessed the reliability of a newly developed kit procedure for measuring 2OHE-1. Although under certain conditions the assay would not distinguish 2OHE-1 from estriol, or possibly 2-methoxyestrone, steroids such as 17β-estradiol, estrone and 16OHE-1 should not interfere with the test. Our study evaluated the precision of this enzyme immunoassay (EIA) kit for measuring 2OHE-1 levels in serum obtained from healthy men and women. As a result of several replicate analyses of specimens obtained from 18 men and 20 women, we found that the within-run coefficients of variation (CVs) were approximately 20% and the among run CVs, 30%. Because the SD for the procedure is high, the limit of detection (LOD) was also high (130 ng/l). Nonetheless the assay could distinguish between 2OHE-1 levels in men (128 ng/l) and women (332 ng/l) because we performed a large number of analyses on each specimen. Improving the reproducibility of the assay would reduce the: 1. LOD; number of replicates needed to obtain reliable estimates of 2-OHE-1 levels; amount of time, effort, and cost for each analysis; and greatly improve the reliability of the method. Because the within-run variability is relatively smaller than the total variability (among run + within run), use of the assay for determining differences among groups could be justified only when measurements were made in a single run.

A new method for rapid measurement of lactate in fetal and neonatal blood.

A prospective trial to determine the accuracy and precision of the Boehringer Mannheim Accusport handheld lactate meter in measuring plasma lactate levels in umbilical cord blood and neonatal blood microsamples was performed in the labour ward and the neonatal intensive care unit of the NepeanHospital. Specimens were collected from the umbilical artery of 160 consecutive deliveries covering gestations from 26 to 42 weeks, and from 110 umbilical artery catheters covering a range of gestations from 26 to 41 weeks. Serum was also obtained from an exchange transfusion for coefficient of variation analysis. Blood was simultaneously tested for lactic acid concentration on the Boehringer Mannheim (BM) Accusport held lactate meter and the Radiometer ABL 625 blood-gas machine. Clinical data from the mother and baby were recorded together with the full blood-gas analysis for comparison with the lactate measures. Coefficients of variation for the BM Accusport lactate meter were established by a further 120 samples of plasma lactate at 6 concentrations from 1 to 20 mmol/L. The stability of measurements with the BM lactate meter over a wide range of temperatures was ascertained by repeated sampling of known concentrations of plasma lactate from 0.5 degrees C to 37 degrees C. The BM Accusport lactate meter was found to be accurate from 1 mmol/L to 20 mmol/L with a Passing Bablok regression line y = 0.004 + 0.915 x (95% CI of slope of 0.889 to 0.946 and intercept -0.138 to 0.094) for whole blood, and y = 0.200 + 1.000 x (95% CI of slope 0.989 to 1.018 and intercept 0.080 to 0.222) for plasma. Between run coefficient of variation (CV) was calculated to be 1.23% to 5.53% over the clinically significant range (2.2-19.3 mmol/L). The BM lactate meter was accurate from 5 to 37 degrees C. At 0.5 degrees C the BM lactate meter significantly underestimated the plasma lactate concentration. There was no significant effect of haematocrit (41.5 to 62%), gestation, or operator on the accuracy of the BM lactate meter. The Accusport handheld lactate meter is an accurate, commercially available, method of measuring plasma lactate levels in only 60 seconds at the point-of-care. It requires only 15 microL of blood and is significantly cheaper than other assay methods. The BM lactate meter is well suited to assess lactic acidaemia of fetal scalp and neonatal blood samples to help quantify hypoxic stress in the perinatal period.

Validation of human haptoglobin immunoturbidimetric assay for detection of haptoglobin in equine and canine serum and plasma.

The Incstar(R) SPQ II human haptoglobin (Hpt) (Incstar Corporation, Stillwater, MN) immunoturbidimetric assay was validated for the determination of serum and plasma Hpt concentrations in dogs and horses. The anti-human Hpt antiserum supplied with the assay, displayed monospecificity to both dog and horse serum Hpt by immunoelectrophoresis and Western blotting techniques. The automated immunoturbidimetric assay results correlated well with the cyanmethemoglobin binding assay (r=0.953 for canine serum and r=0.941 for equine serum), and had excellent precision at both high and low serum Hpt concentrations (within run and between run coefficients of variation near or less than 5%). The assay was linear in both species by serial dilution of pooled-high serum with pooled-low serum, saline and with Hpt-free serum. Interference from hemolysis (> 25 mg/dl hemoglobin) and lipemia greater than 100 mg/dl caused a false decrease and false increase respectively in Hpt yield with the immunoturbidimetric assay. The anti-Hpt antibody supplied with the assay kit, once diluted with polymer diluent and stored at 4 degrees C, was stable for up to 6 days and gave consistent results.

MODIFIED BROMOPHENOL BLUE DYE BINDING METHOD FOR QUANTITATION OF MICROALBUMINURIA IN DIABETES MELLITUS

Albumin excretion in microalbuminuria range is one of the earliest manifestation of nephropathy, specially in diabetes mellitus. The modified dye binding method using bromo-phenol blue was studied in 27 healthy controls and 54 patients of diabetes mellitus, negative for albuminuria by albustix test. The analytical recovery (99.4 to 104.0%), within run coefficient of variation (0.8 to 0.36%) and day-to-day coefficient of variation (2.39 to 0.82%), for low and higher range were within acceptable limits. The values in controls ranged as follows: urinary albumin concentration (mg/L) 7.7 - 28.4 in 2-hour specimen and 10.3 - 29.2 in overnight specimen; albumin excretion rate (µg/min) 7.8 - 29.7 in 2-hour and 9.2 - 29.6 in overnight specimen; and albumin creatinine ratio (mg/g) 10.6 - 29.6 in 2-hour and 11.9 - 29.6 in overnight specimens. Correlation analysis of various albumin excretion parameters revealed excellent correlation between estimations from overnight and 2-hour samples for albumin-creatinine ratio (r = 1.00) and albumin excretion rate (r=0.96). Equally good correlation was observed between 2-hour albumin-creatinine ratio and albumin excretion rate (r=0.95). In 10 of 54 patients excretion rate was more than 200 µg/min and could have been detected by repeat albustix test. Of the 36 positive for microalbuminuria, 21 had one or more target organ involvement. There was no target organ involvement in 8 patients negative for microalbuminuria. Screening for microalbuminuria by this simple and economic method, using 2-hour albumin-excretion rate or albumin-creatinine ratio could be one of the earliest investigations in diabetic patients.

Enzyme immunoassay and immunochemical characterization of pancreatic stone protein in human serum.

Monoclonal antibodies were raised against human pancreatic stone protein (PSP) and used for one-step enzyme immunoassay (EIA). PSP-S2-5 was employed as the standard in the assay. The assay's measurable range was 25-1,500 ng/ml and within run coefficient of variation was 3.7-6.4%. Analytical recovery of the assay was 101.5 +/- 5.65% (mean +/- SD). The results of experiments in which serum was fractionated by Mono S (cation exchange chromatography) suggested that most of immunoreactive material in human serum is PSP-S2-5. The EIA offers simple, rapid, and specific analysis of serum PSP level for clinical diagnosis.

Evaluation of the Abbott ADx Amphetamine/Methamphetamine II Abused Drug Assay: Comparison to TDx, EMIT, and GC/MS Methods

Although the legitimate clinical use of amphetamine and amphetamine congeners is declining, the illicit use of these drugs remains high. There is a need for a rapid and conclusive method for detecting these compounds in routine urine drug testing, drug screening in drug rehabilitation centers, and as an aid in the diagnosis and treatment of potential overdoses. The Abbott ADx Amphetamine/Methamphetamine II assay (A/M II), a fluorescence polarization Immunoassay (FPIA), was compared to the Abbott TDx Amphetamine/Methamphetamine II assay (FPIA), the Syva enzyme-multiplied immunoassay technique (EMIT) and a gas chromatograph/mass spectrometry (GC/MS) method. Precision of the A/M II assay was evaluated on the ADx analyzer over a 14-day period in each of three modes of operation (batch, combination, and panel) and was based on within-run and between-run coefficients of variation (CVs). Within-run CVs for all three controls (low [L], medium [M], and high [H]) ranged from 0.40% to 10.60% and between run CVs ranged from 3.96% to 7.92%. Data indicated that the calibration curve was stable for 16 days. Each of the six calibrators and three controls were within 10% of their labeled concentrations when analyzed by GC/MS. Fifty routine clinical specimens from our laboratory and 74 specimens screened as positive for amphetamine or related compounds from a rehabilitation center were screened by ADx, TDx, and EMIT. Any specimen yielding a positive result by any of these three methods was confirmed by GC/MS. In-house controls, as well as clinical samples, which contained both amphetamine and methamphetamine in the same sample produced results greater than two times the expected response on the ADx and TDx.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the Behring Nephelometer for Detection of Low Level Urinary Albumin

Mildly increased urinary albumin excretion rates and concentrations, below the quantity normally detected by conventional urinary protein and albumin methods, have prognostic significance for the development of nephropathy in patients with diabetes mellitus. The authors evaluated the automated Behring Nephelometer using Behring reagents for the detection of low level urinary albumin. Within run coefficients of variation (CVs, N = 20) are 1.7%, 1.3%, and 2.4% at mean urinary albumin levels of 16, 70, and 217 mg/L, respectively. Between run CVs (N = 20) are 4.5%, 2.6%, and 4.4% at mean albumin levels of 19, 71, and 239 mg/L, respectively. The method is sensitive to 3 mg/L. Hemoglobin, immunoglobulins, bilirubin, urea, and radiographic contrast media beyond a few hours of injection show no significant interference at levels normally expected from clinical specimens. Analysis is unaffected by pH within the physiologic range. Most urine specimens are stable for at least eight days when refrigerated at 4 degrees C. Specimen centrifugation before analysis is essential to avoid a negative bias that occurs when analyzing uncentrifuged refrigerated samples. Preanalytical freezing produces results higher than those observed in fresh or refrigerated samples. The authors conclude that automated nephelometry using the Behring Nephelometer is a convenient, simple, and accurate technique for the determination of low level urinary albumin.

Analytical and clinical evaluation of a new one-step non-analogue radioimmunoassay for serum-free thyroxine

We evaluated analytically and clinically the new one-step non-analogue free thyroxine (FT4) assay (Amerlex-MAB from Amersham), using a labelled monoclonal thyroxine-specific antibody as tracer, in comparison with the Gammacoat two-step FT4 kit (Baxter). Analytical performances of the new kit were excellent: within and between run coefficients of variation were less than 5% in the working range. Clinical sensitivities for hypo- and hyperthyroidism were comparable for both kits (FT4 Amerlex-MAB 95% confidence interval: 12-25 pM). When serum was supplemented with albumin we observed a slight decrease in FT4 values measured by both kits. When oleate was added to serum we noted a moderate increase with the Amerlex-MAB kit up to 10 mM oleate added and a much more marked increase with the two-step kit. Results obtained with patients from particular euthyroid populations, known to have low albumin or high free fatty acids concentrations or to have perturbed FT4 results when measured by an analogue-based method, agreed with those of the in vitro studies. With these patients the specificity of the Amerlex-MAB FT4 results was good but slightly decreased compared with the two-step FT4 method, except for heparin-treated patients who were all classified according to their euthyroidal status (17/17 instead of 13/17 with the two-step kit).

A Simple Assay of 3-Methoxy-4-hydroxyphenylethyleneglycol in Cerebrospinal Fluid by High Performance Liquid Chromatography

Abstract An improved method for the quantitation of 3-methoxy-4-hydroxyphenylethyleneglycol in cerebrospinal fluid is described. Sample cleaning was done by SEP PAK C18 Cartridge prior to the MHPG assay by high-performance liquid chromatography with electrochemical detector. The results are in good agreement wilth the GC/MS method. The average recovery is 68.3±4.1%, within run coefficient of variation of 3.8% and day to day of 7.2%. The method is simple, sensitive and accurate and can be used for routine work.

A solid-phase fluoroimmunoassay for human IgE

A fluoroimmunoassay (FIA) for the measurement of immunoglobulin E (IgE) is described. the method involves a sandwich technique in which antiserum to human IgE is adsorbed on a cellulose acetate/nitrate disc which is attached to a plastic StiQ TM sampler. The prepared sampler is reacted with serum and antigen is bound specifically. After buffer wash and treatment with goat serum to block non-specific binding, the samplers are reacted with monospecific fluorescein conjugated antisera to human IgE. Two buffer washes remove unbound material and the fluorescence which is directly proportional to the IgE concentration is measured in a FIAX ® fluorometer. Assay standards range from 2 to 400 IU/ml. The method gives within run coefficients of variation (CV) from 3.3 to 8.4% and between run CV from 6.2 to 16.1% being less precise at low analyte concentrations. IgE concentrations of a group of 74 sera determined by FIA using StiQs TM prepared with antisera from 2 sources correlated well with results found by radioimmunoassay.

Dual-isotope separation technique for radioassay of Cobalamin

Cobalamin is assayed by a dual-isotope separation method using sodium [ 125I]iothalamate as a marker. Two systems are used: one in which the incompletely-separated bound fraction is counted and compared with the single-isotope method in which the bound fraction is separated by washing (Phadebas radiosorbent assay); and one in which an aliquot of the free fraction is counted. In the dual-isotope method counting bound fractions, about 97% of the supernatant is removed by pouring from silicone fluid separators. The results for serum samples obtained using dual- and single-isotope methods were similar (between run coefficients of variation 5–7%). Experimental errors were smaller in the dual-isotope method. A factor in the kit standards, presumably the absence of proteins, was found to affect the separation technique, resulting in relatively large experimental errors for standards in the single-isotope method. Washing the solid phase in the single-isotope method apparently resulted in a loss of bound isotope. In the dual-isotope method counting free fractions reasonable precision was obtained (coefficient of variation of serum samples 6.6%) even though only about 56% of the supernatant (free fraction) was counted.