Mean Interassay Coefficients Of Variation Research Articles

Quality Assurance in Lactation: Reliability of OM-6050 Station System to Test Mother's Milk Osmolality.

Mother's own milk does not provide enough nutrients to feed a preterm baby born before 32 weeks' gestation; therefore, human milk fortifiers are needed. However, human milk fortifiers increase the osmolality, and enteral administration of high osmolality fluids has been associated with gastrointestinal symptoms. For this reason, it is necessary for laboratories to have a validated system in order to measure human milk osmolality. The aim of this study was to validate the OM-6050 Station System for measuring the osmolality of fortified mother's milk samples. Osmolality was measured using the osmometer OM-6050 Station System. Milk samples from healthy mothers (N = 3) unfortified and with two fortifiers (Almirón Fortifier® or NAN FM85®), as well as a nutritional supplement (Duocal MCT®) were used in the validation study through precision and linearity analysis. In the precision study the mean intra-assay coefficient of variation was 1.2% and 1.7% for mother's milk and fortified mother's milk, respectively. The mean inter-assay coefficient of variation was ≤ 1% in both cases. In the linearity study the regression analysis had a linear response to fortified mother's milk osmolality between 294 mOsm/kg and 539 mOsm/kg. The osmometer OM-6050 Station was reliable for determining the osmolality of fortified and unfortified mother's milk. It may be useful in the clinical practices within Neonatal Intensive Care Units.

Development of a Novel Multiplex Bead-based Assay for Measuring Autoantibodies on Flow Cytometric Platform

ABSTRACT Background Autoantibodies (AAbs) are important biomarkers for the diagnosis of Autoimmune Diseases (ADs). The detection of AAbs performed by current methods (indirect immunofluorescence test (IIFT)/Immunoblot (dot/line)/enzyme-linked immunosorbent assay ELISA) which have limitations in terms of performing multiple assays to arrive at laboratory diagnosis. We validated a novel multiplex bead-based assay (NMBA) that could quantify five common antibodies, simultaneously, on a flow-cytometry platform. Methods A total of five recombinant antigens (SS-A Ro60, CENP B, RNP 70, Scl 70 and Histones) were covalently coupled onto beads and tested using known positive sera (positive for AAbs) and analyzed using flow cytometer. Results The sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were obtained for each antigen, analyzed by both assays (NMBA and IIFT). It showed comparable or higher values for the NMBA. The Spearman’s rank correlation coefficient (Rho) were ≥ 0.97, (P < .05), indicating that multiplexing of the five autoantigens did not alter the results obtained when antigens were tested individually. The mean intra-assay precision measured by coefficient of variation (CV) was7.56 ± 1.6% and the mean inter-assay CV was 10.03 ± 1.34%. The time taken from sample receipt to reporting of results was 90 minutes in NMBA as compared to 150 minutes of IIFT Conclusion The NMBA could quantitatively measure antibodies against five autoantigens, simultaneously in patient’s sera. The assay is faster, objective, reproducible, requires low sample volume, and stable. Moreover, the flow cytometer in diagnostic laboratory settings for hematological and transplant immunology tests, can also be used for testing AAbs.

Quantitative Detection of Antibodies to Aleutian Disease Virus in Dried Blood Spots as an Estimation of Hypergammaglobulinemia in Mink

Infections with Aleutian disease virus (ADV) cause a progressive hypergammaglobulinemia, immune complex formation and plasma cell infiltrations in internal organs which induce a multi-systemic disease with high mortality. Serum ADV antibodies have traditionally been diagnosed with counter immunoelectrophoresis (CIEP) as a gold standard for qualitative assessment separating infected from non-infected animals, but less laborious ELISA methods have been confirmed to be equally sensitive. A way to simplify the diagnostics further could be to demonstrate antibodies in Dried blood spot samples (DBS). However, quantitative analysis of ADV antibodies in DBS and its correlation to the degree of hypergammaglobulinemia have not been scientifically published. The aim of this paper was to describe the adaptation and validation of the VP2 ELISA to ADV antibody detection in DBS and compare the estimated antibody levels in DBS to CIEP results, the estimated antibody levels and albumin: gamma globulin ratio in serum. The VP2 ELISA worked technically well when transferred from serum to DBS with mean intra-assay and mean inter-assay coefficients of variation within ± 20%. The DBS VP2 ELISA had a sensitivity of 97.3% and specificity of 93.2% compared to CIEP. Further, we found a correlation coefficient between the level of antibodies in DBS and the A:γG ratio of -0.81. The correlation between the A:γG ratio and the OD450 value was superior in DBS compared to serum samples from the same mink with the most pronounced difference at low A:γG ratios. Our results confirmed that the VP2 ELISA could detect ADV antibodies in DBS with a high sensitivity and specificity when employing CIEP as gold standard. The antibody titers estimated with DBS VP2 ELISA were well correlated to the antibody titters and A:γG ratios in serum, and the DBS VP2 ELISA could be an applicable and preferable tool for estimating AD progression in mink.

High-Resolution HbA1cSeparation and Hemoglobinopathy Detection With Capillary Electrophoresis

The CAPILLARYS HbA(1c) kit used on the CAPILLARYS 2 Flex Piercing system is designed for separating and quantifying the HbA(1c) fraction of hemoglobin while detecting hemoglobin variants and HbA(2). The aim of this study was to assess the performance of this new capillary electrophoresis kit for routine HbA(1c) quantification for patients with diabetes. The reliability of the system for HbA(2) measurement was also assessed. Precision, correlation, and linearity studies were performed. The effect of hemoglobin concentration and the presence of coexistent interfering substances were evaluated. The results were found to be accurate and linear in the clinically significant analytic range with excellent precision (mean interassay coefficient of variation of 0.7%). HbA(1c) measurement is independent of the presence of common hemoglobin interferences for HbA(1c) quantification and the total concentration of hemoglobin. Moreover, the technique provides a rapid and reliable separation of HbA(2). The measurement is reproducible and the results in β-thalassemia carriers were statistically different from those in noncarriers. Thus, this technique might be used for β-thalassemia detection. Given the short time of the analysis and the high throughput, this is a suitable system for the control of diabetes and the detection of hemoglobinopathies in laboratories with high workflow.

Estimating Intra- and Inter-Assay Variability in Salivary Cortisol

Investigators commonly assess intra- and inter-assay coefficients of variation (CVs) to estimate the precision of salivary cortisol enzyme immunoassay (EIA). However, little guidance is available as to which samples to use for CV assessment. The purposes of this methodological study were to compare differences in intra- and inter-assay CVs (a) among controls, standards, and/or unknown samples; and (b) between fresh and previously frozen saliva. A total of 174 duplicates (controls = 58, standards = 48, and unknowns = 68) were tested. The unknowns were from 34 students; all student saliva was assayed as both fresh and frozen samples. All samples were assayed in duplicate, using a commercial salivary cortisol EIA kit, by the same technician with the same equipment. A priori criteria for intra- and inter-assay CV, respectively, were ≤ 4% and ≤ 7%, and a was .05 for CV differences. Mean intra-assay CVs for controls, standards, unknowns, and combined samples were ≤ 2.5%, and mean inter-assay CVs were ≤ 2.8%. Mean intra-assay CVs were 2.2% for fresh saliva and 1.5% for frozen samples. Comparisons showed no significant differences in intra- or inter-assay CV among controls, standards, and/or unknown samples. Inter-assay CV was significantly different between fresh and previously frozen saliva (p = .043), with fresh saliva CV higher than frozen; the difference was not meaningful because all evaluations showed minimal measurement error. In conclusion, results indicate that estimation of precision can be achieved by testing of controls, standards, or unknowns and with either fresh or frozen saliva in this population.

Development and in-house validation of allergen-specific ELISA tests for the quantification of Dau c 1.01, Dau c 1.02 and Dau c 4 in carrot extracts (Daucus carota)

Even though carrot allergy is common in Europe, the amount of different allergens in carrots is still unknown due to a lack of methods for quantitative allergen measurements. The current study aimed at the development of quantitative ELISA tests for the known carrot allergens, namely Dau c 1.01, Dau c 1.02, and Dau c 4 in pure carrot extracts. Monoclonal antibodies targeting the major carrot allergen isoforms Dau c 1.01 and Dau c 1.02 were generated and combined in sandwich ELISA with rabbit antisera against Api g 1, the celery homologue of Dau c 1. A competitive ELISA for the carrot profilin Dau c 4 was based on a polyclonal rabbit antiserum. The three ELISA tests were allergen-specific and displayed detection limits between 0.4 and 6ng allergen/ml of carrot extract. The mean coefficient of variation (CV) as a means of intraassay variability of the Dau c 1.01, Dau c 1.02 and Dau c 4 ELISA tests was 8.1%, 6.9%, and 11.9%, and the mean interassay CV 13.3%, 37.1% and 15.6%, respectively. Target recovery ranged between 93 and 113%. In conclusion, the specific, accurate and reproducible quantification of three important carrot allergens may help to identify less allergenic carrot varieties, as well as to standardize the amount of allergens in extracts used for carrot allergy diagnosis.

Development and evaluation of a fluorogenic real‐time RT‐PCR for the detection of dengue 3 virus

A dengue-3-specific real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was developed using the novel Light Upon eXtension (LUX™) fluorogenic technology. A labeled forward primer and a standard reverse primer that target a conserved region within the non-structural 1 (NS1) gene of dengue 3 strains were designed. The dengue-3-specific assay did not recognize other dengue serotypes and related flaviviruses. Using a tenfold serial dilution of plasmid DNA containing the dengue 3 NS1 gene as standards, the range of dengue virus detection was determined to be from 10(3) to 10(9) copies/ml or from 80 to 8 × 10(7) copies/reaction with an average correlation coefficient of ≥ 0.99. The mean intra-assay coefficient of variation (CV) at 2.01% and the mean inter-assay CV at 2.68% suggest the repeatability of the procedure. Moreover, the fluorogenic assay was evaluated by using clinical specimens and comparing test results with historical data obtained from conventional RT-PCR, which served as the criterion standard. Using patient sera as test samples, the assay demonstrated 95.45% sensitivity and 100% specificity, respectively. These results reveal that the real-time RT-PCR assay may be utilized as a rapid, convenient, and sensitive tool for the detection of dengue 3 in clinical and laboratory specimens.

Sensitive analytical methods for quantification of novel DNA and protein in bovine milk – first results from a long- term feeding study in dairy cows

In several short-term studies, genetically modified (GM) plants registered in the EU were considered as safe. However, possible effects of a long-term feeding have not been studied. Hence, 36 lactating Simmental cows were fed on GM maize MON810 or on the isogenic counterpart for 24 months. Samples from several matrices (milk, blood, feces, urine) were taken monthly and analyzed using sensitive DNA and protein quantification techniques. A method for DNA isolation, previously published by the FOPH Berne (Schweizer Lebensmittelhandbuch), was optimized and validated. Down to 1000 copies of the recombinant cry1Ab gene per 300 μl milk could be detected after DNA extraction with a mean intra-assay coefficient of variation (CV) of 0.15% and a mean inter-assay CV of 0.78%. An ELISA was developed for the surveillance of the Cry1Ab protein. The assay allowed Cry1Ab protein determination in skim milk in an analytical range of 0.1–25ng/mL with a CCβ of 0.25ng/mL and a CCβ of 0.38ng/mL.

Abnormal circulating levels of metalloprotease 9 and its tissue inhibitor 1 in angiographically proven peripheral arterial disease: relationship to disease severity

Peripheral arterial disease (PAD) is associated with adaptive changes in the vascular and muscle extracellular matrix (ECM) in response to reduced blood flow. Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), are key modulators of ECM turnover. We hypothesized that patients with intermittent claudication (with low ankle-brachial blood pressure index, <0.8), and critical ischaemia would have raised circulating levels of MMP-9, TIMP-1 and TIMP-2 compared with healthy controls, reflecting an increase in proteolytic activity which may be related to ECM turnover in PAD. We studied 36 patients (23 males; 65 +/- 9 years) with intermittent claudication and 43 (25 males; 68 +/- 12) patients with critical ischaemia. All patients had angiographic evidence confirming significant PAD. Circulating levels of MMP-9 and TIMP-1 were higher (both P < 0.0001) in the PAD patient groups compared with the controls. Patients with critical ischaemia had MMP-9 and TIMP-1 levels that were significantly higher than those with intermittent claudication. There were no differences in circulating TIMP-2 levels between patients and controls. There was a modest positive correlation between the white cell count (WCC) and MMP-9, both patients with intermittent claudication (Spearman, r = 0.398, P = 0.016) and critical ischaemia (r = 0.378, P = 0.014). We demonstrate higher levels of circulating MMP-9 and TIMP-1 in patients with intermittent claudication and critical ischaemia. Circulating concentrations of both markers can be related to disease severity, being higher in critical ischaemia compared with levels in intermittent claudication.

Determination of the adhesive capability of canine polymorphonuclear neutrophil granulocytes using a fluorometric microtiter plate cellular adhesion assay

A rapid fluorometric cellular adhesion assay in microtiter plates was evaluated for canine polymorphonuclear neutrophil granulocytes (PMN) using the fluorescent indicator calcein acetoxymethyl ester (Calcein AM). Optimum adhesive responsiveness occurred in bovine serum albumin (BSA)-coated plates after 30 min incubation at 37°C with 5 × 10 5 PMN in 100 μl per well using phorbol myristate acetate (PMA, 100 ng/ml) as stimulant. The adhesive responsiveness for 15 laboratory beagle dogs was determined and the mean percentage of adherence for unstimulated (2.9%) and stimulated (74.3%) PMN and corresponding 95% confidence intervals was determined. The mean intra-assay coefficient of variation (CV intra) for unstimulated and stimulated adhesion in the assay was: CV intra = 12.9% and CV intra = 4.1%, respectively. The mean inter-assay coefficient of variation (CV inter) for unstimulated and stimulated adhesion, respectively, in the assay was: CV inter = 22.9% and CV inter = 5.6%. The assay was highly reproducible and labelling with Calcein AM neither reduced PMN viability nor activated the PMN.

Evaluation of a new reagent for preserving fresh blood samples and its potential usefulness for internal quality controls of multichannel hematology analyzers.

We describe a new, easy-to-use reagent, Cyto-Chex (Streck Laboratories, Omaha, Neb), that preserves fresh whole blood in a non-cross-linking, nonformalin manner. Target values assigned to fresh blood were essentially met after preservation and storage of up to 31 days. Respective mean analytic inaccuracies and short-term intra-assay coefficients of variation (n = 30) were as follows: WBCs, 6.7% and 1.99%; RBCs, 0.7% and 0.76%; hemoglobin, -1.8% and 0.79%; hematocrit, -0.3% and 0.75%; mean corpuscular volume, -1.0% and 0.78%; and platelets, 6.9% and 3.12%. Linearity of dilution-sensitive analytes was satisfactory over a wide range of dilutions after preservation of blood samples. Ten independent laboratories using 10 different instruments determined day-to-day interassay imprecision during four 7-day periods after blood preservation. Mean interassay coefficients of variation for participating laboratories were WBC, 1.92%; RBC, 1.00%; hemoglobin, 1.29%; hematocrit, 2.00%; and platelets 3.29%. Cyto-Chex enables long-term monitoring of instrument accuracy and precision with retained blood specimens of healthy persons. Blood from patient cohorts with various hematologic disorders and with a wide range of numeric abnormalities and/or parameter aberrations can be preserved satisfactorily with this reagent. The reanalysis of preserved patient blood samples is an important adjunct to the use of commercial control material in quality control programs of multichannel hematology analyzers.

A method for accurate measurement of GFR in conscious, spontaneously voiding rats

Renal function measurement by clearance methods relies on accurately timed urine collection. In small experimental animals, renal function measurement is usually performed under anesthesia and/or with the application of bladder catheters to ensure accurate urine collection. To avoid both anesthesia and the need for bladder catheters we developed a method to measure glomerular filtration rate (GFR) in spontaneously voiding conscious rats. GFR was measured as the urinary clearance of constantly infused 125I-iothalamate. To correct for incomplete bladder emptying urinary clearance of 125I-iothalamate was multiplied by the ratio of plasma and urinary clearance of simultaneously infused 131I-hippuran, a correction method that has been previously validated in humans. Reproducibility of the technique was evaluated by analysis of the results of four consecutive clearance periods during the day (intra-assay variation) in a group of 17 rats and of two consecutive clearance periods on two or three separate days in a group of 20 rats (inter-assay variation), all with normal renal function. Application of the correction method reduced the intra-assay coefficient of variation (mean +/- SD) from 37.4 +/- 14.3 to 5.4 +/- 2.3% (P < 0.05). The mean inter-assay coefficient of variation fell slightly from 23.4 +/- 10.3 to 11.0 +/- 7.2% (P < 0.10). In rats with moderately impaired renal function (N = 8) the intra-assay variation fell from 27.9 +/- 20.7 to 2.7 +/- 1.6% (P < 0.05). Our data show that this correction method is a useful technique to assess renal function in conscious, spontaneously voiding rats.

Rapid Solid-Phase Extraction of Dopamine, Serotonin, and Their Acidic Metabolites for High-Performance Liquid Chromatography with Electrochemical Detection

Abstract A rapid solid-phase extraction procedure utilizing C18 reverse-phase mini-columns has been established for dopamine, 3,4-dihydroxyphenylacetic acid, serotonin, and 5-hydroxyindoleacetic acid in tissue perfusate samples. The method rapidly separates the analytes from protein in albumin-containing tissue perfusates. The analytes which are recovered in high yield are then determined by high-performance liquid chromatography with electrochemical detection. The mean interassay coefficient of variation for the recovery of dopamine, 3, 4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid was 12, 7, 13 and 8 percent, respectively.

Objective analysis of stallion sperm motility

An image-analysis system utilizing a microcomputer and CellSoft computer-assisted semen analysis software package was evaluated to assess stallion sperm motility characteristics. Analyses were performed at 37°C on a 6 μl drop of diluted semen placed on a glass slide and covered with an 18 mm 2 coverslip. Four groups of 25 cells each per slide, four slides per ejaculate and four ejaculates from each of three stallions were analyzed in a nested model. The percentage of motile sperm cells, mean velocity (μm/sec), mean linearity, and mean angular head displacement (μm) were measured. Statistical analysis of variance components showed that within ejaculates, more variation was accounted for in the differences among groups of 25 cells than among slides. Predicted standard deviations calculated for combinations of slides and groups of cells showed that a combination of two slides from which a total of 400 cells were analyzed resulted in a mean intra-assay coefficient of variation (CV) of 5.7% for the four measured variables. The following are individual coefficients of variation: percentage of motile cells (7.8%), mean velocity (6.4%), mean linearity (1.9%) and mean angular head displacement (6.6%). When ejaculate differences were included in the model and predicted standard deviations were calculated for a single ejaculate, the mean inter-assay CV was 9.2%. Mean velocity (6.4%) and mean linearity (4.7%) were more repeatable among ejaculates than either the percentage of motile sperm (14.4%) or angular head displacement (11.2%). It was concluded that this system is precise enough to determine differences in motility characteristics of stallion semen samples.

Improved high-performance liquid chromatographic assay for the quantification of 5-bromo-2′-deoxyuridine and 5-bromouracil in plasma

A sensitive and specific procedure using high-performance liquid chromatography (HPLC) was developed for the quantification of 5-bromo-2′-deoxyuridine (BUdR) and 5-bromouracil (BU) in plasma. BUdR and BU were first extracted with a mixture of ethyl acetate and 2-propanol from plasma presaturated with solid ammonium sulfate. Following evaporation of the organic extract, the remaining residue was reconstituted in saturated ammonium sulfate solution, washed with a mixture of n-pentane—methylene chloride and re-extracted with the original solvent mixture. The organic extract was evaporated, reconstituted in mobile phase and chromatographed on a regular-bore ODS HPLC column using ultraviolet absorbance detection. The BUdR and BU quantification limits were both 0.1 μ M, the mean intra-assay coefficients of variation were 5.0 and 5.6%, respectively, and the mean inter-assay coefficients of variation were 5.4 and 10.7%, respectively. This method was used to determine steady-state femoral arterial and hepatic venous plasma concentrations of BUdR and BU in a patient receiving a continuous intravenous infusion of BUdR (20 mg/kg per day).

Radioenzymatic determination of phenylpropanolamine in plasma

We modified a norepinephrine radioenzymatic method ( C. R. Lake, M. G. Ziegler, and I. J. Kopin, 1976, Life Sci. 18, 1315–1326) for determination of plasma phenylpropanolamine (PPA) concentrations. PPA is converted to N[ methyl- 3H]ephedrine ([ 3H]EPD) by the enzyme phenylethanolamine N-methyltransferase (PNMT) and S-[ methyl- 3H]adenosyl- l-methionine ([ 3H]AdoMet). The product, [ 3H]EPD, is isolated from unreacted [ 3H]AdoMet and labeled side products by an organic extraction and a TLC procedure. In addition, a preincubation organic extraction procedure is included to remove inhibitors of PNMT from plasma and to concentrate the sample for enhanced enzymatic conversion. In order to accurately quantitate PPA across the wide range of possible concentrations, the assay is conducted at two plasma volumes. PPA concentrations between 0.3 and 50 μg/liter can be detected with 1 ml of plasma, while concentrations between 4 and 1500 μg/liter can be detected with 0.1 ml of plasma. The intra-assay coefficients of variation (CVs) are 9.3 and 5.7% at 0.5 and 1500 μg/liter, respectively, while the mean interassay CV is 13.8%.