Mean Intra-assay Coefficient Of Variation Research Articles

Hypocretin-1 measurements in cerebrospinal fluid using radioimmunoassay: within and between assay reliability and limit of quantification.

Study ObjectivesThe most sensitive and specific investigative method for the diagnosis of narcolepsy type 1 (NT1) is the determination of hypocretin-1 (orexin-A) deficiency (≤110 pg/mL) in cerebrospinal fluid using a radioimmunoassay (RIA). We aimed to assess the reliability of the Phoenix Pharmaceuticals hypocretin-1 RIA, by determining the lower limit of quantification (LLOQ), the variability around the cutoff of 110 pg/mL, and the inter- and intra-assay variability.MethodsRaw data of 80 consecutive hypocretin-1 RIAs were used to estimate the intra- and inter-assay coefficient of variation (CV). The LLOQ was established and defined as the lowest converted concentration with a CV <25%; the conversion is performed using a harmonization sample which is internationally used to minimize variation between RIAs.ResultsThe mean intra-assay CV was 4.7%, while the unconverted inter-assay CV was 28.3% (18.5% excluding 2 outliers) and 7.5% when converted to international values. The LLOQ was determined as 27.9 pg/mL. The intra-assay CV of RIAs with lower specific radioactive activity showed a median of 5.6% (n = 41, range 1.6%–17.0%), which was significantly higher than in RIAs with higher specific activity (n = 36; median 3.2%, range 0.4%–11.6%, p = .013). The CV around the 110 pg/mL cutoff was <7%.ConclusionsHypocretin-1 RIAs should always be harmonized using standard reference material. The specific activity of an RIA has a significant impact on its reliability, because of the decay of 125I radioactivity. Values around the hypocretin-1 cut-off can reliably be measured. Hypocretin-1 concentrations below 28 pg/mL should be reported as “undetectable” when measured with the Phoenix Pharmaceuticals RIA.Clinical Trial InformationThis study is not registered in a clinical trial register, as it has a retrospective database design

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.

Validation of endotoxin-core antibodies in dried blood spots as a measure of environmental enteropathy and intestinal permeability.

To validate a method for measuring endotoxin-core antibodies (EndoCAb) from dried blood spots (DBS)-drops of capillary whole blood collected and dried on filter paper-as an indicator of environmental enteropathy (EE) in infancy and early childhood. A commercially available enzyme immunoassay kit was adapted for use with DBS, with optimized steps for sample elution. Technical validation included analysis of linearity/recovery, precision and reliability, lower limit of detection, and correspondence between matched plasma and DBS samples. Validation in a field-based setting was implemented with samples from Peruvian infants (n = 82; age = 2-33 months) collected at two time points six months apart. A high correspondence between plasma and DBS levels of EndoCAb was observed (R2 = 0.93, P < .001). The lower limit of detection was found to be 0.01 GMU/mL. Interassay coefficient of variation (CV) was 10.9% and 8.06% for low and high controls, respectively. Mean intra-assay CVs were 3.22% and 1.83%, respectively. In a sample of Peruvian infants, EndoCAb levels increased with age as expected (P < .001). Age explained nearly 34.6% of the variance in EndoCAb across the sample. These findings demonstrate the validity and feasibility of measuring EndoCAb in remote field settings using minimally invasive DBS sampling.

Evaluation of an Automated CLIA System for the Determination of Carbohydrate Antigen 19-9.

To compare analytical characteristics between two different methods, a automated chemiluminescent immunoassay (CLIA) versus a radioimmunometric assay (RIA), for the determination of carbohydrate antigen 19-9 (CA19-9). One hundred and eighty-five blood samples from consecutively enrolled individuals (87 males and 98 females, aged 22-89 and 29-89 years, respectively) were evaluated. For both assays, a cut-off of 37 U/ml was used. Comparison between assays was analyzed using Passing-Bablok regression, which showed a high inter-assay correlation (r=0.926). The mean intra-assay coefficient of variation was 17.6% (range=6.1-50.7%) for the RIA (n=10 samples) and 5.2% (range=0.4-17.0%) for the CLIA assay (n=26 samples). This study showed that CLIA for CA19-9 has a good reliability on all samples analyzed and should be preferred to RIA with the aim of reducing costs, greater standardization and more harmonized results.

Filter paper blood spot enzyme linked immunoassay for adiponectin and application in the evaluation of determinants of child insulin sensitivity.

BackgroundAdiponectin is an adipocyte-derived hormone that acts as a marker of insulin sensitivity. Bloodspot sampling by fingerstick onto filter paper may increase the feasibility of large-scale studies of the determinants of insulin sensitivity. We first describe the validation of an enzyme-linked immunoassay (ELISA) for quantifying adiponectin from dried blood spots and then demonstrate its application in a large trial (PROBIT).MethodsWe quantified adiponectin from 3-mm diameter discs (≈3 µL of blood) punched from dried blood spots obtained from: i) whole blood standards (validation); and ii) PROBIT trial samples (application) in which paediatricians collected blood spots from 13,879 children aged 11.5 years from 31 sites across Belarus. We examined the distribution of bloodspot adiponectin by demographic and anthropometric factors, fasting insulin and glucose.ResultsIn the validation study, mean intra-assay coefficients of variation (n = 162) were 15%, 13% and 10% for ‘low’ (6.78 µg/ml), ‘medium’ (18.18 µg/ml) and 'high’ (33.13 µg/ml) internal quality control (IQC) samples, respectively; the respective inter-assay values (n = 40) were 23%, 21% and 14%. The correlation coefficient between 50 paired whole bloodspot versus plasma samples, collected simultaneously, was 0.87 (95% CI: 0.78 to 0.93). Recovery of known quantities of adiponectin (between 4.5 to 36 µg/ml) was 100.3–133%. Bloodspot adiponectin was stable for at least 30 months at −80°C. In PROBIT, we successfully quantified fasting adiponectin from dried blood spots in 13,329 of 13,879 (96%) children. Mean adiponectin (standard deviation) concentrations were 17.34 µg/ml (7.54) in boys and 18.41 µg/ml (7.92) in girls and were inversely associated with body mass index, fat mass, triceps and subscapular skin-fold thickness, waist circumference, height and fasting glucose.ConclusionsBloodspot ELISA is suitable for measuring adiponectin in very small volumes of blood collected on filter paper and can be applied to large-scale studies.

Initial Validation of Cytokine Measurement by ELISA in Canine Feces

Measurement of fecal cytokines has been used as a marker of intestinal inflammation in people and correlates with endoscopic findings. The aim of this study was to evaluate the use of canine-specific enzyme-linked immunosorbant assays (ELISAs) for quantification of cytokines in canine fecal samples as a non-invasive biomarker. Interleukin (IL)-6, -8, -10, -23/12p40 and TNF-a were assessed by using spiked fecal samples from 3 healthy dogs. Standard curve validation was performed, and the impact of time to freeze, duration of storage and number of freeze-thaw cycles on cytokine concentration were also examined. All the cytokines assayed could be detected, with varying accuracy. The mean coefficient of variation (CV) for all standard curves ranged from 2.95%-9.8%. The mean intra-assay CV ranged from 3.1%-11.14%, and inter-assay CV from 4.36%-18.83%. Recovery of IL-23 was poor (7.23%-17.12%), precluding further interpretation of stability studies. Mean recovery did not appear to be affected by time to freeze and repeat freeze-thaw cycles in all cytokines investigated. Recovery for all cytokines after short-term storage of 30 days at -80℃ showed a recovery of 70% or >130%. In conclusion, although fecal IL-6, -8, -10, and TNF-a could be used as biomarkers of intestinal inflammation in the dog, the quality of laboratory performance and poor recovery at lower concentrations limit their application. Bench-top and freeze-thaw stability was acceptable, and samples should ideally be analyzed within a week. Investigation involving dogs with acute and chronic inflammatory intestinal disease is required to determine the role of this methodology in a clinical setting.

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 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.

A highly sensitive electrochemiluminescence immunoassay for the neurofilament heavy chain protein

Background The loss of neurological function is closely related to axonal damage. Neurofilament subunits are concentrated in neurons and axons and have emerged as promising biomarkers for neurodegeneration. Electrochemiluminescence (ECL) based assays are known to be of superior sensitivity and require less sample volume than conventional ELISAs. Methods We developed an ECL based solid-phase sandwich immunoassay to measure the neurofilament heavy chain protein (NfH SMI35) in CSF. We employed commercially available antibodies as previously used in a conventional ELISA (Petzold et al., 2003; Petzold and Shaw, 2007). The optimised and validated assay was applied in a reference cohort and defined patient groups. Results Analytical sensitivity (background plus three SD) of our assay was 2.4 pg/ml. The mean intra-assay coefficient of variation (CV) was 4.8% and the inter-assay CV 8.4%. All measured control and patient samples produced signals well above background. Patients with multiple sclerosis (MS) (median 46.2 pg/ml, n = 95), amyotrophic lateral sclerosis (ALS) (160.1 pg/ml, n = 50), mild cognitive impairment/Alzheimer's disease (MCI/AD) (65.6 pg/ml, n = 20), Guillain–Barre syndrome (GBS) (91.0 pg/ml, n = 20) or subarachnoid hemorrhage (SAH) (345.0 pg/ml, n = 20) had higher CSF NfH SMI35 values than the reference cohort (27.1 pg/ml, n = 73, p < 0.0001 for each comparison). Conclusion The new ECL based assay for NfH SMI35 in CSF is superior in terms of sensitivity, precision and accuracy to previously published methods (Petzold et al., 2003; Shaw et al., 2005; Teunissen et al., 2009). The improved performance and small sample volume requirement qualify this method in experimental settings and clinical trials designed to perform a number of tests on limited amounts of material.

Rapid method for cholesterol analysis in bovine milk and options for applications

The adaptation of a colorimetric technique for the analysis of cholesterol in raw milk is presented. Performance quality was satisfying (mean intra-assay coefficient of variation (CV) 4.8, inter-assay CV 9.1%, linearity between 0 and 7 mm, recovery of spiked cholesterol into raw milk 98.1 and 106.3%). However, the milk fat extraction must be carried out within the 48 hours following milk sampling. When performing sampling, the significant variation of milk cholesterol composition during the milking process has to be taken into account.

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.

Multicenter evaluation of the hemolysis index in automated clinical chemistry systems

In vitro hemolysis, the prevailing cause of preanalytical error in routine laboratory diagnostics, might influence the reliability of several tests, affect the quality of the total testing process and jeopardize patient safety. Although laboratory instrumentation is now routinely equipped with systems capable of automatically testing and eventually correcting for hemolysis interference, to our knowledge there are no reports that have compared the efficiency of different analytical platforms for identifying and classifying specimens with hemolysis. Serum from a healthy volunteer was spiked with varying amounts of hemolyzed blood from the same volunteer, providing a serum free hemoglobin concentration ranging from 0.0 g/L to 2.0 g/L as measured by the reference cyanmethemoglobin assay. The spiked serum samples were shipped to seven separate laboratories and the hemolysis index (HI) was tested in triplicate on the following analytical platforms: Roche Modular System P (n=4) and Integra 400 Plus (n=1), Siemens Dimension RxL (n=3), ADVIA 2400 (n=1) and ADVIA 1800 (n=1), Olympus AU 680 (n=1) and Coulter DXC 800 (n=1). Satisfactory agreement of HI results was observed among the various analytical platforms, despite a trend toward overestimation by the ADVIA 2400 and 1800. After normalizing results according to the instrument-specific alert value, discrepancies were considerably reduced. All instruments except for the Dimension RxL gave values normalized to the instrument-specific alert value, <1.0 for the sample with 0.048 g/L free hemoglobin, and >1.0 for the sample with 0.075 g/L free hemoglobin. The results of the four Modular System P tests were also highly reproducible among the different facilities. When evaluating instruments that provided quantitative HI results, the mean intra-assay coefficient of variation (CV) calculated for the triplicate determinations was always between 0.1% and 2.7%. The results of this multicenter evaluation confirm that efficiency of different analytical platforms to correctly identify and classify unsuitable samples is satisfactory. However, more effort should be placed on the standardization of reporting HI. All the instruments that we tested provide either quantitative or qualitative results that are essentially comparable, but which should always be compared with the instrument-specific alert values to harmonize their efficiency.

Evaluation of the Cardiac reader® as a point-of-care instrument for measurement of fibrin D-dimers in dogs

Summary Objective: The aim of this study was the evaluationof the automated point-of-care analyzer Cardiac reader® for quantitative fibrin D-dimer measurement compared to the Liatest™ D-Di™ run on the bench top analyzer STA Compact (all Roche). Material and methods: 41 fresh canine blood specimens submitted from the Clinic for Small Animals, Justus-Liebig-University Giessen were included. Measurement of fibrin D-dimers with the Liatest™ D-Di™ immunoturbidimetric assay was performed in citrated blood plasma stored at –80 °C until analysis. Using the Cardiac reader, D-dimer plasma concentrations were measured with an immunological assay in heparinized fresh (&lt; 1 hour) whole blood specimens. Results: Mean intra-assay coefficient of variation (CV) was 6.13% (range: 2.2–10.0%) for the Liatest™ D-Di and 19.8% (range: 20.1–19.6%) for the Cardiac reader. Fibrin D-dimers detected with the STA Compact exceeded the upper limit of the reference interval (0.4 μg/ml; n = 62 healthy dogs) in 20/41 samples. There was a fair correlation between D-dimer measurements with the Cardiac reader and the comparison method (r = 0.78). Generally, results were underestimated with a mean proportional bias (0.45 μg/ml). The area under the Receiver Operating Characteristic (ROC) curve was 0.895 regarding the detection of increased fibrin D-dimers with the Cardiac reader. At a cut-off value of &gt; 0.2 μg/ml, sensitivity/specificity were 85%/91% and positive/negative likelihood ratios were 8.93/0.17. At a lower cut-off value of 0.15 μg/ml sensitivity and specificity were 90% and 66%, respectively. Conclusion and clinical relevance: The Cardiac reader can be used for quantitative measurement of canine D-dimers, however, due to the odds of false negative results and the high imprecision the comparison method should be preferred.

Clinical Validation of a New Triplex Real-Time Polymerase Chain Reaction Assay for the Detection and Discrimination of Herpes simplex Virus Types 1 and 2

A new triplex real-time polymerase chain reaction (PCR) assay for Herpes simplex virus (HSV) (artus HSV-1/2 TM PCR kit, QIAGEN) was evaluated. This assay simultaneously uses three differently labeled probes targeted to HSV-1 (FAM), HSV-2 (NED), and to the manufacturer's Internal Control (VIC). HSV-1/2 typing capability and quantitation accuracy were determined using HSV stocks and quality control panels. Performance in routine clinical testing was compared with a nested HSV-1/2 PCR assay. Dilution series and quality control panel testing revealed an approximately 10-fold higher HSV-2 sensitivity in real-time PCR compared with an in-house nested PCR assay. The sensitivity for HSV-1 was comparable in both assays. All HSV-positive proficiency panel samples (n = 21) and virus stocks were typed correctly as HSV-1 or HSV-2 using real-time PCR. Quantitation correlated well with reference values (HSV-1, r = 0.98; HSV-2, r = 0.88), and 95% detection limits were determined as 9.4 HSV-2 copies/reaction and 18 HSV-1 copies/reaction. Based on C(t) values, the mean intra-assay coefficient of variation was 1%, whereas the interassay coefficient of variations were 2.7% and 2.5% for HSV-1 and -2, respectively. Testing of 309 clinical samples resulted in 100% specificity and 97% sensitivity. In conclusion, the artus HSV-1/2 TM PCR kit represents an excellent tool for the detection and differentiation of HSV-1 and -2 in clinical samples.

A flow cytometric method for detection and enumeration of low‐level, residual red blood cells in platelets and mononuclear cell products

Conventional automated cell counters cannot accurately count residual red blood cells (rRBCs) that are often present in various blood products. A two-color flow cytometric method (FC) was validated for detecting and enumerating rRBCs in platelets (PLTs) and mononuclear cell (MNC) products. PLT and MNC products for PLTs (CD61-fluorescein isothiocyanate) and rRBC (anti-glycophorin A-phycoerythrin) were double stained, and data were acquired with a flow cytometer. Assay linearity, accuracy, and precision were assessed with a standard-dilution series of rRBCs. This assay was used to determine the rRBCs of apheresis PLTs collected with Trima Accel (Gambro BCT) and MNC products collected with the COBE Spectra (Gambro BCT). The linear range of this assay in PLT and MNC products is 10 to 2000 RBCs per microL (R2=0.994). FC had a mean intraassay coefficient of variation of 11.8 percent at 34 RBCs per microL. A standard clinical hematology analyzer overestimated rRBCs in MNC products by 1.59x10(5)+/-0.7x10(5) RBCs per microL. Apheresis PLTs had a median of 17.4 RBCs per microL, with 99.0 percent containing fewer than 90.0 RBCs per microL. This method for determining rRBCs in blood products is accurate and repeatable with a lower limit of detection adequate to assess currently available blood products. FC should be considered for determining rRBCs in MNC products.

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.

Application of the Comet Assay for Investigation of Oxidative DNA Damage in Equine Peripheral Blood Mononuclear Cells

Oxidative stress occurs when antioxidant defense mechanisms are overwhelmed by free radicals and may lead to DNA damage, which has been implicated in processes such as aging and diseases such as cancer. The two main techniques presently used to quantify DNA damage are measurement of 8-hydroxydeoxyguanosine and the Comet assay (also known as single-cell gel electrophoresis). The aim of this study was to apply the comet assay to equine peripheral blood mononuclear cells (PBMCs) and identify two conditions in which we hypothesized that oxidative DNA damage would be increased in PBMCs: aging and equine recurrent airway obstruction (RAO, a condition similar to human asthma). The images obtained were similar to those previously published for humans, cats, and dogs. The optimum concentration of H(2)O(2) to estimate susceptibility to exogenous damage was 50 microM. Mean intraassay coefficients of variation were 4.7 and 9.7% for endogenous and exogenous tail-DNA quantities, respectively, and 7.3 and 8.3%, respectively, for interassay coefficients. There was no significant difference in either endogenous or exogenous percentages of tail DNA for samples collected from six ponies on three consecutive days. There was no significant difference in endogenous, exogenous, or exogenous (corrected for endogenous) oxidative DNA damage between mature and aged ponies. However, young pony foals had significantly less endogenous DNA damage than mature or aged ponies (P < 0.05). RAO-affected horses without airway inflammation (i.e., in clinical remission) had significantly greater endogenous damage compared with non-RAO-affected control animals (P = 0.009). There was a significant correlation between endogenous percentage of tail DNA in PBMCs and red blood cell hemolysate glutathione concentration (r = 0.720; P < 0.001). In conclusion, the comet assay appears to be suitable for investigating DNA damage in equine PBMCs.

Effect of a common reference plasma on the inter-laboratory variation of the measurement of total and free protein S: a collaborative study of the Dutch Working Group on Haemostasis Laboratory Diagnosis

The comparability of test results for protein S between laboratories is hampered by a high inter-laboratory variability. The effect of the use and type of common reference plasma on the inter-laboratory variability of the total and free protein S measurement was evaluated. The results of 10 plasma samples measured against a centrally distributed frozen plasma and a centrally distributed lyophilized plasma were compared with those of various local reference plasmas regularly used by the 11 participating laboratories. The mean intra-assay coefficient of variation for total protein S in each laboratory varied from 3.8 to 12.8% (mean intra-assay CV of all laboratories: 7.4 - 2.3%); for free protein S this range was 3.1 to 13.3% (mean intra-assay CV of all laboratories: 6.6 - 2.7%). We confirmed the high inter-laboratory coefficient of variation (mean - SD) with the several local reference plasmas for both total protein S (13.4 - 5.6%; n = 10) and free protein S (17.1 - 7.5%; n = 11). For total protein S, the inter-laboratory CV was reduced to 11.5 - 4.8% (p = 0.005) by using a common frozen reference plasma, while it was increased to 16.8 - 3.4% (p = 0.022) using a common lyophilized reference plasma. For free protein S, these values decreased only statistically significantly for the common lyophilized reference plasma, to 15.1 - 6.0% (p = 0.008). For free protein S, the dilution factor used was identified as a factor influencing the inter-laboratory variability. This study shows that, for both types of protein S measurements, using one frozen reference plasma shows a slight decrease in inter-laboratory variability, while a common lyophilized plasma shows inconsistent results. It is concluded that further investigation is necessary to examine other sources of variability to increase the comparability of laboratory results for both total and free protein S.

In vivo antioxidant effect of green tea.

The object of this study was to investigate the in vivo antioxidant effect of green tea and dosage effect of green tea on antioxidant effect. We tested 10 healthy subjects (aged 23-25 y, five women and five men) with overnight fasting. The total antioxidant capacity of plasma was measured at baseline and 60 min and 120 min after ingestion of 150 ml green tea. Green tea was prepared by infusing 2.5 g of dried green tea leaves for 2 min at 80 degrees C in 150 ml of water. In the second week, they took 300 ml of tea (5.0 g of green tea leaves) and, in the third week, 450 ml of tea (7.5 g of green tea leaves). The total antioxidant capacities of plasma were determined with a Total Antioxidant Kit (Randox Laboratories Ltd, UK) using a Cobas Mira analyser (Roche Diagnostic Systems Inc., Switzerland). The mean intra-assay coefficient of variation was 1.2%. The total antioxidant capacity of plasma increased by 1.1% at 60 min and 2.1% at 120 min over baseline value in subjects consuming 150 ml of green tea, which was statistically not significant. However, total antioxidant capacity of plasma after consuming 300 ml of green tea showed a significant increase of 7.0% after 60 min and 6.2% after 120 min (P<0.0001), and after consuming 450 ml 12.0% after 60 min and 12.7% after 120 min over baseline value (P<0.0001). Total antioxidant capacity of plasma was significantly increased after taking green tea in amounts of 300 and 450 ml. A positive increment according to green tea dosage was also observed. This work was funded by the Pacific Corporation (Korea).

A rapid, automated enzymatic fluorometric assay for determination of D-arabinitol in serum.

A rapid enzymatic fluorometric assay for measuring D-arabinitol in serum was developed using recombinant D-arabinitol dehydrogenase from Candida albicans (rArDH). rArDH was produced in Escherichia coli and purified by dye-ligand affinity chromatography. rArDH was highly specific for D-arabinitol, cross-reacting only with xylitol (4.9%) among all polyols tested. A Cobas Fara II centrifugal autoanalyzer (Roche) was used to measure NADH fluorometrically when rArDH and NAD were added to serum extracts, and D-arabinitol concentrations were calculated from standard curves derived from pooled human serum containing known amounts of D-arabinitol. The method was precise (mean intra-assay coefficients of variation [CVs], 0.8%, and mean interassay CVs, 1.6%) and rapid (3.5 min per assay) and showed excellent recovery of added D-arabinitol in serum (mean recovery rate, 101%). The mean and median D-arabinitol/creatinine ratios were 2.74 and 2.23 microM/mg/dl, respectively, for the 11 patients with candidemia compared to 1.14 and 1.23 microM/mg/dl, respectively, for 10 healthy controls (P < 0.01). These results confirm earlier studies showing that serum D-arabinitol measurement may help to promptly diagnose invasive candidiasis. The technique shows a significant improvement in terms of accuracy, cost, simplicity, specificity, and speed compared with gas chromatography, mass spectrometry, and earlier enzymatic assays.