Inter-assay variation

In a protocol about collaborative studies [10] it is also considered what is called preliminary estimates of precision. Among these the protocol defines the total within-laboratory standard deviation . This includes both the within-run or intra-assay variation (= repeatability) and the between-run or inter-assay variation. The latter means that one has measured on different days and preferably has used different calibration curves. It can be considered as a within-laboratory reproducibility. These estimates can be determined prior to an interlaboratory method performance study. The total within-laboratory standard deviation may be estimated fi-om ruggedness trials [10]. 

The intra- and inter-assay variation is determined in tenfold analyses of a pool plasma sample. Table 3.4.2 shows the results of these measurements. The linearity of this method should be assessed for all analytes. Pristanic acid and the C26 0 fatty acid were linear up to 16 pmol/1, phytanic acid to 100 pmol/l and the C22 0 and C24 0 fatty acid to 200 pmol/1. The lower detection limit for all analytes was at a level of less than 0.01 pmol/l, for the lower reporting levels (LOQ), an analysis of a blank solution was taken into account. The blank levels of the analytes phytanic acid, pristanic acid, and fatty acids C22 0, C24 0 and C26 0 were 0.04, 0.01, 0.41, 0.68 and... 

Lab B reported its inter-assay variation as total variation of Daytrol only at the higher therapeutic level. Respectively, Lab C reported variation only at the lower therapeutic level (Table 1). As the national target coefficient of variation (CV%) for analytical variation is 2% for S-Li, all methods fulfilled this quality specification, when the long-term control, Daytrol, was used. Inter-assay variation of the lower level system control was 3.5 times higher, than quoted by the manufacturer. This stresses the point that every laboratory should check the performance specifications given by the manufacturer. The variation of the higher therapeutic level showed equality with the obtained validation results. 

Table 1 Inter-assay variation, CVmeas % of the three ion-selective electrode (ISE) applications during method validation and at the 6-month checkpoint for the lower and higher therapeutic levels and the toxic level of serum lithium (S-Li)...

The third advantage of quantitative real-time RT-PCR is that it allows one to monitor the treatment effect in individual patients as a potential surrogate marker after adjuvant chemotherapy (K3) or immunotherapy (S4) against micrometastatic diseases. For the purpose of monitoring, a relative quantification method which is designed to determine exact, PCR efficiency-corrected mRNA concentration, normalized to a calibrator, might be desirable to overcome the inter-assay variation from run to run (SI3). This may make it possible to directly compare the mRNA values at different time points. 

The ID-GC-MS-SIM method of Adlecreutz and coworkers (1993) is perhaps the most frequently used in clinical and medical studies, although intra- and total-assay variabilities of all analytes are considered to be high, probably reflecting low-analyte concentrations in biological fluids (Wilkinson et al., 2002). Other GC-MS methods report lower inter-assay variation of 6%-ll% isoflavones in serum. The latest method of measuring urinary excretion of isoflavones (daidzein, genistein, equol. 

Rao et al.20 demonstrated a fluorescence polarization immunoassay for evaluating serum concentrations of tricyclic antidepressants (amitriptyline, imipramine, clomipramine, and doxepin) with respect to nonresponse, compliance, therapeutic window, and influences of age, sex, substance abuse, and toxicity. Abbott Laboratories TDx/TDxFLx Toxicology Tricyclic Assay FPIA (fluorescence polarization immunoassay) was used. This assay of 50 /uL samples contained tricyclic antidepressant antibodies raised in rabbits and fluorescein-labeled tricyclic antidepressant as a tracer. The assay was calibrated with imipramine in the range of 75 to 1000 fig/L (268 to 3571 nmol/L). Intra-assay and inter-assay coefficients of variation for internal quality control samples from the manufacturer were 4.2 and 4.7%, respectively. The limits of detection were 72,71,64, and 72 nmol/L for amitriptyline, imipramine, clomipramine, and doxepin, respectively. This high-throughput immunoassay was easy to use although amitriptyline, dosulepine, desipramine, and nortriptyline showed cross-reactivities ranging from 74 to 100%. 

A calibration curve was constructed over a concentration range of 1 to 50 fig/mL with a correlation coefficient of 0.998. Intra-assay and inter-assay coefficients of variation were less than 7.9 and 9.5%, respectively. Mean absolute recoveries at 10 and 20 fig/mL were 72 and 76%, respectively. Limit of detection was 0.3 fig/mL. Limit of quantification was 1.0 fig/mL. 

Linear 1/y2 regression analyses of the ratio of the peak area of lercanidipine to the concentration compared with the ratio of the IS were constructed over the range of 0.05 to 30.00 ng/mL. Correlation coefficients exceeded 0.995. Intra-assay and inter-assay coefficients of variation were less than 7.3 and 6.1%, respectively. The limit of detection was calculated to be 0.02 ng/mL, and the limit of quantitation was 0.05 ng/mL. 

The hydrogel coatings achieved the lowest limits of detection (LTD) 1300 to 1600 amoles/spot, but exhibited significant assay variation (22% intra-slide to 37% inter-slide CV). LLD levels of surface-modified polysfyrene slides (Maxisorb black. Nunc) equaled 1500 amole/spof af 15 to 32% CV, while reflective (mirror-like) slides coated with 3-aminopropyltriethoxysi-lane (Amersham) showed the lowest variahon with CVs at 11 to 14%. 

Taylor and colleagues [98] at the Mayo Clinic published a method for the simultaneous analysis of urinary cortisol and cortisone. They used 2H4 cortisol as an internal standard and took a 0.5-ml urine sample. An API 2000 with Turboion-spray source was used in the positive-ion mode. Chromatography was conducted on a standard-bore C18 column with Q8 precolumn filter. MRM was conducted in the positive-ion mode monitoring m/z 363—>121 for cortisol, 367—>121 for 2TL, cortisol, and 361— -121 for cortisone. Cortisol and cortisone were separated and both were eluted within 2 min. Inter- and intra-assay variation for both compounds was < 9% for amounts above 2 pig/dl. The values obtained agree well with those of other studies, such as ours (Table 5.3.2) [62]. They found a range for cortisol for adult males of 4.2-60 pg/24 h and for adult females 3.0-43 pg/24 h. In summary, the 3-min run time of their method has allowed the Mayo group to completely transfer their cortisol and cortisone workload from RIA and HPLC to MS/MS. 

Kim et al. [48] developed a rapid, sensitive, and selective LC-ESI-MS/ MS method for fhe deferminafion of lornoxicam in human plasma. Lornoxicam and isoxicam (infernal standard) were extracted from human phasma wifh efhyl acefafe at acidic pH and analyzed on a Sunfire Cis column with the mobile phase of methanol-ammonium formate (10 mM, pH 3) (70 30). The analyte was detected using a mass spectrometer, equipped with electrospray ion source. The instrument was set in the multiple-reaction-monitoring mode. The standard curve was linear (r = 9998) over the concentration range of 0.5-500 ng/ml. The coefficient of variation and relative error for the intra- and inter-assay at four QC level were 0.7% fo -4.2% and —4.5% to 5%, respectively. The recoveries of... 

Two immunosensors developed by O Regan et al. [89,90] have demonstrated their usefulness for the early assessment of acute myocardial infarction (AMI). Human heart fatty-acid binding protein (H-FABP) is a biochemical marker for the early assessment of AMI. The authors constructed an amperometric immunosensor for the rapid detection of H-FABP in whole blood. The sensor is based on a one-step, direct sandwich assay in which the analyte and an alkaline phosphatase (AP) labelled antibody are simultaneously added to the immobilized primary antibody, using two distinct monoclonal mouse anti-human H-FABP antibodies. The substrate p-amino-phenyl phosphate is converted to p-aminophenol by AP, and the current generated by its subsequent oxidation at +300 mV vs. Ag/AgCl is measured. The total assay time is 50 min, and the standard curve was linear between 4 and 250 ng ml . The intra- and inter-assay coefficients of variation were below 9%. No cross-reactivity of the antibodies was found with other early cardiac markers, and endogenous substances in whole blood did not have an... 

Assay Development and Validation. Reproducibility of this ELISA assay was determined based on a set of clomazone standards that were run on different plates on the same day (intra-assay) and on different days (inter-assay). The intra-assay coefficient of variation of the standards changed from 1.5 at the highest clomazone concentration (250 ppb) to 22 at the lowest concentration of 1.4 ppb. The coefficient of variation(CV) at clomazone rate of 12.5 ppb was 10. Similar values were obtained for the inter-assay variability, with the CV of the 1.4 ppb concentration being 22.5, and the CV for the 250 ppb concentration being 2.7. The CV for the 10 ppb concentration of clomazone was about 5 between tests. Analysis of the data for this range of clomazone concentrations indicates that there is good correlation (r =0.97) between the log of the concentration of clomazone and percent inhibition in the assay when the linear regression equation was used. Based on these results, the limit of the test s sensitivity was defined as 2 ppb (10 ppb in soil) and the limit of detection was set at 1 ppb. 

Precision describes the variability in a set of measurements, i.e. how closely repeated measurements on a single sample agree. Precision can be calculated for samples within an individual itin (intra-assay precision) or across multiple runs (inter-assay precision). Precision is customarily expressed as the percentage coefficient of variation (%CV, or simply CV), which is the standard deviation expressed as a percentage of the mean ... 

Precision defines the scatter of repeated analysis, or the coefficient of variation of analytical results. Both intra-assay and inter-assay precision must be investigated. Intermediate precision describes the influence of different analysts, equipment, days and other intra-laboratory variabihty. Inter-laboratory comparison is also of interest in establishing the precision of the method. AU testing on accuracy and precision must be carried out by replicate analyses of a statistically relevant number of samples. Depending on the use of the method, it may be necessary to estabhsh both parameters over the measurable range, or in the case of content determination simply in the range of 80-120% of the nominal value. 

The analytical part of this study confirmed the adequate imprecision of holoTC determination, with mean intra- and inter-assay coefficients of variation (CVs) ranging from 2.9% to 4.1% on assay controls and from 6.0% to 7.7% on our local pooled control serum, good holoTC linearity from 8.8 to 143.3 pmol/L (r = 0.99), good recovery in spiked specimens (mean 95%, interval 90 100%), mean recovery determined by dilution (100%, range 93-111 %) and detection limit (0.07 pmol/L). 

Fig. 11. Narrow inter-assay and intra-assay variations are critical parameters for successful high-throughput screens using FLIPR technology. Some final advice...

A similar HPLC method with a postcolumn derivatization system was used for the analysis of total thiamine in human whole blood as well as in serum, cerebrospinal fluid, and milk (26). The HPLC system consisted of a pBondapak column and the mobile phase was a mixture of methanol-50 mM sodium citrate buffer pH 4.0 (45 55, v/v) plus 10 mM sodium 1-octanesulfonate. Two milliliters of blood was needed. The minimum detectable amount was 60 fmol of thiamine. The intra-assay and inter-assay coefficients of variation were 2.3% and 3.9%, respectively. The recovery of TPP added to blood samples was 98.7%. 

Immunoassay is one of the most sensitive analytical techniques described for phytoestrogen measurements in human biological fluids. Detection limits for target analytes are below 1 nmol/L for most methods with an average recovery of 92% and inter-/intra-assay variation less than 10% for most methods (WiUdnson et al., 2002). Immunoassays have been validated against other analytical techniques and excellent correlation has been demonstrated. Immunoassay can be a powerful alternative to GC-MS- or HPLC-based methods for studying the bioavaUabflity and metabolic fate of dietary isoflavonoids because of its ease, relatively low cost, availability for... 

SMITH p, FLEMING GTA and CARROLL c (2008), Reducing inter-operator variation in disc diffusion assays by the inclusion of internal controls in a standard susceptibility test protocol . Aquaculture, 285,273-276. 

Although DC maturation can be used to detect sensitizing capacity, major concerns remain on this assay (i) the limited reproducibility within and between laboratories due to inter-donor variability and variations in cell isolation and culture techniques (ii) the lack of sensitivity and dynamic range [122]. To circumvent interdonor variability cell lines such as THP-1, U937, KG-1 and MUTZ-3 have been used. 

---