Microsoft word - t4-report smart

Evaluation of the EUROLYSER-SOLO
Total T4 assay
for veterinary samples
Dr. rer.-nat. Volker Schlüter
Zacherlweg 18
D-82061 Neuried
Date: 2011 April 27th
Scope of the study
Goal of this study was the validation of the new veterinary Total T4 assay assignedfor analysis with the Eurolyser-SOLO analyzer. For this, the system was evaluatedfor:  Precision at different concentrations  Method comparison to the present gold standard Materials and methods
Serum samples from dogs and cats with and without thyroid disorders were collectedat two different sites in Germany. The animals need not to be fasting, and no specialpreparations are necessary. Blood samples taken by venipuncture were collected inplain tubes without anti-coagulants. Samples were stored at room temperature for atleast 10 minutes to allow a complete cell aggregation. Afterwards, serum wasseparated by centrifugation. Serum samples were subjected to direct analysis orfrozen at -20°C until they were subjected to analysis. Before testing samples wereallowed to come to room temperature and mix by gentle swirling or inversion.
EUROLYSER-SOLO analyzer system
The EUROLYSER-SOLO analyzer (single method automated reading technology) isa fully automated instrument allowing a simple quantitative determination of analytesin a point-of-care format. The EUROLYSER-SOLO analyzer is designed formeasurement of veterinarian samples.
The system comprises from the analyzer itself and the respective reagents. Thereagents are provided in form of “Single-use units” avoiding the usual open stabilityissue of wet chemistry reagents. The smart Total T4 kit includes cuvettes and ERScaps containing the reagent and in addition RF-ID cards containing all importantassay information like the test programming and the calibration data.
The RF-ID card is supplied to the analyzer and the instrument is programmedaccording the instruction for use. Before measurement, cuvette and ERS cap areequilibrated to room temperature for at least 10 minutes. Afterwards, the sample(volume of 20 µl or 5 µl, respectively) is transferred into the cuvettes’ reagent usingan appropriate pipette. The ERS cap is placed onto the cuvette and the combineddevice is applied to the smart instrument. The fully automated measurement takesabout 6 minutes.
When using a sample volume of 20 µl, the linearity is limited to 8 µg/dl. In order toextend the linear range by a factor of 4 (up to 32 µg/dl), the EUROLYSER-SOLOsystem offers an option for pipetting a sample volume of 5 µl.
Immulite 1000/canine Total T4
For method comparison analysis, the Siemens Immulite 1000 system with theSiemens canine Total T4 test kit was chosen. The canine Total T4 assay is a solid-phase, chemiluminescent competitive immunoassay. The system is the usually usedfor veterinarian T4 measurements and therefore selected as the best referencesystem on market today.
Samples described above were used in a parallel analysis according to themanufacturers’ instruction for use. A volume of 30 µl serum was used2,6.
For higher concentrated samples with Total T4 values above 6 µg/dl, dilutions wereused. A dilution of 1:4 (3 parts diluents/1 part sample) was made with a pooled serumsamples (5 samples) having a low total T4 concentration. To estimate the Total T4concentration the pooled sample was analyzed three times (value: 0.68 µg/dl).
Results were corrected prior its usage for data analysis.
Reference values
Since the evaluation of the EUROLYSER-SOLO system was scope of this study,normal values had not been evaluated in detail. Several publications are availabledefining the reference values of common species using different immunologicalmethods. Table 1 show that normal values of dogs and cats are in a similar rangingfrom 1.0 to 4.5 µg/dl. The data were derived from publications as well as fromcompanies offering veterinarian Total T4 assays.
Table 1: Reference values for Total T4 from published sources [µg/dl].
Immulite
Laboklin7
canine T46
Snap
Test8
Limitations
The assay is optimized for the determination of Total T4 in serum and plasma. Theuse of whole blood is not possible. In rare cases, animals may have autoantibodieswhich interfere with the assay and result in low test values.
Specificity and Interferences
Test system had been analyzed for various interferences. Components with chemicalstructure similar to that of thyroxine and certain concurrently used components weretested for possible cross reactivity in the thyroxine assay. The % cross reactivity wasdetermined as the percent of equivalent Total T4 concentration observed whentested concentration of the cross reactant was added to a T4 negative serum.
Compound
Conc. Tested [µg/dl]
% cross reactivity
* The tested concentrations greatly exceed the normal serum concentrations of thesecomponents. Therefore the cross reactivity is not clinically significant. In addition, following probably causes of interference had been analyzed and foundnot to interfere significantly: Results and discussion
Precision
Since the EUROLYSER-SOLO system comprises from single-use-units, only a run-to-run precision analysis is suitable. Assay precision was determined by assayingthree clinical serum samples derived from cats or dogs. At least 10 replicates hadbeen performed. The samples had been selected that different concentration levelswere covered.
Table 2: Precision of EUROLYSER-SOLO Total T4 assay.
Replicates
volume [µl]
[µg/dl]
[µg/dl]
The result of the run-to-run assay precision is shown in table 2. The variationcoefficient (CV) ranges from 3.8-4.4 % when using 20 µl sample volume.
To further analyze whether samples with a concentration of around 8 µg/dl werefound reproducibly using both instrument modes, sample number 1 was reanalyzedusing the reduced sample volume of 5 µl. 5 replicates were found with a standarddeviation of 3.5%. Mean vales were 7.98 µg/dl (20 µl sample volume) and 7.79 µg/dl(5 µl sample volume), respectively. The difference between the mean values wasfound to be 2.44 % demonstrating the high assay precision at the junction point ofboth assay modes. In conclusion table 1 show that the EUROLYSER-SOLO Total T4assay is highly reproducible with different concentrations and precise using bothmodes of the system.
Method comparison
In total, serum samples derived from 48 dogs and from 43 cats with Total T4concentration ranging from approximately 0.5 to 20 µg/dl had been chosen for thismethod validation. As reference system for the EUROLYSER-SOLO analyzer,Siemens Immulite canine Total T4 assay had been chosen. According to the scopeof the EUROLYSER-SOLO system, 20 µl volume had been used for samplesconcentrated below 8 µg/dl, 5 µl sample volume had been used for samples with aconcentration of higher than 8 µg/dl. Similarly for measurements with the SiemensImmulite system, samples having concentrations higher than 6 µg/dl had been diluted1:4 (3 parts diluent/1 part sample) before measurement. For dilution a lowconcentrated pooled serum sample had been used, results were corrected beforedata analysis.
Data were subjected to a linear regression analysis, which is shown in Figure 1. Fig1A shows the analysis of the cat-specific data, Fig. 1B the analysis of data obtainedwith the dog samples. Finally, Fig 3C combines all data.
Total T4-Cats
Total T4-Dogs
EUROLYSER-SOLO [µg/dl]
EUROLYSER-SOLO [µg/dl]
Total T4- Cats and Dogs
l]
/d
g
[µ 10
EUROLYSER-SOLO [µg/dl]
Figure 1: Method comparison of EUROLYSER-SOLO Total T4 assay. A: Dogs; B:
Cats; C: Dogs and Cats.
The analysis shows that both assays correlate very well for cats as well as for dogs.
In all cases the value of correlation coefficient R2 was higher than 0.95 demonstratinga high correlation between both assay systems. Mean values of all samples was 2.38µg/dl for both groups, respectively.
Linearity
Three samples derived from two cats and one dog were diluted to the lower detectionlimit of the EUROLYSER-SOLO Total T4 assay. All samples were measured induplicates using 20 µl sample volume (with one exception). The result (mean of theduplicates) is shown in table 3.
Table 3: Linearity of EUROLYSER-SOLO Total T4 assay.
Dilution
Sample 1: Cat
Sample 2: Cat
Sample 3: Dog
Recovery
Recovery
Recovery
[µg/dl]
[µg/dl]
[µg/dl]
The data shown in table 2 demonstrate recovery rates of 95-105 %. If samples werediluted to concentration of 0.7 µg/dl or lower to the end of the measuring range of 0.5µg/dl recovery values are slightly higher. In summary the data show that the Total T4assay is linear with respect to the indicated measuring range.
Goal of this study was the validation of the new veterinary Total T4 assay assignedfor analysis with the EUROLYSER-SOLO analyzer. In summary the studydemonstrates that the EUROLYSER-SOLO total T4 assay  is precise at different concentrations
 is linear over the wide range of 0.5 – 32 µg/dl
 has an excellent correlation when compared to the present gold standard
Literature
1. Solter PF, Farner S. 2000. Correlation of two non-radioactive immunoassays to a radioimmunoassay technique for thyroxine measurement in equineserum. J Vet Invest 12:51-56 2. Singh AK, Jiang Y, White T, et al. 1997. Validation of non-radioactive chemiluminescent immunoassay methods for the analysis of throxine andcortisol in bloo samples obtained from dogs, cats and horses. J Vet DiagnInvest 9:261-268.
3. Thoresen SI,Wergeland R et al. 1966. Evaluation of an enzymic immunoassay for free thyroxine determination in canine serum. Vet Res Comm 20: 411-420.
4. Lien Y.-H., Wu T.-J., Huang H.-P. 2008. evaluation of a point-of-care enzyme- linked Immunosorbant assay for determination of basal serum total thyroxineconcentration in cats. JVCS 1:86-89.
5. Petersen ME, Kintzer PP, Cavenagh PG. 1983. Feline hyperthydoidsm: pretreatment clinical and laboratory evaluation of 131 cases. J Am Vet MedAssoc 183:103-110.
6. Immulite(Immulite 1000 canine Total T4 (PILKCT-e, 2006-12-29.
http://www.medical.siemens.com/siemens/en_GLOBAL/gg_diag_FBAs/files/package_inserts/immulite/Veterinary_n/pilkct-5.pdf 7. Laboklin, reference value table for clinical diagnostic.
www.laboklin.de/pdf/de/leistungsspektrum/referenzwerttabelle.pdf

Source: http://veterina.bigblue.hr/fileadmin/slike/Slike_Solo/T4-Evaluation_report_16_05_2011.pdf