The BAP test and the global assessment of oxidative stress ... - Medial

The FREE system is an “open” systemconsisting of a photometer and a drythermostating block. Both the tools aremanaged by a computerized system able toreceive, to elaborate and to export the data.The initials FREE means Free Radical ElectiveEvaluator, i. e. system for the elective study offree radicals.The dedicated kit of the BAP test for FREEcontains cuvettes ready-to-use with a solutionof a thiocyanate derivative (R 1 reagent) and avial of ferric chloride (R 2 reagent).According to the procedure for the FREEsystem, at least 10 min before the analyticalsession, it is preferable to seat the cuvettes,ready to use for analysis, in the thermostatedhousing of instrument. It is also very importantto bring the reagents to working temperaturebefore the use.When ready, the operator must select,through the keyboard of instrument, the presetchannel.For each analytical series, the procedureinvolves, before testing plasma or serum, thezeroing and the subsequent automaticsubtraction of reagent blank absorbance.Practically, after having adjusted the zeroagainst distilled water in the photometer, 50 µLof R 2 reagent (ferric chloride) is transferredinto the cuvette containing the R 1 reagent (athiocyanate derivative). The resulting coloredsolution, after gently mixing by inversion,undergoes photometric reading. Then, 10 µL ofsample is added in the same cuvette and theobtained solution is gently mixed andincubated into the thermostatic block for 5 minat 37 °C. After incubation, the sample can beread for absorbance.FRAS 4 is a new integrated analyticalsystem consisting of a dedicated photometerwith incorporate centrifuge designed to allowfor the global assessment of oxidative stresson whole blood, by means of two tests, i. e. d-ROMs test and BAP test. The initials FRASmeans Free Radical Analytical System, i. e.system for the analytical study of free radicals.The most innovative technological featureof FRAS 4 is the integration of centrifuge withthe analytical module. This system enables theoperator to perform not only thecentrifugation but also the photometricanalysis.The dedicated kit of BAP test for FRAS 4 issimilar to that one of FREE system; moreover,it contains also the tools for taking the wholeblood from a finger.Practically, a small amount of plasma (10µL), which has been obtained after centrifugingthe whole blood sample (100-150 µL), isdissolved in the colored solution, previouslyprepared by mixing the R 2 reagent (FeCl 3 ) withR 1 reagent (a thiocyanate derivative). After 5min of incubation at 37 °C, the solutionundergoes photometric reading.To periodically calibrate FREE and FRAS 4systems, a lyophilized human control serum isavailable with a known antioxidant titleexpressed as µmol/L.Both devices directly provide the results ofthe BAP test – that is the “biologicalantioxidant potential” of plasma – asµEquivalents/L (µEq/L) (by using ascorbate asiron reducing agent).Data of Producer from healthy individualsindicated initially that the optimal value of BAPtest should be higher than 2.200 µEq/L. Thestudies performed in the last 3 yearssubstantially confirmed this statement (seebelow), although every laboratory shoulddetermine its own reference value.5. Analytical performancesAnalytical performances were initiallyevaluated with a first set of experiments onstandard solutions of ascorbic acid (AsA) withknown values, by using a FREE equipment(Diacron International, Grosseto, Italy), inorder to establish the lowest limits of detectionand quantification, respectively, and linearity.The lowest detection limit, i. e. the lowestconcentration of a signal that can bedistinguished by the blank (as calculated bymultiplying 3 times the standard deviation, asobtained by the average of 10 replicatedanalyses of the blank) was 118 (mean value).The lowest quantification limit, i. e. the lowest10www.fras4evolvo.it