preanalytical problems are the major source of laboratory errors , accounting for 6070% of total errors encountered within the total testing process .
preanalytical errors not only may lead to spurious test results , but also , more importantly , may influence patient care , even dramatically .
the immunosuppressive drugs tacrolimus ( tac ) and cyclosporine a ( csa ) are used in organ transplantation .
these drugs are calcineurin inhibitors that block interleukin-2 ( il-2 ) transcription and t lymphocyte signal transduction .
they are used for prophylaxis against tissue rejection after liver , heart , and kidney transplantation . in addition
, csa is used in the treatment of graft - versus - host reactions , in severe psoriasis , and after bone marrow transplantation .
tac is a lipophilic drug that has variable absorption and is extensively metabolized in the liver [ 4 , 5 ] .
approximately 95% of tac is eliminated by the biliary route , while 5% is excreted unchanged in the urine .
csa is a substrate of the p - glycoprotein ( abcb1 ) efflux transporter and primarily metabolized by cyp3a4 and cyp3a5 .
it is nephrotoxic and causes damage to the kidney vasculature , glomerulus , and proximal tubule at high dose .
csa and tac cause numerous side effects , including neurological , hepatic , renal , and immunological complications .
dose adjustment or discontinuation may be necessary , because these complications occur in a significant number of patients [ 9 , 10 ] . a significant consequence of overdosage of csa and tac is nephrotoxicity , while underdosage of these drugs can result in transplant rejection .
therefore , in order to reduce the risk of organ rejection and toxicity , therapeutic drug monitoring plays a key role in maintaining therapeutic blood levels of these drugs .
immunoassays methods ( such as microparticle enzyme immunoassay and cloned enzyme donor immunoassay ) and liquid chromatography based methods ( such as high - performance liquid chromatography ( hplc ) with ultraviolet detection , lc - mass spectrometry ( lc - ms ) , and lc - tandem mass spectrometry ( lc - ms / ms ) ) are used for determination of concentrations of immunosuppressive drugs .
overestimation of concentrations is a major problem because cross reactions can occur with some metabolites .
the lc - ms / ms method is more sensitive and specific than immunological methods and has significant advantages over other methods .
therefore this method is considered to be the gold standard in therapeutic drug monitoring [ 1315 ] .
these drug levels are not analyzed in every hospital laboratory , but rather the samples are transferred to laboratories in other cities . therefore samples can not be analyzed immediately .
it is known that prolonged storage time and inappropriate temperature may cause incorrect results for many analyses . in this study
, we investigated the effect of storage time and temperature on drug concentrations in whole blood samples in order to determine the optimal storage conditions for accurate measurement of these drugs .
the research protocol was approved by the local ethics committee of dicle university school of medicine .
blood samples from 15 patients using tac and 15 patients using csa were included in the study .
nine patients using tac had received kidney transplants , and the 6 patients had received liver transplants .
all the patients using csa were patients who had received kidney transplants . patients using any other drug were excluded from the study .
blood samples ( 2  ml ) were drawn into k2edta tubes ( becton dickinson company , usa ) from the subjects in the morning after an overnight fast and were transferred to laboratory within 1 minute .
the second aliquot was immediately analyzed and kept for 24 hours at room temperature ( 24c26c ) and measured again .
the third aliquot was kept at + 4c and analyzed 24 and 48 hours later .
0.1  m znso4 precipitation solution containing the internal standards cyclosporin d ( 1234/1217 ) and ascomycin ( 809.6/756.6 ) was prepared before sample preparation .
whole blood samples were put into a tube as 100  l volume and 300  l precipitation reagent was added .
after vortexing for additional 5  sec , the tubes were centrifuged for 10 minutes at 10000  rpm at 4c .
three level quality control serums ( qcs ) were purchased from utak ( valencia , ca ) .
the lc - ms / ms was performed using an ionics 3q 120 triple quadrupole mass spectrometer ( bolton , on canada ) with a shimadzu ultra - fast liquid chromatograph ( uflc ) system .
20  l of supernatant was loaded on a porous r1/20 pretreatment column ( 30  2.1  mm ) for on - line washing with water for 0.25 minutes at a liquid flow rate of 3  ml / min and then eluted by an imtakt cadenza cd - c18ht analytical column ( 50  2.0  mm , 3  m ) at flow rate of 0.6  ml / min using solvent a ( water  :  methanol = 98  :  2 , v / v , with 0.1% formic acid and 10  mm ammonium acetate ) and solvent b ( water  :  methanol = 2  :  98 , v / v , with 0.1% formic acid and 10  mm ammonium acetate ) .
the sensitivity of tac and csa was 0.1  ng / ml and 1.0  ng / ml respectively .
repeated measurements made from the same samples kept at different storage conditions were tested using the wilcoxon test .
we did not find a significant difference between samples analyzed immediately and those kept at + 4c for 24 hours ( p = 0.241 ) ( percent change 13.78% ) and 48 hours ( p = 0.285 ) ( percent change 10.4% ) , and there was no significant difference between samples analyzed immediately and those stored at 20c for one month ( p = 0.646 ) ( percent change 0.91% ) .
however , there was a significant difference between samples analyzed immediately and those kept at room temperature for 24 hours ( p = 0.005 ) ( percent change 32.89% ) ( figure 1 ) . 

cyclosporine a. we found significant differences between csa samples that were immediately analyzed and those stored under different conditions ( figure 2 ) .
the results of the samples that were kept at + 4c for 24 hours ( p = 0.003 ) ( percent change 19.47% ) and 48 hours ( p = 0.002 ) ( percent change 15.38% ) and those kept at room temperature ( p = 0.011 ) ( percent change 9.71% ) for 24 hours were significantly different .
however , there was no significant difference between samples analyzed immediately and samples that were stored at 20c for one month ( p = 0.173 ) ( percent change 3.82% ) .
in this study we evaluated the effects of different storage conditions on the detected concentrations of tac and csa that were measured in the same samples .
our results showed that the stability of tac was affected by storage at room temperature for 24 hours ( p = 0.005 ) , but the samples were stable if they were stored at + 4c for one day or two days , or at 20c for 1 month ( p = 0.241 , p = 0.285 , and p = 0.646 , resp . ) .
however , csa levels were decreased in the samples that were kept at room temperature for one day or two days and in the samples that were kept at + 4c for 1 day ( p = 0.003 ,  p = 0.002 , and  p = 0.011 , resp . )
therefore , the drug levels of samples that are left at room temperature and not stored properly will be inaccurate .
organ transplant recipients must take immunosuppressant drugs to prevent rejection . to maximize drug efficacy and minimize adverse events , accurate measurements of blood immunosuppressant concentrations and dose adjustments are important .
therapeutic drug monitoring must deliver a sensitive and accurate measurement of an administered drug to be a successful diagnostic instrument in patient care .
therefore , the optimization of sample processing especially the run - time and storage conditions in the preanalytical period is important for the accuracy of measurements .
taylor and sethi evaluated 27 biochemical analytes in their study and determined that some results of biochemical parameters changed in different conditions and storage time .
reported that tumor markers ( afp , cea , and ca125 ) are significantly affected by long - term frozen storage .
however in another study repeated freezing to 70c and thawing had no meaningful effects at the plasma and serum concentrations of hormones .
stored 6 whole blood samples at room temperature , + 4c and 20c for one week , and they observed that there was no change in the levels of tac .
specimens were analyzed on the day of collection and repeatedly for nine to 13 days after storage at room temperature ( 2024c ) and 37c with radioimmunoassay method and they observed no significant difference at the results .
using 21 tac and 13 csa specimens stored the samples at ambient temperature and + 4c for 7 days and reported that there was no decrease in csa concentrations over a seven - day period for specimens stored at either ambient temperature or + 4c , while the concentrations of tac in whole blood stored at ambient temperature for seven days had a slight downward trend ( a mean decrease of 5% ) .
the methods were fluorescence polarization immunoassay ( fpia ) for csa and monoclonal antibody microparticle enzyme immunoassay for tac .
observed that concentrations of tac were stable at room temperature , + 4c , and 70c for the two - week study period in their study using the microparticle enzyme immunoassay method .
they also reported that the samples stored at 70c for almost one year and then reanalyzed were found to be stable . in our study
, we observed that the stability of tac did not change over a two - day period for samples stored at + 4c or a one month period at 20c ( table 1 ) .
however , concentrations of tac were decreased in samples that were left out at room temperature .
they were unstable when they were kept at room temperature or + 4c ( table 1 ) .
we used lc - ms / ms in our study because it is a rapid , specific , sensitive , and accurate reference method .
studies showed that lc - ms / ms had significant reproducibility and accuracy advantages compared to fluorescence polarization immunoassay , microparticle enzyme immunoassay , and conventional hplc - uv methods for the quantitation of tac and csa concentrations in whole blood [ 26 , 27 ] .
based on these results , to accurately determine the level of csa samples should be measured immediately or stored at 20c until analysis . in conclusion ,
the stability of tac and csa depends on storage conditions , which must be considered for accurate measurement .
furthermore , these findings can give an idea about the conditions under which the serum samples should be kept for tac and csa measurement .
 tacrolimus and cyclosporine a are immunosuppressant drugs with narrow therapeutic windows . the aim of this study was to investigate the stability of tacrolimus and cyclosporin a levels in whole blood samples under different storage conditions . 
 whole blood samples were obtained from 15 patients receiving tacrolimus and 15 patients receiving cyclosporine a. samples were immediately analyzed and then stored at different conditions ( room temperature ( 24c26c ) for 24 hours , + 4c for 24 and 48 hours , and 20c for one month ) and then analyzed again . for tacrolimus 
 , there was a significant difference between samples analyzed immediately and those kept 24 hours at room temperature ( p = 0.005 ) ( percent change 32.89% ) . 
 however , there were no significant differences between the other groups . for cyclosporine 
 a , there was a significant difference between samples analyzed immediately and those kept 24 hours ( p = 0.003 ) ( percent change 19.47% ) and 48 hours ( p = 0.002 ) ( percent change 15.38% ) at + 4c and those kept 24 hours at room temperature ( p = 0.011 ) ( percent change 9.71% ) . samples of tacrolimus should be analyzed immediately or stored at either + 4c or 20c , while samples of cyclosporine a should be analyzed immediately or stored at 20c . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion