acommon problem in clinical laboratories is maintaining the stability of serum analytes during sample storage .
samples are usually stored in the door ( 48c ) of a refrigerator for short durations or in a deep freezer ( 20c ) for longer time periods .
thus , the temperature at which the samples are stored constitutes an important preanalytical variable that may affect analysis results in the clinical biochemistry laboratory setting .
these laboratories face many challenges including equipment breakdown and the lack of reagents , which can prevent same - day processing of samples . in such cases ,
the only option is to preserve the samples in a deep freezer ( 20c ) .
in addition , samples are sometimes stored for an extended duration until subjected to routine batch analysis for research purposes .
previous studies have provided information regarding the stability of analytes in serum using a number of methods that have since become obsolete .
although many blood analytes have been shown to deteriorate within hours in unseparated samples kept at ambient temperature , the few studies examining unseparated samples stored at low temperatures involved prolonged contact between serum and cells , which could have caused erroneous test results . moreover ,
the stability of 72 analytes following prolonged serum - cell contact has been previously described .
the effects of prolonged storage on the stability of 31 analytes in plasma and serum separated from cells with a gel barrier have also been reported .
a number of studies have described approximately thirty analytes in serum immediately separated from cells .
in addition , several studies have examined the effect of storage conditions on the stability of various serum components . however , limited information is available regarding the stability of commonly used clinical biochemical analytes in human serum including the effect of storage temperatures as low as 20c on blood - separated serum .
therefore , the present study examined the stability of 17 routine chemistry analytes in immediately cell - separated serum following storage at a designated temperature ( 20c ) for different periods ( 0 , 7 , 15 , and 30 days ) using the previously described standard guidelines for blood sample handling and separation .
this hospital - based study included ten random samples from outpatients being treated at the hospital clinics .
the samples collected from each patient were for physician - ordered laboratory testing ; no additional blood was taken from the subjects .
the institutional ethical committee approved the study , and informed consent was obtained from all the participants .
all procedures were conducted in accordance with the guidelines of the helsinki declaration on human experimentation .
fasting venous blood ( total of 6 ml blood ) was collected in the morning using a vacuette standard tube holder and vacuette 22ga  1  ( 0.7 mm  25 mm ) multisample needle ( becton , dickinson and company , usa ) .
the blood specimens were drawn into 7.5 ml plastic vacuette serum tubes ( bd vacutainer serum ; bd , franklin lakes nj , usa ) .
the sample tubes were left in an upright position for 30 min at room temperature followed by centrifugation at 3500 rpm for 10 min .
the serum samples of each subject were pooled into a plain tube and then aliquoted into 1.5 ml eppendorf tubes ( eppendorf , milano , italy ) ; four aliquots per patient samples were kept ( three for storage at 20c ) and the remaining serum was used for the baseline measurement ( t1d ) .
the serum aliquots were stored frozen at 20c for either 7 ( t7d ) , 15 ( t15d ) , or 30 ( t30d ) days and then analyzed separately for stability .
the following analytes were examined : [ table 1 ] methods used for measuring biochemical parameters 
 metabolites : na , k , urea , creatinine , uric acid , total calcium , phosphorus , direct bilirubin , and total bilirubinproteins : total protein and albuminlipids : total cholesterol and triglyceridesenzymes : alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , alkaline phosphatase ( alp ) , and amylase . 
  metabolites : na , k , urea , creatinine , uric acid , total calcium , phosphorus , direct bilirubin , and total bilirubin proteins : total protein and albumin lipids : total cholesterol and triglycerides enzymes : alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , alkaline phosphatase ( alp ) , and amylase .
all measurements were performed at the hospital laboratory services using an olympus au 400 auto analyzer , except for serum electrolyte levels , which were obtained with a roche avl electrolyte analyzer . to determine time - dependent changes in cell - separated serum analytes ,
the mean value from all ten subject samples was calculated for each analyte at each time point .
clinically significant changes were determined using the significant change limit ( scl ) approach , defined as : scl = initial value  3.0 usual standard deviation ( usd ) .
this is based on the assumption that the usd is representative of the inherent day - to - day variability of the method . in our study ,
the usd was obtained by averaging the standard deviation of the quality control data of the previous 2 months for each analyte .
the quality - control reference serum with the target mean most closely matching the t1d mean for each analyte was used to determine the usd . for simplicity ,
the scl was computed for each analyte by establishing the range ( 3.0 usd ) from the subject mean at t1d .
this hospital - based study included ten random samples from outpatients being treated at the hospital clinics .
the samples collected from each patient were for physician - ordered laboratory testing ; no additional blood was taken from the subjects .
the institutional ethical committee approved the study , and informed consent was obtained from all the participants .
all procedures were conducted in accordance with the guidelines of the helsinki declaration on human experimentation .
fasting venous blood ( total of 6 ml blood ) was collected in the morning using a vacuette standard tube holder and vacuette 22ga  1  ( 0.7 mm  25 mm ) multisample needle ( becton , dickinson and company , usa ) .
the blood specimens were drawn into 7.5 ml plastic vacuette serum tubes ( bd vacutainer serum ; bd , franklin lakes nj , usa ) .
the sample tubes were left in an upright position for 30 min at room temperature followed by centrifugation at 3500 rpm for 10 min .
the serum samples of each subject were pooled into a plain tube and then aliquoted into 1.5 ml eppendorf tubes ( eppendorf , milano , italy ) ; four aliquots per patient samples were kept ( three for storage at 20c ) and the remaining serum was used for the baseline measurement ( t1d ) .
the serum aliquots were stored frozen at 20c for either 7 ( t7d ) , 15 ( t15d ) , or 30 ( t30d ) days and then analyzed separately for stability .
the following analytes were examined : [ table 1 ] methods used for measuring biochemical parameters 
 metabolites : na , k , urea , creatinine , uric acid , total calcium , phosphorus , direct bilirubin , and total bilirubinproteins : total protein and albuminlipids : total cholesterol and triglyceridesenzymes : alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , alkaline phosphatase ( alp ) , and amylase . 
 
metabolites : na , k , urea , creatinine , uric acid , total calcium , phosphorus , direct bilirubin , and total bilirubin proteins : total protein and albumin lipids : total cholesterol and triglycerides enzymes : alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , alkaline phosphatase ( alp ) , and amylase .
all measurements were performed at the hospital laboratory services using an olympus au 400 auto analyzer , except for serum electrolyte levels , which were obtained with a roche avl electrolyte analyzer .
to determine time - dependent changes in cell - separated serum analytes , the mean value from all ten subject samples was calculated for each analyte at each time point .
clinically significant changes were determined using the significant change limit ( scl ) approach , defined as : scl = initial value  3.0 usual standard deviation ( usd ) .
this is based on the assumption that the usd is representative of the inherent day - to - day variability of the method . in our study , the calculated mean for each analyte at t1d represented the initial value .
the usd was obtained by averaging the standard deviation of the quality control data of the previous 2 months for each analyte .
the quality - control reference serum with the target mean most closely matching the t1d mean for each analyte was used to determine the usd . for simplicity ,
the scl was computed for each analyte by establishing the range ( 3.0 usd ) from the subject mean at t1d .
the analysis results for 17 biochemical parameters measured in serum samples under different storage conditions are shown in table 2 .
statistical analysis of selected analytes ; serum samples were stored at -20c for 7 ( t7d ) , 15 ( t15d ) , or 30 ( t30d ) days and then analyzed at room temperature no significant statistical or clinical differences were found among most of the metabolites ( na , k , urea , creatinine , uric acid , total calcium , phosphorus , direct bilirubin , and total bilirubin ) under the different storage conditions .
albumin also demonstrated statistically significant variation on t30d compared with fresh sera on t1d ; however , this variation was not clinically significant . aside from amylase , none of the enzymatic parameters exhibited clinically significant reduction inactivity .
sera stored for different periods at 20c showed a statistically significant decrease in alp ; however , this trend was not clinically significant .
the effect of storage at lower temperatures on serum amylase was statistically as well as clinically significant .
the changes in serum cholesterol and serum triglyceride were statistically significant on t15d and t7d , respectively , compared to t1d ; however , they were not clinically significant .
the results of our study indicate that nearly all of the examined metabolites were stable even after 30 days of storage at 20c . in agreement with the findings of zhang et al .
, no clinically significant differences were found for sodium levels following different storage durations compared to fresh samples ; however , our serum na findings are not consistent with the findings of studies investigating serum with prolonged contact with cells at room temperature .
serum potassium was found to be stable up to t30d when the serum samples were separated from cells and stored in aliquots , whereas previous studies have demonstrated an increase in k after 24 h due to serum - cell contact at room temperature .
the increase in k after 24 h is most likely caused by malfunction of the na / k atpase pump , resulting in diffusion of k from the erythrocytes driven by the intracellular  extracellular concentration gradient .
moreover , our results showed a clinically insignificant increase in na level at t15d and in urea , calcium , total bilirubin , and direct bilirubin at t30d .
however , no significant statistical or clinical differences were observed between the levels of the metabolites ( na , k , urea , creatinine , uric acid , total calcium , phosphorus , direct bilirubin , and total bilirubin ) in fresh samples and in samples stored at 20c for 7 , 15 , and 30 days .
these results are in agreement with those of a previous study reporting that serum calcium , total bilirubin , and direct bilirubin showed clinically equivalent levels , but that serum urea levels exhibited an appreciable increase in bun values over time ; however , bun instability , indicated by a substantial decrease ( 15.6% on average ) in levels , has been reported for samples stored at 20c .
consistent with a previous study , we did not detect any statistically or clinically significant change in creatinine levels .
however , according to boyanton and blick , the increase in serum creatinine levels after 24 h is due to serum - cell contact at room temperature .
in contrast to previous studies demonstrating that serum uric acid concentrations were unstable after 48 h of storage at 4c , our results showed a serial decrease in uric acid concentration over time ; however , these changes were neither statistically nor clinically significant .
furthermore , our results demonstrating a slight increase in phosphorus concentration ( not statistically or clinically significant ) disagree with previous studies showing that serum phosphorus concentrations increased after 24 h. no differences in serum protein ( total protein and albumin ) were detected at any of the three time points compared to fresh samples .
moreover , our results are similar to a previous study reporting clinically equivalent levels of total protein and albumin .
total cholesterol and triglyceride concentrations were stable up to t30d , in agreement with the findings of cuhadar et al . and paltiel et al .
a decrease in the concentration of serum alt , ast , and alp was observed ; however , these changes were not statistically or clinically significant .
in addition , according to paltiel et al . , ast activity remains stable for 1015 freeze - thaw cycles following storage at 80c .
significant statistical or clinical differences were observed between serum amylase levels in fresh samples and samples stored at 20c for 7 , 15 , and 30 days ; serum amylase levels decreased with prolonged storage . to the best of our knowledge ,
this is the first report regarding the effects of storage at low temperatures on serum amylase levels .
the results of our study indicate that nearly all of the examined metabolites were stable even after 30 days of storage at 20c . in agreement with the findings of zhang et al .
, no clinically significant differences were found for sodium levels following different storage durations compared to fresh samples ; however , our serum na findings are not consistent with the findings of studies investigating serum with prolonged contact with cells at room temperature .
serum potassium was found to be stable up to t30d when the serum samples were separated from cells and stored in aliquots , whereas previous studies have demonstrated an increase in k after 24 h due to serum - cell contact at room temperature .
the increase in k after 24 h is most likely caused by malfunction of the na / k atpase pump , resulting in diffusion of k from the erythrocytes driven by the intracellular  extracellular concentration gradient .
moreover , our results showed a clinically insignificant increase in na level at t15d and in urea , calcium , total bilirubin , and direct bilirubin at t30d .
however , no significant statistical or clinical differences were observed between the levels of the metabolites ( na , k , urea , creatinine , uric acid , total calcium , phosphorus , direct bilirubin , and total bilirubin ) in fresh samples and in samples stored at 20c for 7 , 15 , and 30 days .
these results are in agreement with those of a previous study reporting that serum calcium , total bilirubin , and direct bilirubin showed clinically equivalent levels , but that serum urea levels exhibited an appreciable increase in bun values over time ; however , bun instability , indicated by a substantial decrease ( 15.6% on average ) in levels , has been reported for samples stored at 20c .
consistent with a previous study , we did not detect any statistically or clinically significant change in creatinine levels .
however , according to boyanton and blick , the increase in serum creatinine levels after 24 h is due to serum - cell contact at room temperature .
in contrast to previous studies demonstrating that serum uric acid concentrations were unstable after 48 h of storage at 4c , our results showed a serial decrease in uric acid concentration over time ; however , these changes were neither statistically nor clinically significant .
furthermore , our results demonstrating a slight increase in phosphorus concentration ( not statistically or clinically significant ) disagree with previous studies showing that serum phosphorus concentrations increased after 24 h.
no differences in serum protein ( total protein and albumin ) were detected at any of the three time points compared to fresh samples .
interestingly , zhang et al . noted similar observations for albumin and total protein in serum specimens .
moreover , our results are similar to a previous study reporting clinically equivalent levels of total protein and albumin .
total cholesterol and triglyceride concentrations were stable up to t30d , in agreement with the findings of cuhadar et al . and paltiel et al .
a decrease in the concentration of serum alt , ast , and alp was observed ; however , these changes were not statistically or clinically significant .
in addition , according to paltiel et al . , ast activity remains stable for 1015 freeze - thaw cycles following storage at 80c .
significant statistical or clinical differences were observed between serum amylase levels in fresh samples and samples stored at 20c for 7 , 15 , and 30 days ; serum amylase levels decreased with prolonged storage . to the best of our knowledge ,
this is the first report regarding the effects of storage at low temperatures on serum amylase levels .
all common clinical chemistry analytes examined , aside from serum amylase , showed adequate stability following up to 30 days storage at 20c .
these results indicate that deep freezing at 20c could serve a useful tool for additional analyses at later time points as well as for research purposes , which require that samples be stored for longer periods until batch analysis can be conducted .
 background : storage of serum and other blood products is often necessary in laboratories because of technical issues or to preserve samples for subsequent research purposes . 
 the aim of this study was to determine whether the stability of biochemical analytes is affected by storage conditions.materials and methods : a total of 17 biochemical analytes in the sera of ten patients were examined following storage . 
 subsequent to determining the baseline measurements , the serum of each patient was aliquoted and stored at 20c for 7 , 15 , and 30 days and then analyzed for stability . 
 the results were compared with the initial analysis measurements obtained from fresh samples . 
 mean changes compared to baseline ( t0 ) concentrations were evaluated both statistically and clinically.results:our results show that sodium , potassium , urea , creatinine , uric acid , total calcium , phosphorus , direct bilirubin , total bilirubin , aspartate aminotransferase , alanine aminotransferase , alkaline phosphatase , total protein , albumin , cholesterol , and triglyceride levels were stable under all conditions . 
 serum amylase was the only analyte demonstrating instability following prolonged storage ; amylase levels changed significantly ( both statistically and clinically ) at 7 , 15 , and 30 days ( p < 0.05).conclusion : most common biochemical analytes , except for amylase , showed adequate stability in serum following 30 days of storage at 20c . 
 serum amylase analysis should be conducted on the same day that the sample is received in the laboratory . 