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| Biochemical analytes in centrifuged blood samples could be affected by the age of subjects in different time periods and storage temperatures |
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Mahsa Yazdanbakhsh1, Mohammad Reza Deyhim2, Hanieh Jafary1, Mohammad Hessam Rafiee2, Mohammad Moradi3
1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
2 Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
3 Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran; Department of Biotechnology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran
Click here for correspondence address and email
| Date of Submission | 07-Dec-2021 |
| Date of Decision | 17-Jul-2022 |
| Date of Acceptance | 17-Jul-2022 |
| Date of Web Publication | 26-Sep-2022 |
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 Abstract | | |
BACKGROUND AND OBJECTIVES: Collection and providing standard blood samples for examination is critical for all medical laboratories. Currently, for all blood samples taken from the elderly and young people, the same preparation protocol is applied. We hypothesized that the blood taken from older individuals is more vulnerable to preparation conditions compared with the younger ones. This study aimed to assess the effect of storage duration and temperature on biochemical analytes in serum samples taken from old and young participants.
MATERIALS AND METHODS: Blood samples from 30 healthy participants including 15 young people (eight males and seven females, 20–35 years old) and 15 elderly people (eight males and seven females, 60–75 years old) were collected. For each participant, serum sample tubes stored at different time intervals (2 and 24 h) and temperatures (4°C, 25°C, and - 20°C). The biochemical analytes were examined by an automated analyzer. Total antioxidant capacity was assayed by a commercial kit. Nitric oxide metabolites were measured by Griess reagent.
RESULTS AND CONCLUSION: Intragroup analysis in young and older participants showed that lactate dehydrogenase levels were significant in 2 h incubation time at 20–24°C and in 24-h incubation times at 20–24°C and 4°C compared with the baseline time (T0) (P < 0.05). Intergroup analysis showed that glucose and urea levels were higher among the older participants than the youth group at baseline and 24 h times (P < 0.05). Furthermore, the magnesium and nitric oxide levels were higher in the youth group than the elderly ones at baseline and 24 h times (P < 0.05). Keywords: Age, biochemical analyte, blood sample, incubation time, temperature
How to cite this URL:
Yazdanbakhsh M, Deyhim MR, Jafary H, Rafiee MH, Moradi M. Biochemical analytes in centrifuged blood samples could be affected by the age of subjects in different time periods and storage temperatures. Asian J Transfus Sci [Epub ahead of print] [cited 2023 Mar 24]. Available from: https://www.ajts.org/preprintarticle.asp?id=356873 |
| Introduction | |  |
The results of laboratory tests are critical for providing daily clinical care. In all medical diagnostic laboratories, different strategies are used to prepare the standard blood samples. The main goal for all of them is to control the conditions before the analysis and minimize the interfering factors in the final test result. Despite advances in medical science, various factors cause errors during the assay procedure.[1],[2] Medical diagnostic laboratories receive whole blood samples. After centrifugation, the clots are removed from the serum, and they are examined. Usually, in the standard mode, samples are stored at room temperature (22°C-25°C) for up to 30 min to clot, and they are centrifuged at intervals of up to 2 h from the time of collection (maximum time limit of 2 h from the time of collection). After centrifugation, the serum is separated. The NCCLS recommends keeping the plasma/serum at room temperature for <8 h. If the test is not performed within 8 h, the sample should be stored at refrigerator temperature (2°-8°C). If the refrigerator sample is not tested for up to 48 h or long-term storage of samples, freezing temperatures below-20°C are recommended. This is the main responsibility of laboratories to use the existing authorities to measure the specific stability of the analytes to determine them in terms of temperature conditions and time delay.[3]
Biologically, biochemical changes in the blood samples during the preparation process (such as not providing temperatures of 22°-25°C) are associated with changes in the level of many biochemical parameters. The effect of time and temperature on the values of analytes has already been proven.[4] Apart from the effect of time and temperature variables on the values of analytes, another influential factor could be the age of patient. The primary indicator for assessing the potential of oxidative stress in aging is the plasma antioxidant capacity. The imbalance between antioxidants and oxidants creates oxidative conditions. Earlier researches show that changes in oxidative stress are also associated with aging.[5],[6]
In some studies, malondialdehyde levels and lipid peroxidation have been shown to increase in elderly people. Free radicals oxidize lipids and membrane proteins of red blood cells (RBCs), which can damage RBCs, and this process is an essential factor for RBC hemolysis during the storage or preparation process.[7],[8] Other studies have also reported that older people's RBC membranes are more sensitive toward oxidative stress, and they show more vulnerability than the young people's blood cells.[9] According to the process of preparing blood samples,[2] variables such as temperature of blood sample storage can trigger oxidative damage, RBCs membrane damage, and change biochemical properties. Therefore, blood samples collected from the elderly people and younger individuals could have a different antioxidant capacity that creates a different response toward oxidative stress. Accordingly, it affects the quality of the blood sample during preparation.[10]
Although there are numerous studies on the effects of temperature and storage duration on sample preparation conditions, so far in none of the reports the samples were evaluated based on the age of the patient/donor.[11],[12] However, today for all blood samples taken from the older people and young people, a constant preparation protocol is considered for the storage temperature of the serum/plasma sample (Kiechle, 2010). We hypothesized that the blood taken from elderly people is more vulnerable to preparation conditions compared to younger ones, and their sample analytes are more unstable in response to temperature and storage conditions.[10] If it could be proven that the blood samples taken from older people are more affected by the sample preparation variables such as temperature conditions, further research on the blood sample preparation and the storage temperature of the serum/plasma sample must be considered according to the age of the individuals.
| Materials and Methods | | |
Study design and participants
The blood samples were collected from 30 healthy paricipants divided into two groups: 15 young people (eight males and seven females, 20–35 years old) and 15 older people (eight males and seven females, 60–75 years old). Informed written consent was taken from all participants or their guardians, prior to their contribution to this study.
Sample collection
Fasting venous blood was collected in the morning from each volunteer in the laboratory of Iranian Psychiatric Hospital from November 2018 to December 2018. Thirty milliliters of blood were taken from each person by syringe and poured into six gel tubes (Hebei Xinle Technical Co. Ltd) and a complete blood count (CBC) tube containing ethylenediaminetetraacetic acid (Hebei Xinle Technical Co. Ltd).
Sample preparation
CBC tube was analyzed with the Sysmex-k × 21n apparatus. Each of the six tubes was allowed to clot for 30 min at real-time (RT), then was centrifuged (Hettich Rotofix 32A, Tuttlingen, Germany, settings: 3000 g, 10 min, 22°C). All samples from each person were categorized according to storage temperatures and time intervals for analysis:
- One tube Immediately analyzed after centrifugation as baseline value (T0)
- Two tubes stored at 20°C –24°C at different time intervals (2 and 24 h)
- Two tubes stored at 4°C at different time intervals (2 and 24 h)
- One tube stored at -20°C for 24 h.
In order to avoid evaporation, sample tubes were kept closed until analysis completion. All samples were immediately analyzed after incubation time. The incubation time and temperature were following the routine conditions used in the laboratory.
Measurement of biochemical parameters
The biochemical factors examined in this study included: fasting blood sugar, creatinine, uric acid, urea, serum glutamic-pyruvic transaminase, serum glutamic-oxaloacetic transaminase, bilirubin direct, bilirubin total, magnesium, potassium, sodium, triglycerides, cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, iron, and lactate dehydrogenase (LDH) which were measured by the automated analyzer (Chemistry Analyzer BT-1500, Italy) using the commercial kits (Pars Azmoon, Iran). Control serum was used at the beginning of each run to measure all tests. Also, to ensure the accuracy of the tests prior to starting with each device, quality control was performed using valid commercial controls and following the device catalog.
Total antioxidant capacity
A commercial kit (ZellBio GmbH, Germany) was used to measure the total antioxidant capacity (TAC) of the serum. The basis of the oxidation reaction is 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). The antioxidants in the sample had an inhibitory effect on this reaction. The reduction in the amount of cation radical (+ABTS) was formed by increasing the number of antioxidants in the sample, which reduced the amount of light absorption. In this method, the standard watersoluble analog of vitamin E (Trolox) was used as an antioxidant. The concentration of the samples was measured by enzyme-linked immunosorbent assay (ELISA) Reader (Stat fax 4300, USA).
Measurement of nitric oxide metabolites (nitrite/nitrate)
The determination of nitric oxide metabolites was performed by Griess reagent.[13] First, the serum was deproteinized. For this purpose, 150 μL of 0.3 molar NaOH (German-Merck), and 150 μL of 5% ZnSO4 (German-Merck) were added to 300 μL of serum. Samples were incubated at room temperature for 20–25 min, then centrifuged in a refrigerated centrifuge at 3000 g for 20 min. In order to measure nitrite, serum nitrates were converted to nitrite with a reductant chemical vanadium trichloride (German-Merck). Then the total nitrite was measured using the Griess reagent at room temperature. Optical absorption of the desired color was read by ELISA Reader (Hiperion, MPR4 Plus, Germany) at 540 nm, and the concentration of metabolites was obtained based on the standard sodium nitrate curve (German-Merck) which was already prepared.
Statistical analysis
All data were analyzed using version 23 of SPSS software (IBM company, Armonk, New York, United States). Kolmogorov–Smirnov test showed that the data were not normally distributed. For this reason, the nonparametric Mann–Whitney test was used to compare data during incubation times between two groups of young and elderly people. Kruskal–Wallis variance tests were used to evaluate data changes for intragroup incubation times in both groups. To ensure the interpretation of the results, all data were reanalyzed in parallel by parametric t-test and analysis of variance (ANOVA). P < 0.05 was considered statistically significant.
| Results | | |
The results of hematology parameters including white blood cell, Neu, Lym, Mon, Eos, Bas, RBC, hemoglobin, hematocri, mean corpuscular volume, mean corpuscular hemoglobin concentration, red blood cell distribution width (RDW)-CV, RDW-standard deviation, platelet, mean platelet volume, platelet distribution width, plateletcrit (PCT), platelet larger cell count (P-LCC), and platelet–large cell ratio were checked. Those volunteers who had normal baseline blood tests for hematology were included in this study. [Table 1] shows the results of measured parameters for the biochemical analytes in two groups of young and elderly participants according to the different times and temperatures of incubation in the study condition. | Table 1: Results of measured biochemical parameters for the study in two groups of young and elderly participants, according to different times and incubation temperatures
Click here to view |
Intragroup analysis in young and elderly participants
In the group of younger participants, the evaluation of analytical changes with parametric t-test and nonparametric Mann–Whitney test between incubation times showed that LDH levels were significant in 2 h incubation time at 20°C-24°C and in 24-h incubation times at 20°C–24°C and 4°C compared to the baseline time (T0) (P < 0.05) [Table 2]. Other parameters with a P value near to significant level are presented, too [Table 2]. However, intragroup analysis with Kruskal-Wallis for defferent incubation times was only significant for LDH (P = 0.04) and near to significant level for cholesterol (P = 0.186). A significant increase was only observed with ANOVA analysis in 24-h incubation times at 4°C compared to T0 (P = 0.019) | Table 2: Intragroup analysis of biochemical analytes in young and elderly participants
Click here to view |
In the elderly group, LDH levels were significant at 24-h incubation times at 20°C-24°C and 4°C compared to the baseline time (T0) with the Mann–Whitney. LDH were significantly high at 24-h incubation time at 20°C-24°C with a t-test (P < 0.05) [Table 2]. Also, in the elderly group, changes in calcium levels and, to some extent, cholesterol at 24-h incubation time at 4°C compared to baseline time (T0) was close to the significant level of 0.05 [Table 2]. No significant changes were detected with Kruskal–Wallis and ANOVA. Intragroup analysis with Kruskal–Wallis was close to significant for LDH (P = 0.125) [Table 2].
Intergroup analysis of young and elderly participants
According to [Table 3], the analysis was performed between young and elderly groups with t-test and Mann–Whitney test for each time of incubation, and the results showed that glucose and urea levels were higher in the older people than the young ones at baseline and 24 h times (P < 0.05). Furthermore, the magnesium and nitric oxide levels were higher in the young group than in the elderly participants at baseline and 24 h period (P < 0.05). In addition, iron levels at 2 and 24-h incubation times at 20°C-24°C and 4°C were higher in young participants than the elderly group. However, it was statistically close to a significant level. | Table 3. Intergroup analysis of of biochemical analytes in young and elderly participants
Click here to view |
| Discussion | | |
The most important finding of the current study was to observe the significant intragroups changes in LDH compared to the baseline. However, no significant difference was observed for other studied analytes. The changes in LDH levels were increasing, and they appear to be higher among young participants' samples than the those of the elderly group. The onset of this instability in the young group from 2 h incubation at room temperature was the same as changes in the older participants from 24-h incubation. A 24-h delay in measuring LDH at room and refrigerator temperatures was not desirable for both young and old groups. The stability of this analyte appears to be higher in samples stored at frozen temperatures for older participants than the younger ones. According to the results of the current study, a 2 h delay in refrigerator temperature could be suitable to measure the LDH in both young and elderly groups. As well, a 24-h delay in measuring LDH is not recommended for younger indivuals. Even though, a 24-h delay is needed to measure the sample of older people, the frozen storage temperature could be considered.
Besides LDH changes, some analytes, such as calcium and cholesterol, showed near-significant changes in elder participants, and their samples appeared to be more sensitive to a delay in measurement and storage temperature. Elevated levels of calcium were observed for all temperatures at 24 h, and it appears a 24-h delay in measurement is not appropriate for this analyte. The changes in cholesterol were sinusoidal during the incubation time. Its values were decreased at 24 h times compared to the baseline and continued for only 24 h at refrigerator temperature. The reduction did not appear at room and frozen temperatures. This trend was observed in both young and elderly groups. Although it is making the results difficult to interpret, we believe that it could be addressed in future studies with a focus on more sample size and replicates.
Although there have been no report on the comparison of the stability of the analytes between the young and older people, several studies have been conducted with delays in measuring serum samples during different temperatures and incubation time.[14],[15] Tanner et al. examined the effects of different temperatures (15°C, 25°C, or 35°C) on biochemical analyses at 4, 8, and 24 h. Similar to our research, they found that LDH levels were increased at 24°C after 24 h. At their study, Calcium was reduced at room temperature during different incubation times, but the mechanism of this change was not known.[16] In another study, Kift et al. studied analytes at the temperatures of 25°C and 4°C until 4 days. LDH stability was reported at 25°C for 6 h and at 4°C for 24 h. Calcium and cholesterol levels were also unstable at 4°C.[17] Similar to the study of Kift et al., Oddoze et al. reported the stability of LDH within SST tubes at 25°C for 6 h and 4°C for 24 h, but within glass tubes at both temperatures for 24 h. Furthermore, the stability of glucose levels was observed at 25°C and 4°C for up to four and 6 h, respectively.[18]
Marjani reported that the stability of glucose, urea, creatinine, cholesterol, triglycerides, and calcium at room and refrigerator temperature for the incubation time of 2 and 24 h. However, in this study, glucose and creatinine were reported to decrease in room temperature over 24 h.[14] van Balveren et al. showed that calcium levels rise at 4°C and 8°C, which is in agreement to the results by the current report.[15] Besides, the results of a meta-analysis have showed that LDH is unstable at RT for 12 h with an increasing trend. The authors recommended a refrigerated storage temperature.[19]
At this study, the intergroup analysis between the elderly and young participants showed significant differences in baseline (T0) levels for glucose, urea, nitric oxide, and magnesium. Similar differences from four analytes were detected only at-20°C and were not observed in other incubation times and temperatures. As expected, the intragroup analysis also showed that the level of analytes stored at freezing temperature compared to the other temperatures had the least changes in the stability of the analytes compared to the baseline concentrations (T0). The freezing temperature was used as a reference in this study to assess the optimal long-term storage conditions with the least probability of stability changes in the analytes.
Dirar et al. and Cuhadar et al. analyzed the analytes for up to 72 h at room and refrigerator temperatures, reported that the temperature did not affect the stability of the biochemical analytes.[4],[20] Oddoze et al., in a 24-h stability study, reported that storage temperature did not have much effect on the stability of biochemical analytes.[18] In contrast, in a stability study of analytes at refrigerator and freeze temperatures, Flores et al. reported the freezing temperatures as a more appropriate way to store glucose, creatinine, and uric acid for longer periods.[2]
It is not surprising that some of the results of the earlier studies do not agree with the findings in the current study. The reason for these deviations could be difference in sampling tubes, whether the tubes are capped during the incubation, sample evaporation and increase in sample concentration, degradation of analytes during incubation, differences in storage conditions, statistical techniques studied and the number of different samples.[17],[21] Lack of water during freeze or sample freezing could also increase the concentration or further changes the results.[22] Also, in most studies, the samples were kept frozen until the analysis time.
In our research, to eliminate the intervention factors like the freezing temperature, and the storage time of serum samples, all measurements were performed immediately after the incubation time, which could be one of the reasons for the difference between our results and others. In order to minimize the possible variation effects of analytical runs on the results, it was also tried to have an equal number of samples from the older people and young people every day. To furthur prevent the misinterpretation of results due to statistical tests, it was tried to report both parametric and nonparametric analyzes in parallel.
Finally, a comparison of analytes values between the elderly and young participants showed that the older individuals had a higher glucose and urea level and a lower nitric oxide, iron, and magnesium level compared with the younger ones at the baseline and other incubation times. Similar results have been reported in the other studies,[23],[24],[25] and there are many possible reasons for this deviation. For example, decrease in the muscle mass, digestive problems, less activity, decrease in the normal activity of gonadal hormones, impaired renal function, and the use of dietary supplements in the older people could be the reasons for this deviation.
In the current study, we expected to obtain a significant difference in TAC between the elderly and younger participants, which could justify the difference in the stability of some analytes during storage conditions, but there was no difference. However, we still believe in TAC changes and their potential effects on the stability of analytes in older people. An increase in the sample size and longer incubation times could resolve the mentioned issue.
| Conclusion | | |
According to the results of the current study, the stability of serum analytes such as LDH, glucose, urea, magnesium and nitric oxide are likely affected in both young and elderly groups in response to temperature and storage conditions. It seems that the more and comprehensive studies must be considered according to the age of the individuals.
Informed consent
Written informed consent was taken from all participants or their guardians, prior to their contribution to this study.
Research involving human and/or animals participants
Helsinki protocol for medical researches was followed accordingly, and the moral aspects of this study was supervised and accepted by the moral committee of Science and Research Branch of Islamic Azad University, Tehran, Iran.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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Correspondence Address:
Mohammad Hessam Rafiee,
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran
Iran
Mohammad Moradi,
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran; Department of Biotechnology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan
Iran
 Source of Support: None, Conflict of Interest: None DOI: 10.4103/ajts.ajts_178_21
[Table 1], [Table 2], [Table 3] |
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