|Year : 2018 | Volume
| Issue : 2 | Page : 33-36
Influence of freeze –thaw and storage time on some specific human hormones
Promise Nwankpa1, Celestine N Ekweogu1, Festus C Emengaha1, Patrick Ugwuezumba2, Obinna G Chukwuemeka3, Chinedu C Etteh1, Jude N Egwurugwu2, Sunday A Ezekwe1, Desmond I Izunwanne2
1 Department of Medical Biochemistry, College of Medicine, Imo State University, Owerri, Nigeria
2 Department of Human Physiology, College of Medicine, Imo State University, Owerri, Nigeria
3 Department of Biochemistry, Michael Okpara University of Agriculture, Umudike, Nigeria
|Date of Web Publication||25-Feb-2019|
Dr. Promise Nwankpa
Department of Medical Biochemistry, College of Medicine, Imo State University, Owerri
Source of Support: None, Conflict of Interest: None
Background: The objective of this study was to examine the effect of freeze–thaw and storage time on stability of some specific human hormones. Materials and Methods: Follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, estrogen, progesterone, and testosterone were carried out immediately after sample collection, after undergoing freeze–thaw at −4°C, −20°C, and −70°C, at day 0 and after 7-day storage at −4°C, −20°C, and −70°C. A total of 100 healthy participants (50 males and 50 females) were used for the study, and blood serum was used for the analysis. Results: From our results, there was no statistically significant difference (P > 0.05) between FSH, LH, prolactin, estrogen, progesterone, and testosterone levels obtained after freeze–thaw at −4°C, −20°C, and −70°C at day 0 when compared with the control for both males and females. Furthermore, there was no statistically significant difference (P > 0.05) in the levels of FSH, LH, prolactin, estrogen, progesterone, and testosterone levels obtained after freeze–thaw at 7-day storage at −4°C, −20°C, and −70°C in both males and females when compared with the control. Conclusion: The results showed that the specific hormones studied were most stable when stored at −70°C for 7 days assuming sample analysis is not carried out shortly after sample collection.
Keywords: Freeze, hormones, storage, temperature, thaw
|How to cite this article:|
Nwankpa P, Ekweogu CN, Emengaha FC, Ugwuezumba P, Chukwuemeka OG, Etteh CC, Egwurugwu JN, Ezekwe SA, Izunwanne DI. Influence of freeze –thaw and storage time on some specific human hormones. Niger J Exp Clin Biosci 2018;6:33-6
|How to cite this URL:|
Nwankpa P, Ekweogu CN, Emengaha FC, Ugwuezumba P, Chukwuemeka OG, Etteh CC, Egwurugwu JN, Ezekwe SA, Izunwanne DI. Influence of freeze –thaw and storage time on some specific human hormones. Niger J Exp Clin Biosci [serial online] 2018 [cited 2019 Sep 22];6:33-6. Available from: http://www.njecbonline.org/text.asp?2018/6/2/33/252840
| Introduction|| |
Time of sample collection and sample handling/processing as well as storage/preservation,, are some of the preanalytical factors that affect the result of most laboratory biochemical assays. In most clinical and research laboratories, analyses are carried out over an extended period of time and more often than not, there may be need for the researcher to perform different tests on the same sample. It may, therefore, not be practicable to carry out all the analyses in a particular sample in 1 day. There is, therefore, the need to store samples usually in the refrigerator door (4–8°C) or deep freezer (−20°C) for subsequent investigations at different days so as to ensure stability of biochemical analytes. In developing countries like Nigeria, equipment breakdown, power failure, lack of reagents, and acquisition of samples after work hours or over the weekends are some of the factors that may prevent sample analysis from being carried out on the day of specimen collection., Such longtime storage of samples and subsequent repeated freeze–thaw cycles may adversely affect the results of clinical chemistry analysis.,
Hormones are signaling molecules produced by endocrine glands and transported to distant organs through the circulatory system to regulate physiology and behavior. Hormonal assay of blood samples and those appropriately matched control provide useful information in the diagnosis of diseases ranging from Graves disease to prostate and other malignancies as well as infertility. Studies by other authors showed either stability,, or some degradation,, over time of some steroidal hormone stored at temperature as low as −20°C and −80°C over a period of 3 to 7 years. In the research carried out by Bielohuby et al., storage temperature and the number of freeze–thaw cycles can be critical for accurate measurement of metabolic hormones.
Some of the most frequently assayed hormones are steroidal hormones, follicle-stimulating hormone (FSH), prolactin, luteal hormone, and thyrotropin. Although available data suggest that long-term storage and repeated freeze–thaw may affect the results of hormonal assay,, power failures and other logistical problem in developing countries necessitate unintended storage of samples. Sequel to the above and the paucity of data in developing country like Nigeria on the effects of storage and repeated freeze–thaw on the stability of some specific human hormones necessitated this study.
| Materials and Methods|| |
A total of 100 healthy participants consisting of 50 males and 50 females who presented at the Outpatient Department of Imo State University Medical Centre between April 2018 and July 2018 were randomly selected for this study. The samples collected from each participant were strictly for laboratory investigations. Informed written consent was obtained from all the participants, and Imo State University Ethical Committee approved the study. All procedures were conducted in accordance with the guidelines as stipulated in the Helsinki declaration on human experimentation.
Sample collection and analysis
10 mL of fostering venous blood was collected in the morning using the standard blood collection technique. Venipuncture of the median cubital vein in the antecubital fossa was done with a 10 mL syringe and needle (21 g × 1½ inches −0.8 mm × 40 mm) manufactured by Anhui Kangning Industrial (Group) Co. Ltd., Anhui Province, China. The blood sample was transferred into 10 mL plain plastic specimen containers. Samples were allowed to stand at room temperature for about 30 min followed by centrifuge at 6000 revolutions per minute for 5 min before harvesting of serum. Serum sample of each patient was divided into seven aliquot. The first aliquot of the female participants was immediately assayed for the following: FSH, luteinizing hormone (LH), prolactin, estrogen, and progesterone, while the male participants were assayed for FSH, LH, prolactin, and testosterone. This first aliquot served as the control. Another three samples were analyzed after a single freezing–thawing within 24 h of sample collection at −4°C, −20°C, and −70°C and served as the basal fresh values (day 0). The remaining aliquots were stored at −4°C, −20°C, and −70°C for 7 days before analysis. Samples were left to stand at room temperature to thaw and repeatedly inverted to allow for proper mixing before analysis. All hormonal assays were done using competitive enzyme-linked radioimmunoassays using commercial kits (manufactured by Nichols Institute, San Juan Capistrano, CA) at the laboratory of Imo State University Centre, Owerri.
Data obtained from the study were analyzed using the Statistical Package for the Social Science (IBM-SPSS), Software for windows version 21.0, New York, USA. A test of significance was done using Student's t-test and analysis of variance at 0.05 level of significance. Results were presented in tables and expressed as mean ± standard deviation.
| Results|| |
[Table 1] shows the values of serum hormonal levels (FSH, LH, prolactin, estrogen, and progesterone) obtained from fresh female blood samples analyzed shortly after collection (control) and then within 24 h of sample collection (day 0) after undergoing freeze–thaw at −4°C, −20°C, and −70°C, whereas [Table 2] shows hormonal levels obtained from the analysis of female blood samples after 7-day storage at different temperatures of −4°C, −20°C, and −70°C. There was no statistically significant difference (P > 0.05) between the serum hormonal levels obtained from analysis shortly after sample collection and after undergoing freezing at −4°C, −20°C, and −70°C and thawing within 24 h of sample collection. Furthermore, there was no significant difference (P > 0.05) between the values obtained after 7-day storage at −4°C, −20°C, and −70°C and the control.
|Table 1: Effect of freezing–thawsing on hormonal profile of female blood samples within 24 h of collection|
Click here to view
|Table 2: Effect of 7-day storage at different temperatures on hormonal profile of human female blood samples|
Click here to view
[Table 3] and [Table 4] show the values of hormonal levels obtained from the analysis of freshly collected male blood sample shortly after collection (control) and with 24 h of sample collection after undergoing freeze–thaw at −4°C, −20°C, and −70°C and the values of hormonal levels obtained from analysis of male blood samples after a 7-day storage at −4°C, −20°C, and −70°C, respectively. The pattern of variation of results obtained from the analysis of male blood sample at days 0 and 7 and at various temperatures is similar to those of female samples.
|Table 3: Effect of freezing-hawing on hormonal profile of male blood samples within 24 h of collection|
Click here to view
|Table 4: Effect of 7-day storage at different temperatures on hormonal profile of human male blood samples|
Click here to view
| Discussion|| |
In most clinical and research laboratories, there is always need to carry out analysis of specimens at times different from their time of collection. This gives rise to the need to store such samples for a specific period of time mostly at sub-zero temperatures followed by thawing before analysis. Our study was designed to evaluate the effect of freezing–thawing and storage time on some specific human hormones.
The result showed a slight decrease in the concentrations of FSH, LH, prolactin, progesterone, and testosterone in both males and females after analysis of both fresh samples (analyzed within 24 h of sample collection) and samples stored at different temperatures of −4°C, −20°C, and −70°C for 7 days. However, these changes were statistically and clinically insignificant (P < 0.05).
This result is consistent with those of Comstock et al., who found that three cycles of freeze–thaw have no effect on the levels of human and androstenedione, FSH, LH, progesterone, estrogen, estradiol, prolactin, and testosterone even after 6 or 10 cycles.
The study by Taieb et al. demonstrated that levels of E2, FSH, and LH were relatively stable when human serum sample was stored at −4°C and −25°C for up to 72 h. Furthermore, in the studies carried out by Rosario Reyna et al., no significant changes in hormonal assay results were seen with repeated freezing and thawing or with storage at −20°C or −70°C for insulin, LH, FSH, prolactin, and androstenedione. However, Rosario Reyna et al., demonstrated a significant decrease (P < 0.05) in the levels of sex hormone-binding globulin and progesterone stored at −20°C and −70°C, respectively, with a respective 3.3% and 1.1% drop per freeze–thaw cycle. Furthermore, hormone levels in gelada baboons (Theropithecus gelada) fecal extracts showed a significant decrease after two freeze–thaw cycles for glucocorticoid metabolites and six freeze–thaw cycles for testosterone metabolites which are at variance with our findings. The variation in this reports may be alluded to sample handling before harvesting of serum, storage temperature, and methods of analysis.,
| Conclusion|| |
This present study shows that there is no significant or predictable change in human hormonal assay results when samples are stored for 7 days at −4°C, −20°C, and −70°C for FSH, LH, prolactin, progesterone, and testosterone. Overall, hormonal assay should be done shortly after sample collection; otherwise, storage for 7 days at −4°C, −20°C, and −70°C does not adversely affect result outcomes.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Turriani M, Bernabò N, Barboni B, Todisco G, Montini L, Berardinelli P, et al.
Circadian rhythm and stress response in droppings of Serinus canaria
. Vet Med Int 2016;2016:3086353.
Kalbitzer U, Heistemann M. Long-term storage effects in steroid metabolite extracts from baboon (Papiosp
) faeces-a comparison of three commonly applied storage methods. Methods Ecol Evol 2013;4:493-500.
Shutt K, Setchell JM, Heistermann M. Non-invasive monitoring of physiological stress in the western lowland gorilla (Gorilla gorilla gorilla
): Validation of a fecal glucocorticoid assay and methods for practical application in the field. Gen Comp Endocrinol 2012;179:167-77.
Nugraha TP, Heistermann M, Agil M, Purwantara B, Supriatna I, Gholib G, et al.
Validation of a field-friendly extraction and storage method to monitor fecal steroid metabolites in wild orangutans. Primates 2017;58:285-94.
Kachhawa K, Kachhawa P, Varma M, Behera R, Agrawal D, Kumar S, et al.
Study of the stability of various biochemical analytes in samples stored at different predefined storage conditions at an accredited laboratory of India. J Lab Physicians 2017;9:11-5.
] [Full text]
Boyanton BL Jr., Blick KE. Stability studies of twenty-four analytes in human plasma and serum. Clin Chem 2002;48:2242-7.
Divya PD, Jayavardhanan KK. Effect of time and temperature on the storage stability of hepatobiliary enzyme activities in cattle serum. Indian J Anim Res 2014;48:129-33.
Simundic AM, Lippi G. Preanalytical phase – A continuous challenge for laboratory professionals. Biochem Med (Zagreb) 2012;22:145-9.
Bolelli G, Muti P, Micheli A, Sciajno R, Franceschetti F, Krogh V, et al.
Validity for epidemiological studies of long-term cryoconservation of steroid and protein hormones in serum and plasma. Cancer Epidemiol Biomarkers Prev 1995;4:509-13.
Ito Y, Nakachi K, Imai K, Hashimoto S, Watanabe Y, Inaba Y, et al.
Stability of frozen serum levels of insulin-like growth factor-I, insulin-like growth factor-II, insulin-like growth factor binding protein-3, transforming growth factor beta, soluble Fas, and superoxide dismutase activity for the JACC study. J Epidemiol 2005;15 Suppl 1:S67-73.
Henderson BE, Bernstein L, Ross RK, Depue RH, Judd HL. The early in utero
oestrogen and testosterone environment of blacks and whites: Potential effects on male offspring. Br J Cancer 1988;57:216-8.
Garland CF, Friedlander NJ, Barrett-Connor E, Khaw KT. Sex hormones and postmenopausal breast cancer: A prospective study in an adult community. Am J Epidemiol 1992;135:1220-30.
Cauley JA, Gutai JP, Kuller LH, Dai WS. Usefulness of sex steroid hormone levels in predicting coronary artery disease in men. Am J Cardiol 1987;60:771-7.
Phillips GB, Yano K, Stemmermann GN. Decrease in serum estradiol values with storage. N
Engl J Med 1984;311:1635.
Phillips GB, Yano K, Stemmermann GN. Serum sex hormone levels and myocardial infarction in the Honolulu Heart Program. Pitfalls in prospective studies on sex hormones. J Clin Epidemiol 1988;41:1151-6.
Bielohuby M, Popp S, Bidlingmaier M. A guide for measurement of circulating metabolic hormones in rodents: Pitfalls during the pre-analytical phase. Mol Metab 2012;1:47-60.
Rosario Reyna BS, Kyle D, Traynor MD, Gene H, Boots LR, Ricardo Azziz MD. Repeated freezing and thawing does not generally alter assay results for several commonly studied reproductive hormones. Fertil Steril C2001;76:823-5.
Comstock GW, Burke AE, Norkus EP, Gordon GB, Hoffman SC, Helzlsouer KJ, et al.
Effects of repeated freeze-thaw cycles on concentrations of cholesterol, micronutrients, and hormones in human plasma and serum. Clin Chem 2001;47:139-42.
Livesey JH, Hodgkinson SC, Roud HR, Donald RA. Effect of time, temperature and freezing on the stability of immunoreactive LH, FSH, TSH, growth hormone, prolactin and insulin in plasma. Clin Biochem 1980;13:151-5.
Taieb J, Benattar C, Birr AS, Lindenbaum A, Frydman R, Olivennes F, et al.
Delayed assessment of serum and whole blood estradiol, progesterone, follicle-stimulating hormone, and luteinizing hormone kept at room temperature or refrigerated. Fertil Steril 2000;74:1053-4.
Pappano DJ, Roberts EK, Beehner JC. Testing extraction and storage parameters for a fecal hormone method. Am J Primatol 2010;72:934-41.
[Table 1], [Table 2], [Table 3], [Table 4]