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Letters to the Editor |
Departments of1
Obstetrics and Gynecology and2
Public Health Science and General Practice University of Oulu Oulu, Finland
3 National Public Health Institute Oulu, Finland
4 Department of Clinical Chemistry University of Oulu Oulu, Finland
5 Department of Epidemiology and Public Health Imperial College London London, United Kingdom
aAddress correspondence to this author at: Department of Obstetrics and Gynecology, University of Oulu, PO Box 5000, 90014 Oulu, Finland. Fax 358-8-3154310; e-mail Tuija.Mannisto{at}oulu.fi.
To the Editor:
Data concerning the effects of freezing, thawing, and storage on serum thyroid-stimulating hormone (TSH) and thyroid hormones [free thyroxine (fT4) and free triiodothyronine (fT3)] are scarce (1)(2)(3), and we can find no data on their effects on thyroid autoantibodies [thyroid-peroxidase antibody (TPO-Ab) and thyroglobulin antibody (TG-Ab)].
We evaluated the effect of freezing, thawing, and storage of samples for up to 23 years on TSH, fT4, fT3, TPO-Ab, and TG-Ab concentrations in human serum (Architect i2000, Abbott Diagnostics). Whole blood samples for the Finnish Maternity Cohort (FMC) were collected without preservative from pregnant women, most of whom were in the 1st trimester of pregnancy. Each sample was stored in a single serum aliquot in a polypropylene cryovial (Nunc GmbH & Co.) at –25 °C. The ethics boards of the Finnish National Public Health Institute and Oulu University Hospital approved this study.
The effect of freezing and thawing was evaluated by comparing frozen serum samples (n = 50) with fresh samples (n = 50) from FMC. Samples stored for 6 months vs 2–23 years at –25 °C (50 different samples collected from FMC at every time point, total n = 645) were analyzed to evaluate the effect of storage time. Short-term storage of up to 6 days at 4 °C was studied in fresh sera (n = 8) from nonpregnant women. Details of the methods are available on request.
After 0, 1, 3, and 6 days at 4 °C, no statistically significant effect was seen on any tests studied. There were no differences in TSH, fT4, TPO-Ab, or TG-Ab concentrations when 50 frozen and thawed serum samples were compared with 50 fresh serum samples. fT3 concentrations were significantly higher (Student t-test, P <0.001) in frozen samples but remained within reference intervals.
We analyzed 645 samples from pregnant women with a mean gestational age of 11.9 weeks (range 6.7–18.0) to evaluate the effect of storage time. Storage time had no effect on TSH or fT3 concentrations (Fig. 1
). Concentrations of fT4 remained within reference intervals at all time points, although they differed significantly from baseline after 10, 12, and 20–23 years of storage (Fig. 1
). Because fT4 concentrations were comparable between 0 and 14–18 years, the difference at 10 and 12 years might be the result of random variation. Storage time explained only 5.5% of the total variation in fT4 concentrations (linear regression analysis, P <0.001), which was smaller than the interassay variation of the method.
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The concentrations of TPO-Ab increased steadily with extended storage time, and a significant difference compared with baseline was seen when storage time was >2 years (Dunnett test, P = 0.047). Thereafter, TPO-Ab concentrations steadily increased with increasing storage time (Dunnett test, P <0.001) until 14 years, at which point a more marked increase was seen (Dunnett test, P <0.001; Fig. 1
). Storage time explained 19.7% of the total variation in TPO-Ab concentrations (linear regression analysis, P <0.001). Although median TPO-Ab concentrations stayed under the upper limit of the reference interval (5.61 kIU/L), considerable change was seen after 14 years of storage (Fig. 1
).
The concentrations of TG-Ab increased with extended storage time, differing significantly from the baseline after storage for >6 years (Dunnett test, P <0.05), with the exception of the 10th year of storage. Storage time explained 19.4% of the total variation seen in TG-Ab concentrations (linear regression analysis, P <0.001). Median TG-Ab concentrations exceeded the upper limits of the reference interval (4.11 kIU/L) after 14 years of storage (Fig. 1
).
Higher thyroid autoantibody concentrations were likely to be the result of storage time, because preanalytic conditions (sampling, tubes, transportation, sample processing, freezing, thawing, or storage conditions) did not differ in our study. The causes of changes in thyroid autoantibody instability during long-term storage are unknown and probably of complex character.
We conclude that TSH, fT4, and fT3 can reliably be analyzed in samples stored for 23 years at –25 °C, and that TPO-Ab and TG-Ab are also stable for 14 years of storage. Cross-sectional comparison of samples is possible at every time point, but comparison between samples with different storage times, at least when exceeding 14 years, should be carried out cautiously.
Acknowledgments
Grant/funding support: This study was supported in part by grants from Alma and K.A. Snellman Foundation, Oulu, Finland; the Jalmari and Rauha Ahokas Foundation, Finland; and the Lilly Foundation, Finland.
Financial disclosures: None declared.
Acknowledgments: We thank Jouni Sallinen and Frank Quinn from Abbott Laboratories for providing laboratory kits.
References
The following articles in journals at HighWire Press have cited this article:
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  T. Mannisto, M. Vaarasmaki, A. Pouta, A.-L. Hartikainen, A. Ruokonen, H.-M. Surcel, A. Bloigu, M.-R. Jarvelin, and E. Suvanto-Luukkonen Perinatal Outcome of Children Born to Mothers with Thyroid Dysfunction or Antibodies: A Prospective Population-Based Cohort Study J. Clin. Endocrinol. Metab., March 1, 2009; 94(3): 772 - 779. [Abstract] [Full Text] [PDF]
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