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The Effects of Temperature as a Preanalytical variable on Sample Stability
A brief review of the literature
Research is constantly being conducted on the effects of freeze procedures and storage conditions
for samples. In the current biobanking industry, much of what we know or assume about sample
storage is based on anecdotal knowledge or the concept of "this is the way I/we have always done
it". In the past 10 years, call for standardization of biobanking protocols has exploded coinciding
with the growth in personalized medicine and research utilizing banked samples.
In many cases, our understanding of proper storage has lagged behind the demand for these
valuable specimens, which has left researchers with numerous questions about sample suitability
and stability.
Bluechiip took a look at the state of current research into the concept of temperature as a key preanalytical variable. Many of the papers mention temperature in the context of the following areas
(listed in order of frequency from papers reviewed):
•
•
•
•
•
Temperature at sample collection
Shipping Temperature
Centrifugation Temperature
Storage Temperature
Temperature During Handling
While we think everyone would agree that all of these represent areas where temperature can
have significant impact on sample stability, we have chosen to focus on the last two areas in this
summary. For this article we reviewed 35 papers written primarily in the last 12 years across a
variety of research domains. All of them involved studies that attempted to elucidate the proper
storage temperature for optimum sample stability. The majority of the studies looked at blood,
serum and plasma as their preferred sample type.
Temperature as a pre-analytical variable 0.05, March 2014
Eric Hall and Jason Chaffey, Bluechiip Ltd, © Copyright 2014.
It is clear from the review articles that standardization is a goal that many people are seeking to
achieve. Two of the most promising steps in that direction are the two proposed coding sets for
biospecimens; SPREC (Betsou et al, 2010) and BRISQ (Moore et al, 2011). Both of these address
temperature as a pre-analytical variable and include temperature at various points in the coding
schema. This is a tremendous step toward standardization and it will be interesting to follow the
adoption of one or both of these over the near term.
Review of the Literature
Sample
type/analyte of
interest
PBMC
Recommended
Storage
Special Considerations/Comments
Reference
-190°C Vapor
Phase
Key indicator for success is better control of
shipping temperature.
Olsen et al, 2001
"biofluid specimens"
Vapor phase LN
Hubel et al, 2011
Amyloid B and tau in
CSF
-80°C
If vapor phase storage is not available, -80°C
is next best option.
20% decrease in AB42 after 3 freeze/thaw
cycles.
folate in human
serum
-25°C
Samples stored for up to 29yrs at time of
study without statistically significant
reduction in folate.
Hannisdal et al 2010
MMP7 in human
serum
-80°C
Significant deceases seen in MMP7 after 20
f/t cycles and completely degraded at 30
cycles.
Chaigneau et al, 2007
CD40L in human
serum
-80°C
Lengelle et al, 2008
"Biological fluids and
Tissues"
-80°C
Multiple conditions prior to freezing and
multiple sets of freeze thaw cycles. Storage
at -20°C showed 50% loss of
immunoreactivity, storage at 37°C showed
complete loss
If the biomarker of interest is know to degrade
at -80 then it should be stored in LN).
PBMC
-135°C
Cryopreserved at a rate of -1/min and then
stored at -80°C for 24 hrs before being
transferred to LN.
Germann et al, 2013
serum, whole blood,
tissues
-80°C
For maintaining maximum recovery of RNA
and proteins.
Beyer et al, 2011
paraffin blocks
-80°C
Pest and humidity control also essential.
NCI
-80°C to -196°C
Recommended storage
NCI
Tissues
Temperature as a pre-analytical variable 0.05, March 2014
Eric Hall and Jason Chaffey, Bluechiip Ltd, © Copyright 2014.
Schoonenboom et al,
2005
Schrohl et al, 2008
Serum, plasma
-25°C or -80°C
Both were found to be acceptable for the
metabolites of interest.
Hustad et al, 2012
Serum, plasma
Lymphocytes and
other cellular
specimens
Rat serum
Serum and urine
-80°C
Vapor phase LN
Recommended storage
Recommended storage
Vaught, 2006
Vaught, 2006
Lower than -70°C
-80°C
Recommended storage
Storage and shipping at 4 for up to 24 hrs prior
to freezing was found to be acceptable.
Bielohuby et al, 2012
Dunn et al , 2008
Whole blood
Vapor phase LN
Cryopreserved to -90°C before being place in
LN.
Hayes et al, 2002
Serum
-20°C
Found to actually be detrimental for
epidemiologic research.
Ocke et al, 1995
Serum
-80°C
Lee et al, 2010
Plasma, urine and
bile
37°C
-80°C is the preferred storage condition for
samples collected for proteomic analysis.
goal was to stabilize pH, this was the ideal
temperature for a narrow range with 10% CO2
Serum for proteomic
biomarkers
-80°C
Changes in peak intensity were seen even in
samples stored at -80 but much less than
those stored at -20°C.
S. Ahmad et al, 2009
A. Furra et al, 2003
Serum proteins
-25°C was
temperature
studied
Non significant variations in protein
concentrations when stored for 25 years
Giselfoss et al,
2009
Serum components
-25°C was
temperature
studied
Significant variations in stability were noted in
long term storage
Giselfoss et al,
2008
Multiple sample
types
Varies
Recommended storage
Elaine Gunter, Specimen
Solutions LLC
Full references for papers in the above table are available at the end of this document
Discussion
Maintaining the optimal temperature conditions prevents the slow degradation of the useful
content of the samples over the period of their storage12. Below a sample’s critical transition
1
Engel KB, Moore HM. Effects of preanalytical variables on the detection of proteins by immunohistochemistry in formalin-­‐fixed, paraffin-­‐embedded tissue. Arch Pathol Lab Med. 2011;135(5):537-­‐43. 2
Xie R, Chung JY, Ylaya K, Williams RL, Guerrero N, Nakatsuka N, Badie C, Hewitt SM. Factors influencing the degradation of archival formalin-­‐fixed paraffin-­‐embedded tissue sections. J Histochem Cytochem. 2011;59(4):356-­‐65. Temperature as a pre-analytical variable 0.05, March 2014
Eric Hall and Jason Chaffey, Bluechiip Ltd, © Copyright 2014.
temperature the integrity of the sample can be maintained, however above this temperature all
storage achieves is to delay the process of degradation. It is necessary to maintain temperature
uniformity and avoid fluctuations. The samples require careful management to guarantee sample
conditions over their lifetime while also providing a high integrity of sample tracking to ensure
reliable retrieval.
Specimen collection and storage conditions performed by different laboratories create variations in
specimen quality, and little is known about the effects on test results when the samples are used.
For large-scale biobanking two challenges exist, the first is to prepare and ship samples in
controlled environments within short time periods and secondly the storage of biological samples
where low-temperature requirements, coupled with concerns such as avoiding unnecessary
thawing/temperature increases, are generally seen as key3.
Like any process, the lack of adequate controls in cryopreservation has been demonstrated as
being detrimental. In particular, several investigators have reported difficulties associated with
liquid nitrogen freezer systems, specifically the potential for sample contamination and the
formation of temperature gradients resulting in storage temperatures above -130°C4.
A second problem observed in liquid nitrogen freezers is the lack of temperature homogeneity
within the chamber. Liquid nitrogen freezers operate by filling the lower part of the storage vessel
with liquid nitrogen and allowing the vapor to cool the upper chamber. For smaller laboratory dewar
type vessels, the entire tank is filled and then allowed to evaporate over a period of time. During
this period, the cells stored in the upper racks of the chamber start at -196°C and as the liquid
nitrogen vapor level drops, a temperature gradient develops from the liquid upward. In larger liquid
nitrogen freezers, vapor phase gradients have been documented to span the glass transition
temperature of water, at times reaching -70°C to -95°C567. The wide temperature ranges observed
with liquid nitrogen storage systems is inherent to their operation, while automatic filling attempts
to address this issue.
For composite tissue samples, a key factor in the cryopreservation process is the warming rate8. A
successful cryopreservation technique for composite tissues would help meet this demand by
increasing the shelf life of donated tissues. This technique would be supported with reliable
measurement of temperature during the thawing process.
3
Biobanking Needs More Standardized Procedures Jan 15, 2008 (Vol. 28, No. 2), Genetic Engineering & Biotechnology News. 4
Rowley S. and D. Byrne. 1992. Low-­‐temperature storage of bone marrow in nitrogen vapor-­‐phase refrigerators: Decreased temperature gradients with an aluminum racking system. Transfusion 32: 750-­‐754. 5
White W and K. Wharton. 1984. Development of a cryogenic preservation system. American Laboratory Oct. 65-­‐76. 6
Wolfinbarger, L., V. Sutherland, L. Braendle, and G. Sutherland. 1996. Engineering aspects of cryobiology, in Advances in Cryogenic Engineering, 41: 1-­‐12. 7
Rowley S. and D. Byrne. 1992. Low-­‐temperature storage of bone marrow in nitrogen vapor-­‐phase refrigerators: Decreased temperature gradients with an aluminum racking system. Transfusion 32: 750-­‐754. 8
Cui X, Gao DY, Fink BF, Vasconez HC, Rinker B. Cryopreservation of composite tissues and transplantation: preliminary studies. Cryobiology. 2007 Dec;55(3):295-­‐304. Epub 2007 Sep 18. Temperature as a pre-analytical variable 0.05, March 2014
Eric Hall and Jason Chaffey, Bluechiip Ltd, © Copyright 2014.
A recent study demonstrated that for products exposed to elevated temperatures (i.e., >24°C),
rapid loss of viable cells and colony-forming cells that is out of proportion to the decline in trypan
blue or acridine orange/propidium iodide viability (which are more easily measured) led the
researchers to a conclusion that justified sufficient rationale for monitoring temperature during
transport in cases where necessary pre-processing steps need to be delayed9.
Sample preparation is often a complex process demanding high recoveries and tight control over
multiple key variables such as heat, processing time and the introduction of chemicals. Controlling
sample preparation, beginning at the tissue collection point, can provide significant improvements
to downstream analytical results. Ideally, the different temperature requirements for the stability of
each biomarker in the tissue would be addressed but this is not practicable in a large-scale
project10.
On thawing, cryopreserved hepatocytes often have reduced viability and metabolic function in
comparison with fresh cells11. Cryopreservation affects the viability and metabolic function of
hepatocytes on thawing, with the result that the thawed cells are often not suitable for clinical use.
An important of step in the cryopreservation process that can influence the function of the thawed
hepatocytes was the methods used for the freezing and the thawing of the cells. Being able to
measure temperature during these steps would allow an optimized protocol to be produced with
the final result assist with good manufacturing practice. Many researchers use visual assessment
of thawed samples12. In many instances studies that report high success cryopreserved samples
are both small and conducted with limited number of samples. The success rate is related to the
cryopreservation and thawing cycle procedure used in downstream analysis13.
References
Olson Walter C, Smolkin M, Farris E, Fink R, Czarkowski A, Fink J, Chianese-Bullock K, Slingluff Jr,
C. Shipping blood to a central lab in multicenter clinical trials: effects of temperature on
mononuclear cell yield, viability and immunologic function. J Transl Med. 2011 Mar 8;9:26
Hubel A, Aksan A, Skubitz A, Wendt C, Zhong X. State of the art in preservation of fluid
biospecimens. Biopreservation and Biobanking. 2011 9(3):237-244.
9
Solomon M, Wofford J, Johnson C, Regan D, Creer MH. Factors influencing cord blood viability assessment before cryopreservation. Transfusion. 2010 Apr;50(4):820-­‐30. Epub 2009 Nov 16. 10
The UK Biobank sample handling and storage protocol for the collection, processing and archiving of human blood and urine Int. J. Epidemiol. (2008) 37(2): 234-­‐244 11
Terry C, Dhawan A, Mitry RR, Lehec SC, Hughes RD. Optimization of the cryopreservation and thawing protocol for human hepatocytes for use in cell transplantation. Liver Transpl. 2010 Feb;16(2):229-­‐37. 12
De Santis L, Cino I, Coticchio G, Fusi FM, Papaleo E, Rabellotti E, Brigante C, Borini A, Ferrari A. Objective evaluation of the viability of cryopreserved oocytes. Reprod Biomed Online. 2007 Sep;15(3):338-­‐45. 13
De Santis L, Cino I, Coticchio G, Fusi FM, Papaleo E, Rabellotti E, Brigante C, Borini A, Ferrari A. Objective evaluation of the viability of cryopreservedoocytes. Reprod Biomed Online. 2007 Sep;15(3):338-­‐45. Temperature as a pre-analytical variable 0.05, March 2014
Eric Hall and Jason Chaffey, Bluechiip Ltd, © Copyright 2014.
Schoonenboom N, Mulder C, Vanderstichele H, Van Elk EJ, Kok A, Van Kamp G, Scheltens P,
Blankenstein M. Clinical Chemistry. 2005 51(1):89-195
Hannisdal R, Giselfoss R, Grimsrud T, Hustad S, Morkrid L, Ueland P. Analytical recovery of folate
degredation products in human serum stored at -25°C for up to 29 years. J Nutr. 2010
Mar;140(3):522-6.
Chaigneau C, Cabioch T, Beaumont K, Betsou F. Serum biobank certification and the establishment
of quality controls for biological fluids: examples of serum biomarker stability after temperature
variation. Clin Chem Lab Med 2007;45(10)1390-1395
Lengelle J, Panopoulos E, Betsou F. Soluble CD40 ligand as a biomarker for storage-related
preanalytic variations of human serum. Cytokine 2008;44:275-282
Schrohl A, Wurtz S, Kohn E, Banks R, Nielsen H, Sweep F, Brunner N. Banking of biological fluids
for studies of disease-associated protein biomarkers. Mol Cell Proteomics. 2008 Oct;7(10):2061-6
Germann A, Oh Y-J, Schmidt T, Schon U, Zimmerman H. Temperature fluctuations during deep
temperature cryopreservation reduce PBMC recovery,viability and T-cell function. Cryobiology.
2013 Oct;67(2):193-200
Beyer C, Distler J, Allanore Y, Aringer M, Avouac J, Czirjak L, cutolo M, Damjanov N, Del Galdo F,
Fligelstone K, Guiducci S, Kowal-Bielecka O, van Laar J, Martucci-Cerinic M, Muller-Ladner U,
Riemekasten G, Tarner I, Tyndall A, Kennedy A, Valentini G, Vettori S, Walker U, Denton C, Distler
O. EUSTAR biobanking: recommendations for the collection, storage and distribution of
biospecimens in scleroderma research. Ann Rheum Dis. 2011;70:1178-1182.
NCI Best Practices for Biospecimen Resources. Section B.2.1.1 Pre analytical Variables.
http://biospecimens.cancer.gov/bestpractices/2011-NCIBestPractices.pdf
Hustad S, Eussen S, Midttun O, Ulvik A, van de Kant P, Morkrid L, Gislefoss R, Ueland P. Kinetic
modeling of storage effects on biomarkers related to B vitamin status and one-carbon metabolism.
Clinical Chemistry 2012 58(2):402-410.
Vaught, J. blood: collection, shipment, processing, and storage. AACR 97th annual Meeting April
2006.
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 Aug 9;1(1-2):47-60.
Dunn W, Broadhurst D, Ellis D, Brown M, Halsall A, O'Hagan S, Spasic I, Tseng A, Kell D. A GC-TOFMS study of the stability of serum and urine metabolomes during the UK Biobank sample collection
and preparation protocols. Int J Epidemiol. 2008 Apr;37 Suppl 1:i23-30.
Temperature as a pre-analytical variable 0.05, March 2014
Eric Hall and Jason Chaffey, Bluechiip Ltd, © Copyright 2014.
Hayes R, Smith C, Huang W, Read Y, Kopp W. Whole blood cryopreservation in epidemiological
studies. Cancer Epidemiol Biomarkers Prev. 2002 Nov;11(11):1496-8.
Ocke M, Schrijver J, Obermann-DeBoer G, Bloemberg B, Haenen G, Kromhout D. Stability of blood
(pro)vitamins during four years of storage at -20°C: consequences for epidemiologic research. J
Clini Epidemiol. 1995 Aug;48(8):1077-85
Lee DH, Kim J, Jeon S, Park B, Han B. proteomic analysis of the effect of storage temperature on
human serum. Ann Clin Lab Sci. 2010 Winter;40(1):61-70.
Fura A, Harper T, Zhang H, Fung L, Shyu W. Shift in pH of biological fluids during storage and
processing: effect on bioanalysis. J Pharm Biomed Anal. 2003 Jul 14;32(3):513-22
Ahmad S, Sundaramoorthy E, Aora R, Sen S, Karthikeyan G, Sengupta S. Progressive degradation
of serum samples limits proteomic biomarker discovery. Anal Biochem. 2009 Nov 15;394(2):23742
Gislefoss R, Grimsrud T, Morkrid L. Stability of selected serum proteins after long-term storage in
the Janus Serum Bank. Clin Chem Lab Med. 2009;47(5):596-603
Gislefoss R, Grimsrud T, Morkrid L. Long term stability of serum components in the Janus Serum
Bank. Scand J Clin Lab Invest. 2008;68(5):402-9.
Further reading
Balasubramanian R, Muller L, Kugler K, Hackl W, Pleyer L, Dehmer M, Graber A. The impact of
storage effects in biobanks on biomarker discovery in systems biology studies. Biomarkers. 2010
Dec;15(8):677-83.
Banks, RE. Preanalytical influences in clinical proteomic studies: raising awareness of fundamental
issues in sample banking. Clin Chem. 2008 Jan;54(1):6-7.
Betsou F, Barnes R, Burke T, Coppola D, DeSouza Y, Eliason J, Glazer B, Horsfall D, Kleeberger C,
Lehman S, Prasad A, Skubitz A, Somiari S, Gunter E; International Society for Biological and
Environmental Repositories (ISBER) Working Group on Biospecimen Science. Human biospecimens
research: experimental protocol and quality control tools.
Cancer Epidemiol Biomarkers Prev. 2009 Apr;18(4):1017-25.
Betsou F, Lehman S, Ashton G, Barnes M, Benson E, Coppola D, DeSouza Y, Eliason J, Glazer B,
Guadagni F, Harding K, Horsfall D, Kleeberger C, Nanni U, Prasad A, Shea K, Skubitz A, Somiari S,
Gunter E; International Society for Biological and Environmental Repositories (ISBER) Working
Group on Biospecimen Science. Standard preanalytical coding for biospecimens: defining the
sample PREanalytical code. Cancer Epidemiol Biomarkers Prev. 2010 Apr;19(4):1004-11.
Temperature as a pre-analytical variable 0.05, March 2014
Eric Hall and Jason Chaffey, Bluechiip Ltd, © Copyright 2014.
Brisson AR, Matsui D, Rieder MJ, Fraser DD. Translational research in pediatrics: tissue sampling
and biobanking. Pediatrics. 2012 Jan;129(1):153-62.
Carraro P, Plebani M. Errors in a stat laboratory: types and frequencies 10 years later. Clin
Chem. 2007 Jul;53(7):1338-42.
Clement C. Biological sample storage and management. Lab Manager, October 7, 2009.
Elliott P, Peakman T. The UK biobank sample handling and storage protocol for the collection,
processing and archiving of human blood and urine. Int J Epidemiol. 2008 Apr;37(2):234-44.
Ferguson R, Hochstrasser D, Banks RE. Impact of preanalytical variables on the analysis of
biological fluids in proteomic studies. Proteomics Clin Appl. 2007 Aug;1(8):739-46.
Holland T, Pfleger L, Berger E, Ho A, Bastaki M. Molecular epidemiology biomarkers-sample
collection and processing considerations. Toxicol Appl Pharmacol. 2005 Aug 7;206(2):261-8.
Moore H, Kelly A, Jewell S, McShange L, Clark D, Greenspan R, Hainaut P, Hayes D, Kim P,
Mansfield E, Potopova O, Riegman P, Rubinstein Y, Seijo E, Somiari S, Watson P, Weier HU,
Vaught J. Biospecimen reporting for improved study quality. Biopreservation and biobanking.
2011;9(1)57-70.
Temperature as a pre-analytical variable 0.05, March 2014
Eric Hall and Jason Chaffey, Bluechiip Ltd, © Copyright 2014.