BIOPRESERVATION AND BIOBANKING Volume 9, Number 3, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/bio.2010.0034

State of the Art in Preservation of Fluid Biospecimens Allison Hubel,1,2 Alptekin Aksan,1,2 Amy P.N. Skubitz,1,3 Chris Wendt,4 and Xiao Zhong5

Fluid biospecimens (blood, serum, urine, saliva, cerebrospinal fluid and bronchial lavage fluid) contain not only cells and subcellular components, but also proteins, enzymes, lipids, metabolites, and peptides, which are utilized as biomarkers. Availability of high-quality biospecimens is a requirement for biomarker discovery. The quality of the biospecimens depends upon preanalytical variables (ie, collection and processing techniques, freeze/thaw stability, and storage stability), which account for > 60%–90% of the diagnostic errors. Currently, millions of fluid biospecimens are stored in hundreds of biorepositories across the nation, and tens of thousands of new biospecimens are added to the pool daily. Specimen stabilization is imperative, because fluid biospecimens degrade quickly when kept untreated at room temperature. Achieving a high-quality fluid biospecimen requires understanding the effects of storage processing parameters (eg, freezing and thawing as well as cryo-/ lyoprotectant additives) and storage conditions on biomarkers contained within the biospecimens. In this article, we will discuss the main issues related to the stabilization of specific biofluids by reviewing (a) the current preservation and storage practices applied in biobanks/biorepositories and (b) the sensitivity of certain biomarkers to current storage techniques.

Introduction

F

luid biospecimens (blood, urine, saliva, cerebrospinal fluid (CSF) bronchial lavage fluid, tear fluid, seminal fluid, and ascites fluid) contain not only cells and subcellular components, but also proteins, enzymes, lipids, metabolites, and peptides, which are utilized as biomarkers. At the forefront of personalized medicine, proteomic, peptidomic, lipidomic (in general, ‘‘-omics’’) research is discovering increasingly more biomarkers for risk assessment, diagnosis, management, and treatment of various diseases. Newly developed microfluidic technologies can detect, capture,1 and quantify circulating tumor cells2 and conduct single-cell molecular analysis.3,4 Availability of high-quality biospecimens is a requirement for biomarker discovery and for determining the specificity and sensitivity of the discovered biomarkers. The quality of the biospecimens depends upon preanalytical variables (ie, collection and processing techniques, freeze/thaw stability, and storage stability), which account for > 60%–90% of the diagnostic errors.5–7 Hypothetically speaking, a freeze/thaw stable fluid biospecimen is one that is not affected by the thermal, mechanical, and chemical stresses induced during freezing and thawing. For these biospecimens, post thaw cell viability and function would be high and the macromolecules in the solution would regain their native states in terms of structure

and function. Aggregation and precipitation of proteins and other macromolecules would not be observed in the thawed biospecimen. Proper stabilization of a fluid biospecimen would ensure that potential issues associated with cryo-/lyoprotectant toxicity, ice damage, repeated freeze/thaw damage, osmotic damage, and recrystallization damage are carefully managed and minimized and/or accurately predicted. On the other hand, storage stability implies that the biochemical degradation of the biospecimen is halted or minimized. Currently, millions of fluid biospecimens are stored in hundreds of biorepositories across the nation (in freezerfarms), and tens of thousands of new biospecimens are added to the pool daily (*500 million biospecimens are stored in biobanks as of 2010). Most people assume that these cryogenically stored biospecimens are stable with biomarker integrity retained. However, in most biorepositories, fluid biospecimens are stored by freezing without following any preservation protocol. In most cases, samples are directly placed into - 20C to - 80C freezers, in the absence of any cryoprotectant, where they experience very slow cooling ( - 1C–2C/min). These conditions often destroy the cells and also inflict serious damage on macromolecules,8–16 altering their characteristics (eg, structure and activity), often irreversibly.8,17–24 This directly affects the quality of the stored biospecimens, thus altering the biomarkers in the stored biospecimens. Proteomic studies can be biased by storage conditions25 and preanalytical variables affect the

1 Biopreservation Core Resource, University of Minnesota, Minneapolis, Minnesota. Departments of 2Mechanical Engineering, 3Laboratory Medicine and Pathology, 4Medicine, and 5Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota.

237

238 results, even overwriting the real biological variations.5–7 The low quality of the existing biospecimens in biobanks has been identified as one of the major issues inhibiting scientific progress.26 Specimen stabilization is imperative, because fluid biospecimens degrade quickly when kept untreated at room temperature. Degradation rates and biomarker susceptibility differ considerably and those susceptible cannot be easily predicted based on whether the biomarker is a small molecule, a metabolite, or a large macromolecule or whether it serves a specific function or belongs to a certain family. For example, urine hydrogen peroxide levels change by 3–4-fold within 24 h at room temperature,27 whereas the urinary levels of a similar oxidative stress marker, 8-hydroxy-2’-deoxyguanosine, are perfectly stable under identical conditions.28 The success of biomarker research depends not only upon the availability of the tools (eg, proteomic, peptidomic, lipidomic, and metabolomic technologies) to extract information from biospecimens, but also upon the availability of ‘‘highquality’’ biospecimens.29 Achieving a high-quality fluid biospecimen requires understanding the effects of storage processing parameters (eg, freezing and thawing as well as cryo-/lyoprotectant additives) and storage conditions on biomarkers contained within the biospecimens. Establishing standard protocols for sample collection, handling, and storage is the key to having high-quality biospecimens that yield reproducible and reliable results. Without establishing the manner by which storage conditions influence candidate biomarker stability, meaningful methods of biomarker detection cannot be developed. Most of the proteins present in fluid biospecimens are affected by freezing and storage at cryogenic temperatures, with some of the proteins being more extensively affected than others.8,12,17–19,21–24 The UK Biobank has acknowledged that different serum biomarkers (eg, TNF, IFN-a, IFN-g, IL-la, IL-Ib, and IL-6) require different storage conditions for stabilization.30 Some of the most promising cancer proteomic biomarkers are very susceptible to freeze/thaw and frozenstate storage.31,32 For example, in sera from cancer patients, the levels of albumin, fibrinogen, and C3a significantly decrease in correlation with the length of time spent in storage.33 At one time, C3a was considered as a potential biomarker for breast cancer,34 until it was discovered to be too sensitive to storage conditions.33 Similarly, freezing lactate dehydrogenase (LDH)35 under any condition is detrimental. LDH is a clinical biomarker for patients with sickle cell disease36 and is also being evaluated as a testicular cancer biomarker. In addition, matrix metalloproteinase-9 (MMP-9) starts to degrade at - 80C, dropping by 65% in activity within 2 years of storage.37 More recent studies that utilize highly sensitive techniques such as LC-MALDI-TOF and MALDI-FT-ICR mass spectrometry (MS) have reported significant effects of repeated freeze/thaw on the proteome.38,39 Other proteins that are very susceptible to freeze/thaw include the MMP family (MMP-1, MMP-7, MMP-9, MMP-13)37 and a related family, ADAMs (a disintegrin and metalloprotease), which are considered to be diagnostic and prognostic biomarkers in all major cancers including breast, pancreas, lung, bladder, colorectal, ovarian, prostate, and brain40; tissue inhibitors of metalloproteinases41; polymeric proteins such as transthyretin (that forms fibrils leading to amyloidosis)38 as well as glycoproteins22; and even small molecules such as folate,21 ddimer,42 and thyroid hormones.24

HUBEL ET AL. Most of the studies of biomarker storage stability conducted to date have been confined to MS analysis, which is adequate for the measurement of the relative amounts of peptides/proteins or their absence/presence. However, MS does not provide information on changes in the secondary and tertiary structure, state of denaturation, aggregation, or functional activity of proteinaceous biomarkers.38,43 Therefore, MS may underestimate the changes in biomarkers that could be measured using alternative methods such as enzyme linked-immunosorbent assay (ELISA). In the remainder of this article, we will discuss the main issues related to the stabilization of specific biofluids by reviewing (a) the current preservation and storage practices applied in biobanks/biorepositories and (b) the sensitivity of certain biomarkers to current storage techniques.

Storage Stability of Plasma and Serum Plasma and sera are easy to collect and abundant (constituting *7% of the total body water).44 More importantly, this fluid bathes all tissues and organs, picking up macromolecules and solutes from all major organ systems, providing a rich source for biomarkers. One of the main challenges in studying plasma/serum samples is the large dynamic concentration range of the macromolecules it contains. To date, over 10,000 proteins have been identified in plasma/serum, with concentrations spanning 10 orders of magnitude.45 Many of the studies examining the stability of plasma and serum proteins have used MS and examined trends in large numbers of proteins (vs. studying the stability of specific biomarkers of interest). Plasma and serum protein levels obtained from whole blood can be influenced by relatively short periods of holding time (*2 h) prior to processing. Ayache et al. observed that levels of 37 different factors (principally cytokines) changed significantly within 2 h after venipuncture.46 Banks et al.47 determined that significant changes to low molecular weight serum protein profiles occurred between 30 and 60 min after venipuncture and then remained constant up to 4 h postcollection. Ostroff et al.48 determined that the majority of the 498 proteins examined were stable if the sample was centrifuged within 2 h of venipuncture and frozen within 2 h after centrifugation. Holding temperatures also influence protein stability. Significant protein losses were observed when samples were kept at room temperature for > 4 h or at 24 h when stored at 4C.49 Studies have demonstrated the differential response of biomarkers to the temperature of storage. For example, Pieragostino et al. utilized MALDI-TOF MS spectra analysis to show that specimens stored at - 20C demonstrated ongoing biochemical processes resulting in protein modifications such as oxidation, amino acid truncation, and carbonylation, whereas none of these effects was observed during storage at - 80C.50 Proteins found to be specifically susceptible to modification during storage at high temperatures include C3/C4, a1B glycoprotein, a2-macroglobulin, apolipoprotein, and hemopexin.22 Three breast cancer biomarkers (C3a anaphylatoxin, albumin, and fibrinogen) were found to be significantly degraded when stored at - 30C (C3a, months; albumin, 1.4 years; fibrinogen, 6 years),51 whereas free and total prostate-specific antigen in the sera of 160 prostate cancer patients were shown to be stable during frozen-state storage at - 70C for 5 years.52

STATE OF THE ART IN PRESERVATION OF FLUID BIOSPECIMENS Endothelial microparticles obtained from platelet-poor plasma were affected by cryostorage at - 80C for 1 week.53 Coagulation proteins exhibit significant degradation when stored for > 2 years at - 74C.54 In contrast, West-Nielsen et al.49 reported no effect on protein stability during storage at - 20C or - 80C. To minimize protein degradation, a protease inhibitor cocktail may be added to the plasma samples. This can help protect the stability of the proteins during storage at both - 20C and - 70C.55 Moreover, the use of stabilizing agents such as glycerol combined with storage in liquid nitrogen is also recommended for the stability of serum samples.56 Repeated freeze–thaw cycles can also affect protein stability. Rai56 and Mitchell et al.57 observed significant degradation in protein stability with 2 freeze/thaw cycles.

Storage Stability of Urine Urine is a biospecimen that can be collected noninvasively. The large array of proteins present in urine not only reflect the physiology of the kidneys and the urogenital tract but also the blood (plasma proteins contribute *30% of the proteins found in the urine).58 Several biomarkers (such as NMP22, Calreticulin, Clusterin, CystatinB, Proepithelin, UHRF1, a-1B-Glycoprotein, PCA3, and Cathepsin D) for urinary tract carcinoma, prostate cancer, and bladder cancer have been successfully identified in urine.59–62 An interesting phenomenon observed in frozen/thawed urine is the formation of a precipitant.63 The precipitates, which are usually discarded before analysis, are determined to be calcium oxalate dihydrate and amorphous calcium crystals. However, these precipitates have been shown to deplete not only the calcium ions but also urinary proteins during storage at - 20C in as little as 12 h of storage.63 As the normal urinary calcium levels are 1 order of magnitude higher than those in the sera, the effect of this ion on proteins during freezing is amplified and thus becomes easily detectable in the urine.64 Creatinine is a clinically relevant measure of renal function and its stability in storage is important. Multiple reports have found that creatinine is stable for up to 2 years when stored in the frozen state at - 20C or - 80C.65,66 However, it is stable for only 30 days at 4C and 2 days at 55C.67 Albumin is another important urinary biomarker for renal and cardiovascular diseases68 and its increase in urinary secretion mirrors disease progression. However, as detailed by Innanen et al.,69 freeze/thaw and storage stability of albumin is controversial with the reports supporting mainly 2 opposite conclusions: freeze/thaw does not have an effect on albumin content70,71 vs. freeze/thaw causes a significant decrease in albumin content.72,73 Moreover, Brinkman et al.74 studied the stability of urinary albumin up to 24 months using immunonephelometry. They found that samples could be stored at - 20C for 5 months without significant change in the average albumin concentration; however, extended storage at this temperature resulted in decreased levels.75 Similarly, Collins et al.76 reported that urinary albumin concentration remained stable for up to 6 months at - 20C. Parekh et al.66 examined urine samples stored for 8 months at - 70C. Using an immunoturbidimetric method, they found minimal decline in urinary albumin concentration for up to 2.5 years, with a decrease of *0.25% every 30 days over the entire storage period. These contradictory reports on albumin stability in urine are partially explained by Innanen et al.,69 in which the

239

decrease in albumin content is minimized when samples are well mixed after freeze/thaw. These studies demonstrate that freezing-induced albumin aggregation (which is reversible by extensive mixing) is to a degree responsible for the observed differences. Other factors that vary between samples and impact protein stability include relative protein concentrations, pH, and ionic strength.77 It has not yet been determined whether the aggregated albumin presents a ‘‘sink’’ for the low abundance proteins in the solution, irreversibly decreasing their concentrations. Immunoglobulin G (IgG) in urine is found to be very sensitive to storage conditions, showing marked decrease in concentration at - 20C, with relative stability at - 70C.77 This behavior is also mirrored by a1-microglobulin and transferrin.77 Many proteins are vulnerable to various storage techniques; urinary cystatin C, a biomarker for glomerular filtration rate, has been shown to be stable using multiple preservation techniques.78 Herget-Rosenthal et al.79 examined the stability of cystatin C under different storage conditions using a particle-enhanced nephelometric immunoassay. They found that cystatin C remained stable for 7 days at both 4C and - 20C and for 48 h at 20C. Repeated rounds of freezing and thawing for up to 3 cycles did not cause significant protein destabilization. Herget-Rosenthal et al.79 also studied the influence of urine pH on protein stability, as urine pH can be highly variable. Cystatin C was shown to be stable at pH > 5, but it lost its stability at lower pH. Similarly, Kidney Injury Protein 1 was stable when stored at pH 6.7–8; however, its levels decreased when stored outside this range.80 Recently, neutrophil gelatinase-associated lipocalin has emerged as a biomarker for acute kidney injury.81 Grenier et al.82 used a chemiluminescent assay to study the effect of preanalytical variables on protein stability. Storage at 4C for 7 days only brought a < 2% change in protein concentration. It was also reported that long-term freezing at - 75C is better than storage at - 20C, resulting in smaller sample-tosample variation. Additional studies have examined the influence of storage temperature on disease-specific biomarkers using specific populations. Schultz et al.65 investigated the effects of storage temperature on urine proteins from 262 children with type I diabetes using an enzyme-linked assay (ELISA). Urinary disease biomarkers such as albumin, retinol-binding protein, and N-acetyl glucosaminidase were less stable when stored for 6–8 months at - 20C compared with - 70C. Urinary exosomes and exosome-associated proteins have the potential to serve as biomarkers for early diagnosis and treatment of kidney diseases. Zhou et al.83 reported that exosomes in the urine could be recovered after storage if the storage temperature was - 80C and if protease inhibitors were present. They noted that storage at - 20C caused a major loss of exosome-associated proteins (even when compared with urine stored at 4C). Gazzolo et al.84 studied the stability of S100B, an acidic calcium-binding protein used in the clinics to monitor highrisk newborns. Using an immunoluminometric assay, S100B was found to be stable when left for up to 72 h at room temperature. Using high-pressure liquid chromatography analysis, it was shown that the oxidative stress marker, 8-hydroxy-2’-deoxyguanosine, is stable when stored at room temperature for up to 24 h, and for over 2 years when stored at - 80C.28

240 Most studies focused on individual proteins, whereas recent studies have examined the effect of storage on a large number of urinary proteins by MS. Traum et al.85 used SELDI-TOF MS to characterize changes in protein profiles when urine samples were held at 4C prior to freezing. For holding times up to 24 h at 4C, no differences in protein profiles were found. However, when held at room temperature, Papale et al.86 observed (using SELDI-TOF MS) degradation of urine protein profiles. They reported that stability could be increased to 2 h postcollection with the use of protease inhibitor compound (PIC). They also observed that protein profiles were stable for up to 5 freeze/thaw cycles. Fiedler et al.87 used magnetic beads to separate urine peptides from urine and then characterized the peptidome using MALDI-TOF. Differences in the peptide profile were observed between the fresh samples and those that were frozen/thawed once. Additional freeze/thaw cycles (up to 3) did not result in any significant change in the peptide profile. Schaub et al.88 found that urinary proteins were stable for up to 4 freeze/thaw cycles; however, certain proteins were not detectable after the fifth freeze/thaw cycle. Some biomarkers are highly susceptible to storage, whereas some molecules can remain relatively stable in urine over long periods of time. Phthalates and metabolites, such as dimethyl phthalate and mono-methyl phthalate, were shown to be stable for over 20 years when the urine samples were stored at - 20C.89 This shows the differential stability of small molecules and metabolites with respect to large macromolecules in the urine.

Storage Stability of Saliva Proteomic studies of saliva have drawn increasing attention because saliva is easily accessed and noninvasively collected.90 Saliva contains a rich source of proteins and peptides, some of which are plasma proteins. The salivary proteins are not only responsible for oral health, but they can also serve as a useful investigational tool for detection and early diagnosis of other diseases such as cancers91–94 and Sjogren’s syndrome.95 Further, some salivary antibodies have been found to be elevated in patients with infectious diseases such as hepatitis and HIV.96–98 Preanalytical variables such as the methods used for the collection and processing of saliva influence protein stability. Hu et al.93 added PIC to saliva supernatant and stored it at - 80C until further analysis for oral cancer biomarkers. Shpitzer et al.94 performed multiple rounds of centrifugation on saliva samples, then incubated the cell pellets with lysis buffer, and stored them at room temperature. Other studies, including Myers’ research on hepatitis C biomarkers, fail to mention conditions used for sample collection, preparation, or storage.97 The lack of standardized sample handling protocols for saliva samples remains a significant challenge in data analysis and comparisons. Specific salivary antibodies have been used to detect infectious diseases. Gaudette et al.99 studied the stability of IgG and HIV-1 antibody in whole saliva and oral fluid under different storage times and temperatures. Oral fluid was collected on a salt-treated cotton-padded filter and immersed in an antimicrobial solution. After 7 days of storage at room temperature, 93% of IgG in the oral fluid was stable. However, HIV-1 antibody remained stable at - 20C to 37C for up to 21 days in oral fluid.99

HUBEL ET AL. Salivary-reduced glutathione (GSH) and tissue factor (TF) have been used as measures of oxidative stress in patients.100 Emekli-Alturfan et al.101 found that GSH levels were highly variable during storage, with the levels increasing over the first 2 months and then decreasing significantly after 6 months of storage at - 20C. TF activity also decreased significantly over 6 months when stored at - 20C and had a positive correlation with GSH levels. Long-term freezing at - 20C reduced GSH integrity and TF activity, and therefore, storage for < 30 days was proposed as optimum for these 2 markers. Using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Jiang et al.102 evaluated the stability of 2 salivary protein markers, b-actin and cystatin C, by immunoblotting. About 90% of b-actin and 70% of cystatin C remained stable in the presence of a commercial stabilizing agent of unknown composition for up to 6 days at room temperature. However, only 20% of b-actin remained stable under the same conditions in the whole saliva without the stabilizing agent. Some groups have investigated global salivary protein profile stability by varying the preanalytical procedures. Chevalier et al.103 evaluated the effects of storage time, temperature, and the use of PIC on saliva protein stability using 1- and 2-dimensional gel electrophoresis. They reported that salivary protein stability decreased during storage. Schipper et al.104 examined the stability of samples stored at - 20C and - 80C for up to 6 months. Protein degradation was observed for samples stored at - 20C, whereas proteins stored at - 80C were stable (with a couple of exceptions). Additionally, for samples that underwent up to 4 freeze– thaw cycles, they reported no change in the protein profiles as measured using SELDI-TOF MS.

Storage Stability of Cerebrospinal Fluid Cerebrospinal fluid (CSF) is in direct contact with tissues in the brain and spinal cord. CSF proteomic studies are therefore very helpful in the detection and study of disorders in the central nervous system. Schoonenboom et al.105 investigated the effect of storage time and repeated freeze–thaw cycles on amyloid b (I-42) and tau, 2 biomarkers for Alzheimer disease, using ELISA. The tau protein was still stable following 6 freeze–thaw cycles, but the concentration of amyloid b (I-42) decreased by 20% after 3 freeze–thaw cycles, in contradiction to earlier studies.106 Changes in the levels of the 2 proteins were observed at high-temperature storage (4C and 37C), and therefore, the authors recommended - 80C storage. The consensus protocol published by a group of scientists recommended - 80C storage based on the idea that ‘‘liquid nitrogen storage is not feasible’’ for CSF, albeit without offering any data to support the rationale for the idea.107 However, previous studies indicated decreased sample quality during storage at - 20C and - 80C beyond 3 months, especially when PIC was not added.108

Storage Stability of Bronchoalveolar Lavage Fluid A thin layer of epithelium lining fluid (ELF) covers the human airways protecting them from the external environment and helping to maintain normal gas exchange. ELF contains cells and many soluble components of the lung, which have been sampled by a variety of techniques to study the physiology of the respiratory tract. The most commonly

STATE OF THE ART IN PRESERVATION OF FLUID BIOSPECIMENS used sampling procedure is bronchoscopy, with the collection of bronchoalveolar lavage fluid (BALF). Biochemical analysis of BALF has revealed an abundance of proteins in the airways of patients with pulmonary diseases, and therefore, BALF proteomics is being increasingly studied in the hope to identify biomarkers of disease. In addition to the usual preanalytical variation of time in storage, temperature, and processing, there are several unique challenges for the biopreservation of BALF. As the sample is collected via the installation of saline, dilution of ELF proteins and the presence of high salt concentrations are inherent challenges. Therefore, it is imperative to first standardize the processing, handling and storage conditions of the BALF. In addition, many plasma proteins, such as albumin and IgG, are found in abundance in BALF and can interfere with the detection of less-abundant proteins of interest. Mucus is abundant in the respiratory tract and is often removed from the BALF by filtering through a single layer of gauze followed by centrifugation to separate cells and debris from the fluid. This process may result in the loss of many proteins and potential biomarkers. Although many methods to study BALF proteins have been reported to date, no studies have been conducted to examine the effects of different preservation conditions on BALF protein stability. In a recent study, interleukin-8 and neutrophil elastase levels in BALF collected from patients with cystic fibrosis was compared with their initial values after storage at 4C for 7 days and at - 80C for up to 6 years. It was observed that interleukin-8 levels were stable, independent of storage temperature and time. Neutrophil elastase levels were stable for up to 6 months at - 80C but decreased after 7 days at 4C.109

Conclusion Hundreds of biomarkers found in biofluids such as plasma, serum, urine, and saliva are used to monitor health, disease, and response to treatment. Clearly, these fluids contain as-yet to-be identified biomarkers that could be used to inform patient care. The studies described in this review make it apparent that different collection and storage conditions have profound effects on protein stability, bringing artifacts to experimental results and resulting in invalid conclusions. Note that the only ‘‘preservation stability’’ studies conducted on fluid biospecimens focus on the temperature of storage and the duration of storage, and thus, all the current information summarized in this review are therefore limited. In addition, the vast majority of studies are limited to studying relatively few molecules and there is a paucity of studies examining the global effects of storage on proteins, cells, and subcellular particles. The optimal protocols for the collection, processing, and storage of each different type of fluid biospecimen has yet to be determined. However, even with the limited preanalytical data that are currently available, some trends have emerged, which lead us to propose the following advice when one is contemplating the preservation of fluid biospecimens: 1. Biofluid specimens should be stored in the vapor phase of liquid nitrogen or, if liquid nitrogen is not feasible, at a minimum of - 80C. 2. Specimens should experience a constant cooling rate during freezing, using a controlled rate freezer or commercially available containers for smaller samples.

241

3. Temperature fluctuations during storage (due to repeated freezer access) and repeated freeze/thaw should be minimized. 4. Fast thawing methods should be utilized (eg, immersing the sample in a continuously stirred 37C water bath). Thawing rates should be monitored and validated. 5. For biofluid samples that contain high concentrations of proteins that are freeze susceptible (such as albumin or LDH) or high concentrations of specific ions (such as calcium in urine), frozen-state storage should be optimized (potentially by using cryo-/lyoprotectants). 6. If specific biomarkers or family of biomarkers are of interest, their freeze/thaw response, characteristics, and susceptibility should be characterized not only by techniques such as MS, which is based on the charge density of the macromolecules, but also by techniques that determine the secondary and tertiary structural changes (eg, infrared, Raman, and intrinsic fluorescence spectroscopies). 7. More research is required to develop alternative biopreservation techniques that will offer significant advantages over cryopreservation.

Author Disclosure Statement No competing financial interests exist.

References 1. Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 2007;450:1235–1239. 2. Alunni-Fabroni M, Sandri MT. Circulating tumour cells in clinical practice: methods of detection and possible characterization. Methods 2010;50:289–297. 3. Tarnok A, Pierzchalski A, Valet G. Potential of a cytomics topdown strategy for drug discovery. Curr Med Chem 2010;17: 1719–1729. 4. Wang D, Bodovitz S. Single cell analysis: the New Frontier in ’Omics’. Trends Biotechnol 2010;28:281–290. 5. Lippi G, Salvagno GL, Montagnana M, et al. Reliability of the thrombin-generation assay in frozen-thawed platelet-rich plasma. Clin Chem 2006;52:1827–1828. 6. Apweiler R, Aslanidis C, Deufel T, et al. Approaching clinical proteomics: current state and future fields of application in fluid proteomics. Clin Chem Lab Med 2009;47:724–744 (review). 7. Bonini P, Plebani M, Ceriotti F, et al. Errors in laboratory medicine. Clin Chem 2002;48:691–698. 8. Bhatnagar BS, Bogner RH, Pikal MJ. Protein stability during freezing: separation of stresses and mechanisms of protein stabilization. Pharm Dev Technol 2007;12:505–523. 9. Chang L, Shepherd D, Sun J, et al. Mechanism of protein stabilization by sugars during freeze-drying and storage: Native structure preservation, specific interaction, and/or immobilization in a glassy matrix. J Pharm Sci 2005;94:1427–1444. 10. Pikal MJ. Freeze-drying of proteins. In Cleland JL, Langer R, eds. Stability, Formulation and Delivery of Peptides and Proteins. Washington, DC: American Chemical Society; 1994: 120–133. 11. Tang X, Pikal MJ. The effect of stabilizers and denaturants on the cold denaturation temperatures of proteins and implications for freeze-drying. Pharm Res 2005;22:1167–1175. 12. Ragoonanan V, Aksan A. Protein stabilization. Transfus Med Hemother 2007;34:246–252.

242 13. Carpenter JF, Crowe JH. An Infrared spectroscopic study of the ineractions of carbohydrates with dried proteins. Biochemistry 1989;28:3916–3922. 14. Carpenter JF, Crowe JH, Arakawa T. Comparison of soluteinduced protein stabilization in aqueous solution and in the frozen and dried states. J Dairy Sci 1990;73:3627–3636. 15. Crowe JH, Carpenter JF, Crowe LM, et al. Are freezing and dehydration similar stress vectors—a comparison of modes of interaction of stabilizing solutes with biomolecules. Cryobiology 1990;27:219–231. 16. Randolph TW, Carpenter JF. Engineering challenges of protein formulations. AIChE J 2007;53:1902–1907. 17. Strambini GB, Gabellieri E. Proteins in frozen solutions: Evidence of ice-induced partial unfolding. Biophys J 1996;70:971–976. 18. Strambini GB, Gonnelli M. Protein stability in ice. Biophys J 2007;92:2131–2138. 19. Manning MC, Chou DK, Murphy BM, et al. Stability of protein pharmaceuticals: an update. Pharm Res 2010;27:544– 575 (review). 20. Privalov PL, Griko YV, Venyaminov SY, et al. Cold denaturation of myoglobin. J Mol Biol 1986;190:487–498. 21. Hannisdal R, Gislefoss RE, Grimsrud TK, et al. Analytical recovery of folate and its degradation products in human serum stored at-25 degrees C for up to 29 years. J Nutr 2010;140:522–526. 22. Lee DH, Kim JW, Jeon SY, et al. Proteomic analysis of the effect of storage temperature on human serum. Ann Clin Lab Sci 2010;40:61–70. 23. Manning MC, Patel K, Borchardy RT. Stability of protein pharmaceuticals. Pharm Res 1989;6:903–918. 24. Panesar NS, Lit LCW. Stability of serum thyroid hormones following 8–11 years of cold storage. Clin Chem Lab Med 2010;48:409–412. 25. McLerran D, Grizzle W, Feng Z, et al. Analytical validation of serum proteomic profiling for diagnosis of prostate cancer; sources of sample bias. Clin Chem 2007;54:44–52. 26. Silberman S. Libraries of Flesh: the Sorry State of Human Tissue Storage. The Wired. May 24, 2010. 27. Yuen JWM, Benzie IFF. Hydrogen peroxide in urine as a potential biomarker of whole body oxidative stress. Free Radic Res 2003;37:1209–1213. 28. Matsumoto Y, Ogawa Y, Yoshida R, et al. The stability of the oxidative stress marker, urinary 8-hydroxy-2 ’-deoxyguanosine (8-OHdG), when stored at room temperature. J Occup Health 2008;50:366–372. 29. Schrohl AS, Wurtz S, Kohn E, et al. Banking of biological fluids for studies of disease-associated protein biomarkers. Mol Cell Proteomics 2008;7:2061–2066 (review). 30. Elliott P, Peakman TC. The UK Biobank sample handling and storage protocol for the collection, processing and archiving of human blood and urine. Int J Epidemiol 2008;37:234–244. 31. Hsieh S-Y, Chen R-K, Pan Y-H, et al. Systematical evaluation of the effects of sample collection procedures on low-molecular-weight serum/plasma proteome profiling. Proteomics 2006;6:3189–3198. 32. Engewegen JYMN, Alberts M, Knol JC, et al. Influence of variations in sample handling on SELDI-TOF MS serum protein profiles for colorectal cancer. Proteomics Clin Appl 2008; 2008:936–945. 33. Gast MCW, van Gils CH, Wessels LFA, et al. Influence of sample storage duration on serum protein profiles assessed by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS). Clin Chem Lab Med 2009;47:694–705. 34. Li JN, Orlandi R, White CN, et al. Independent validation of candidate breast cancer serum biomarkers identified by mass spectrometry. Clin Chem 2005;51:2229–2235. 35. Greiff D, Kelly RT. Cryotolerance of enzymes: I. Freezing of lactic dehydrogenase. Cryobiology 1966;2:335–341. 36. Kato GJ, McGowan V, Machado RF, et al. Lactate dehydrogenase as a biomarker of hemolysis-associated nitric oxide resistance,

HUBEL ET AL.

37.

38.

39.

40.

41.

42.

43. 44.

45. 46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

priapism, leg ulceration, pulmonary hypertension, and death in patients with sickle cell disease. Blood 2006;107:2279–2285. Rouy D, Ernens I, Jeanty C, et al. Plasma storage at - 80 C does not protect matrix metalloproteinase-9 from degradation. Anal Biochem 2005;338:294–298. Rosenling T, Slim CL, Christin C, et al. The effect of preanalytical factors on stability of the proteome and selected metabolites in cerebrospinal fluid (CSF). J Proteome Res 2009;8:5511–5522. Chaigneau C, Cabioch T, Beaumont K, et al. 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;45:1390–1395 (proceedings paper). Roy R, Yang J, Moses MA. Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer. J Clin Oncol 2009;27:5287–5297. Holten-Andersen MN, Schrohl AS, Brunner N, et al. Evaluation of sample handling in relation to levels of tissue inhibitor of metalloproteinases-1 measured in blood by immunoassay. Int J Biol Markers 2003;18:170–176. Bohm-Weigert M, Wissel T, Muth H, et al. Long- and shortterm in vitro D-dimer stability measured with INNOVANCE D-Dimer. Thromb Haemost 2010;103:461–465. Hanash SM, Pitteri SJ, Faca VM. Mining the plasma proteome for cancer biomarkers. Nature 2008;452:571–579. Guyton A, Hall J. The body fluid compartments: extracllular and intracellular fluids, interstitial fluid and edema, 10th edition. In Guyton A, Hall J, eds. Textbook of Medical Physiology, 10th Ed. Philadelphia: W.B. Saunders; 2000. Anderson NL, Anderson N. The human plasma proteome. Mol Cell Proteomics 2002;1:845–867. Ayache S, Panelli M, Marincola FM, et al. Effects of storage time and exogenous protease inhibitors on plasma protein levels. Am J Clin Pathol 2006;126:174–184. Banks RE, Stanley AJ, Cairns DA, et al. Influences of blood sample processing on low-molecular-weight proteome identified by surface-enhanced laser desorption/ionization mass spectrometry. Clin Chem 2005;51:1637–1649. Ostroff R, Foreman T, Keeney TR, et al. The stability of the circulating human proteome to variations in sample collection and handling procedures measured with an aptamer-based proteomics array. J Proteomics 2010;73:649–666. West-Nielsen M, Hogdall EV, Marchiori E, et al. Sample handling for mass spectrometric proteomic investigations of human sera. Anal Chem 2005;77:5114–5123. Pieragostino D, Petrucci F, Del Boccio P, et al. Pre-analytical factors in clinical proteomics investigations: impact of ex vivo protein modifications for multiple sclerosis biomarker discovery. J Proteomics 2010;73:579–592. Gast M-CW, van Gils CH, Wessels LF, et al. Influence of sample storage duration on serum protein profiles assessed by surfaceenhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS). Clin Chem Lab Med 2009;47: 694–705. Scaramuzzino D, Schulte K, Mack B, et al. Five-year stability study of free and total prostate-specific antigen concentrations in serum specimens collected and stored at - 70C or less. Int J Biol Markers 2007;22:206–213. van Ierssel SH, Van Craenenbroeck EM, Conraads VM, et al. Flow cytometric detection of endothelial microparticles (EMP): effects of centrifugation and storage alter with the phenotype studied. Thromb Res 2010;125:332–339. Woodhams B, Girardot O, Blanco MJ, et al. Stability of coagulation proteins in frozen plasma. Blood Coagul Fibrinolysis 2001;12:229–236. Hulmes JD, Bethea D, Ho K, et al. An investigation of plasma collection, stabilization, and storage procedures for proteomic analysis of clinical samples. Clin Proteomics J 2004;1:17–31.

STATE OF THE ART IN PRESERVATION OF FLUID BIOSPECIMENS 56. Rai AJ, Gelfand CA, Haywood BC, et al. HUPO plasma proteome project specimen collection and handling: towards the standarization of parameters for plasma proteome samples. Proteomics 2005;5:3262–3277. 57. Mitchell BL, Yasui Y, Li CI, et al. Impact of freeze-thaw cycles and storage time on plasma samples used in mass spectrometry based biomarker discovery projects. Cancer Inf 2005;1:98–104. 58. Rembert P, Christine LG, Andrew MM, et al. Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots. Proteomics 2004;4:1159–1174. 59. Soloway MS, Briggman JV, Carpinito GA, et al. Use of a new tumor marker, urinary NMP22, in the detection of occult or rapidly recurring transitional cell carcinoma of the urinary tract following surgical treatment. J Urol 1996;156:363–367. 60. Sanchez-Carbayo M, Urrutia M, de Buitrago JMG, et al. Evaluation of two new urinary tumor markers: bladder tumor fibronectin and cytokeratin 18 for the diagnosis of bladder cancer. Clin Cancer Res 2000;6:3585–3594. 61. Efthimiou I, Ferentinos G, Tsachouridis G, et al. Determination of the association of urine prostate specific antigen levels with anthropometric variables in children aged 5–14 years. Int Braz J Urol 2010;36:202–207; discussion 7–8. 62. Thomas CE, Sexton W, Benson K, et al. Urine collection and processing for protein biomarker discovery and quantification. Cancer Epidemiol Biomarkers Prev 2010;19:953–959 (review). 63. Saetun P, Semangoen T, Thongboonkerd V. Characterizations of urinary sediments precipitated after freezing and their effects on urinary protein and chemical analyses. Am J Physiol Renal Physiol 2009;296:F1346–F1354. 64. Stern JE, Guinjoan SM, Cardinali DP. Correlation between serum and urinary calcium levels and psychopathology in patients with affective disorders. J Neural Transm 1996;103:509–513. 65. Schultz C, Daltont R, Turnert C, et al. Freezing method affects the concentration and variability of urine proteins and the interpretation of data on microalbuminuria. Diabet Med 2000;17:7–14. 66. Parekh RS, Kao WHL, Meoni LA, et al. Reliability of urinary albumin, total protein, and creatinine assays after prolonged storage: the family investigation of nephropathy and diabetes. Clin J Am Soc Nephrol 2007;2:1156–1162. 67. Spierto FW, Hannon WH, Gunter EW, et al. Stability of urine creatinine. Clin Chim Acta 1997;264:227–232. 68. Basi S, Lewis JB. Microalbuminuria as a target to improve cardiovascular and renal outcomes. Am J Kidney Dis 2006; 47:927–946. 69. Innanen VT, Groom BM, de Campos FM. Microalbumin and freezing. Clin Chem 1997;43:1093–1094. 70. Giampietro O, Penno G, Clerico A, et al. How and how long to store urine samples before albumin radioimmunoassay: a practical response. Clin Chem 1993;39:533–536. 71. Torffvit O, Wieslander J. A simplified enzyme-linked immunosorbent assay for urinary albumin. Scandinavian J Clin Lab Investig 1986;46:545–548. 72. Osberg I, Chase H, Garg S, et al. Effects of storage time and temperature on measurement of small concentrations of albumin in urine. Clin Chem 1990;36:1428–1430. 73. Elving L, Bakkeren J, Jansen M, et al. Screening for microalbuminuria in patients with diabetes mellitus: frozen storage of urine samples decreases their albumin content. Clin Chem 1989;35:308–310. 74. Brinkman JW, de Zeeuw D, Duker JJ, et al. Falsely low urinary albumin concentrations after prolonged frozen storage of urine samples. Clin Chem 2005;51:2181–2183. 75. Brinkman JW, de Zeeuw D, Gansevoort RT, et al. Prolonged frozen storage of urine reduces the value of albuminuria for mortality prediction. Clin Chem 2007;53:153–154 (letter). 76. Collins ACG, Sethi M, Macdonald FA, et al. Storagetemperature and differing methods of sample preparation in

77.

78. 79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90. 91.

92. 93.

94. 95.

96.

97.

243

the measurement of urinary albumin. Diabetologia 1993;36: 993–997 (proceedings paper). Klasen IS, Reichert LJM, de Kat Angelino CM, et al. Quantitative determination of low and high molecular weight proteins in human urine: influence of temperature and storage time. Clin Chem 1999;45:430–432. Uchida K, Gotoh A. Measurement of cystatin-C and creatinine in urine. Clin Chim Acta 2002;323:121–128. Herget-Rosenthal S, Feldkamp T, Volbracht L, et al. Measurement of urinary cystatin C by particle-enhanced nephelometric immunoassay: precision, interferences, stability and reference range. Ann Clin Biochem 2004;41:111–118. Pennemans V, De Winter LM, Faes C, et al. Effect of pH on the stability of kidney injury molecule 1 (KIM-1) and on the accuracy of its measurement in human urine. Clin Chim Acta 2010;411:2083–2086. Devarajan P. Neutrophil gelatinase-associated lipocalin-an emerging troponin for kidney injury. Nephrol Dial Transplant 2008;23:3737–3743 (editorial). Grenier FC, Ali S, Syed H, et al. Evaluation of the ARCHITECT urine NGAL assay: assay performance, specimen handling requirements and biological variability. Clin Biochem 2010;43: 615–620. Zhou H, Yuen PST, Pisitkun T, et al. Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery. Kidney Int 2006;69:1471–1476. Gazzolo D, Lituania M, Michetti F. Effects of temperature on pre-analytical stability of S100B protein concentrations in urine of healthy full-term infants. Clin Chim Acta 2004;350:231–232. Traum AZ, Wells MP, Aivado M, et al. SELDI-TOF MS of quadruplicate urine and serum samples to evaluate changes related to storage conditions. Proteomics 2006;6:1676–1680. Papale M, Pedicillo MC, Thatcher BJ, et al. Urine profiling by SELDI-TOF/MS: monitoring of the critical steps in sample collection, handling and analysis. J Chromatogr B 2007;856:205–213. Fiedler GM, Baumann S, Leichtle A, et al. Standarized peptidome profiling of human urine by magnetic bead separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Clin Chem 2007;53:421–428. Schaub S, Wilkins J, Weiler T, et al. Urine protein profiling with surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry. Kidney Int 2004;65:323–332. Baird DD, Saldana TM, Nepomnaschy PA, et al. Within-person variability in urinary phthalate metabolite concentrations: measurements from specimens after long-term frozen storage. J Expo Sci Environ Epidemiol 2010;20:169–175. Aps JKM, Martens LC. Review: The physiology of saliva and transfer of drugs into saliva. Forensic Sci Int 2005;150:119–131. Maring JG, Wachters FM, Maurer M, et al. Gemcitabine and Epirubicin plasma concentration-related excretion in saliva in patients with non-small cell lung cancer. Ther Drug Monit 2010;32:364–368. Brooks MN, Wang JG, Yang L, et al. Salivary protein factors are elevated in breast cancer patients. Mol Med Rep 2008;1:375–378. Hu S, Arellano M, Boontheung P, et al. Salivary proteomics for oral cancer biomarker discovery. Clin Cancer Res 2008;14: 6246–6252. Shpitzer T, Hamzany Y, Bahar G, et al. Salivary analysis of oral cancer biomarkers. Br J Cancer 2009;101:1194–1198. Ryu OH, Atkinson JC, Hoehn GT, et al. Identification of parotid salivary biomarkers in Sjogren’s syndrome by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and two-dimensional difference gel electrophoresis. Rheumatology 2006;45:1077–1086. Pomarico L, de Souza IPR, Castro G, et al. Levels of salivary IgA antibodies to Candida spp. in HIV-infected adult patients: a systematic review. J Dent 2010;38:10–15 (review). Myers T, Allman D, Xu KY, et al. The prevalence and correlates of hepatitis C virus (HCV) infection and HCV-HIV co-infection

244

98.

99.

100.

101.

102.

103.

104.

105.

HUBEL ET AL. in a community sample of gay and bisexual men. Int J Infect Dis 2009;13:730–739. Arrieta JJ, Rodriguez-Inigo E, Ortiz-Movilla N, et al. In Situ Detection of Hepatitis C Virus RNA in Salivary Glands. Am J Pathol 2001;158:259–264. Gaudette D, Griffin K, North L, et al. Stability of clinically significant antibodies in saliva and oral fluid. J Clin Immunoassay 1994;17:171–175. Arana C, Cutando A, Ferrera MJ, et al. Parameters of oxidative stress in saliva from diabetic and parenteral drug addict patients. J Oral Pathol Med 2006;35:554–559. Emekli-Alturfan E, Kasikci E, Alturfan AA, et al. Effect of sample storage on stability of salivary glutathione, lipid peroxidation levels, and tissue factor activity. J Clin Lab Anal 2009; 23:93–98. Jiang J, Park NJ, Hu S, et al. A universal pre-analytic solution for concurrent stabilization of salivary proteins, RNA and DNA at ambient temperature. Archiv Oral Biol 2009;54: 268–273. Chevalier F, Hirtz C, Chay S, et al. Proteomic Studies of saliva: a proposal for a standarized handling of clinical samples. Clin Proteomics 2007;3:13–21. Schipper R, Loof A, de Groot J, et al. SELDI-TOF-MS of saliva: methodology and pre-treatment effects. J Chromatogr B 2007; 847:45–53. Schoonenboom NSM, Mulder C, Vanderstichele H, et al. Effects of processing and storage conditions on amyloid beta (I-42) and

106.

107.

108.

109.

tau concentrations in cerebrospinal fluid: implications for use in clinical practice. Clin Chem 2005;51:189–195. Sjogren M, Vanderstichele H, Agren H, et al. Tau and A{beta}42 in cerebrospinal fluid from healthy adults 21–93 years of age: establishment of reference values. Clin Chem 2001;47:1776–1781. Teunissen CE, Petzold A, Bennett JL, et al. A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking. Neurology 2009;73:1914–1922 (review). Berven FS, Kroksveen AC, Berle M, et al. Pre-analytical influence on the low molecular weight cerebrospinal fluid proteome. Proteomics Clin Appl 2007;1:699–711. Berry LJ, Sheil B, Garratt L, et al. Stability of interleukin 8 and neutrophil elastase in bronchoalveolar lavage fluid following long-term storage. J Cystic Fibrosis 2010;9:346–350.

Address correspondence to: Dr. Allison Hubel Department of Mechanical Engineering University of Minnesota 111 Church St. SE Minneapolis, MN 55455 E-mail: [email protected] Received 23 December, 2010/Accepted 4 February, 2011