Obtaining High-Quality Blood Specimens for Downstream Applications: A Review of Current Knowledge and Best Practices

Abstract

Blood is a biological fluid that contains multiple blood fraction and cellular components. High-quality blood specimens are essential prerequisites for various downstream applications such as molecular epidemiology studies, genomics, and proteomics studies. Currently, protocols and research publications concerning the collection, handling, preservation, and stability of blood or blood fractions are constantly emerging. Moreover, standardized guidelines are a requirement for biorepositories to tightly control preanalytical variables originating from these procedures and obtain high-quality blood specimen for downstream analyses. In this review article, we summarize the best practices and fit-for-purpose protocols regarding blood collection, processing, storage, and stability. In addition, we present some typical quality biomarkers, which could be used to evaluate the integrity of blood specimens.

Introduction

Blood is a biological fluid that is a valuable resource that contains not only multiple blood fractions and cellular components but also nucleic acids, proteins, and metabolites, which play a crucial role in current omics study, clinical, and translation reseach.¹ Moreover, blood is relatively accessible and less invasive to obtain in terms of clinical samples, which is particularly true when dried blood spots (DBS) technology is introduced.² DBS technology further minimizes the possible discomfort caused by venipuncture. Therefore, these unique advantages make blood one of the most favored biological specimens archived in biobanks.³

With the gradual improvement of biobanks and biospecimen science, there will be an increased demand for high-quality biological specimens.⁴ Moreover, the availability of high-quality biological specimens is of major importance when conducting downstream analyses. However, obtaining high-quality blood samples is becoming increasingly problematic. This has been mainly due to the complex mixture of blood fractions and blood components. Secondly, physical environment and underlying physiological factors may also have a significant influence on the quality of blood samples.⁵ In addition, various preanalytical variables, including blood collection methods, processing techniques, storage time, and freeze/thaw cycles can all have a potentially adverse effect on the final quality analyses of blood samples.⁶ In practice, unnoticed differences in operation procedures can lead to quite distinct results and poor reproducibility. As a result, minimizing of preanalytical variables has created a need for standardized operation guidelines and best practices for blood samples.⁷ Furthermore, in the early stage, it is unlikely for biorepositories to foresee future analytical requirements. As new biospecimens are archived in biorepositories daily, biospecimens are often directly cryogenically frozen and stored without proper processing. The condition directly affects the quality of the biospecimens, and the downstream analyses can fail as a consequence.¹

Therefore, fit-for-purpose operating procedures and best practices for collection, processing, storage, and stability of blood-derived specimens should be established.⁸ On this basis, preanalytical variables and potential bias could also be moderately controlled by research groups through validated methods and standardized operations. However, due to the limited ability of the approach to control preanalytical variables directly, an approach to accurately assess the quality of biospecimens should be performed. In this case, identification of evidence-based quality biomarkers or quality indicators is critical.⁹ For blood fraction specimens, several representative quality biomarkers have been reported. These quality biomarkers could indicate blood specimen quality and preanalytical conditions directly or indirectly.

In this review, we will provide an overview of current knowledge and best practices for collection, processing, storage, and stability of blood-derived specimens. More importantly, the protocols and best practices for blood-derived specimens are applicable to downstream analyses. Meanwhile, potential preanalytical variables are also discussed in the review. Furthermore, we also highlight typical quality biomarkers that have originated from different blood matrices or different preanalytical conditions.

Blood Collection Procedure

Safe blood drawing and standardized blood collection are important in producing high-quality blood specimens. A well-established guideline concerning blood drawing has been launched by the World Health Organization, and the guideline should be highly recommended and properly followed in the entire collection process.¹⁰ Before blood sampling, study participants should be well instructed about the additional considerations such as fasting and avoidance of medications. These factors are determined by the specific aim of the research. Moreover, informed consent should be obtained from participants from an ethical perspective.

During blood collection, unexpected release of cellular components into extracellular fluid as a result of hemolysis poses a big threat to downstream analyses of serum or plasma.¹¹ Hemolysis may introduce additional interference factors in quantitative assays and cause significant fluctuation in metabolomic or proteomic profiles. Hemolysis is determined by the free hemoglobin concentrations in blood fraction samples. Guder defined the upper reference limit of free hemoglobin concentration for blood samples.¹² Generally, hemolysis could be observed visually when a pink to red tinge emerges in a blood sample. However, when hemolysis is minor and not visible, researchers could measure the concentration of free hemoglobin to assure accuracy.¹³ Based on the previous research, hemolyzed samples may not be suitable for metabolomics and proteomics analyses.^14–16 To reduce the incidence of hemolysis, straight needles, partial vacuum tubes, and the correct gauge of hypodermic needle are strongly recommended to use for venipuncture.¹⁷

Frequently, the dilemma facing many biospecimen repositories is the choice between plasma and serum for future analyses. Whole blood is a complex mixture of various blood fractions. During sampling, whole blood is typically mixed with an anticoagulant in a specified collection tube before further processing. Then whole blood is processed to blood fractions for the expected laboratory uses. After consuming inherent clotting factors, a clot forms and serum is acquired by centrifugation. Plasma is obtained from plasma collection tubes containing different anticoagulants, so clotting factors are retained in plasma.⁸ After centrifugation of anticoagulated blood fraction, clear and straw-colored aqueous plasma is separated from a buffy coat layer and a red fluid layer containing erythrocytes. The buffy coat layer is an important source of mononuclear lymphocytes that can be cryopreserved to maintain viability. The availability of live cells consistently provides higher purity nucleic acids which are applicable to a series of cell-based assays. However, in this review, we place less emphasis on the establishment of blood cell lines. In this study, we mainly focus on the other blood-derived samples and summarize the basic features of the blood fractions (Table 1).

Table 1.

Characteristics and Applications of Blood Fraction Sample

Blood fraction	Collection	Preferred downstream applications	Advantages	Limitations
Whole blood	Using EDTA-containing tubes¹⁸	Suitable for hematology. A source of cellular DNA and RNA for genomics and transcriptomics.^6,19	Minimal processing;	Limited downstream analyses.
Whole blood	Using EDTA-containing tubes¹⁸		Economical collection	Elevated cytokine levels resulted from additive effects.²⁰
Plasma	Using anticoagulant-containing tubes	Proteomics (Detection of a broad-spectrum proteins and peptides), metabolomics, biomarker discovery, a source of miRNA and ccfDNA^2,21,22	Quick processing;	Matrix effects produced by different anticoagulants.¹³
Plasma	Using anticoagulant-containing tubes		High reproducibility²³	Potential hemolytic effect.²⁴
Buffy coat	Using anticoagulant-containing tubes	Main source of lymphocytes and monocytes for establishment of cell lines.^25,26 Main source of cellular DNA and RNA for omics analyses^27,28	Availability of live cells	Requirement of proper handling and processing.²⁹
Serum	Collect coagulated whole blood using plastic or glass blood tubes with or without clotting activator.³⁰	Proteomics and metabolomics research, a source of miRNA^6,22,31	Higher sensitivity for biomarker-based studies.²³	Clotting time would introduce variation and influence future analysis.^32,33
Red blood cell clot	Isolated from coagulated whole blood.³⁴	A source of DNA for genotyping and some other DNA related studies³⁵	Easy processing;	Large DNA yield variation; costly DNA isolation²⁹
Red blood cell clot	Isolated from coagulated whole blood.³⁴		No need for additives.²⁹	Large DNA yield variation; costly DNA isolation²⁹

Although both serum and plasma are common candidates for metabolomics and proteomics, the question of which one performs better in practice still remains unresolved. Large differences exist between the metabolite profiles of serum and plasma. Objectively, plasma offers a distinct advantage over serum for elimination of long clotting time (about 30–60 minutes). Teahan et al. demonstrated that varied clotting times altered the metabolite composition of serum.³² On the contrary, different plasma collection tubes contain different anticoagulants, which make a difference in the metabolite and protein composition of plasma. Rai et al. processed whole blood with several anticoagulants and compared the proteomics profiles of the final plasma samples. The final results revealed that different anticoagulants affected downstream proteomics analysis differently.³⁶ Yu et al. concluded that plasma demonstrated better reproducibility than serum, primarily due to a consistent operation procedure.³⁷ However, this study also revealed that concentrations of metabolites in serum were generally higher than in plasma.³⁷ Therefore, in terms of biomarker detection assays, serum samples are less vulnerable to background noise and show better sensitivity than plasma. Both plasma and serum samples are suitable for proteomics and metabolomics research, and plasma performs better for broad detection of proteins and peptides.

Considerations for Blood Fraction Processing

ISBER released a third edition of best practices guidance for repositories in April 2012. The best practices collected the most effective practices for the management of biological material and biorepositories.³⁸ The guidance recommended that blood samples should be processed within 4 to 24 hours of blood drawing, while processing time variability was correlated to the endpoints of subsequent analyses.³⁹ Depending on the intended downstream analyses, whole blood is further processed into various blood fractions. The availability of commercial collection tubes with built-in functions ensures lot-to-lot consistency and provides convenience for isolation of targeted samples. For instance, Serum Separator Tubes or Cell Preparation Tubes (BD Biosciences) are designed for convenient isolation of serum or mononuclear cells. However, preanalytical variables such as additives in collection tubes, time lag before processing, and real-time temperature could potentially interfere with downstream analyses.

Serum Processing Considerations

The major risks in terms of preanalytical variables involved in serum processing procedures are clotting time and room temperature. Clotting time is normally controlled in a range of 30–60 minutes; a slightly longer time is acceptable when participants are treated with anticoagulants. When clotting time is less than 30 minutes, cellular components or other contaminants remain within serum and further interfere with downstream analyses. However, when clotting time is longer than 60 minutes, clotted cells would lyse and undesired cellular components would be released.⁷ Previous investigations demonstrated that the metabolite composition of serum was influenced by clotting time variation.³²

Commercial collection tubes packaged with a clot activator are now available, which facilitate blood clotting and serum isolation. The emergence of serum-separated tubes could decrease the incidence of hemolysis. Both types of tubes may standardize the processing procedure and minimize clotting time variables. However, Barri et al. found that tube materials could potentially influence metabolomics analysis and lead to inaccurate results.³⁰ We recommend using plastic or glass collection tubes without additives for mass spectroscopy-based analyses.²³ When archiving blood specimens in biorepositories, silicone-coated tubes with clot activators are recommended due to their capacity for rapid separation. These tubes have good reproducibility and reduce the occurrence of hemolysis. It is very important to maintain consistency of the serum collection tubes and operation procedures. Systematic bias can be strictly controlled accordingly.

Lag time and holding temperatures before serum processing will have a negative influence on serum quality. West-Nielsen et al. observed significant protein losses in a mass spectrometric proteomics study of human serum when serum samples were stored at room temperature for more than 4 hours and stored at 4°C for about 24 hours.⁴⁰ Therefore, immediate processing and rapid freezing are prerequisites for high-quality serum samples. To be specific, a 30–45 minute lag time at room temperature before centrifugation is acceptable.⁴¹ To achieve optimal centrifugation at room temperature, centrifugation of serum for about 10 minutes at 2000 g has been validated.⁴¹ The preferred processing temperature for serum samples is 4°C.

Plasma Processing Considerations

Whole blood samples can be processed into plasma when processed with anticoagulants, and then, plasma is isolated from the cellular components. However, different anticoagulants have different performance characteristics, and are fit-for-purpose for different downstream applications. The selection of anticoagulants remains to be standardized, making it a critical preanalytical variable during plasma processing. To standardize the application of these anticoagulants to future analyses, the characteristics of the anticoagulants are detailed in Table 2.

Table 2.

A Summary of Commonly Used Anticoagulants

Anticoagulant	Description	Preferred uses	Notes
Sodium citrate	A calcium chelator. Liquid form in collection tubes²³	Functional clotting factor assay and coagulation tests.^18,42	Ionic strength and pH adversely influence LC-MS analysis.⁴³ Risk of varied concentration ratio between blood and sodium citrate.⁴³
Lithium-heparin	Stimulating thrombin inactivation. Solid form in collection tubes.²³	Metabonomic studies; better quality of DNA and RNA extraction; Higher yield of lymphocytes¹⁸	Recommended for LC-MS-based metabolomics study by the HUSERMET.⁴⁴ Used by the U.K. biobank as a universal additive for a broad purpose.¹⁸ Potentially interact with cellular proteins and interfere with downstream analysis.^45,46
EDTA	A calcium chelator. Inhibit Mg²⁺-dependent enzymes.¹⁴ Solid form in collection tubes.²³	Suitable for a wide range of DNA-based and protein assays¹⁸	Used by EDRN and the U.K. biobank as a universal additive for a broad purpose.^7,18 Potentially influence Mg²⁺ concentration and interfere with cytogenetic analysis.^18,45

Heparin effectively activates antithrombin, thereby promoting the inactivation of thrombin and other clotting factors. EDTA and citrate work as chelators by chelating calcium ions. Kamlage et al. concluded that EDTA could also inhibit Mg²⁺ -dependent enzymes and lead to better metabolomics profile.¹⁴ In addition, the EDRN (Early Detection Research Network, U.S. National Cancer Institute) recommended EDTA as a universal anticoagulant for standard operating procedures. In addition, EDTA is a better candidate for DNA-based assays than cytogenetic analyses. Sodium citrate may be ideal for high-quality DNA and RNA isolation. Moreover, Banks et al. revealed that sodium citrate-treated plasma generated more stable protein profiles when compared to EDTA-treated plasma.⁴⁷ However, it should be noted that the dilution factor of citrate needs to be strictly controlled. When variable volumes of blood are drawn into plasma collecting tubes containing a constant volume of citrate, additional variation will be introduced. Heparin may interact with different cellular proteins and inhibit T cell proliferation and activity of antithrombin. However, Denery et al. and Townsend et al. demonstrated that no significant differences were observed in mass spectrometry-based metabolic profiles when using heparin, EDTA, and citrate.^48,49 In addition, the HUSERMET (Human Serum Metabolome) recommended using heparin for MS-based metabolic analyses. Obviously, anticoagulants should be selected according to downstream analyses and be used for one single study. When using a specific anticoagulant, possible preanalytical variables and all limitations of the anticoagulant should be taken into consideration.

Even though processing of plasma samples is free from the influence of clotting time, immediate processing and rapid freezing are still required. Ayache et al. determined that cytokines were altered dramatically within 2 hours at room temperature.⁵⁰ Moreover, the lag time before centrifugation of anticoagulated blood fractions should be minimized as much as possible. To be specific, a 0–2 hour lag time at room temperature before centrifugation is acceptable for plasma samples.⁴¹ To achieve optimal centrifugation at room temperature, centrifuging plasma for about 20 minutes at 2000 g has been validated.⁴¹ The preferred processing temperature for plasma samples is 4°C.

Nucleic Acids Processing Considerations

Whole blood and blood fractions are the main sources of nucleic acids. Serum and plasma provide limited amounts of DNA at the nanogram level. Lymphocytes from buffy coat is a source of immortal cells, however, the yield of nucleic acids is relatively low. Moreover, the isolation of nucleic acids from lymphocytes is time-consuming and laborious. Blood clots are also a source of DNA, but DNA isolation from clots is difficult and costly. In terms of biospecimen archiving in a biorepository, whole blood is the most productive biospecimen as a source of nucleic acids.

DNA Processing Considerations

PAXgene blood DNA tubes (PreAnalytiX) stabilize DNA immediately after whole blood sampling. Citrate and EDTA are also used for blood DNA stabilization. Moreover, EDTA is commonly used for a variety of DNA-based studies. It should be noted that, however, EDTA may alter Mg²⁺ concentrations and potentially influence downstream analysis. Heparin is not recommended because it could inhibit PCR-based analyses.⁵¹

In addition, Wahlberg et al. proposed a fit-for-purpose and easy-handling blood storage method for biobanks.⁵² They devised a citrate-phosphate-dextrose (hereafter as CPD) buffer for short-term storage of blood for subsequent DNA analysis. The results revealed that CPD-preserved whole blood was stable at 4°C for ∼2 days and stable at −80°C for 4 weeks. CPD buffer is easily accessible and economical. In resource-limited areas, DNA of the whole blood samples could be preserved in CPD buffer temporarily. Nevertheless, longer storage duration remains to be further investigated. PAXgene blood DNA tubes allow for long-term preservation, which preserve DNA for subsequent analyses. Therefore, PAXgene blood DNA tubes would be more suitable for many applications. Prolonged storage time leads to lysis of cellular components, and further causes DNA losses. In this respect, immediate processing is required. Moreover, hemolysis could have a negative effect on future PCR analyses, so whole blood should be processed before being frozen at −80°C.

Although the gold standard of DNA extraction by phenol/chloroform extraction could be used for DNA isolation from blood samples, more efficient and labor-saving methods are commercially available.²⁹ Large amounts of high-quality DNA could be obtained by using commercial kits such as PAXgene Blood DNA Kit (PreAnalytiX) and Maxwell^® 16 LEV Blood DNA Kit (Promega).

RNA Processing Considerations

High-quality RNA is an essential prerequisite for gene expression research. However, inappropriate processing and handling of whole blood samples may have a significant impact on the quality of RNA. Similarly, PAXgene blood RNA tubes (PreAnalytiX) and Tempus blood RNA tubes (Applied Biosystems) are two commercially available systems for immediate stabilization of RNA originating from whole blood.

Previous research indicated that EDTA could be used to stabilize RNA. But currently, that conclusion is in doubt. Malentacchi et al. concluded that the gene expression of blood–mRNA from blood stored in EDTA tubes was altered significantly.⁵³ Rainen et al. and Tanner et al. demonstrated that EDTA was not effective as a RNA stabilizer even for short-time storage.^54,55 In addition, Wahlberg et al. also evaluated the quality of RNA extracted from CPD-stored whole blood.⁵² Their findings demonstrated that CPD buffer was not suitable for storing RNA in whole blood samples.

Therefore, PAXgene blood RNA tubes and Tempus blood RNA tubes represent reliable options to avoid RNA degradation. Whole blood RNA stabilized in PAXgene blood RNA tubes could be stored at room temperature for 3 days and at 4°C for 5 days before further processing. Compared to DNA, RNA is less stable and more vulnerable to inappropriate handling risks. It is imperative to prevent degradation of RNA during processing.⁵⁶ Currently, commercially available kits for RNA isolation, including PAXgene Blood RNA kit (PreAnalytiX) and the Tempus™ 6-Port RNA Isolation Kit (Life Technologies), have been developed for high-quality RNA isolation.

Considerations for DBS Samples

DBS is a method of blood collection in the dry state.⁵⁷ Small volumes of capillary blood are collected onto a pretreated filter card such as Whatman FTA card (Whatman), Perkin Elmer 226 spot saver card (Perkin Elmer), and the like.⁵⁸ Compared with conventional blood sampling by venipuncture, DBS provides several prominent advantages. These advantages include lower cost, easier sampling and handling, easier transportation, and less space requirement.⁵⁹ In addition, absorption and the solid matrix of DBS make blood components less reactive and fragile in ambient conditions when DBS samples are packed in sealed bags with desiccants.⁶⁰ Moreover, a relatively less invasive procedure and less requirement for professional blood collection qualifications reduce the risks of preanalytical variables in blood sampling and handling.

DBS samples may be a cost-effective choice for downstream omics-based studies, including metabolomics, proteomics, genomics, and transcriptomics.^61–63 It should be noted, however, that a relatively small amount of sample material is available from DBS.⁶⁴ Assarsson et al. found that some protein levels increased abnormally when DBS samples were measured.⁶⁵ Therefore, DBS samples make greater demands on assay validation and sensitivity of analytical methods.

Preanalytical variables, including paper specifications, spot heterogeneity, and drying conditions will have potential influences on analytical results. Michopoulos et al. recommended using untreated paper instead of pretreated paper cards in untargeted metabolomics studies because pretreated paper cards might introduce background noise.⁶⁶ In practice, spots are recommended to be punched into 3.2 or 6 mm discs to keep the spots homogenous.² In addition, the drying condition of DBS is independent of ambient humidity and paper substrate type, and a drying time of about 150 minutes at room temperature is enough to maintain steady moisture content. After drying, DBS cards could be stored in sealed envelopes or packed in sealed bags with desiccants.

Optimal Storage Condition

Long-term storage at room temperature can cause rapid degradation of blood specimens. A series of investigations proposed that some biomarkers degraded dramatically when serum samples were stored at −20°C.⁶⁷ Pinto et al. observed that some metabolites components were altered significantly when plasma was stored at −20°C for 1 month.⁶⁸ Overall, most of the blood-derived biospecimens, including whole-blood samples, plasma, serum, DNA, and RNA, are recommended to be cryopreserved in −80°C freezers for safe and long-term preservation.

However, at remote sites or in resource-limited areas, instant and timely preservation in −80°C freezers may not be available. Under these circumstances, consideration for temporary storage of blood-derived specimens is critical. Plasma and serum samples are stable at room temperature for no more than 4 hours and at 4°C for no more than 24 hours.⁴¹ For plasma samples intended for metabolomics study, no significant changes are observed for short-term storage at −20°C within 7 days.⁶⁸ For common clinical chemistry analyses, serum samples are stable at −20°C for 3 months.⁶⁹ DNA extracted from blood or blood fractions can be stored at 4°C for several weeks, at −20°C for months and at −80°C for years.⁷⁰ When whole blood is stabilized in PAXgene blood RNA tubes, RNA is stable at room temperature for 3 days, and at 4°C within 5 days before further handling. Purified RNA could be flash-frozen with liquid nitrogen and then stored at −80°C for long-term storage.⁷¹ DBS samples can be stored at 4°C for no more than 2 years and at −80°C for long-term storage.⁴¹

Generally, serum and plasma samples to be analyzed for metabolite composition and protein profiling are well preserved when stored at −80°C for up to 4 years.^72–74 When specific biomarkers are taken into consideration, storage temperature and duration should be reassessed. Details about the stability of quality biomarkers are discussed in the following section.

Considerations for Freeze-Thaw Cycles

Blood-derived specimens are vulnerable to repetitive freezing and thawing cycles when they are preserved at −80°C for long-term storage, especially when the specimen is under continuous inspection over a period of time. Freeze–thaw cycles can have a significant influence on integrity of blood-derived specimen.

Rai et al. observed that protein degraded significantly in proteomic investigation when serum and plasma samples were subjected to two freeze/thaw cycles.³⁶ In addition, Mitchell et al. suggested that the integrity of the plasma proteome was strongly influenced after two freeze/thaw cycles when plasma samples were used in mass spectrometry-based research.⁷² Charde et al. pointed out that yields of DNA from plasma and serum both declined after a single freeze–thaw cycle. They also revealed that mRNA from serum rapidly degraded after three freeze–thaw cycles.⁷⁰

Glinge et al. observed significant reduction of miRNA levels in plasma and serum after four repetitive freeze–thaw cycles.⁷⁵ Zhao et al. also demonstrated that levels of miRNA were significantly decreased as the number of freeze–thaw cycles increased.⁷⁶ Quite the opposite, Farina et al. showed that the expression level of miRNA in serum was increased after two freeze–thaw cycles.⁷⁷ These results suggest that further investigations should be performed to evaluate the influence of freeze–thaw cycles on miRNA expression levels of blood fractions.

It should be noted that the number of freeze–thaw cycles should be strictly monitored and minimized. Freeze–thaw cycles should be limited to no more than one, and should not exceed an absolute limit of twice to ensure the high quality of blood-derived samples. Furthermore, samples may be aliquoted into small volumes to avoid freeze–thaw cycles. The optimal protocol for sample aliquots should be designed according to the intended analyses, and extra sample aliquots can also be prepared for future reference.

Considerations for Quality Biomarkers in Blood-Derived Samples

Many preanalytical variables may affect all processes before final analyses. Preanalytical variables can influence the integrity of samples, which then can lead to inaccurate results. Controlling of preanalytical variables is extremely challenging and complex. Thus, some researchers have proposed to apply appropriate tests to evaluate the integrity status of biospecimens, further address the issue of controlling preanalytical variables.⁹ Quality biomarkers therefore become a useful tool to predict sample quality and assess possible preanalytical variables. For blood-derived samples, several possible quality biomarkers have been identified in the past decade. In this review, we summarize some typical quality biomarkers with higher applicability grade and accessibility grade, as listed in Table 3.

Table 3.

A Summary of Quality Biomarkers Identified from Diverse Specimen

Specimen origin	Type	Quality biomarker	Comments
K₂EDTA(EDTA) tubes	mRNA	FOSB	Dysregulate after only 2 hours at RT⁷⁸
K₂EDTA(EDTA) tubes	mRNA	LMNA	Dysregulate after 24 hours at RT⁷⁸
K₂EDTA(EDTA) tubes	mRNA	TNRFSF 10C	Degrade after 24 hours at RT⁷⁸
PAXgene blood RNA tubes	mRNA	USP32	RNA degradation after blood was stored in PAXgene tubes at 30°C for 48 hours⁷⁸
Citrated plasma	Protein	Protein S	Begin to decrease after 8 months at −74°C and decrease to lower detection limit after 9 years at −80°C⁷⁹
Citrated plasma	Protein	MMP-9	Degrade completely after 25 month at −80°C⁸⁰
Heparin plasma	Protein	IL-1β; IL-10; IL-15	Degrade completely after 4 years at −80°C⁸¹
Heparin plasma	Protein	IL-15; IL-17; IFN-γ	Degrade completely after four freeze–thaw cycles⁸¹
Serum	Protein	CD40L	Degraded completely after 12 hours at 37°C and after 48 hours at room temperature⁸²
Serum	Protein	VEGF	Degrade significantly after six freeze–thaw cycles and degrade completely after 11 month at −20°C and after 4.5 years at −80°C⁸³
Serum	Protein	MMP-7	Degrade significantly after 20 freeze–thaw cycles.
Serum	miRNA	miR-451	Degrade completely after 30 freeze–thaw cycles⁸⁴
Serum	miRNA	miR-451	miRNA contamination resulted from hemolysis⁸⁵

Discussion

In this review, we summarize the common considerations and best practices in the collection, processing and long-term storage of blood-derived samples. In addition, we also present some typical quality biomarkers, which have been thoroughly studied. These quality biomarkers play an important role in primary assessment of sample quality and can be applied to track preanalytical variables in the future.

Currently, the optimal protocol for the proper collection as well as processing and storage of blood and blood-derived samples remains to be established. More importantly, one single protocol or operating procedure may not be applicable for all blood-derived sample analyses. It's impractical to set up a universal protocol for all biospecimens, and therefore, biorepository researchers should initially determine the expected downstream applications. Then, the best practices for the specified analytes should be applied for downstream analyses. A pilot study should be performed to investigate the stability of samples and effects of preanalytical factors on samples before large-scale testing. Biospecimen science requires identification of preanalytical variables and the controlling of potential bias. Therefore, monitoring of these variables with promising quality biomarkers will help researchers achieve reliable results.

Footnotes

Acknowledgments

The authors thank the colleagues in China National GeneBank for their assistance and review.

Author Disclosure Statement

No conflicting financial interests exist.

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