The Early Detection Research Network (EDRN), a National Cancer Institute (NCI) initiative, was funded for systematic biomarker discovery and validation for early detection of multiple types of cancers. A consortium of investigators around the U.S. and other countries collaborate on epidemiology, sample collection and processing, biomarker discovery and validation in organ-specific cancers. The EDRN is organized into functional units responsible for the overall direction of the initiative. These units have specific tasks and scientific objectives that contribute to the overall aims and successes of the EDRN. A hallmark of the EDRN is the collaboration on all aspects of the group’s mission. To further the mission of the EDRN, prospectively designed biomarker discovery and validation requires utilization of many types of human samples. Because of their frequent use in early cancer detection, establishment of standard operating procedures (SOPs) for serum and plasma were identified as the first priority of the EDRN.
The quality of human sample collection impacts analytical outcomes.1,3,5,6
There is significant discussion taking place in the field of biomarker discovery, particularly on the issues of sources of bias in sample collection and handling,1,3,7,8
but little in the way of specific instructions or SOPs that might provide points for discussion of sources of bias in study design. Here, we outline the EDRN approach to Standard Operating Procedures for collection, handling, and management of human biosamples, specifically serum and plasma.
Defining SOPs in a Multi-Institutional Consortium Environment
Standard operating procedures must provide systematic, detailed, logical instructions. They should provide enough detail to allow someone new to the procedure to understand the steps and perform the procedure consistently. Ideal SOPs are recipes, easy to follow once the right ingredients or supplies are available. They should not be teaching or “laboratory” manuals with extensive discussions and directions on theory, but rather concise step-by-step instructions.
Contributing members of the Standard Operating Procedure Internal Working Group (SOPIWG) and other EDRN members had developed their own procedures based on their individual experiences and expertise; however, there were significant variations between these methods. We acknowledged that there were numerous variables to be considered in optimum specimen management, so our first step as a group was to identify the critical variables to be considered in defining the SOPs that would be useful in our work. Important considerations were that the SOPs were generalizable, feasible and cost-effective for most groups. We addressed those variables that were thought to have the most significant impact on current proteomic platforms used by EDRN researchers. Important considerations were also the potential impact on -omic analyses and the future use of existing sample sets collected under varying protocols in combination with prospectively collected samples under the new SOPs.
General Blood Handling Considerations
The routine use and collection of blood samples for clinical diagnostics has provided general information on optimal methodologies and potential pitfalls. One such consideration is hemolysis of the specimen as it impacts the accuracy of laboratory tests, particularly chemistry tests.9
The release of cellular material due to hemolysis into the serum or plasma may introduce additional confounding factors in downstream analysis of such samples. Hemolysis can be prevented by careful handling techniques. We have provided instructions on optimum needle size, proper handling of the tubes, and pipetting techniques to reduce the incidence of hemolysis. Proper phlebotomy technique is also critical, but that is beyond the scope of our SOPs. We also recommend that if hemolysis (pink to red tinge in sample) is observed, this information should be recorded. It is likely that hemolyzed samples would not be used for proteomics analysis, but destroying them may be unnecessary; it is worth saving any samples, unless there are storage space constraints. Annotation of this and other pertinent information about the samples allows for a thorough discussion of potential factors that influence outcomes.
Serum Handling Considerations
Serum provides the liquid portion of the blood without cells and clotting factors and, therefore, should contain proteins and other molecules that represent the whole body system. The cells and clotting factors must be removed from the blood sample by allowing adequate time for a clot to form. Most manufacturers of collections systems for serum samples recommend 30–60 min at room temperature for a clot to form and longer if the subject was taking any kind of anticoagulant at sample collection.9
The selection of a collection tube was left to an individual’s discretion as long as it is without additives and designated for serum isolation by the manufacturer. This matches standard practice in clinical diagnostics. Serum samples that are allowed to sit less than 30 min are likely to retain cellular elements and other contaminants impacting future analysis. Samples that sit longer than 60 min are likely to experience lysis of cells in the clot, releasing cellular components not usually found in serum samples.2
Several investigators have tested various preanalytical sample handling parameters for proteomic serum samples.2,6,10
Their findings correspond well with decisions the SOPIWG made regarding SOPs for serum including time for clot formation,temperatureconsiderations,maximumnumberoffreeze-thaw-cycles and hemolysis.
Plasma Handling Considerations
Plasma includes cellular material, providing different analytes. The dilemma facing the EDRN and any prospective biosample collection for a repository is that of unknown future analytical requirements. Plasma collection tubes contain different anticoagulants such as EDTA, heparin, or sodium citrate, and each of these additives can impact the protein makeup in the plasma and, therefore, can influence potential uses of these samples in proteomics or genomics analysis.5
To proceed with SOPs, the EDRN chose EDTA as the additive deemed most universally usable for our work. There was concern that heparin could interfere with some types of assays,11
and was therefore not selected. Other tubes were considered including Cell Preparation Tubes (CPTs) (BD Diagnostics, Franklin Lakes, NJ) in order to isolate the buffy coat and plasma (using sodium citrate) in the same tube. We did not recommend the use of these tubes for our SOPS due to their cost and the limited need for isolating and collecting white blood cells from every subject’s sample. Additional challenges with plasma include the need to properly fill the tubes during collection. The additives are calibrated to provide the optimum blood/additive ratio. Therefore, the volume of blood collected in each tube and adequate mixing of the sample into the additive are critical steps.5,10
The HUPO Plasma Proteome Project5
noted that there are too many variables to consider to make a universal statement on the best plasma SOPs for everyone to use, but the consideration of these variables in study design, along with thorough documentation of all steps of handling the samples, can minimize or even mitigate some of these pitfalls.
Temperature is a major variable in specimen management. Everything from the temperature at collection and transport to long-term storage temperature may have major impact on the quality of the samples.4,5,7
As discussed above, the serum samples require ambient temperatures to form clots. Protein stability and enzyme activity are temperature-dependent as well as temperature-sensitive. The use of ice or cold packs for transport and handling steps, and quick and efficient processing of samples can minimize the degradation of proteins.1,4,5
The long-term storage temperature should be at least −80 °C. Rai et al. suggest that liquid nitrogen storage is optimal for protein stability,5
but often that is not as feasible as −80 °C. Several investigators1,4,5
have shown that the number of freeze–thaw cycles a specimen undergoes has a dramatic negative effect on the quality of the specimen. Serum and plasma specimens are of better quality for analysis if smaller volume aliquots are initially prepared rather than larger ones that have to be thawed, handled, and refrozen, perhaps multiple times.1,4
Indeed, the ability to provide ready-made aliquots without additional handling steps facilitates the sharing of samples and provides multiple replicates that were handled in an identical manner.
Potential temperature issues arise in the transportation of samples within and between facilities. Care must be taken to ensure that the serum and plasma samples have sufficient dry ice for the expected duration of the trip, whether it is across the hall or across the country. Samples that have thawed (e.g., due to rerouted flights or left out too long during relabeling) should not be used, so steps must be taken to protect the specimens from delays that can and do happen. To address this potential pitfall, EDRN SOPs require the addition of sufficient dry ice to keep samples frozen for an additional 24 h beyond the expected shipment duration and discuss the need to have backup plans for freezers.
Some analytes can be broken down by exposure to natural or fluorescent light.10
Examples in clinical chemistry include bilirubin and beta-carotene,10
but there are many other compounds that are potentially adversely affected by light exposure. Blood tubes wrapped in aluminum foil during transport and handling, and the use of opaque boxes for storage can minimize the potential damage to photosensitive analytes.
To begin this process, EDRN collaborative groups focusing on different organ systems (Breast & Gynecological Cancers, Colorectal and Other Gastrointestinal Cancers, Lung and Upper Aerodigestive Cancers, and Prostate and Other Urologic Cancers) provided SOPs for their blood samples. One set of SOPs was selected as the starting point. Conference calls were held monthly to discuss decisions to be made on the first set of SOPs for serum and plasma. Similarities and differences between the serum/plasma SOPs were charted. While every step was reviewed and discussed, those variables deemed to be critical generated more significant debate.
Major SOP differences can be summarized as times, temperatures, and types of additives. Times varied for clot formation (range 30 min to 5 h), time allowed at various temperatures until aliquoting and freezing, and time for centrifuging. Temperatures varied (in conjunction with times) from room temperature to 4 to −80 °C at stages from collection, clotting, holding, centrifuging, and long-term storage. Finally, all 3 types of common anticlotting additives (EDTA, heparin and sodium citrate) were used for plasma collection. We consulted with end-users and commercial sources (such as BD.com) to help develop consensus on the critical issues. Specifically, we sought the input of various EDRN units such as Biomarker Reference Laboratories (BRLs), members of each organ site collaborative group, and proteomic and genomic experts in the EDRN. Several drafts were reviewed until consensus through compromise was achieved. A final SOP for plasma processing and a final SOP for serum processing were circulated for approval by all EDRN members. The SOPs are posted on the EDRN Web site (http://edrn.nci.nih.gov/resources/standard-operating-procedures/biological-specimens
). Additional discussions reviewed the implementation process for each group based on their existing and planned studies.
One difference between serum methods was in the processing time and temperature of the blood specimens. As noted above, in order to obtain serum of high quality, blood samples should be allowed time to form a clot at room temperature for 30–60 min.10
The contributed SOPs had a range of time to allow clot formation that was reasonably consistent at 30 min minimum to 60 min maximum (with one at up to 5 h), and the final SOP directs users to allow 30–60 min to clot, then process in a centrifuge or hold at 4 °C for no more
than 4 h. The contributed SOPs had various times allowed for holding at 4 °C until processing (range 0–26 h). The consensus SOP reflects a compromise between the most optimal specimen processing (processing immediately after clot formation) and the reality of collecting and processing samples in clinical settings with busy staff. In cases where samples can be processed immediately, the SOPs allow for that, while setting a maximum acceptable time limit. Data regarding the time and temperature during processing need to be recorded, as well as specific handling steps. These data are critical for the complete analysis of the collected samples.
The most significant differences among the different collaborative groups were noted for plasma collection. Contributing groups used different types of blood collection tubes, different holding temperatures (e.g., room temperature or 4 °C) and different time periods prior to centrifugation (from 2 h up to 26 h after collection). The wide array of differences in processing the samples limits the generalizability of assays within and between organ groups. Our final SOPs settled on EDTA tubes with immediate processing or holding no more than 4 h at 4 °C prior to processing. The decisions on time and temperature variables for plasma were much easier after the serum processing parameters were agreed upon. The decision on which anticoagulant to use was more difficult. Each anticoagulant has advantages and disadvantages depending on the intended uses for the plasma, and there is no universal agreement on the tube of choice. EDTA was selected as it was determined to be the least likely to interfere with the majority of assays in use currently.
Additional variables, albeit seemingly minor details, can impact the success of an assay. The use of glass versus plastic blood collection tubes is one such variable.12
In the interest of personnel safety, suppliers provide plastic blood collection tubes for routine clinical use instead of glass tubes;10
however, it is not known if there is a potential for leaching of plastic ingredients into the specimen during collection that could impact sensitive assays. Stankovic and Parmar12
discuss the potential negative impact on clinical assays due to additional materials such a silica particles or polyvinylpyrrolidone to plastic collection tubes. Clinical laboratories and the corresponding common laboratory tests have established protocols, quality assurance steps, and significant testing by suppliers to minimize the damage from a major change such as from glass to plastic.10,12
Existing samples were most likely to have been collected in glass blood collection tubes; switching to plastic could introduce potential bias. Our SOPs recommend the use of glass tubes due to the potential problems of leaching from plastic or switching from glass.
While the EDRN strongly encourages the use of these SOPs for developing repositories of member groups, it was understood that there would be reasons why this would not be feasible for every group on every study. The implementation process acknowledged this constraint, and groups agreed to implement the necessary changes to their own SOPs as they could. Studies ongoing under particular specimen SOPs did not change methods midstream, as that would clearly cause significant detrimental differences between samples collected on the same protocol. Groups that are implementing new studies are using the EDRN SOPs so that samples collected from different groups going forward can be assayed together. The process of developing these SOPs also encouraged continued communication between those groups collecting samples and those groups using samples within the EDRN. This discussion served to re-emphasize the need for detailed documentation of the specimen handling steps from collection to assay and strict adherence to the sample processing protocols in use. Attention to these two critical details should yield quality, well-annotated samples for biomarker discovery and validation work.