After excision of tissue from the patient, it should be placed in chilled Tis-U-Sol®
(Baxter Healthcare, Deerfield, IL) immediately to inhibit degradation, and transferred as rapidly as possible to the pathology department [41
]. The time in Tis-U-Sol®
until immersion into preservative or snap freezing should ideally be kept under 15 minutes, to minimize the alteration of cellular state due to ischemia and because of the sensitivity to degradation, especially of RNA. A realistic time from excision to preservative immersion could be up to 15 minutes for RNA or 30 minutes if only DNA is to be studied, but any delay should always be tracked and recorded.
The first priority of the pathologist is diagnosis and only if there is residual tissue, should it be used for research purposes. The average weight of the research samples should be about 200 mg, with a minimum weight of 200 μg needed if sample size is limited and only DNA isolation is possible. An effort should be made to harvest the samples away from necrotic, hemorrhagic, or fibrotic capsular tissue since these factors risk the purity and abundance of tumor cells. When a large tumor specimen is available, serial sectioning should be done, with the sections or slices not more than 5–7 mm thick. If the slices are wider than 1 cm, it should be cut into 5mm horizontal strips. The ends of each strip should be trimmed off and placed into 10% neutral buffered formalin in separate cryovials to be embedded into paraffin for permanent sections. The remaining middle portion of each sample is then put into a pre-labeled cryovial with RNA later, proteinase (see below), or snap frozen in liquid nitrogen. () Labeling must distinguish which top and bottom histology sections correspond to which frozen sample. This is a critical quality control measure because the pathologist cannot accurately distinguish tumor from surrounding fibrosis or pancreatitis on gross examination at the time of specimen procurement. Specimens should be handled with gloves, and preferably sterile instruments and dishes changed between tumor and matched normal tissue, to prevent contamination with foreign DNA, RNA etc. The processing of tissue samples should not be left to the pathologists, but should at least be overseen and preferably done by a member of the research team to assure adequate sample collection.
Fig. 2 Specimen Collection. Samples of tumor and normal tissue are collected as soon as possible to minimize warm ischemia time. Sites with tumor and normal tissue are selected by gross examination of the surgical specimen. Each margin of the research sample (more ...)
Preservation techniques for DNA, RNA, protein, and tissue architecture need to be considered. For example, it is believed by some that protein structure is better preserved by formalin fixation and paraffin embedding (FFPE) rather than fresh freezing in OCT [42
]. On the other hand, formalin fixation is known to cause cross linking of DNA and RNA and results in very short fragments that may not be appropriate for sequencing and mutation discovery. However, these samples could be useful for genotyping, or validation of known mutations. RNA and DNA can be isolated from FFPE specimen using commercially available kits but fresh freezing is still considered best for RNA and DNA isolation [43
Recently developed preservation solutions like RNAlater
(Ambion, Austin, TX), have facilitated long term storage of tissue and eliminated the need for immediate RNA, allowing flexibility without compromising RNA quality [46
]. Irrespective of the exact preservation solution, the time that elapses between the procurement of the specimen until fixation or immersion into preservative and freezing is the most important factor. The temperature at which the sample is kept, transported, and processed also needs to be considered and standardized. These parameters need to be standardized since both proteins and nucleotides are subject to enzyme degradation and chemical modification. This is particularly true for a pancreatic cancer tissue resource since pancreatic specimens have an abundance of pancreatic enzymes which may be active during warm ischemia [49
The specimen for DNA study should be fresh frozen using an isopentane bath readily available in most hospital frozen section facilities. The specimen is then transferred to a −80C freezer. The specimen for RNA study should be incubated overnight in RNAlater solution at 4°C so that the solution penetrates the tissue, the fluid drained, and then frozen at −80°C. The specimen for protein study should be immersed in a proteinase inhibitor solution (Roche) and then frozen in an isopentane bath and stored at −80°C. Screw cap cryovials should be used for all specimens because they are leak proof and stable to freezing. In cases where the tissue quantity is ample, extensive study can be achieved using a sample piece for immunohistochemistry after freezing in OCT and tissue microarrays can be constructed from FFPE tissue.
Each tumor sample should also have a matched peripheral blood sample which will be used to isolate germline DNA. Normal matched DNA, RNA, and protein can also be obtained from surrounding normal pancreatic tissue (usually at the margin of resection) that is not infiltrated with tumor. However, areas of pancreas around the tumor may contain genetic changes associated with the development of the tumor; therefore, blood samples are preferred for determining the germline DNA sequence. Blood component samples can be stored as whole blood, although frozen separated peripheral blood lymphocytes (PBLs) or frozen separated PBLs in Dimethyl Sulfoxide (DMSO) are preferred.
We recommend use of PAXgene blood collection tubes, which are commercially available and simplify storage of blood samples without the need to separate blood constituents. These tubes contain a proprietary blend of reagents that can stabilize the cellular constituents of blood for up to 14 days at room temperature, 28 days at 4°C, or indefinitely at −80°C allowing some time until the DNA is extracted. However, caution should be exercised as these blood collection tubes do have expiration dates.
Patient serum and pancreatic juice are also very valuable and should be collected for future studies that will concern possible clinical correlations with soluble biomarkers. These studies may lead to much needed diagnostic tests for pancreatic cancer. Genetic changes, such as KRAS
mutations, are already beginning to be studied in pancreatic juice [52
]. Pancreatic juice can be obtained in the operating room at the time of resection by aspiration from the pancreatic duct at the transection margin using a small syringe and plastic angiocath. The juice is placed in a cryovial, frozen in an isopentane bath, and stored at −80°C.