BIOBANKING


The basis of translational research is the correct collection and storage of human tissues. The development of a tissue bank with optimal preservation of the morphology and of the quality of DNA, RNA and proteins in the sample is an absolute prerequisite. To this end, standard operation procedures for tissue, blood and bone marrow sampling and storage, with integrated quality control, have been developed by the Translational Cancer Research Group Antwerp. All patients are treated in the General Hospital Sint-Augustinus and follow-up data are obtained prospectively.


1. Anonimisation and data collection: TCRG DATABASE

Sample information, experiment results, clinical and histopathological data, therapy, relapse and survival information from each patient are entered in a database. The TCRG database is a secured database made up of different tables, queries and forms with a personnel accessibility depending on function and position.


2. Blood sample collection

Peripheral blood samples are collected for three purposes:
(i) Detection of circulating tumour cells with quantitative real-time RT-PCR in human whole blood.
(ii) Determination of useful clinical parameters such as white blood cell count, platelet count, concentration of d-dimers and fibrinogen.
(iii) Storage of serum and plasma samples for proteomics: e.g. quantification of angiogenic cytokines (VEGF, bFGF, IL6, IL8) and other biomarkers (HER2) in serum samples.
At each sampling mission, 40 ml of peripheral blood is collected.

TCRG standard operation procedures for peripheral blood sampling and serum storage:

  • Anticoagulans: none
  • Collection tube: SST (serum separator tube)
  • Volume: 8.5 ml
  • Sample processing: after minimal 20 minutes and maximum 120 minutes of blood clothing
  • Centrifugation: 2000g, 5 minutes, room temp.
  • Storage: -80°C

TCRG standard operation procedures for peripheral blood sampling and plasma storage:
  • Anticoagulans: Na-citrate
  • Collection tube: BD 9NC 0.129M
  • Volume: 4,5 ml x 2
  • Sample processing: maximum 120 minutes after blood sampling
  • Centrifugation: 2000g, 10 minutes, room temp.
  • Storage: -80°C


3. Bone marrow collection

In general mononuclear cells (MNC) and metastatic cancer cells are separated from the bone marrow samples using a density-gradient like Ficoll or Lymphoprep. After centrifugation, the interface cells are removed and washed. For ICC analyses, known quantities of cells are deposited onto glass slides by cytocentrifugation. Preparing cytospins has the advantages that only a single layer of cells is present which allows quantification of tumour cells in relation to the number of MNC. The cell pellets can also be resuspended in RNA stabilising buffers for molecular down stream applications.

TCRG standard operation procedures for bone marrow sampling and storage:
  • Time of aspiration: pre operative, prior to the invasive surgical procedure
  • Puncture site: anterior or posterior iliac crest
  • Number of sites: 1
  • Needle: Yamshidi, 16g
  • Anticoagulant: Heparin 5000 I.U./20 ml syringe
  • Anaesthesia: locale or complete
  • Incision: before puncture with surgical blade
  • Collection tube: BD NH 170 I.U.
  • Volume: +/- 20 ml (first 2 ml discarded)
  • Sample processing: within 1 hour after sampling
  • Method: gradient centrifugation over Ficoll-Paque and isolation of mono nuclear cells (MNC)
  • RT-PCR analysis: suspension of the cell pellet in a guanidine isothiocyanate containing buffer (RLT + β-mercaptoethanol) and stored at -80°C.
  • ICC analysis: preparation of cytospins containing 500 000 cell per spot.


4. Tissue collection

After removal of the tumour sample a stopwatch is started and the pathologist is informed about the arrival of a tissue for the biobank. The surgical specimen is immediately transported to the lab whereas the pathologist first collects sections for routine histopathological and cytopathological routine analyses. From the remaining tumour specimen, small sections are collected for research purposes and immediately frozen in liquid nitrogen. As soon as the samples are placed into liquid nitrogen, the stopwatch is stopped and the time is registered on the study application form. Other useful information like date, name surgical, name pathologist, and others are also registered on the application form. If possible, sections of the surrounding normal tissue are also collected in the same way. Samples placed in liquid nitrogen are transported to the research lab and retained in a cryovial. These samples are stored in the gas phase of liquid nitrogen.