Plan nacional de logística y transporte de Brasil PNLT

2.2.1. Human blood

2.2.1.1. Fresh blood

For the blood collection tube comparison, two blood samples were collected from one healthy donor into lithium heparin or ethylenediaminetetraacetic acid (EDTA) blood collection tubes (BD Becton Dickinson, Oxford, UK, cat. 366643). 100 µl aliquots were sham irradiated or exposed to a dose of 4 Gy X-ray and collected 24 h post exposure.

For biomarkers identification 20 ml of peripheral blood samples were obtained anonymously from three healthy female working individuals (age range 37–53 years old) on three independent occasions with informed consent and ethical approval from

Berkshire Research Ethics Committee (reference number 09/HO505/87). Peripheral blood was collected via venipuncture into anticoagulant EDTA vacutainer tubes (Becton Dickinson) and kept on ice for a maximum of two hours until 30 minutes prior to irradiation. 5 ml of blood from each donor was aliquoted into two 5 ml cell culture tubes and placed at 37 °C 30 min prior irradiation. The rest of blood was used to set up T-lymphocyte cultures. Blood samples were sham irradiated or exposed to 2 or 4 Gy of X-ray and collected 2 or 24 h post exposure.

For assessment of inter-and intra-individual variability peripheral blood samples were obtained anonymously on three independent occasions from 32 healthy blood donors with informed consent and ethical approval from Berkshire Research Ethics Committee (reference number 09/HO505/87). Peripheral blood was collected via venipuncture into anticoagulant EDTA vacutainer tubes (Becton Dickinson) and kept on ice for a maximum of two hours until 30 minutes prior to irradiation. 500 μl of blood from each donor was aliquoted into two 2 ml screw-cap tubes and placed at 37 °C 30 min prior to irradiation. Samples were sham irradiated or exposed to a dose of 2 Gy X- ray and collected 2 h post irradiation.

2.2.1.2. Cultured blood

The sample preparation was performed in the laboratory of Prof. Michael Abend at Bundeswehr Institute of Radiobiology, (Munich, Germany). Briefly, 2-3 ml of whole blood from one healthy male individual (age 29) was collected into heparinized tubes (Becton Dickinson, cat. 367874) on two separate occasions. Blood was taken with informed consent and the approval of a local ethics committee. The blood was diluted 1:2 with Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10 % foetal bovine serum (FBS) (all from GIBCO/Life Technologies Ltd., Paisley, UK, cat.

22409-015 and 10500-064 respectively) and incubated for 24 h at 37 °C before irradiation. One batch of blood was used to construct a calibration curve, the second batch represented “unknown” samples. Seven calibration curve samples were irradiated with a sham dose, 0.25 Gy, 0.5 Gy, 1 Gy, 2 Gy, 3 Gy and 4 Gy and collected 24 h post irradiation. Ten unknown samples were irradiated with doses ranging from 0 to 6.4 Gy and collected 24 h post irradiation.

2.2.1.3. Cancer patients

Blood collection from cancer patients was performed at University Hospital Hradec Kralove (Hradec Kralowe, Czech Republic) by Dr Ales Tichy. Peripheral blood samples were obtained anonymously from two cancer patients (two females, age 65- 72) undergoing radiotherapy for endometrial cancer with informed consent and ethical approval from University Hospital Hradec Kralove Ethics Committee (Hradec Kralove, Czech Republic, reference number 201401-S15P). Neither of the patients had previous radiotherapy or chemotherapy treatment. 2.5 ml of peripheral blood was collected via venipuncture into PAXgene RNA blood tubes (Becton Dickinson, cat. 762165) before first dose of radiotherapy and 24 h after first 1.9 Gy fraction. After blood collection, the blood samples were mixed several times by inversion and incubated at room temperature for 2 h, then frozen at -20 °C and shipped to our lab on dry ice.

2.2.2. Mouse blood

For the RT validation experiments, blood from a C57Bl/6 mouse was collected by cardiac puncture into EDTA blood collection tubes (Becton Dickinson) and then 5 µl, 10 µl, 25 µl, 50 µl, 75 µl and 100 µl aliquots were transferred to 2 ml screw-cap tubes containing 0.5 ml of RNALater and stored at -80 °C until further analysis. The experiment was repeated twice.

2.2.3. Human stimulated T-lymphocytes

2.2.3.1. RNA extraction kit comparison

For the RNA extraction kit comparison, stimulated human T-lymphocytes obtained from healthy donor C2 were used. Cells were sham irradiated or exposed to a dose of 4 Gy X-ray (6 x 106 cells per dose) and collected 4 h post exposure.

2.2.3.2. Biomarkers identification

T-lymphocytes obtained from three blood donors described in chapter 2.2.1.1 were used. Cells were sham irradiated or exposed to 2 or 4 Gy of X-ray and collected 2 or 24 h post exposure. Three independent repeats were performed.

2.2.3.3. Time course and dose-response experiments

For time course and dose response experiments, human stimulated T- lymphocytes from two healthy donors (C1 and C2, both females, age range 39-41) and one Ataxia – Telangiectasia patient (AT, a gift from Dr. Colin Arlett, University of Sussex, UK) were studied; 2 x 106 cells were used for each sample.

For time course experiments cells were sham irradiated or with a dose of 2 Gy X-ray and samples were collected at 15 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h and 24 h post irradiation. The experiment was repeated three times, samples for the first repeat were cultured and prepared by Ms Claudine Raffy.

For high dose-response experiments cells were sham irradiated or exposed to a doses 0.1 Gy, 0.2 Gy, 0.3 Gy, 0.4 Gy, 0.5 Gy, 1 Gy, 2 Gy, 3 Gy, 4 Gy and 5 Gy of X-ray and collected at two different time-points – 2 h and 24 h. The experiment was repeated two times, samples for the first repeat were cultured and prepared by Ms Claudine Raffy.

For low dose-response experiments only T-lymphocytes obtained from donor C1 were used. Cells were sham irradiated or exposed to a doses 5 mGy, 10 mGy, 20 mGy, 30 mGy, 40 mGy, 50 mGy, 75 mGy and 100 mGy of X-ray and collected at two different time points – 2 h and 24 h. The experiment was repeated four times, samples for the first repeat at 2 h were cultured and prepared by Ms Claudine Raffy, two other repeats were performed by Ms Grainne Manning.