The Micronucleus Assay on Cryopreserved Whole Blood

Our research mainly focuses on examining the impact of ionizing radiation on healthy tissues. We are studying the chromosomal radio sensitivity of individuals with cancer and those with DNA repair disorders. Additionally, we are involved in biological dissymmetry where we assess the DNA damage induced by exposure to ionizing radiation.

To advance research in our field, we use automated microscopic systems for high throughput analysis. Threshold blood samples are the most preferred sample type for the micronucleus assay, as it is easy to obtain with minimal discomfort for the patient. However, the need for immediate processing can be logistically challenging during time constraints.

Freezing of full blood samples allows repeated analysis without the further need for extra sampling. This procedure is an easy and time efficient way to store blood samples in large scale and multicenter studies. The protocol of the cryo-preservation of whole blood will not only offer advantages for the micronucleus assay, but also for other cytogenetic assays or functional assays that require the proliferation of lymphocytes.

To prepare the culture wells mark the lid of a 24 well plate with the donor code A or B, the date, and the dose. Remove the cryo vials containing human blood from the liquid nitrogen and partially thaw them in the warm water bath until small ice crystals are seen. Next in a laminar flow hood, carefully open the vial to prevent air bubbles from spilling inside the tube.

Re-suspend and transfer the contents of the vial to a 15 milliliter centrifuge tube. While gently swirling the tube dropwise, add one milliliter of pre-warmed PBS. Use a P 1000 pipette to mix PBS and blood.

For dilution and uniform washing, add seven milliliters of pre-warmed PBS to the 15 milliliter tube and mix by pipetting. Centrifuge the blood at 180 G for eight minutes. Meanwhile, fill the unlabeled wells of a 24 well plate with 500 microliters of PBS and prewarm the plate at 37 degrees Celsius.

After centrifugation, carefully remove the supernatant leaving approximately one milliliter in the tube without disturbing the pellet. Using a pipette, re-suspend the pellet in the remaining supernatant. Then gradually add eight milliliters of warm PBS into the tube as demonstrated previously.

Centrifuge the re-suspended blood. In the meantime, add 200 microliters of RPMI culture medium to the marked wells, and prewarm the plate as demonstrated. After centrifugation, remove the supernatant leaving approximately 80 microliters of the supernatant.

Then add 280 microliters of fetal calf serum and re-suspend the pellet. Transfer 180 microliters of the cell suspension to the marked wells. Incubate the plate in a carbon dioxide incubator at 37 degrees Celsius for 10 minutes.

After culturing thawed, cryo-preserved blood sample, irradiate it with the required X-ray doses at room temperature. Then add 1.62 milliliters of warm RPMI culture medium to each well. To stimulate the cell division in T lymphocytes, add 40 microliters of phytohemagglutin to each well and re-suspend thoroughly.

Incubate the plate at 37 degrees Celsius and five percent carbon dioxide for 23 hours. The next day, add eight microliters of cytochalasin B per well to block the cytokinesis. After 70 hours, remove the plate from the incubator and transfer the cell suspension to a 15 milliliter tube.

Rinse each well with two milliliters of PBS and add this PBS to the 15 milliliter tube. Centrifuge the cell suspension at 180 G for eight minutes and discard the supernatant, leaving 500 microliters over the pellet. While vortexing the pellet, add two milliliters of cold potassium chloride into the tube.

Again, centrifuge and discard the supernatant as demonstrated previously. Vortex the pellet by slowly adding two milliliters of cold fixative one and incubate overnight at four degrees Celsius. The next day, centrifuse and discard the supernatant.

While vortexing the pellet dropwise, add two milliliters of cold fixative two to the tube. Leave the tube overnight at four degrees Celsius. Centrifuge the sample and transfer the supernatant to another tube to concentrate the cells according to the pellet size.

Clean the slides with isopropanol and label them properly. After vortexing the pellet, add 40 microliters of fixed cell suspension onto a slide. After drying, immerse the slide in acridine orange stain for one minute.

Then wash the slide quickly with distilled water before placing it in a phosphate buffer for one minute. Dry the back of a slide and place it on clean tissue paper. Add 20 microliters of phosphate buffer onto the slide and cover it with a clean cover slip avoiding air bubbles.

Seal this slide with silicon cement. Place the slide under a fluorescent microscope and examine binucleated cells. Finally, manually count the micro nuclei.

The presence of rounded binucleated cells indicated successful retrieval of healthy viable cells from cryopreserved whole blood samples. With increased doses of radiation a linear quadratic increase in micronucleus yield was observed. The background micronucleus yields in sham irradiated control samples primarily resulted from lagging chromosomes.