thawing and culturing cryopreserved blood for cytokinesis assay

Examining Micronuclei Yields in Irradiated Whole Blood Samples

Ever since its discovery, the use of ionizing radiation (IR) has been a matter of debate among researchers because of its adverse effects on living beings. The detrimental effect is usually manifested by DNA damage such as double-stranded breaks (DSBs), and the failure to repair these DSBs leads to chromosomal aberrations and mutations, which are important hallmarks of cancer1,2. Such chromosomal aberrations can be examined by cytogenic assays such as the cytokinesis-block micronucleus (CBMN) assay. Micronuclei are lagging chromosomal fragments that cannot be incorporated into daughter nuclei and hence, are left behind during mitosis.
CBMN is a commonly used, reliable cytogenetic technique to assess chromosomal damage in individuals exposed to in vivo or in vitro ionizing radiation. Either fresh whole blood or isolated peripheral blood mononuclear cells (PBMCs) can be used in the CBMN assay. Fresh whole blood is mostly the biological material of choice since the isolation and processing of PBMCs can be time-consuming and is accompanied by a loss of serum plasma that acts as the supporting medium for cell survival and growth. To achieve a good binucleated cell yield, fresh whole blood should be processed immediately after collection. However, the need for immediate processing can be logistically challenging during time constraints. Moreover, when many samples are supposed to be acquired over an extended period or collected at points away from the processing centers, storage of fresh blood samples can be a limiting factor3,4.
Further, to allow repeated MN analysis in the same individual/patient, freezing of blood samples would be beneficial. One way to store lymphocytes for later application of the CBMN assay is by freezing isolated PBMCs5,6. This technique, however, requires several processing steps before the PBMCs can be frozen. Therefore, cryopreservation of whole blood would represent a simple and time-efficient alternative to the cryopreservation of isolated PBMCs. Little information is available concerning the use of frozen whole blood for cytogenetic assays or assays that require the proliferation of lymphocytes. Only one paper reports the use of cryopreserved whole blood for metaphase analysis7.
As cryopreservation of whole blood would offer many advantages in the field of biomonitoring, biodosimetry, and radiosensitivity assessment, our group optimized a cryopreservation protocol for whole blood that allows the application of the CBMN assay8. We demonstrated that lymphocytes present in cryopreserved whole blood cultures retain their genomic integrity and proliferation capacity for at least 1 year. In this methods paper, we describe in detail the cryopreservation procedure and CBMN assay protocol, which was optimized by Beyls et al.8, and report findings obtained for frozen blood samples of 30 healthy individuals. For the CBMN assay, the blood cultures were irradiated in vitro with doses of 0.5, 1, and 2 Gy to assess the MN response in lymphocytes of cryopreserved whole blood samples.

Author Spotlight: Cryopreservation of Whole Blood Samples for High Throughput Analysis 

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. Fresh whole 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 whole 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 cryopreservation 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.

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Thawing and Culturing Cryopreserved Blood for Cytokinesis Assay  

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JoVE Journal

Cancer Research

587 vues.  Ghent University. Pour préparer les puits de culture, marquez le couvercle d’une plaque à 24 puits avec le code du donneur A ou B, la date et la dose. Retirez les flacons cryogéniques contenant du sang humain de l’azote liquide et décongelez-les partiellement au bain-marie jusqu’à ce que de petits cristaux de glace soient visibles. Ensuite, dans une hotte à flux laminaire, ouvrez soigneusement le flacon pour éviter que des bulles d’air ne se répandent à l’intérieur du tube.Remettre e...

Vidéo: Thawing and Culturing Cryopreserved Blood for Cytokinesis Assay  

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, and in vitro radiosensitivity testing. This cytogenetic method is based on the detection of micronuclei in binucleated cells resulting from chromosomal fragments lagging during cell division. Fresh whole blood samples are the most preferred sample type for the CBMN assay. However, the disadvantages of working with fresh blood samples include immediate processing after blood collection and the limited number of repeated analyses that can be performed without extra blood sampling. 
As the need for fresh blood samples can be logistically challenging, CBMN assay on cryopreserved whole blood samples would be of great advantage, especially in large-scale patient studies. This paper describes a protocol to freeze whole blood samples and to perform the CBMN assay on these frozen blood samples. Blood samples from healthy volunteers have been frozen and thawed at different time points and then, subjected to a modified micronucleus assay protocol. The results demonstrate that this optimized procedure allows the performance of the CBMN assay on frozen blood samples. The described cryopreservation protocol may also be very useful for other cytogenetic assays and a variety of functional assays requiring proliferating lymphocytes.

Here we present an optimized protocol for the cytokinesis-block micronucleus assay on cryopreserved whole blood samples. This optimized method of cryopreservation of whole blood for micronucleus analysis is a reliable technique for large-scale sampling and multi-center studies and can be used for other blood-related assays as well.

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, ...