Additional information may be necessary to consider your observation.

For dosimetric consideration because an electron beam has a limited penetration depth. Did you irradiate cells in suspension? If so, what was the depth of culture medium in the dish or flask? What is the penetration range in water for the electron beams you used?

For biological consideration to know the dose dependence of the observed phenomena given no dosimetric issue. Do you mean that [1] irradiation with 6 Gy and 8 Gy caused 43.4% and 70% increase in the number of leukocytes at Day 3 after irradiation relative to that at Day 0, or [2] Irradiation with 6 Gy caused 43.4% increase and 8 Gy caused 30% decrease in the number of leukocytes at Day 3 after irradiation relative to that at Day 0?

Respected Sir

Thank you so much for your concern and consideration on my doubt. Exactly sir as you said i observed  irradiation with 6 Gy and 8 Gy caused 43.4% and 70% increase in the number of leukocytes at Day 3 after irradiation but compare to control sample where there was no irradiation. Total number of cells per culture of Control have been found (9.67*107 ). Where irradiated culture have been found 13.87*107 and 16.85*107 respectively for 6 gray and 8 gray irradiation after 3 days. 

Sir i have used culture vial which is made up of glass and usually used for karyotyping culture and it was cell suspension. 1 ml of intravenous blood was cultured in 10 ml of RPMI-1640 media with additional PHA and FBS(20%)

Please clear my doubt regards to this. It will be very helpful for me.

Sincerely

Shashank Kumar

From your feedback to my question for biological consideration, I understood that 8 Gy irradiation was more mitogenically stimulative to cultured leukocytes than 6 Gy irradiation. It will be helpful if you can also rely to my questions for dosimetric consideration as aforementioned.

Respected Sir

As i understood by the concern question,The depth of the culture is approximately 5 cm in liquid suspension.

I am asking a limited penetration depth of electron beams in water.

If cells are at the bottom of the flask or dish, and if a beam penetration depth is less than 5 cm, 6 or 8 Gy irradiation does not actuallty delivers 6 or 8 Gy. 

Respected Sir

Thank you so much for pointing out this point. Sir i have no idea regards to the electron beam penetration depth and i am going through articles related to this. Apart from other details, I have used 6 MeV as energy of electron beam. I will try to find out more details regards to this question. One article has shown that 6 MeV penetration depth of electron beam in water is aroaund 2-3 cm under 0.75 tesla magnetic feild. I suppose as you concern and your point the actual delievered radiation to the cells is much less than what i gave in actual. Please correct me and give me some points regards to this. I am sending you the link of the article please go through it. As a biological student its little difficult to understand the whole term.

http://www.sciencedirect.com/science/article/pii/S1120179715001040?np=y&npKey=5dc0a0cd277ac3bebea474240bd8a0100eaf16c3da9715116dd125a34adcc17b

Sincerely

Shashank Kumar

It is important to know the underlying dosimetry in your experiments because electron dose distribution differs depending on depth. Your supervisors or physicists/engineers taking care of beams should have the information on dose distribution, so please consider asking them.

Respected sir

I will try to find out the details regards to the actual dose. I will try to find out more information regards to this. Thank you so much for your kind consideration sir. It has given me a way to find out answer.

Will look forward to receiving your update.

Although dosimetric uncertainties remain, provided below are some of my thoughts from biological viewpoints.

In your study, peripheral blood lymphocytes (PBLs) were mitogenically stimulated with phytohemagglutinin (PHA), and culture medium contained 20% FBS (I think 10% FBE is more generally used).

Mixed electron beams (80% is 6 MeV electrons and 20% is 12 MeV electrons) have been used in a minipig model to simulate inhomogeneous solar particle event proton dose distribution where higher dose is deliver to the skin and subcutaneous tissue than to internal organs, followed by analysis of changes in leukocye count.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356949/   (PDF freely downloadable)

A series of studies in PBLs exposed to electrons look at cytogenetic changes. Relative biological effectiveness of 8 MeV pulsed electron beam vs 60Co gamma-rays for micronucleation was about 1 in human blood lymphocytes.

https://www.ncbi.nlm.nih.gov/pubmed/19142652

I think it is well accepted that cell numbers in PBLs decrease following irradiation for which apoptosis accounts (although not fully). Nevertheless, I here highlight two papers in relation to increased proliferation after irradiation of PBLs.

[1] A significantly increased proliferation rate was observed following irradiation of human PBLs with 0.75 and 1 Gy of protons (but not with the same dose of X-rays).

https://www.ncbi.nlm.nih.gov/pubmed/25818831

[2] Recombinant human growth hormone increased proliferation of irradiated human PBLs.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1573792/   (PDF freely downloadable)

I do not go into details here, but ionizing radiation is known not only to inactivate but also to stimulate cell proliferation under several conditions in other cell types.

 Thank you so much for this paper sir. PHA treated lymphocytes have shown more proliferation and I guess may be in my case too its somehow mitogenetically enhancing the proliferation. I will try to gain more information and will update sir.

Thank you so much for your kind consideration