CD 34 count is important for Cord Blood Banking and usage of Cord Blood Units for transplantation. Optimum number of CD34 is of paramount importance. Flowcytometer is a standard method for counting CD34 HSCs in cord blood. But, there are financial and logistic constraints. Countess FL2 uses light tubes to detect and count fluorescent markers. Hence, the query that by using fluorescent dyes to stain CD34 cell surface markers on HSCs, can one use the instrument to count the number of HSCs present in cord blood sample.
Hi Sughosh,
Honestly I never use the cell counter in object, but I know the principle on which is based on and I'm experienced in CD34 quantification.
This cell counter is based on hemocytometer slides pretty similar to Burker's or Thoma's ones and applying automatic image analysis software, it performs the counting. Moreover this model is equipped with a fluorescence cube and the manufacturer declares the capability to detect GFP- or RFP-fluorescent protein expressing cells.
Regarding you hypothesis of applying this device for CD34 counting, I do not believe that it could represent a feasible goal to reach, for the following reasons:
i) To detect CD34 you should apply immunofluorescence techniques and the signals produced by fluorescent antibodies are several logs lower than the fluorescence produced by GFP or RFP. I do not believe that this device provide the sufficient sensibility to detect fluorescent antibodies, and probably that is the reason why the manufacturer declare to be able to detect only fluorescent proteins.
ii) CD34 HSC are normally rare events and to perform a valuable quantification a total from 50'000 to 100'000 events are usually analyzed by flow cytometry. Slide-based imaging techniques are normally far to be able to analyze such a huge number of cells.
Take my objections as my personal point of view, you could also contact the Thermofisher application specialist to discuss this possibility.
Hope this could help you,
Good Luck
Simone
Hi Sughosh,
Honestly I never use the cell counter in object, but I know the principle on which is based on and I'm experienced in CD34 quantification.
This cell counter is based on hemocytometer slides pretty similar to Burker's or Thoma's ones and applying automatic image analysis software, it performs the counting. Moreover this model is equipped with a fluorescence cube and the manufacturer declares the capability to detect GFP- or RFP-fluorescent protein expressing cells.
Regarding you hypothesis of applying this device for CD34 counting, I do not believe that it could represent a feasible goal to reach, for the following reasons:
i) To detect CD34 you should apply immunofluorescence techniques and the signals produced by fluorescent antibodies are several logs lower than the fluorescence produced by GFP or RFP. I do not believe that this device provide the sufficient sensibility to detect fluorescent antibodies, and probably that is the reason why the manufacturer declare to be able to detect only fluorescent proteins.
ii) CD34 HSC are normally rare events and to perform a valuable quantification a total from 50'000 to 100'000 events are usually analyzed by flow cytometry. Slide-based imaging techniques are normally far to be able to analyze such a huge number of cells.
Take my objections as my personal point of view, you could also contact the Thermofisher application specialist to discuss this possibility.
Hope this could help you,
Good Luck
Simone
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