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I had a abstract on this topic (265A) which was placed under
Haematological Malignancies. This may have been missed by
'sorters' as the abstracts were not indexed.
We have been selecting minor populations (0.1->3.0%) of labelled
cells using the following strategy.
1) Trigger on fluorescence such that the machine only detects +ve
cells.
2) Adjust the flow rate to place an average of one cell (+ve or -ve)
in each droplet. i.e. total flow rate = drop drive frequency. For
example if the poulation is 1% and the drop drive 30000/sec adjust
the flow rate to give 300 triggers/sec. We now expect every sorted
cell to be accompanied by two unwanted ones (three droplet sort)
giving a purity of 33%
This throughput is close to 100 million cells /hour.
For this example the 'ideal' sort should yield 1 million +ves
with 2 million -ves.
You can think of this as 'slow speed sorting' - at 300/second abort
rates are almost zero and can be ignored. We do not bother turning
the abort off.
3) Purify this enriched sample with conventional
(FCS threshold) sorting. (3 million would take less than 30
minutes).
We can therefore process close to 100 million cells in 1.5 hours.
This would take us 10 hours by conventional one stage sorting.
Our net recoveries are up to 50% of the starting material.
High cell densities are essential (10 million / ml) such that
minimal sample pressure can be used to minimise turbulence.
This strategy has been used to purify CD34 cells from bone
marrow, peripheral blood stem cell harvests, and cord blood.
Cryopreserved mononuclear cells have also been used successfully with
the cord blood samples.
Cells have subsequently been used for
1) Clonogenic assays (Brit. J. Haematol 88 472-480)
2) Differenciation studies
3) Transfection experiments
4) Signalling assays (J.Immunolgical. Methods 179 187-192)
5) Cryopreservation followed by 1) & 2)
This method was introduced in parallel with magnetic sorting for the
first (enrichment) stage. The above is currently our method of choice.
(Not determined by me, my 'magnetic' colleagues are still trying
their best!) About 150 samples have been processed to date. The
failure rate has been very low. Purities are always better than 95%
and commonly reach 98%
This is used routinely on a FACS440, 70 micron nozzle and drop drive
frequency 27,000 (30,000 makes the sums easier!).
A single attempt with another assay indicated the process worked on a
Vantage.
The technology is suitable for sub-sets. i.e. pull out all the wanted
cells at enrichment and select the sub sets at the purification stage.
I would be pleased to hear from anybody who has tried this technique
with CD34's or any other 'rare event' sorting.
Terry Hoy.
Department of Haematology Telephone 44 1222 743458
University of Wales College of Medicine FAX 44 1222 744655
Heath Park E-mail hoy@cardiff.ac.uk
CARDIFF CF4 4XN
U.K.
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