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The lab produces a pheresis sample from human peripheral blood that is then
ficoled to give a mononuclear cell population followed by percol to enrich
for monocytes. The goal is then to sort out the monocytes at high speed
(10,000/sec) on the ELITE ESP. The sample is adjusted to 20M/ml to allow
for the high sort speed. The problem has been that a short time (30-60 sec)
after sample start the monocytes "disappear" from the display.
Prepared Ficoled samples of whole blood taken in three different
anticoagulants - ACD, Heparin, EDTA. All cells, after washing, were
adjusted to 20M/ml.
Under the microscope we could see a significant amount of clumping in all
samples. Also noted were a large number of platelets in the preparation.
When running the samples on the ELITE, after 60-120 seconds, the monocytes
would increase in count rate and very quickly drop to a very low level
(similar to what happens with the pheresis sample). This type of sample had
also been analyzed on an XL, FACScan, and Vantage and the same result was
seen. The effect was irrespective of anticoagulant used. Also of note,
the lymphocytes maintained a steady flow rate throughout.
Adding MO2 or PLT-1 to the preparation produced a slight decrease in the effect.
Since there was a significant number of platelets in the preps and also, as
monocytes tend to bind platelets, we added EDTA (20 ul from EDTA vacutainer)
to the preparations to prevent the platelets from doing their sticky purpose
in life thing.
*The addition of EDTA completely eliminated the drop in monocyte flow rate.*
Since the amount of EDTA we were using was probably in large excess we
resuspended the cells in ISOTON which has an EDTA conc of 0.38g/L. This
also eliminated the monocyte flow rate decrease. In an older manual lysing
procedure, the EDTA conc was 0.039g/L was used to prevent aggregation. The
cells were then resuspended in the lab's PBS with 10% ISOTON. This also
completely eliminated the problem.
We also labeled the sample with CD14(MO2), CD41(PLT-1), and CD14/CD45. The
addition of CD14 did have some effect on preventing the drop of the monocyte
flow rate and the PLT-1 ab delayed the drop to approximately 120 sec, but in
all cases the only remedy was to add EDTA.
A pheresis sample was prepared and the same series of variables was tested.
Essentially identical results were seen as with the ficol samples. The
pheresis sample did contain a large platelet contamination and this was
reduced by low speed centrifugation but also resulted in loss of cellular
material.
Regards,
Dave Lundy.
(Coulter Canada Ltd.)
dlundy@inforamp.net
Work: Coulter Canada Ltd., 905 Century Drive, Burlington, Ontario L7L 5J8
TEL: 905-452-9187 FAX: 905-333-3787
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