Cell clumps after Ficoll PBMC extraction

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K_K
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Cell clumps after Ficoll PBMC extraction

I carried out PBMC extraction using Ficoll paque PLUS. However after using PBS washed for twice, cells clump together and cannot be resuspended. Could anyone help to see what the problem is?

Pic of cell clumps are here (sorry not so clear):
http://imgur.com/wzvhGLK
http://imgur.com/h898A5h

The protocol used are as follows:

Blood from healthy person used was collected in EDTA tube. Fresh blood collected 2-3 hrs before used. Store at room temp.
Before used, blood was diluted by PBS (room temp, pH=7.4, no Ca++ and Mg++, no EDTA or other anticoagulant added, http://www.lifetechnologies.com/order/catalog/product/21600010?CID=search-21600010)
12ml blood diluted with 12 ml PBS, and the 24 ml diluted blood layer on 15ml Ficoll (room temp). Centrifuge for 20 mins, no brake, 2200rpm

Then get the PBMC layer and put it in 15ml tube.
A little bit of plasma and Ficoll are also get together with PBMC (around 2.5 – 3ml), then fill the tube with PBS (same solution used as above). 1800rpm 10mins
After the first wash the cells can still be resuspended

Wash one more time using PBS. 1800rpm 10 mins.
However, after this wash the cell clumped together and cannot be resuspended (I suppose they are cells…)

I have performed two times of PBMC extraction using two different healthy people’s blood but cells clump together in all two trial.

I would like to ask:
why cells clumped together after my second wash?
How can I prevent the cells clumped together?
- Too much blood used and therefore too much cells in one tube?
- Problem of PBS?
- get too much plasma/Ficoll?
- temp of the solution used?
- my technique problem?

Can anyone please help me how to improve the problem. Many many thanks!!