[Histonet] RE: Saponin Technique

[Tony Henwood]

The following has been found useful in my hands:

Removal of Excess Blood 
Haemolysing haemorrhagic specimens cause fewer problems in identifying
individual cells. The removal of red blood cells can be achieved using
Ficoll-Hypaque gradient separation or using a lysis solution such as
isotonic ammonium chloride (Kuenen-Boumiester etal (1996) Acta Cytolog
40:475-479.):

SOLUTIONS:
1.	Lysis solution
Ammonium Chloride        4.5g
Potassium carbonate      0.5g
EDTA                     0.0186g
Distilled water          500mls
2.	Hanks medium
METHOD:
1.	Centrifuge bloody fluid.
2.	Remove supernatant and add equal volume of lysis solution.
3.	Resuspend and incubate for 5 minutes at 4oC.
4.	Centrifuge, if blood still remains, then repeat from step 2.
5.	Resuspend pellet in Hanks.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of McMahon,
Loralee A
Sent: Tuesday, 26 October 2010 2:51 AM
To: Hannen, Valerie; histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: Saponin Technique


It's been a while but I think that we used to add a little diluted
glacial acetic acid to our cytology specimens to lyse the red cells. 
Not sure on the dilution, but maybe someone else knows. 



Loralee McMahon, HTL (ASCP)
Immunohistochemistry Supervisor
Strong Memorial Hospital
Department of Surgical Pathology
(585) 275-7210
________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu
[histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Hannen, Valerie
[Valerie.Hannen <@t> parrishmed.com]
Sent: Monday, October 25, 2010 11:39 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] FW: Saponin Technique

________________________________

From: Hannen, Valerie
Sent: Monday, October 25, 2010 11:39 AM
To: 'histonet <@t> list.utsouthwestern.edu'
Subject: Saponin Technique


Hi folks..
I am hoping someone out there in Histo-land can help. One of our
Pathologists is asking us to check into doing the Saponin Technique to
lyse the red cells in some of our cytology specimens. She gave a copy of
one of the pages from one of her manuals, and it shows that in order for
us to do this we would need to buy alot of reagents that we currently do
not have. Being that we would probably not be doing this technique very
often, and not sure how cost effective it will be for us to buy the
reagents, we are wondering if  anyone knows of a commercial product that
we can buy that would be a cheaper way out.

Thanks in advance!!
Valerie Hannen, Histotechnologist
Parrish Medical Center
Titusville,Florida


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