[Histonet] Cell Block Procedure??

[Tony Henwood]

The following notes might be of help:

Fibrin Clot Method 
Reference:
Furtado (1970)
Principle:

The Thromboplastin cell-block is very gentle on cells and allows a wide
range of routine immunohistochemical stains to be done.

Plasma, treated with EDTA, is mixed with the specimen in the presence of
thromboplastin and calcium ions to form a cell clot.

This method is not suitable for cells fixed in formalin or specimens of
bile fluid (bile enzymes prevent clot formation). In these instances the
agar cell block method is preferred.

Solutions:

1.	Normal Plasma, containing EDTA, use out of date plasma obtained
from Blood Bank. Freeze in 2ml aliquots. This plasma has been tested for
known viruses and is considered safe.

2.	Thromboplastin, use "out of date" thromboplastin obtained from
Haematology.

3.	1% calcium chloride

4.	10% formalin

Method:

1.	Spin fluid using a centrifuge.
2.	Decant off supernatant
3.	Resuspend pellet in 3 drops of plasma.
4.	Add 3 drops of thromboplastin, mix
5.	Add 3 drops of Calcium Chloride, mix gently
6.	Allow to stand undisturbed for 15-20 minutes
7.	Add 5-8ml 10% formalin and allow to fix
8.	Place in a labelled tissue cassette and process as usual.

Egg Albumin Cell Block Method
Reference:
Yamamoto et al (1985)
Principle:

In this method, egg albumin coagulates in the presence of ethanol to
form a cellblock.

Solutions:

1.	Egg albumin stock: 
Dissolve 1g of Egg albumin in a minimum of glycerol (about 1/10 V/V).
2.	95% ethanol

Procedure:

1.	Centrifuge cell suspension and decant supernatant.
2.	Add 250-500ul Egg albumin stock and mix gently.
3.	Add an equal volume of 95% ethanol and allow to gel.
4.	Add 5ml fixative and allow to fix for 4-24 hours.
5.	Process as usual.


Agar Cell Block Method

Reference:
Olson et al (1986)

Solutions:

1.	3% Agar
2.	Fixative

Procedure:

1.	Fix material in preferred fixative, 50% ethanol 30min.
2.	Heat 3% agar in an oven until molten.
3.	Centrifuge specimen and decant supernatant.
4.	Add 0.5ml molten agar
5.	Allow to cool, add fixative, remove block and process.



Mini-processing Cell Block Method
Reference:
Krogerus & Andersson (1988)
Principle:

This is a mini-processing method where cell centrifugation and
processing is undertaken in the same centrifuge tube. The xylene step is
omitted, being replaced by acetone and drying. Acetone evaporates
rapidly at 60oC while fast drying is not achieved with xylene. Samples
containing abundant mucus cause some problems since these were not
efficiently infiltrated by paraffin.

Solutions:

1.	Fixative  
Acetone                   	900ml
Ethanol                   	100ml
Trichloroacetic acid      	0.5ml

or 10% buffered formalin or 50% ethanol can be used.

2.	Acetone
3.	Melted paraffin

Procedure:

1.	Fix cell pellet in fixative for at least one hour.
2.	Centrifuge cell suspension and decant supernatant.
3.	Add acetone, mix gently and leave for 10min.
4.	Centrifuge specimen; remove supernatant and dry tubes in 60oC
oven for one hour.
5.	Add melted paraffin to the warmed cell pellets, gently mix,
centrifuge and leave tube to cool to room temperature.
6.	Remove paraffin cellblock by gently tapping the bottom of the
tube. The blocks are now ready to section.

Notes:  
A similar technique to the above has been reported by Domagala et al
(1990)

Specific Processing Method for Cell Blocks
Reference:
Kung et al (1989)
Principle:

Often the cytological preservation in cellblock sections was far
inferior to that of surgical specimens fixed and processed according to
the standard histological processing schedule. The cells appear
shrunken, nuclei were small and dark rendering assessment of chromatin
pattern difficult, and the nucleoli were less conspicuous. The following
technique uses a shorter time in xylene and a more gradual change over
from alcohol. The use of 7.5% rather than 10% formalin is also
recommended.

Procedure:

1.	7.5% formalin       6-12hrs.
2.	70% ethanol         30min.
3.	95% ethanol         30min.
4.	Absolute ethanol      1hr.
5.	Absolute ethanol      1hr.
6.	Absolute ethanol      1hr.
7.	Ethanol: xylene        1hr.
8.	Xylene                1hr.
9.	Xylene                1hr.
10.	Wax                   2hr.
11.	Wax                  3 1/2 hr.


OCT Cell Block Method

Reference:
Arisio (1989)

Solutions:

1.	Methacarn fixative
Methanol		75ml
Chloroform 		20ml
Acetic acid		5ml

2.	OCT Compound 4583 (Miles)

Procedure:

1.	Centrifuge specimen and add 10ml methacarn fixative.
2.	Mix and allow fixing for 1-2min.
3.	Add 0.5-1ml OCT compound to the fixative.
4.	Cap the tube and gently agitate the tube until the OCT clots.
5.	Centrifuge the tube, remove the supernatant and add fresh
fixative.
6.	Process the cellblock as usual.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of MICHELLE
SEAGLE
Sent: Wednesday, 11 July 2007 12:46 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Cell Block Procedure??


Our  Histo Lab is currently in the process of starting to do cell
blocks. We would be interested in knowing about any procedures that you
may like to share with us about doing these.
  We are interested in knowing the products and companys that you use to
perform these as well as the procedure. Any info that you may be able to
share with uswould be greatly appreciated.
   
  Thanks,
  Michelle Seagle HT(ASCP)
  Rutherford Hospital

 
---------------------------------
Don't pick lemons.
See all the new 2007 cars at Yahoo! Autos.
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