[Histonet] Van Gieson question....

Thu Jul 5 16:58:39 CDT 2007

The VVG ELASTIC Stain is used for the demonstration of
pathologic changes in elastic fibers.  These include
atrophy of the elastic tissue; thinning or loss that
may result from arteriosclerotic changes; and
reduplication, breaks, or splitting that may be used
to demonstrate normal elastic tissue, as in the
identification of veins and arteries, and to determine
whether or not the blood vessels have been invaded by
tumor.

PRINCIPLE OF TEST:									
The tissue is over-stained with a soluble lake of
hematoxylin-ferric chloride-iodine.  Both ferric
chloride and iodine serve as mordants, but they also
have an oxidizing function that assists in converting
hematoxylin to hematein.  The mechanism of dye binding
is probably by formation of hydrogen bonds, but the
chemical groups reacting with the hematoxylin have not
been identified.  Because this method requires that
the section be over-stained and then differentiated,
it is a regressive method.  Differentiation is
accomplished by using excess mordant, or ferric
chloride, to break the tissue-mordant-dye complex. 
The dye will be attracted to the larger amount of
mordant in the differentiating solution and will be
removed from the tissue.  The elastic tissue has the
strongest affinity for the iron-hematoxylin complex
and will retain the dye longer than the other tissue
elements.  This allows other elements to be
decolorized and the elastic fibers to remain stained. 
Sodium thiosulfate is used to remove excess iodine. 
van Gieson solution is the most commonly used
counterstain, but others may be used.

COMMENTS AND PRECAUTIONS:						

NOTE: It is not necessary to remove mercury deposits
if tissues have been fixed in a mercury-containing
fixative, since they will be removed by the staining
solution.

NOTE: To prepare Working ELASTIC Stain, the reagents
must be added in order given.  Prepare in a flask,
swirling as each ingredient is added. This is
critical!  DO NOT JUST ADD IN A GRADUATED CYLINDER AND
THEN POUR INTO A COPLIN JAR.  This technic will give
inconsistent results!! 

Check sections microscopically for adequate
differentiation.  Repeat till desired end-point. 
Sections may be left a little darker than actually
desired, since they become slightly lighter after the
iodine is removed in sodium thiosulfate.  If
differentiation has been carried too far, the sections
may be restained, provided they have not been treated
with alcohol.  ***Do not leave in van Gieson Solution
for extended time.  ***The picric acid component
decolorizes the elastic fibers.

SPECIMEN REQUIREMENTS:							
Specimen consists of any well-fixed tissue.  10%
formalin or Zenker's preferred.  Cut section 3-5
microns.

SOLUTIONS:

1.	Hematoxylin Solution: "A"

2.	Ferric Chloride Solution: "B"

3.	Iodine/Iodide Solution: "C"

Working ELASTIC Stain:
Solution "A"       22.0 ml or 30.0 ml 
Solution "B"	  8.0 ml or 12.0 ml
Solution "C"	  8.0 ml or 12.0 ml

4.	Differentiation Solution:

5.	5% Sodium Thiosulfate Solution:

6.	van Gieson Counterstain:

STORAGE AND STABILITY:
7.	Store Solutions in a dark place at room temperature
(18-26°C).  Hematoxylin Solution: "A", Ferric Chloride
Solution: "B", Iodine/Iodide Solution: "C",
Differentiation Solution are stable for 18 months.  5%
Sodium Thiosulfate Solution and van Gieson
Counterstain are stable for 12 months.

QUALITY ASSURANCE AND CORRECTIVE ACTION GUIDELINES:	
Use a section of aorta embedded on edge or a cross
section of a large artery.  Check control slide
microscopically after differentiating in ferric
chloride for proper result. 

STANDARD STAINING METHOD:							
1.	Deparaffinize and hydrate to water.
2.	Place slides in Working ELASTIC Stain for 15 to 30
minutes.  (Working ELASTIC Stain is good for at least
24hrs).
3.	Wash sections in running water until no excess
stain remains on slides.  Tissue sections should be
intense black and slides will look dirty where they
have been immersed in the staining solution.
4.	Dip sections in differentiation solution 15-30
times and transfer to tap water. Check sections
microscopically for adequate differentiation.  Repeat
till desired end-point.  Sections may be left a little
darker than actually desired, since they become
slightly lighter after the iodine is removed in sodium
thiosulfate.  If differentiation has been carried too
far, the sections may be restained, provided they have
not been treated with alcohol.
5.	Wash well in running water.
6.	Place slides in Sodium thiosulfate Solution for 1
minute.
7.	Wash in tap water.
8.	Counterstain in van Gieson's Solution 2-5 minutes.
*Longer time as solution ages.
9.	Differentiate in 95% alcohol (2 changes), followed
by dehydration in absolute alcohols.
10.	Clear in several changes of clearing agent.
11.	Mount with resinous mounting media.

RESULTS:											

Elastic fibers		    Blue-black to black
Nuclei			    Blue to black
Collagen		            Red
Other tissue elements   Yellow

REFERENCES:										
Sheehan, D.C. and Hrapchack, B.B., Eds. Theory and
Practice of Histotechnology, 2nd Ed. Mosby, St. Louis,
MO. Pp. 196-197

E. B. Prophet, B. Mills, J.B. Arrington, L.H. Sobin,
A.F.I.P. Laboratory Methods in Histotechnology, 1994,
pp134

Histotechnology A Self-Instructional Text, 2nd Ed. F.
L. Carson, pp.138-139, 1996
Modifications by A. Allison, BIOCARE MEDICAL, Walnut
Creek, CA, 2001

Akemi Allison-Tacha BS, HT (ASCP) HTL
President
Phoenix Lab Consulting & Staffing
Specializing in Histology, SS, IHC, & TMA
Tele: (925) 788-0900
E-Mail: akemiat3377 <@t> yahoo.com

--- AGrobe2555 <@t> aol.com wrote:

> Good afternoon,
> We are doing an Elastic Van Gieson stain on FFPE
> tissues and have come up  
> against a bit of a question.  While the elastin
> seems to be staining quite  
> nicely, the nuclei either are very faintly stained
> or not at all.  We  have 
> shortened the differentiation time in the FeCl, with
> little  difference. Any ideas?
> Thanks, 
> Albert  
> 
> Albert C.  Grobe, PhD
> International Heart Institute of Montana Foundation
> Tissue  Engineering Lab, Saint Patrick  Hospital
> 
> 
> 
> 
> 
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