[Histonet] Re: How to troubleshoot a cytology slide withdehydration problems

[Tony Henwood]

Valerie,

The technique you are using is the re-hydration technique of air-dried
slides of Ng et al (Acta Cytolog 38(1):56-64, 1994)- 30 sec in saline
followed by fixation in 95% ethanol (at this stage, slides can remain in
ethanol before staining). Please note that it is a re-hydration, not a
de-hydration step. I have found that slides that have been air-dried for
longer than two days do not rehydrate well. Cells appear pink with large
air-dried, pale staining nuclei - lacking chromatin details.
Unfortunately there is little you can do in these cases.

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 Valerie
R.
Sent: Saturday, 1 May 2010 10:49 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Re: How to troubleshoot a cytology slide
withdehydration problems


I forgot to include the complete protocol that my lab uses so you can
help me better:

1.After dehydrating in sodium chloride for 25 seconds you have to dip
the slides in alcohol 95. 2. 10 dips in water then 1.30 minutes in
hematoxylin, it used to be 1 min, then rinse in water. 3. 1 min in rinse
solution 4.10 dips in water. 5. 1 min in bluing solution 6. 10 dips in
water. 7. 10 dips in alcohol. 8. 10 dips in alcohol. 9. 1 min in orange
solution 10. 15 dips in 95 alcohol. 11. 15 dips in 95 alcohol. 12.  3.15
mins in EA solution 13. The slides are then dipped in 3 containers of 95
alcohol (20 dips each) 14. Slides are dipped in 3 containers of 99
alcohol (15 dips in each) 15. Then they are dipped in Isoxylene which is
a combination of xylene and 99 alcohol (15 dips) 16. And lastly Xylene
10 dips


When the slides do not absorb the h20 saline seems that the slides
absorb more EA than hematoxylin.  I don't know why.

On Fri, Apr 30, 2010 at 5:34 PM, Valerie R. <vrodriguez10 <@t> gmail.com>
wrote:

> I am an HTL but I have to stain pap smears. In the Papanicalou 
> procedure for fine needle aspirations I have to stain the slides in 
> sodium chloride first for 25 seconds in total (15 seconds in one 
> container and 10 in another container). This for me is the trickiest 
> part when staining cytology because if slides are not well dehydrated 
> in saline then the cells are not going to absorb the stains well, and 
> the result will be a pinkish slide, when it is suppose to look 
> blue-greenish.
>
> I had this problem today where I stained an entire rack of slides, and

> all the slides turned blue (which means they stained correctly) except

> 4 slides which turned out pinkish (which is a sign that they did not 
> dehydrated properly in sodium chloride (h20 saline).
>
> Is there a solution to this issue. Can these slides be re-stained?
>
> I know this newsgroup is for histology only but I would like to know 
> if histotech and cytotechs have encountered this issue in their labs 
> and how they ended up solving the problem.
>
> Thanks in advance
>
>
>
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