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

Sat May 1 04:16:27 CDT 2010

Dear Valerie,
I can only agree that Papanicolaou stain is very tricky, having 20 yrs experiencies with it! looks very simple but you need to know some basic rules. 
first of all the slides should be hydrated at the beginning of staining procedure since the hematoxylin is water based stain!
it seems to me thatyour slides are not enough hydrated, I never heard about sodium chloride solution as a hydrating but I guess cannot harm, however then you put slides in alcohol it's a bit confusing! and 25 seconds is a very short time for any incubation!
beginning of a staining procedure depends on a fixation of the slides! in a case of a wet fixed slides you can proceed directly to staining (95% alcohol) if the slides are wet fixed and then dried you need to rehydrate them before staining and if you have spray fixed slides you need to remove the wax from slides before staining!

my suggestions:
1. 
- leave slides in a sodium chloride longer, perhaps 2-3 minutes
- alcohol 95%
- alcohol 70%
- water at least 3 min better 5 minutes, you'll probably need to shorten staining time in hematoxylin to 1 minute because the slides will be better hydrated and ready to accept stain.
- staining time in EA solution is too short to allow proper staining and this is probably the reason to get reddish staining, you should leave the slides in EA 5 minutes since only then the light green will start to stain.
hope this help! do you stain by hand or in a stainer? how often do you change stains and other solutions? you should also be very carefull to have the proper level of all solutions in a dishes! the basic rules is to keep the level of stains bellow the level of rinsing solutions.
let me know about your staining results or even better to send me a picture! sometimes you can get the all redish staining also becouse of improper fixation! you can prepare your proprely fixed slide and stain it to see the results!
best regards!
Irena 

> From: vrodriguez10 <@t> gmail.com
> Date: Fri, 30 Apr 2010 17:48:58 -0700
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Re: How to troubleshoot a cytology slide with dehydration 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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