Variability of histology staining-request for input
Hi everyone, I am the director of a new small outpatient laboratory, and we have had a very persistent and recalcitrant problem with variability of staining (some sections light, others dark, especially on eosin but to a lesser degree hematoxylin). We are soaking slides in xylene for 3 minutes in each of 3 bins with agitation, then staining them on a Varistain 24-4 with a regressive stain. Our staining protocols have been:
1. 100% Alcohol 20 seconds
2. 100% Alcohol 10 seconds
3. 95% Alcohol 10 seconds
4. Tap Water 10 seconds
5. Hematoxylin 7 minutes
(Protocol Harris mercury free non-acidified type)
6. Tap Water 20 seconds
7. Acid Alcohol 2 seconds
8. Tap Water 20 seconds
9. Ammonia Water 2 minutes
10. Tap Water 10 seconds
11. 95% Alcohol 10 seconds
12. Eosin 15 seconds
13. 95% Alcohol 10 seconds
14. 95% Alcohol 10 seconds
15. 100% Alcohol 20 seconds
16. 100% Alcohol 20 seconds
1. 100% Ethanol 40 seconds
2. 100% Ethanol 40 seconds
3. 95% Ethanol 40 seconds
4. Tap water 60 seconds
5. Hematoxylin 10 minutes
( Protocol Harris mercury free non-acidified type)
6. Tap water (sink) 60 seconds
7. Acid Alcohol 2 seconds
8. Tap water (sink) 60 seconds
9. Ammonia water 2.5 minutes
10. Tap water 5 minutes
11. 95% EtOH 1 minute
12. Eosin 10 seconds
13. 95% EtOH 10 seconds
14. 95% EtOH 10 seconds
15. 100% EtOH 40 seconds
16. 100% EtOH 40 seconds
We have had the same problem with both protocols, and process mostly small skin biopsies.
Our acid and ammonia solutions are as follows:
1% acid alcohol
2000 ml of 70% EtOH
5 ml of HCl (hydrochloric acid)
1% Ammonia water
1980 cc tap water
40 ml of ammonium hydroxide
We have tried longer xylene pre-treatment with more agitation, and wiping off alcohol drops before removing the bin from the last alcohol, as well as various staining adjustments. We process most of our tissue on a short 3 hour cycle however believe this problem has persisted even on overnight-processed tissue.
I would very much appreciate any advise any of you can give me on this problem. Thanks very much and have a happy holiday! Alice Neumann M.D. Precision Pathology PC, Arvada CO alineumann@aol.com 303-432-7855
<< Previous Message | Next Message >>