Persistent Problem of Formalin Crystals in the H&E stained tissue sections

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Rajveer Singh P...
Rajveer Singh Pawaiya's picture
Persistent Problem of Formalin Crystals in the H&E stained tissue sections

For last several months I am trying to get rid of formalin crystals in the H&E Stained tissue sections. I have tried Ammonia solutions, washing well in water even for two days continuously under running tap water, but nothing seems to have worked.
I use neutral buffered formaldehyde (10% solution of commercially available formalin; i.e. effectivelt 4% solution). We have a centralized histological laboratory where about 200 to 300 sections blocks are cut daily and stained dialy using automatic tissue processor and stainer.
Persistent presence of formalin crystals in finally stained slides are annoying and mar all the charm of seeing the section under microscope. More so when I am working on cancer slides, these artifacts/formalincrystals totally mask the nuclei and pose problem in identifying mitotic figures, as also to get any photo quality field.
Is anybody more experienced there to help me out of this problem. Because all stake holders are really annoyed getting crystal soiled stained slides. Please help get me some solid solution.
Dr RVS pawaiya
Scentist, Division of Pathology
IVRI, Izatnagar - 243122, Bareilly, U.P., INDIA

Arvind Singh Pundir
Arvind Singh Pundir's picture
rvspawaiya wrote:

rvspawaiya wrote:

For last several months I am trying to get rid of formalin crystals in the H&E Stained tissue sections. I have tried Ammonia solutions, washing well in water even for two days continuously under running tap water, but nothing seems to have worked.
I use neutral buffered formaldehyde (10% solution of commercially available formalin; i.e. effectivelt 4% solution). We have a centralized histological laboratory where about 200 to 300 sections blocks are cut daily and stained dialy using automatic tissue processor and stainer.
Persistent presence of formalin crystals in finally stained slides are annoying and mar all the charm of seeing the section under microscope. More so when I am working on cancer slides, these artifacts/formalincrystals totally mask the nuclei and pose problem in identifying mitotic figures, as also to get any photo quality field.
Is anybody more experienced there to help me out of this problem. Because all stake holders are really annoyed getting crystal soiled stained slides. Please help get me some solid solution.
Dr RVS pawaiya
Scentist, Division of Pathology
IVRI, Izatnagar - 243122, Bareilly, U.P., INDIA

its a common problem at times with the tissue containing blood, Formalin pigment (also refered as acid formol hematin ) will form in bloody tissues in the presence of  an acid (formic acid). generally seen in the blood vessels and where there is blood ,you can confirm that the pigments on sections are formalin pigments by examining the section under polarized light microscope, as Formalin pigment is birefringent they will appear bright on a dark background to get rid of the pigment from the sections, treat the de-waxed sections with saturated alcoholic picric acid for about 30 minutes, then wash the sections very well to remove the yellow  stain , then proceed for regular H&E hope it helps out
other method is
ammonium hydroxide in ethanol,  add 15 mL of 28% ammonium hydroxide to 50 mL of 95% ethanol, add solution for 1 hour.  Wash well in tap water
 refer: Winsome Garvey (J.Histotechnol. 13(3):163-165, 1991)where he  has suggested three methods for its removal:
 
go to the followink link :   us.geocities.com/tonyhenwoodau/h6.htm
 

excalibur
excalibur's picture
I question that the crystals

I question that the crystals are formalin pigment. Formalin pigment is usually only seen in tissue that have sat in formalin for months or longer.
If these "crystals" are on all the slides, the cause is most likely the lack of filtering the hematoxylin. A shimmering film forms on the surface of hemat overnight and it should be filtered daily before the first staining run. As the first racks of slides hit the surface, the film is broken into many pieces and as the rack leaves the dish, the fragments coat the slides and some do not wash off.

peter36
peter36's picture
I have found that in neutral

I have found that in neutral buffered formalin you can get buffer pigment in your sections. Its looks like formalin pigment except more chunky. The buffers used in nbf transfer along the processing schedule and being more insoluble in higher conc's of alcohol tend to precipate out and transfer to the tissue. the only way to eliminate is to replace your sols more often and clean your containers with hot water flushes or u can use non buffered formalin which is no problem unless you fix tissue for extended times. take a sample of your last alcohol in the processing step and add water should be clear right, if it turns cloudy then you have buffer transfer and this will deposit on your sections looking very similar to formalin pigment especially in bloody areas of tissue.
pete