Fixation on Histology

Pros:

• The technique is rapid due to over staining
• Hematoxylin is more concentrated
• Differentiation using acid alcohol requires a few seconds.
• The techniques good for frozen tissues

Cons:

• If differentiation is incomplete or omitted, it can or will prevent the uptake of eosin.
• The technique requires additional step.

In a progressive staining, the cell tissue is left long enough in hematoxylin until it reaches the desired endpoint. In this technique, the chromatin is the primarily stain instead of the cytoplasm. There are 2 common hematoxylins used in progressive staining: Mayer’s and Gills (I, II, III). Here are pros and cons of using this technique.

Pros:

• The hematoxylin only stains to a desired intensity
• There are no additional steps since differentiation is not needed
• Hematoxylin is less concentrated
• The technique works slowly to avoid overstaining
• Technique works well on both frozen and FFPE tissues

Cons:

• The technique is a slower staining process

 Now let’s investigate what makes one hematoxylin different from another, and how these differences play a major part of the decision making.  Gills and Mayer’s is made up of aluminum sulphate 52g, 75% water, and 25% ethylene glycol, which keeps Gills more stable, no precipitation, no filtering. Whereas Harris is made up of aluminum ammonium sulphate 90-100g, 95% water and 5% ethylene alcohol. Harris hematoxylin produces well delineated crisp nuclear staining, however due the components in Harris, it may require weekly or daily filtering to remove precipitation caused by oxidation.  There are other factors that play a role in choosing the best technique. The question of choice is very subjective. Ultimately, it depends on the pathologist’s expectations, preferences, your lab’s workflow and tissue types. I interviewed one of our pathologists, Dr. Gerald Chu, who I worked with to develop an H&E protocol that fit his expectations, preferences, and our lab workflow.  Dr. Chu prefers to see a crisp cell definition, and the color contrast between the components of the tissue.  We started off with regressive technique using a progressive reagent (GillsIII).  We experienced overstaining, and no contrast between some of the tissue components, so we tweaked the timing between the hematoxylin, differentiation, and Eosin to reach our preferred endpoint. However, we still didn’t get the desired result, so we switched to a regressive technique using Harris hematoxylin.  This change worked perfect for Dr. Chu’s preferences BUT did not support our lab’s workflow.  The constant weekly filtering of the Harris hematoxylin caused inconsistency in the staining pattern (see image).  I’m currently in search for a hybrid hematoxylin, which hopefully will help me solve this issue. 

To answer the question – which technique is best?  Well, in the end it is very subjective and depends on the pathologist’s preferences and your laboratory workflow.  My suggestion is to run each technique using multiple different types of tissues and provide your pathologist with several options.  At the end of the day, “Happy Pathologist, Happy Lab”.