I just bought a new H&E stainer. How do I validate the new stainer, per CAP? I already traded in my old stainer, so I cant compare the two machines.

Congratulations on buying a new stainer. Don’t worry, there’s no need to compare the new machine with the old machine. After all, what proof do you have that the old machine was producing high quality H&E stained slides?

The CAP Standard ANP.23045 is a VERIFICATION, not a validation: “The performance of all instruments and equipment is verified before use.” In the Note, it says: The function of all instruments and equipment is verified upon installation and before use to ensure that it will function as intended. Instruments and equipment function should be re-verified after scheduled maintenance, after major instrument repairs, or if it is relocated. Instruments and equipment include tissue processors, microtomes, cryostats, automated strainers (H&E, Histochemical, and IHC), coverslippers, cassettes, and slide label printers, and digital image scanners.

So what you need to do is first write up a policy that states you are going to do all of the above. Then, you need to create a checklist where you are recording doing the verification testing, and what the results are.

The reason for this should be fairly obvious. You should not pour all the solutions into the H&E stainer, and put your rack of patient slides on the machine, WITHOUT HAVING FIRST tested the run, to make certain that the quality of the H&E stain is acceptable for diagnosis.

If you don’t know what an acceptable H&E looks like, grab a Histotechnology reference book (Carson, Bancroft or Brown), and look at the photos. Look at the written descriptions of good quality H&E. Read the explanations of poor staining and the suggestions on how to improve staining. Why do you want “reference book” quality H&E, instead of “well, this is what my pathologist likes”? What if your lab was sued about the quality of your work, and had to go to court? The opposition attorneys would show photos of acceptable quality, as established by ASCP, NSH and/or HistoQIP (proficiency standard for US laboratories). Would your lab be able to meet these standards?

Run some test slides through, made up of several types of tissues. Definitely tissue that you process a lot (e.g., breast, skin, GI biopsy, placenta, whatever), but also tissue that would help you see the quality of the H&E. I like to use small or large intestine. There are several types of nuclei (epithelial, muscle, lymphoid), various WBC (eosinophils, plasma cells), and several types of connective tissue and other tissue components to assess eosin staining (muscle, collagen, epithelial cells, mucin cells).

For your verification sheet, start with a grid that would list all the solutions on the machine for the H&E.
– Write down what solution is in each container.
– Write down the time spent in each container.
– Record the temperature if appropriate, e.g., running water.
– Record the pH of appropriate solutions (e.g., water, hematoxylin, eosin, acid rinse).
– Record date and testing number.
– Have a place to record tech doing the testing, and pathologist doing the assessing.

Next, have some YES and NO questions on your verification sheet, where if everything is looking good, it is recorded as YES, and if there is a problem, it is recorded as a NO. Some examples would be:

OVERALL QUALITY (10 x objective):
– Is the staining even?
– Are the nuclei standing out darker than the background?
– Is there absence of splotches (e.g., water droplets)?

HEMATOXYLIN STAINING (40 x objective):
– Is the nuclear wall dark and crisp?
– Is the chromatin pattern stippled, not smudgy?
– Is the nucleolus (if present) a red to purple color?
– Are other cells expected to stain bluish doing so (e.g., plasma cells, pancreatic ascinar cells)
– Are the mucin cells clear of color (or you may have to phrase this “a pale blue color” if you are using a Gill hematoxylin and/or are not doing a regressive stain)?
– Are the muscle and connective tissue cells free from a bluish color?

EOSIN STAINING (40x objective):
– Are RBC the darkest red?**
– Are eosinophil granules, Paneth cell granules, and xymogen granules as dark, or nearly as dark, as RBC?**
– **(note: if you are using a fixative with acetic acid, these components will be lysed, and this question should be dropped.)
– Is muscle a medium shade of pink, and collagen a light shade of pink?
– Can muscle be differentiated from collagen? (check out medium size blood vessels)

If there are any NO answers, then read up on what could be the problem, make changes to the H&E protocol, run another rack of test slides, and record these results. Continue doing this, until you finally get good quality H&E. This is now your H&E staining schedule.

You might want to continue doing a test run every morning, so that you find out when you need to rotate or change solutions. For example, every 200 or 400 slides.

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