What is glyoxal? And, more importantly, why should you care?

By: Sheila Criswell, PhD, HTL (ASCP), QIHC (ASCP)

“What is glyoxal?” If you ask most histotechnologists, they will look at you blankly, unless, of course, they have recently been cramming for the HT or HTL registry examination. Even then, the response is typically something like, “Isn’t that one of those old fixatives that histology labs don’t use anymore?”

The actuality is that glyoxal does not fall into the same category as mercuric chloride or potassium dichromate, fixatives long used, but now exiled from the routine histology lab due to health concerns. Instead, glyoxal may be considered an “up-and-coming” fixative. In fact, a few labs already use it routinely.

“But we use formalin. That’s all anyone ever uses, right?” For the most part, yes, but if you’ll think back on exactly which permissible exposure limit badges you have to wear occasionally to document acceptable exposure levels to CAP and your safety departments, you’ll note that formalin and xylene are probably the most common chemicals monitored. Formalin must be used with a functional grossing or chemical hood to avoid vapor exposure. Most of us who have loaded a tissue processor with cassettes know how potent those fumes are as we hold our breath and squint our eyes when removing the lid from the formalin container in order to transfer cassettes into the processing well.

The nifty thing about glyoxal is that it does not produce those horrific fumes that formalin does, and in fact does not require any extra ventilation when grossing tissues. A person could use a regular lab benchtop to do grossing. Now, did anyone just hear me say that glyoxal is not harmful at all? Nope, not what I said. Glyoxal is still a fixative, and just because it does not give off vapors does not mean that it won’t try to fix your own skin and mucous membranes if it comes into contact with them. Gloves and eye protection are still 100% necessary when handling this fixative.

“Well, that’s super cool! If we don’t need a grossing hood and it doesn’t stink, why aren’t we using it already? There MUST be some downsides!” Unfortunately, yes, there are a couple of disadvantages.

The H&E and special stains used on glyoxal-fixed tissues are extremely similar to those seen with tissues fixed with formalin, but are not exactly the same. So, you have to make sure your pathologists are comfortable interpreting the tissues with these minor differences. The most striking thing is that tissues fixed with glyoxal display fewer shades of pink on H&E. The reduction in shading is due primarily to the acidic pH of glyoxal. Whereas formalin is buffered near neutral pH of 7, glyoxal functions best at a lower pH between 4 and 5. The low pH causes red blood cells (RBCs) to lyse and changes the affinity of eosin to tissue elements. Eosin is typically attracted to the alkaline components in tissue, but because glyoxal is acidic, tissue basophilia is reduced. In the image below, the top 2 panels demonstrate differences in staining of feline uterus due to lysing of the RBCs in the glyoxal tissues. The RBCs are clearly seen in red in the formalin-fixed tissues. In the glyoxal-fixed tissues, the RBCs are still faintly visible on high power, but due to loss of hemoglobin, no longer take up stain. The middle 2 images are testes/seminiferous tubules. The pink connective tissue between the tubules is more recognizable in the formalin-fixed section than in the glyoxal-fixed section because of altered affinity of collagen for eosin. However, the bottom 2 images of spermatogenic cells captured at 100X demonstrate that chromatin, nuclear membranes, and nucleoli are actually better defined and less smudgy in the glyoxal-fixed tissues.

Our lab recently performed a project titled “Glyoxal: a proposed substitute for formalin in H&E and special stains” by Victoria DeJarnatt and Sheila Criswell and were fortunate to have this worked published online in the Journal of Histotechnology https://doi.org/10.1080/01478885.2020.1830664. We found that the special stains we assayed worked very well with glyoxal-fixed tissues.

The second disadvantage in using glyoxal fixatives is that immunohistochemistry (IHC) assays do not function exactly as they do with formalin-fixed tissues. Most labs rely heavily on IHC assays for tumor diagnosis, pathogen identification, and quantification of protein expression. Because glyoxal fixation does not cross link proteins in the same way or to the same degree that formalin does, antigen retrieval is different, and in many cases may not be needed at all. Richard W. Dapson is reputed to be the leading expert in glyoxal fixation. Work he and colleagues published in 2006 examining IHC assays with glyoxal can be found here: https://doi.org/10.1080/01478885.2006.11800879.

In our recent work, we initially attempted to compare IHC assay results between formalin-fixed and glyoxal-fixed tissues, but because we were using fresh organs obtained from routine feline and canine sterilization procedures, and our antibodies were against human targets, we determined that an objective comparison was not possible at the time. We hope to revisit this question in the future to see if a routine histology lab might be able to easily incorporate glyoxal-fixed tissues into IHC procedures with minimal modifications.

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