Subject: Vacuole artifacts
HELP!
I am processing small mucosal GI biopsies at a reference path lab
with a quick 24 hour turn around time, so we use a quick schedule and have
had strange vacuoles appear in the mucosal and connective tissue of many
specimens. Originally, the vacuoles were identified in our longer-processed
tissues. These intense cytoplasmic and nuclear vacuole artifacts were found
in glandular epithelium and within the stroma. The three cases were from a
thyroid gland, a uterine biopsy, and a stomach biopsy. These large vacuoles
were located everywhere throughout the thyroid and uterine slides, but only
on one level of the stomach biopsy. However, now the vacuoles are found
throughout almost all of the GI specimens which are run on a short three
hour cycle.
It isn't apparently related to reagent changes, since the artifact appears
almost all of the time, regadless of how new the reagents are. Furthermore,
the artifact is showing up in almost all mucosal biopsies at sometime or
another. Below is our processing schedule. All tissue is 10 %
NBF-fixed--usually for at least 12 - 20 hours prior to processing.
If anyone has ideas to alleviate, eradicate, destroy and/or fix this
problem, I'd be grateful. I have included an image on the Histonet.org image
database titled "Vacuole Artifacts" to show you what the problem is. Any
suggestions or orbservations are appreciated.
Thanks also to Dr. Linda Margraf, Dr. Herb Hagler and the University
of Texas Southwestern Medical Center for the website--what a great idea!
Jon Kerr
Routine processing steps all done at Vacuum pressure
15 inches Hg
Station: Reagent Time (min) Temp Pmax (psi)
1 10% NBF 35 40* C. 4
2 10% NBF 30 40* C. 3
3 70 % alcohol 30 ambient 1
4 80 % alcohol 30 ambient 2
5 95 % alcohol 25 ambient 1
6 95 % alcohol 30 ambient 3
7 100 % alcohol 25 ambient 4
8 100 % alcohol 30 ambient 4
9 Xylene 25 ambient 3
10 Xylene 30 40* C. 2
11 Paraffin 30 56* C. 7
12 Paraffin 45 56* C 7
13 Paraffin 45 56* C. 7
End of tissue processing, tissue may be taken out of processor
Rapid processing steps for small GI mucosal/ liver/ prostate biopsies.
Station: Reagent Time (min) Temp Pmax
3 70 % alcohol 10 ambient 1
4 80 % alcohol 15 ambient 1
5 95 % alcohol 10 ambient 1
6 95 % alcohol 15 ambient 1
7 100 % alcohol 12 ambient 1
8 100 % alcohol 12 ambient 1
9 Xylene 15 ambient 0
10 Xylene 20 40* C 3
11 Paraffin 30 56* C 7
12 Paraffin 20 56* C 7
13 Paraffin 20 56* C 7
Dear Jon Kerr: On my opinion there are two possible causes of the vacuolar
artifacts:
Gas bubbles: We've seen these many times in fixed tissues, e.g. samples
that had been collected in too small bottles in which the final ratio
=specimens' volume/ 10 % formalin volume was under 1/20; formalin was
precipitated so having lost its original fixing power; the bottles had been
preserved refrigerated, so banning the pentration of formalin into the
tissues, etc., etc. However, when we see gas bubbles we usually detect other
signs indicating post-mortem autolysis as well, such as rods (=bacilli),
partial or complete loss of histologic structure, absence of infammation,
lack of red blood cells, etc. That's why it is so important that the
technician and the pathologists work together, in my case I started working
as histotechnician for some years and ended exerting Vet Pathology.
Freezing artifacts: We've observed these in tissues processed after
freezing. In these cases the submitted organ samples were already thawed, so
we had to find out by ourselves the origin of the microscopic artifacts.
Tissues that have been frozen show either vacuoles or empty irregular clefts
that may cause an appearance of disrupted or torn material. The remaining
tissue looks disorganised.
I hope having been useful, sincerely yours
Agustin Jose Venzano Halliburton
Veterinary Pathologist
INTA Castelar, Argentina