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IHC and Special Stains To Identify Pathogens
IHC and Special Stains To Identify Pathogens
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Overview
In recent years, there has been an increase of pathogens findings in tissues. This presentation will
discuss some of the reasons for the increase of these pathogens in the past few years. The
presentation will also will give an overview of why some of these pathogens are invasive and the
complications associated with some of these diseases. Due to the histology department being tasked
to produce slides that are of highest quality to identify these pathogens, a discussion of good
histology techniques and stains that can help identify these pathogens will be reviewed.
Learning Objectives:
Webinar Transcription
Types of infections
These are some of the types of bacteria. The shape is important to the pathologists when they're
looking at these organisms under a microscope trying to decide what they are. The shape often helps
them decide what type of bacteria they're looking at.
Fungal infections can be superficial, cutaneous, subcutaneous, systemic, and opportunistic. A lot of
fungal infections aren't that serious, but they can be serious if you get in to the systemic version of a
fungal infection, especially after a surgery or if you're immunocompromised.
Here are your viruses. Viruses are also classified as one of the deadliest pathogen. They're very hard
to fight. There's not a lot of things we can do to fight them, other than vaccinate, and there are some
antivirals out there that can help.
Protozoa are also on the list of pathogens. These can be very deadly as well. They're usually found in
tropical areas, and they can be very dangerous when they infect water supplies. There were cases of
water supplies, like swimming areas, where people dove in to water and got a protozoon that came
in to their body and they ended up dying because of the infection.
So, how do you treat patients that have become infected with pathogens? Well, for fungal infections,
you can treat with antifungal medication. Bacteria and protozoon are often treated with antibiotics.
Viral infections can be treated with antivirals if they're available. The common theme for all the
treatments is that it has to happen quickly, so proper ID of the pathogen and speedy ID of the
pathogen is needed.
How do you get these pathogens? Well, you have to have some sort of contact with it. You can
breathe it in. You can touch it. You can eat it, if it's a contaminated food source. They can spread
from animals to humans or human to human. A very big source of pathogens is mosquitoes and
ticks.
We know that pathogens can be deadly, and complicating the matter is that we have a lot of people
out there that are immunocompromised due to cancer treatments or transplants or HIV infection.
We also have an increase in pathogens that are resistant to antibiotics. So, again, correct diagnosis
and a fast diagnosis helps combat this.
The gold standard for identifying pathogens is microbiology, microbiology culture. It needs to happen
quickly for the best patient outcome. If you're going to treat the patient, you need to find out what
the causative agent is so you can treat it properly, so it needs to happen quickly. Histology can
diagnose an infection when the pathogen cannot be grown in culture or it's difficult to grow or it
takes a long time to grow. If it takes more than a day or two, or up to a week, the patient may not
have that much time, or the infection may get ahead of the medication, so that the medication isn't
helping.
What happens when it takes a culture several days or a week to grow? What do you do? What
happens if the ID of the pathogen needs to happen on the operating room table? Well, here is where
histology can come in to play. Just a couple examples of where histology can help, pneumocystis is
notoriously difficult to grow in culture. You can do a diff quick on a bronchial washing, or you can do
GMS on the tissue that comes in to the lab. If the patient is on the operating room table, and the
surgeon suspects that the patient has some sort of necrotizing fasciitis, which can be very deadly,
the surgeon can leave the patient's surgical wound open to the air, which will help it heal, rather
than close the wound. So, you can do a diff quick on the sample from the operating room within 20
minutes, and then the patient can either have a closed surgical wound and be treated for some
other pathogen, or leave the wound open so it can heal.
Some of the reasons that we are seeing an increase in the incidence of pathogens in pathology, and I
touched on this before, was treatment of patients with cancer, treatments for organ transplant, HIV
positive patients. More and more of these patients are coming in and having biopsies to find out if
there's something wrong with the transplant or if they're being treated for cancer and they present
to the office with some sort of skin rash or some other symptom that they may be come in to contact
with a pathogen that is not normally seen in the human population. Like I said before, leprosy has
kind of re-appeared because of world travel, and tuberculosis, again, also reappearing due to a lot of
world travel. Again, there's this controversy on vaccinations. Should you or shouldn't you? It's
causing some of these once thought eradicated diseases to be seen again in the labs. Pathologists
need to be aware of this, and the lab needs to brush up on those stains that will prove that those
pathogens are infecting the patient, so that they can give a positive identification.
Histology of pathogens – H&E
There are some pros and cons for both culture of a pathogen and histology of a pathogen. Culture is
relatively non-invasive. You can get a specimen from blood, phlegm, anything that can be swabbed
off of a patient. Histology is more invasive. You usually need some sort of surgical biopsy or a
fine-needle aspiration to remove the sample from the patient. The turnaround time for microbial
culture can be one to fourteen days. I've read recently where there is some automation coming to
the microbiology lab, and this can take it from a day to a number of hours. Not being a
microbiologist, I'm not totally abreast of those technologies, but histology has a relatively quick
turnaround time, depending on the size of the specimen. If you get a small biopsy in to the lab, you
can usually turn that specimen around within eight hours. If you have a larger specimen, it might
take overnight. A failed culture in micro means you've missed some time. If you had to culture a
specimen overnight or two days or four days or fourteen days, you've lost that culture, and you've
lost all that time, and you also need to go back to the patient and retrieve a sample. A failed stain in
histology just requires you to re-cut and re-stain, so as long as there's tissue available in the block,
you can still get another sample. One of the cons for processing histology is some of those organisms
may not survive. Leprosy is an example. Leprosy will survive the processing steps, but when you go
to stain the leprosy organisms you have to pretreat it with a very specific solution.
Fungal and bacterial infections in tissue are often detected with a good quality H&E. Frozen sections
you can do a diff quick to get a rapid diagnosis. Infectious agents can be classified by special stains.
You can also use IHC. IHC can help you find out if there's a viral infection.
MODERATOR: Well, it says specimens, but I think she may have meant reagents?
MS. MCMAHON: I guess if you're talking about creating your—I guess it would depend. You run in to
a problem if you're creating your own in-house reagents, because of the classification of IHC. So, if
you're in a research lab, and you can do it, great. But, if you're in a clinical lab, and you're billing for
these, you're going to run in to some issues with billing for antibodies that aren't in vitro diagnostic
or IDD or ASR status. You're just going to run in to that issue. So, if you can create antibodies that are
specific to these organisms, great. But, I would just be careful. I live in New York. We are inspected by
the Department of Health of New York State, and we are not allowed to do any of that. It's
considered fraud in the eyes of New York State for some reason. So, we just have to be careful of the
regulatory and the billing aspects of it. But, if you can do it, I don't see a problem with doing it.
Is there a life expectancy or expiration date for control slides? Do freshly-cut control blocks perform
better than pre-made slides that have maybe been prepared years prior? Is there a difference? So,
the first part was is there a life expectancy or expiration date for control slides?
MS. MCMAHON: Well, I think it depends on who you talk to. I've worked with quite a few
pathologists who have said that, yes, there is an expectancy or life expectancy to these slides,
because they're cut, they're exposed to oxidation, and having to do with the proteins. In the same
sense, I have stained control slides that have been years old and they've still worked. So, I think what
you need to do is consider—do a test yourself. Cut one, stain it, and then cut another one, and just
hide it somewhere for a year or so. Then, stain it again and see if it works. But, if you do have a good
set of control tissue that is stained that you can consult while you're looking at your controls on a
daily basis, you can maybe see if the antibody specificity of the control tissue is starting to fall off. I
know that there's a lot of this when you deal with some of the ERPRs and HER-2s. They don't like the
slides to be very old. Obviously, if you're doing any kind of in situ work you don't want your slides to
be very old, because that RNA/DNA can degrade. But, I guess it depends on who you talk to. I've
stained slides that have been very old, and they've worked just fine. It's probably antibody-specific,
so that's another thing to consider, but again, have a good set of control slides that you can look at
for reference when you're checking your daily control slides to see if maybe this stain isn't as dark as
it used to be when I cut the slide fresh. You can see some trends, maybe some antibody specificity is
dropping off.