What magnifications do pathologists typically work with?

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PeteM
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What magnifications do pathologists typically work with?

#1 Post by PeteM » Sun Jul 25, 2021 1:50 am

A friend's father recently had a skin cancer removed and said the surgeon briefly stopped to check the margins by viewing the sample (per the surgeon) at 10x and decided to take a bit more. This was apparently an old school compound microscope - nothing fancy.

Would this be 100x (a 10x objective) rather than 10x? I have zero experience, other than casually viewing prepared pathology slides. It seems to me that a stain applied to a recently-excised sample might be fairly definitive in terms of margins viewed at 10x - and some sort of gross determination could be made at low magnification even without staining. But it also seems to me (based on that ignorant viewing of pathology slides) that something a lot closer to 100x would be required to see individual cell abnormalities - and 400x even better.

Anyone know what the typical practice is (magnifications, staining etc.) among pathologists?

lorez2
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Re: What magnifications do pathologists typically work with?

#2 Post by lorez2 » Sun Jul 25, 2021 4:49 am

A typical pathology microscope will be equipped with 2X, 4x, 10x,40X, and 100X objectives. Some also use 1X, 20X, 50X, and 60X. All the objectives are of higher quality. When you are considering the caliber of scopes they are using even the base objectives are very good, but many are using apo and fluorite. Every Dr is using what is necessary for the work they are doing.
Nikon 80i

PeteM
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Re: What magnifications do pathologists typically work with?

#3 Post by PeteM » Sun Jul 25, 2021 5:05 am

Would you know what magnification is typically used to distinguish normal from cancerous cells? I was surprised to hear 10x (1x objective, 10 eyepieces?) and would imagine a 1x or 2x would be more for scanning the slide than cell type ID.

Tom Jones
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Re: What magnifications do pathologists typically work with?

#4 Post by Tom Jones » Sun Jul 25, 2021 3:24 pm

I can't say for tissue, but for body fluids, scanning with a 10x objective is often enough to initially identify the presence of malignant cells, although no one I know would stop at that low a magnification, even if they thought the sample was negative. They tend to be larger, have irregular shapes, large irregular nuclei, may be multinucleate, and stain differently from normal cells. Higher magnification is used to confirm and differentiate between reactive and malignant cells as necessary. Actual cell ID might be done by flow cytometry or one of the newer molecular methods. After that comes the hunt for the primary tumor.

PeteM
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Re: What magnifications do pathologists typically work with?

#5 Post by PeteM » Sun Jul 25, 2021 4:45 pm

Thanks, Tom. I was hoping you might see this and reply.

What puzzled me is that the surgeon told my friend that he scanned the bit he carved out "at 10x magnification." I thought that surely must have been at least 100x, unless staining alone was a clear indicator.

Tom Jones
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Re: What magnifications do pathologists typically work with?

#6 Post by Tom Jones » Sun Jul 25, 2021 8:54 pm

I understand dermatopathologists and dermatologists will sometimes use 1x-2x objectives. Remember, if they're only looking for clear margins, there is usually a fairly distinct difference in the stained cells between malignant and normal. If there are no malignant cells at the margin they quit cutting in that direction. They will already have done a diagnosis on a previous sample. Low mag allows a fast, bigger picture. That can be handy if the patient is under anesthesia.

PeteM
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Re: What magnifications do pathologists typically work with?

#7 Post by PeteM » Mon Jul 26, 2021 12:40 am

Thanks, Tom.

JGardner
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Re: What magnifications do pathologists typically work with?

#8 Post by JGardner » Mon Jul 26, 2021 5:27 pm

When my wife had one of these removed from her forehead, the doctor described the technique as "MOHS". I often see microscopes on sites like eBay and various dealers described as MOHS 'scopes, so this must be a fairly common thing.

apochronaut
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Re: What magnifications do pathologists typically work with?

#9 Post by apochronaut » Mon Jul 26, 2021 7:35 pm

Pathology was a principal driver behind the development of wider field microscopes after the war. F.o.v plays as much of a role as magnification and sometimes a lower magnification will be preferred in order to get a more complete f.o.v.

Mohs surgery examines tissue slices in depth in order to bypass the margins in the z axis.

Matador
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Re: What magnifications do pathologists typically work with?

#10 Post by Matador » Tue Jul 27, 2021 8:08 pm

Being a pathologist myself, I thought I can share my experience/observations:

Nowadays pathology scopes are typically equipped with 2X, 4X, 10X, 20X, 40X and some with an additionnal 1X and 63X objectives as well (the advantage is that the 63X is a dry objective, whearas a 100X is typically an oil immersion one, which is not as user friendly when you have to work with a decently fast pace). Of course you have to multiply by the 10X occular magnification to get total magnification.

When the surgeon said 10X, he surely meant 10X objective x 10X occulars (= 100X magnification).
Unless you're dealing with cytology specimens where you try to caracterise individual cells (typically, cytology will require a scope with 10X, 40X, 63X dry /100X oil objectives) rather than whole tissu sections, most of the work of a pathologist will be done at 4X and/or 2X "scanning" objective magnifications (to see the whole forest - aka all the tissu section on the slide) and at 10X ± 20X (and very rarely 40X) "diving" objective magnifications, as needed (to see the tree in the forest - aka to closely examine the tissu in a given area of interest).

At the very bare minimum, a pathology scope will have a 4X, a 10X and a 40X.
A 20X will also be very uselfull, if accessible.
A 2X is usefull at times (for "scanning land from 30 000 feet") and a 1X is awesome if for example you have to assess distriubtion of interstital lung fibrosis in a whole lung lobule/acini (the functionnal unit of the lung) and in a whole lung tissu slice mounted on a slide.
For pathology, a 63X and/or 100X is really just added luxury, unless you also do a lot of cytology (which many pathologists do). Plus, unless you go "oil immersion" with a 100X, the 63X (air) doesn't give you much more info than the 40X anyways, as the resolution is limited at these magnifications since you start to reach maximum possible theoretical numerical apertures in air (max NA in air) with 40X objectives. For example, my 40X plan-apo has a 0,95 NA, whereas my 63X plan-fluorite has a slightly lesser 0,90 NA, so the 63X does not resolve much more than my 40X, it's just the same details (or lack thereof) but enlarged. Kindoff like when you use a digital zoom on a camera... The image is enlarged sure, but the pixels are also larger so you don't really see more details.

At the very bare minimum, a cytology scope will have a 10X, a 40X and a 63X/100X.

For squamous cell carcinoma of the skin (one of the most common skin cancers), what helps is to see small atypical squamous cell foci invading into the undelying skin stroma (aka the dermis and hypodermis). Then you can diagnose invasive squamous cell carcinoma. But the pre-malignant precursor to invasive squamous cell carcinoma must also be excised during surgery (aka: in situ squamous cell carcinoma and/or high grade squamous dysplasia), and to do so, diagnostic criterias rely more on architectural dissaray of the squamous cells within the epidermis (aka loss of cell polarity), dyskeratotic cells, mitotic figures rising high in the superficial epidermis, atypical (eg tripolar) mitotic figures, atypia rising superficially within the epidermis, loss of maturation toward the superficial third of the epidermis. So very high magnification objectives (63X/100X) are not so usefull as you need to compare atypical squamous cells with the normal surrounding squamous cells of the epidermis to see where the dysplasia is located and where it starts and stops to be able to appreciate if they are close of not (or even in contact with) the surgical resection margins. Most likely, we are talking about Moh's skin procedure here.

This is my estimate of the time of my work I spend on each different objectives during a typical day of work (at work, I use a Olympus BX53 with 7 objectives, all Plan-Apochromats and 10X/22mm occulars).
1X - 5 %
2X - 10 %
4X - 30 %
10X - 25 %
20X - 10 %
40X - 15 % (mostly when I do my cytology cases)
63X - 5 %

Finally mostly pathologist only examine stained slides (routine stains are H&E or HPS (for formaline-fixed parrafin-embedded tissue sections) ... for Mohs (unfixed tissue), we can use rapid stains such as toluidine blue). This is because pathologist do not examine live cells but rather fixed (aka dead) tissu. Anatomic pathologists do not use DIC, Darkfield or Phase contrats. We do not need to, as contrast is "built in the tissu" with the staining process. These DIC/DF/PhC techniques or most usefull to microbiologist who sometimes have to examine live bacteria. Some pathologists (ex: breast pathologists, nephropathologists, dermatopathologists) sometimes also use special fluorescence microscopy techniques in addition to the routine brightfield technique. Neuropathologist and nephropathologist aslo us electron microscopy for some diseases. Finaly neuropathologist also use enzymohistochemical stains. All pathologist also use immunohistochemisty (IHC) techniques (staining of tissu coupled with chromophore antibodies) to check for specific cell markers in tissu. For exemple, squamous cells will be positive for the "p40 antibody", wheras most lung adenocarcinoma will be positive for the "TTF-1 antibody" by IHC. There must be about 500 types of IHCs stains available to us nowadays, and new IHCs with newer antibodies are constantly developped to aid in difficult diagnostic cases.

Hope this helps,

Matador

PeteM
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Re: What magnifications do pathologists typically work with?

#11 Post by PeteM » Tue Jul 27, 2021 10:29 pm

Matador, thanks for that very comprehensive answer. Like you, I thought the surgeon must surely have meant 10x by 10x = 100x when saying he determined the margins at 10x magnification.

With your help, now I understand a bit more when looking at prepared slides. Thanks.

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