Clinical Weeks 1-2

I recently started a clinical rotation at a VA hospital in my area, and will log my experiences.

My first two days were shadowing, first the cytologist and then one of the histotechs.

Adventures in cytology

This was my first exposure to cytopreparation.

Cytology is very similar to histology (the cytologist calls us "cousins"), with the main difference being screening and diagnosis. Cytologists are able to analyze slides for abnormalities and note those down for the pathologist, while histotechs (whether HT or HTL) do not analyze slides.

The preparation of cytologic samples is also a little different. During my second week, I was able to help process and stain gyn and nongyn specimens (Pap tests). Most of these specimens were from urine, cervix for HPV testing, and sometimes peritoneal fluid.

Instead of going through a regular tissue processor like in histology, cytology samples are prepared using either the ThinPrep or SurePath system. This lab uses the ThinPrep processor, which prepares samples using a filtration method.

So with the ThinPrep processor, everything is pretty much automated. All you have to do is slip in the empty slide, slip in the filter, put the vial of sample cells beneath the filter, and put in a small container of 95% ethanol for fixation (since 95% ethanol allows for the specific nuclear chromatin patterns needed that other fixatives can't produce).

Before going through the processors, the samples are first spun in the centrifuge for 10 minutes at 1700-1800 rpm. This allows the samples to evenly distribute, so a monolayer of cells is easily spread later. Once centrifuging is done, the samples move on to the ThinPrep processor.

The processor spins the sample to once again evenly distribute the cells and break up any blood or mucus. Then the cells go through the filter and are smeared onto the slide. The slide is then dropped in the 95% ethanol; remember that slides must be put in ethanol fixative ASAP to prevent air-drying. When samples air dry, it can lead to "cornflaking" (brownish, granular, flaky artifacts) and also compromise nuclear quality.

Once the ThinPrep processing is done, you are ready to move on to staining! I was able to help with manual Pap staining. It's very similar to H&E staining, except the rehydration process starts with 95% ethanol instead of xylene. Then you go through hematoxylin to stain the nuclei, OG-6 to stain the keratinized cells, and eosin to stain the cytoplasm--just like in the textbook!

I was also able to observe how a cell block was made. The cytologist will usually opt to create a cell block if enough samples are left after making the smears. Cell blocks are then able to be processed, cut, and stained by the histotechs. This is usually done to provide extra diagnostic information that you otherwise wouldn't get with just a cytology stain; cell blocks include the tissue, so a "bigger picture" diagnosis can be performed.

There is currently only one cytologist in this lab, so they do everything: processing, staining, screening. Other labs will have cytopreparation techs handle the ThinPrep processing and staining, while the cytologist focuses on screening the slides for abnormalities.

Good news for histotechs: a lot of labs are having the histotechs help with cytopreparation, since they already have that technical knowledge and skill. And with the creeping of AI into cytology screenings, I have heard that some cytologists even shift paths and get licensed as histotechs!

Cutting in a professional histo lab

I was very nervous being in a "real" lab. I was afraid to touch anything; not for health reasons, but I irrationally thought myself clumsy and feared messing things up. Being a mere student, I felt out of place.

But once I started cutting and not just shadowing/following people around, I quickly fell into my old routine at my student lab. The microtome at the VA is a different model than "my" microtome 😁, so it took some getting used to, but I was able to get the hang of things after a few ribbons.

I cut five blocks (per the supervisor) that included skin, brain, and two other tissues I could not identify just from the block. (Need to get better at this.)

Skin was very easy to cut. Great ribbons right out the gate.

The brain...it took me a while to realize it was brain I was cutting. I could not get a good section at 4 microns, and barely got one at 5 microns. Then I took a second to look at the block. It looked very fatty. In fact, it looked a lot like the brain tissue I had cut in class. I also remembered the histotech I shadowed mentioning that fatty tissues are easier to cut at larger microns. In class, we cut brain at 8 microns. And once I moved the dial to 8, I instantly got a beautiful ribbon full of beautiful sections!

To note: this lab does not use gelatin in their water baths. I asked where the gelatin was, and was surprised that they don't use it. The supervisor explained that gelatin is a protein, and this can lead to false-positives when doing IHC.

But without that gelatin, I was having such a hard time getting the sections to stick to the slides! I think I spent more time chasing after tissue in the water bath than cutting! I later found out that student slides are not charged; the staff, however, use charged slides, making it much easier to get sections (but once the sections are on, they are impossible to get off if you want to reposition them, so you need to position them perfectly in one go). What I eventually figured out was, you need to angle the slide down towards the section, and "pull" the section up that way. Just like how it's described in the textbook!

I also appreciated the PARAGard! It makes cleaning the microtome so much easier! In the student lab we use paper towels and 70% alcohol, and it was so hard getting the paraffin off. Now that I've experienced PARAGard, I can't go back to regular alcohol! 😆

Things I learned in weeks 1-2:

• When tissue is pressed down (firmly but gently) during embedding, you can get an even face more quickly when trimming.

• To see if you have trimmed enough, hold the block up to the light and look at the tissue surface. Is it all exposed and ready to section, or are some areas still covered in paraffin? If the latter, continue trimming until all areas are exposed, especially the inked spots (if applicable); the ink spots are how the pathologists can tell how far a cancer has spread.

• Fatty tissue is usually cut at 6 microns (in this lab, at least) to minimize holes; however, if your fatty tissue sections come out with holes, this is usually acceptable to the pathologists because they know it's the tissue and not a microtomy artifact.

• For cyto:

  • Nucleus 3x bigger than normal = dysplasia

  • A clear halo around squamous cell nuclei = HPV

  • Urine degrades quickly, so must add CytoLyt prefixative to preserve the urothelial cells

Thoughts on the first two weeks

I didn't feel very confident in my microtomy skills after finishing my first five blocks. Mainly, my speed. From those five blocks, I cut 22 slides in 1.5 hours (the greater number of slides was due to cutting at different microns from the same block). Professional labs want you to be able to cut at least 30 blocks in one hour. I'm hoping that I will be able to work up to that number during my time at clinicals.

Brain from class (not clinicals)

Skin from class (not clinicals)