Help - Making a permanent blood smear slide

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glabelle
Posts: 8
Joined: Mon Feb 02, 2015 2:41 pm

Help - Making a permanent blood smear slide

#1 Post by glabelle » Tue Jan 10, 2023 10:02 pm

I've looked all over for a process to make a blood smear permanent with no success.

If anyone has done it successfully can you please post how it was done.

Thanks, George Prineville Oregon

PeteM
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Joined: Tue Dec 06, 2016 6:22 am
Location: N. California

Re: Help - Making a permanent blood smear slide

#2 Post by PeteM » Tue Jan 10, 2023 11:17 pm

Try searching "how to make a blood smear microscope slide." Drop of blood, thinned out with the edge of a slide, then fixed using methanol etc. if you want to keep it a long time.
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Screenshot 2023-01-10 at 3.13.36 PM.png (259.67 KiB) Viewed 2086 times
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glabelle
Posts: 8
Joined: Mon Feb 02, 2015 2:41 pm

Re: Help - Making a permanent blood smear slide

#3 Post by glabelle » Tue Jan 10, 2023 11:25 pm

Thanks for the reply, but all those sources only tell you how to make a smear, but not how to make it permanent. i.e. what process, what mountant?

PeteM
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Joined: Tue Dec 06, 2016 6:22 am
Location: N. California

Re: Help - Making a permanent blood smear slide

#4 Post by PeteM » Wed Jan 11, 2023 1:29 am

George - the sites I've looked at in the past suggest fixing the smear with something like methanol (as noted above) or EDTA. Check the CDC site in the links above as an example. Cover slips and mountants over smears are not typically used - just a waste of time for a lab and one more piece of glass and another medium to mess up the image. Oil immersion with no-cover smears is also not a problem.

If you want to view dry - say with a 40x objective and no cover slip - then "NC" and epi objectives are available.

Someone like Tom Jones would be the expert here, but I've had no-cover smears in use for several years. Even a plain blood smear (no dip to fix it but just left to dry) will image for a long time.

Alexander
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Joined: Fri Jun 11, 2021 7:10 pm

Re: Help - Making a permanent blood smear slide

#5 Post by Alexander » Wed Jan 11, 2023 4:22 am

To make blood smears permanent I use the following process.

Make your smear the usual way.
Let it dry in air. This takes only a minute or so.
Fix in methanol.
Stain following the usual process for your chosen staining.
Wash thoroughly in distilled water.
Let it dry in air.
Wash three times in isopropanol.
Let it dry in air.
Mount in Histokitt.

For fixing and staining I use a kit called DiffQuick which gives results closed to Pappenheim. It consists of 3 bathes. First is fixation, second is red color and third is blue color. The slide is dipped 5 times for one second in each bath. Quick and easy. The washing in water and isopropanol removes most color artifacts and the isopropanol dehydrates the smear as well. Histokitt needs only 30 minutes to dry. From taking the blood to the finished permanent slide is less than one hour.

I attached a picture from a smear I mounted in Histokitt awhile ago. Then I did not wash as thoroughly as I do today. That is why you may recognize a good amount of color artifacts.
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BLOOD_SMEAR.jpg
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glabelle
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Joined: Mon Feb 02, 2015 2:41 pm

Re: Help - Making a permanent blood smear slide

#6 Post by glabelle » Wed Jan 11, 2023 4:37 am

Thanks very much Alexander. That is what I was asking for.

Tom Jones
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Joined: Wed Apr 13, 2016 3:47 pm

Re: Help - Making a permanent blood smear slide

#7 Post by Tom Jones » Wed Jan 11, 2023 4:43 am

It's easy. The mountant really isn't important as long as it lasts. I've had to re-coverslip slides I made in the 70's because the mountant dried up and pulled back from the edges of the coverslip. More modern versions work better and last a very long time.

You can use Canada Balsam, Permount, Euparal, Cytoseal and lots of others I don't remember. If you're curious as to what is used locally, call up the local hospital histology or cytology lab and ask what they're using for tissue mounting. Use the same stuff if you can get it. You might even be able to con them into letting you watch them mount tissue sections, or showing you how to do blood smears.

As for process, it's pretty simple. I like to wet the dried blood smear with whatever the diluent is for the mountant, xylene, toluene, or one of the citrus-based solvents like CitriSolv. Then add a drop or two of the mountant to the center of the slide and lay the coverslip onto the drop one end first so it doesn't create bubbles. Just like you'd put a coverslip onto a drop of pond water. You should use just enough mountant to fill to the edges of the coverslip. Too much and it may ride high enough you can't get enough depth of field on a high quality oil immersion lens to actually focus on the cells. You can draw off excess with a KimWipe or paper towel touched to the edge. Wipe the excess from around the coverslip, and set it aside for a couple of days to dry. I like the large coverslips as they allow for a larger useable area of the smear.

You can even coverslip slides that have had immersion oil on them by rinsing or soaking the oil off with xylene, CitriSolv, or toluene and then cover slipping it.

About the only time a clinical lab will make a permanent blood smear is for teaching purposes. But to do it they treat it just like a tissue section.

Alexander
Posts: 402
Joined: Fri Jun 11, 2021 7:10 pm

Re: Help - Making a permanent blood smear slide

#8 Post by Alexander » Wed Jan 11, 2023 8:43 pm

Tom Jones wrote:
Wed Jan 11, 2023 4:43 am
It's easy.
Very much so!

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