Hello,

when i worked with blood platelets, i used thin 400µL microtubes (45mm long, 7mm diam). The ones i used are referenced 391178 on this site https://www.dutscher.com/frontoffice/product?produitId=0H-05-09

They are supposed to be the cheaper version of the beckman tubes 342867 (never used these so i may be wrong).

If you don't have the specific rotor, you can use a classic rotor and 1.5mL microtubes filled with paper as reductors.

Once the centrifugation is done, carefully open the tubes, draw off most of the erythrocytes and add the fixative solution very slowly. When the fixation is done, cut the tube above and below the position where your cells should be and proceed as usual.

Dear Sarah Richert *) , dear Aymeric Neyret :

[*) Dear Sarah, would you mind to send me the Yabuki 2014 - article (see below) as a pdf if you are in possession( unfortunately as retiree I do not have rights to access for free Wiley online articles any more)...Thank you! ]

Do you have any suggestion or a practical imagination how to process the "buffy coat" out from 400µl or "0.5 eppi"

without alteration of "spun down" cell layers, or ROS-changes of mitochondria in your blood cells?

As to the relative volumes which can be expected:

cf.:

https://i.pinimg.com/474x/79/7c/87/797c876612ba36506f4e5057ce0c334b--vet-tech-student-vet-med.jpg

Just for convenience of further reading: Short Communication :

"A Practical Technique for Electron Microscopy of Buffy Coats in Dogs and Cats" by

A. Yabuki M. Sawa H.‐S. Chang O. Yamato First published: 03 September 2014 https://doi.org/10.1111/ahe.12143

=

Article A Practical Technique for Electron Microscopy of Buffy Coats...

= original journal's website (NB: prior to linking in your browser, delete the 2 UNDERLINE-CHARACTERS __ in between https and ://onlinelibrary.....):

https__://onlinelibrary.wiley.com/doi/pdf/10.1111/ahe.12143

(NB: access for me only PPV = Pay Per View)

I have done in my former active times about 200-250 buffy coats from peripheral blood (4-7 ml) by own benchwork due to very special tentative / suspected diagnoses (e. g. regarding NCL, mycosis fungoides / Sezary's syndrome, bacterial / viral infection, etc.) and always had good results regarding ultrastructural morphology... but it may depend on the whole procedure (from blood drawing into EDTA coated tubes - careful centrifugation - primary fixation - secondary fixation - separation of the fixed buffy coat layers out of the specially fabricated 10 ml-(glass) tube(vial) - postfixation 3 and 4, correct dehydration - until embedding into resin....

If you want to see my former standard protocol just send me an e-mail ([email protected]) or send your request via an RG-Message.

Regards and compliments, W.H.M.

Sarah Richert , again:

you might have a look into the RG-Archives by a full text research for

| buffy coat |

which finds some interesting results:

• https://www.researchgate.net/search.Search.html?type=publication&query=Buffy%20coat
• https://www.researchgate.net/search.Search.html?type=question&query=Buffy%20coat

in RESULT-Section Publications

(=https://www.researchgate.net/search.Search.html?type=publication&query=Buffy%20coat) I found:

KAPLOW, 1969:

https://academic.oup.com/ajcp/article-abstract/51/6_ts/806/4817243?redirectedFrom=PDF

Buffy Coat Preparatory Tube

Leonard S. Kaplow, M.D.

American Journal of Clinical Pathology, Volume 51, Issue 6_ts, 1 June 1969, Pages 806–807, https://doi.org/10.1093/ajcp/51.6_ts.806

Published:

01 June 1969

and I added there in the RG-page the following comment

[Article Buffy Coat Preparatory Tube

"One can guess that the technical article on a elegantly modified

"Buffy coat Preparatory tube" will not be available from the author any more.

Nevertheless I would like to point to

https://academic.oup.com/ajcp/article-abstract/51/6_ts/806/4817243?redirectedFrom=PDF

where one can find at least the sketches for the tube itself on the FIRST PAGE of publication ( Article PDF first page preview ) I include this publication not only beacuse of the anniversary of my 18th birth day: the article was published 1st June 1969...(:-))

I have done a lot of 'buffy coats' during my active work in an EM-Lab and my intention in the 1990ies (due to heavy workload) was to design a "buffy coat preparatory vial from glass" which - as of my present knowledge - had a quite similar design and therefore would have been ONLY "re-inventing the wheel".....

For the sake of fairness I nevertheless would like to recommend this paper "posthumously" and deplore the fact that this kind of "preparatory tube" didn't find its way to Europe / European Labs for TEM performing routinely at that time diagnostic ultrastructural blood cell analysis.

If there is anybody out there "in the wild" in possession of a copy of this article I greatly should acknowledge receiving a pdf to my e-mail-address or via RG-message system. THANK YOU!

Best and compliments, W.H.M

Dear Wolfgang Muss,

Thank you very much for your answers! I will send the Yabuki publication to your provided email address.

I am an experienced fluorescence microscopist, but do not have any experience in preparing samples for EM. What I have read so far is that most people take off the supernatant of the centrifuged blood and fix by carefully putting 2,5% glutaraldehyde directly on the buffy coat. After fixation for up to 48h they cut the eppis around the buffy coat layer and precede with their fixation/embedding protocol.

I would also very much like to know if it is possible to freeze the cells before processing for EM as we expect to collect samples every other day over 1 year or so.

Thank you for the further recommendation which I will look at in more detail.

Thank you very much for your help,

Sarah

Dear Sarah Richert

Thank you so much for your offer and your "good deed of the day"....

and, BTW, thank you: in my previous e-mail to you I forgot to mention that I received the pdf of the YABUKI 2014 article properly !

Regarding the "cryofixation/freezing of blood cells I am not convinced this to be a goal-oriented, making sense or feasible possibility of method. and - naturally - the use of such a special method of (cryo) preservation (until further processing) conducivly depends on all the technical and apparative possibilities the researcher(s) find and can use on site. We shall discuss that problem of "sampling" for sure too... but I warn you bluntly: TEM-preparation of tissues of fluids / fluidous substrates like blood cells for sure is no or ever will become a 'sunny promenade'! (you for sure know the 'GIGO'-principle!) .

But perhaps someone else with practical expertise will chime in to this thread and discuss how one can overcome sample flood ((? samples / day x 365 days ))....

Do you know at the moment, how many people you have at hand in the Lab to do the preparation(s)?

Compliments and my best regards, Wolfgang