Hmmmm. I have never worked with Polymorphoprep, only Lymphoprep. There was always some "contamination" of polymorphonuclear cells and monocytes in the lymphocyte rich fraction. So you may have some lymphos in your PMN rich fraction? Are *all* the cells staining for CD3/CD19? Have you made an attempt to gate on singlets only? Have you tried "racking" the samples after the final rinse, before staining (technique of running the test tube across a rack to disaggregate cells)? Are you only examining by fluorescent microscope? Can you take them to a flow cytometer for analysis and compare?

You might try CD15. It's less sensitive to left shift. I am assuming you tried more than one donor... CD16 is a GPI-linked protein and is absent in PNH. It would be quite rare for you to stumble on a PNH patient for your work, but Murphy never rests.

Best of success to you.

CD16 and CD15 markers are highly expressed on mature, peripheral blood neutrophils in humans. However, in patients with aplastic anemia and those with neutropenia, you can find neutrophil sub-population with low CD16. Moreover, human peripheral blood neutrophils when subjected to short range heat stress, the expression of CD16 can be sharply reduced.

Its unusual that all ur neutrophils get stained for CD3/CD19. If there is some, that could be due to possible contamination. You can try staining the same population with other human neutrophil specific markers. CD177 is a neutrophil-specific antigen. In addition, CD66b/CEACAM-8, CD11b/Integrin alpha M, CD33, and the cytoplasmic marker, myeloperoxidase, are other common markers that are used to identify human neutrophils.

Also worth reading this paper in Blood

https://ashpublications.org/blood/article/133/13/1395/261473/Human-neutrophils-express-low-levels-of-Fc-RIIIA

Hi Anirudh, I ran right into this too, there are two types of CD16, CD16a and CD16b, the websites that sell the antibodies are often not clear on this. Neutrophils express CD16b, while NK cells and some monocytes have CD16a. Some antibodies bind both, but I bought one (BD Clone B73.1) that only worked on NK cells and monocytes - even though the website said it worked for both (though less affinity for neutrophils). As for the CD3 and 19, neutrophils are large and granular therefore highly autofluorescent. Without looking at the data I cant tell for sure, but they should be in a similar position of the flow cytometry plot in an unstained sample, but they will look positive if you try standard quadrant gating and compare them to monocytes for example. CD16b is important because along with CD15 you can stain the unique CD16low population that Vinod refers to, but if you aren't getting any CD16 in your neutrophils, then the antibody may be CD16a preferring one.

Vinod Nadella could you please explain what you mean when you said that CD3/CD19 expression could be due to contamination. Actually, the purity of neutrophils in my sample is very high (~98%) so I am not sure contamination is significant here.

Luke C Davies Hi Luke, thank you for sharing your experience. It is very interesting that you mention that the antibody may be specific to CD16a and not CD16b which I need for neutrophils. When you faced issues with the antibody you mentioned, did you change to another one ? If so, would you mind sharing product details ?

Hi Anirudh, This is the one I've been recommended and ordered a few months ago: https://www.biolegend.com/en-us/products/pe-anti-human-cd16-antibody-569 , though I haven't yet tested it it because of COVID restrictions where I work - the clone 3G8 is known to block neutrophil phagocytosis - so I'm fairly sure this means it binds CD16b well

As far I remember well, I have used the words "possible contamination" as you haven't given any info. about cell purity in your question earlier. Moreover, Degel & Shokrani, Clin Lab Sci. 2010 have shown that the percentage of contaminating cells in neutrophil preparations isolated by Polymorphprep varied between 1–17% across individual blood samples from healthy controls. So, that's what I mean !!

https://cvi.asm.org/content/cdli/early/2012/04/30/CVI.05715-11.full.pdf

Few lines from above printed paper talks about similar outcomes as of your....

PolymorphprepTM isolation method resulted on average in 76±19% purity, largely due to a higher amount of contaminating debris particles and lymphocytes (Figure 1C). Also, in our hands, the highest variability in purity between isolations was observed when using PolymorphprepTM, suggesting that this method is somewhat more sensitive to small day to day variations. We also noticed a donor to donor variation in adherence of the lymphocyte ring to the plastic disposables after centrifugation, which may account for some of the contamination variability observed. When analyzing the nature of this contaminating mononuclear cell fraction, these were found to be mainly CD3-positive T- cells, with little or no CD19+ B-cell or CD14+ monocytes (not shown).

Dear Anirudh Gangadhar ,

I agree with Luke C Davies about this problem. You have to use CD16 b antibodies to screening neutrophils. Additionally, eosinophils are CD16 negative poly-morphs and some patients have not CD16 b on their neutrophils (this is a very rare situation).

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Hi Luke C Davies I checked with the company and they say that there is a 95% match between CD16 "a" and "b" so they don't think its likely to be a selectivity issue. Again this could depend on the specific antibody you have.

Thank you for your suggestions everyone. I think I have managed to solve the problem. It turns out that the expression loss was a result of the permeabilization step. When I bypassed this step, I could see that neutrophils stained for CD16.

This is quite possible as this step could be damaging the binding sites especially in the case of cell surface proteins.

Wow Anirudh, a permeabilization step in from left field. It is useful to know this happens, neutrophils loose all their granulation with permeabilization, but I thought CD16 was well anchored to the surface. There are so many answers to this question that I think this conversation thread will be useful as a trouble-shooter for many for years to come.

Thats very informative. Thanks Anirudh

Luke C Davies I felt confident that it could be because of permeabilization when I came across a few articles that actually skipped this step for surface antigens. In general, I believe it is good to try with and without this step and check what works better. These things really depend on the specific antibodies you work with.

You can easily stain CD16 on human neutrophils and detect by flow cytometry using the mouse monoclonal clone 3G8. This clone is commercially available from many suppliers conjugated or unconjugated. You will not require any permeabilisation step because this is a cell surface receptor. You should find a lot of CD16 on mature neutrophils, which will be negative for the B and T cell markers. If you wish to look at the nuclear morphology an easy thing to do is a cytospin which you can stay with DiffQuik and view with a light microscope. Lymphocytes will look very different from neutrophils, which have very characteristic nuclei. If you do a good neutrophil prep from human blood you should not find any contamination with lymphocytes.