2/ First things first, ANCA stands for anti NEUTROPHIL cytoplasmic antibodies. Remember how for ANA we had those fancy HEp-2 cells? (#science) Well here, we just have good ol’ fashioned neutrophils.
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3/ ANCAs are antibodies to things in the CYTOPLASM of NEUTROPHILS. Now remember, lots of things are in the cytoplasm of cells! One thing neutrophils have a lot of is enzymes, and these enzymes are stored in the “azurophilic granules”, remember those?
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4/ These enzymes are the neutrophil’s antimicrobial weapons, as remember, the neutrophil is a warrior, on the front lines of battle! Proteinase 3 (PR3) and myeloperoxidase (MPO) are the two most common enzymes associated with ANCA vasculitis...
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5/ ...and so these are the enzymes routinely tested for on confirmatory testing after screening (
#ELISA). These aren’t the only enzymes in there though, remember catalase, elastase, beta-glucuronidase, etc? They’re in there too!Prikaži ovu nit -
6/ So how do we screen? Immunofluorescence of course! Just like with ANA, we have a buncha neutrophils on a a plate, and we dump our patient’s blood on there. If it lights up – great, this is a + SCREEN, but what does it mean?
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7/ A lot of us are taught that c-ANCA=PR3 and p-ANCA=MPO, is that true? What the heck is a c and p anyway? c is for Cytoplasmic and p is for Perinuclear. This is simply the IF pattern. Remember, these may hint at a specific antigen target, but are not confirmatory.
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8/ So why even differentiate? Probs bc we didn’t used to have confirmatory testing, so we figured out a way to make a best guess:pic.twitter.com/JxkHwBKCWM
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9/ These aren’t just ANY neutrophils on a plate, they’re “ethanol fixed” neutrophils. What this does, is make all of the positively charged proteins in the cytoplasm migrate toward the nucleus as the nucleus is negatively charged. Like MOTHS TO A FLAME, MPO becomes “perinuclear”!
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10/ PR3 is not positively charged, so it remains chillin’ in the cytoplasm, hence it’s “cytoplasmic”.pic.twitter.com/06an2aLqkr
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11/ While this is a great idea, it’s not always correct, and there are “in between” patterns as well, making things even more confusing. So, you’ve done your IF, and you got a positive p-ANCA using ethanol-fixation…but remember! There are antigens in the nucleus too (
#ANA)...Prikaži ovu nit -
12/ So how do we know if these are really perinuclear or nuclear? UGH.pic.twitter.com/tySyuttpRL
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13/ Welp, turns out, formalin fixation does NOT make things positive and cuddle up next to the negative nucleus. SO if you’re REALLY feeling savvy, what you can do is dump your patient’s blood on some ethanol-neutrophils AND some formalin-neutrophils, and see what happens!
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14/ If it’s perinuclear on ethanol but NOT on formalin, there’s a good chance that it’s an ANCA. If it’s “perinuclear” on both, then we might have an ANA on our hands, ie it’s actually in the nucleus. DISCLAIMER: I am deffo not addressing the cytoplasmic "ANAs" right now bc…pic.twitter.com/WrEYg0Hldy
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15/ Well, what the heck do we do now?! Okay, either way, if you have a positive ANCA (c or p) you should do an ELISA test which will specify if it’s really PR3 or MPO. If you have a positive p-ANCA specifically, see if your lab does formalin comparison?
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16/ But you MIGHT think about ordering an ANA (IF CLINICALLY INDICATED ONLY AS ALWAYS, rheumies plz don’t hate me). As if things couldn’t get worse, there is a long list of things other than just AAV (GPA/MPA) and +ANA associated diseases that can cause positive ANCAs, remember:
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17/ These are abs to basically ANY of those neutrophilic enzymes, things like EGPA, drug-induced/associated vasculitis, Goodpasture’s, Buerger’s, CF, IBD, autoimmune hepatitis, (pre)eclampsia, subacute bacterial endocarditis, cGVHD, malaria, and even leprosy can cause +ANCAs!pic.twitter.com/9vzjg6AZro
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18/ SO, when you order an ANCA (and please do this wisely), remember what you’re doing. Just dumping your patient’s blood on neutrophils that have all the things of a neutrophil (and also a cell) and seeing if any antibodies stick.
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19/ And in this case, if they do, ELISA is the next best step for differentiating PR3 vs MPO, but just remember – vasculitis isn’t the only thing that can cause a positive ANCA so BE WARY, and as always,
#clinicallycorrelate.Prikaži ovu nit -
20/ Alright, I hope you found this helpful, please feel free to drop questions below, and if you’re interested in learning more here is a super helpful website!
https://www.clinlabint.com/detail/clinical-laboratory/anca-iif-still-the-screening-method-of-choice/ …Prikaži ovu nit
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