all right uh youtube viewers this is going to be an interesting um experiment my guest today is dr abhimanyu to share he just um he's a pathology applicant from canada who's finished medical school and is hoping to join pathology at some point and we are i just um impromptu started this talk today and just put a message out on twitter seeing who would join and he volunteered so thank you avi manu for doing this thank you very much dr sanjay sure so we are going to talk about immunohistochemistry okay so if you ever start pathology this will be super helpful for you in terms of uh you know what to expect so here let me give you a little introduction to immunohistochemistry okay so on this panel this thing here is the can you see my my arrow moving around yes okay so this thing that i made here is a cell obviously and this thing is the nucleus the gray thing is the cytoplasm makes sense right so on the next panel what i've shown you is an antigen on the cell and an antibody in green so that obviously the antibody antibodies attaching to the antigen so in immunohistochemistry what happens is you are trying to find a particular antigen that's really the goal of immune chemistry and for that you have to have an antibody that goes and attaches to that antigen so the antibody is something you have to buy from a commercial vendor or you have to you know make your own in your lab or whatever but eventually you have to have an antibody to make your menistrochemistry work and so what happens is the antibody attaches to the antigen of interest and then you use the second antibody to attach to that antibody right right so that's the orange thing there so that's the secondary antibody and then how do you see it because you can't really see an antibody under a microscope right so you need a chromogen something to make it get a color and that is the brown thing on top so the chromogen attaches to the secondary antibody so it's a whole link cell antigen antibody another antibody and something that makes it brown kind of like lego blocks right like lego blocks exactly so does that make sense everyone knew for how that works yes yes so at the end of the day what you get is you get a brown color on where the antigen is is located in the cell isn't that a cool thing i mean the idea is pretty cool isn't it yep yeah most of you say i want to look at this antigen you make an antibody against that and you turn everywhere that that antigen is turned brown with amino acid chemistry i'm saying brown but sometimes you can change the chromosome to be red and so you can get red you know you can have a red chromosome and everything with a particular antibody will turn red makes sense right yep yeah so whatever is positive turns brown whatever is negative stays unstained so it stains with the color that your counter stain had which is let's say you counter stain everything blue everything else stays blue and only the thing with the antigen turns brown right makes sense all right so let's go to the next part of this so let us assume your antigen of interest is cd20 now cd20 is an antigen that's found on b lymphocytes and that's found on the membrane of the cell it's sitting on the membrane so the antibody attaches to the cd20 the secondary antibody attaches to that the chromogen attaches to that and then the entire membrane turns brown so that's how it works the entire membrane turns brown and so you'll see when you do immunoso chemistry for cd20 the surf the membrane of the cells with cd20 turns brown makes sense all right so that's one example let's take another example let's say the antigen of interest is not on the membrane it's in the nucleus a typical example of that would be thyroid transcription factor ttf1 right so the same sequence of events happens except the thing that turns brown is the nucleus now there's no brown on the membrane no brown in the cytoplasm only the nucleus will turn brown because only the nucleus has ttf1 in it yep so we call things like these nuclear markers things that where the antigen is only in the nucleus so this the antibody only marks the nucleus and so the color only shows up in the nucleus similarly we have things like hmb45 which is a marker of melanomas where the antigen is in the cytoplasm so only the cytoplasm turns brown so you have membrane antigens you have cytoplasmic antigens and you have nuclear antigens right pretty straightforward yep now let's so how i divided this dark abi manual is by what it's staining so can you tell whether it's nucleus cytoplasm or membrane in this particular case yeah it seems to be nuclear nuclear correct so this is a nuclear stain which means that the antigen that we are looking for is only in the nucleus and the classic one there is thyroid transcription factor i love that because i'm a lung pathologist you know so i use it a lot in my practice so the thyroid transcription factor stains all those things that have ttf1 in their nucleus so that's alveolar pneumocytes normal cells of the alveolus bronchular lining cells lung adenocarcinomas because they arise from that small cell carcinomas of the lung there's an um unusual tumor called sclerosing pneumocytoma and also thyroid carcinomas as you might guess from the name thyroid transcription factor right actually it's it's more difficult to guess from the name that it stains lung then that it stays thyroid you would think it would stay inside right right true so in this particular example the tumor i've stained here is sclerosing pneumocytoma and in sclerosing pneumocytoma the tumor cells are positive for ttf1 [Music] does that make sense oh yes i think you also made a video on sclerosing yes i we made a video on sclerosignocytoma because matt cheney and myself um have explained that in our book in two different chapters in the biopsy chapter and in the reception chapter so that's a tumor that you can get tripped up by in lung pathology all right so let's go to another very common nuclear marker in lung pathology that is called p63 and that's a marker also nuclear of normal basal and myoepithelial cells and it also stains squamous cell carcinomas of of everywhere no matter where the squamous cell carcinoma arises it stains for p63 so lung or oropharynx you know wherever oral skin all are p63 positive and another unusual tumor that stands for p63 is this is the so called nut carcinoma which is a very trendy thing nowadays so in this particular case this is a nut crystal and you can see strong p63 straining in all the nuclei right everyone you're pretty clear right right and i think the difference between p40 and p63 is this nut carcinoma yes yes you're very much ahead of the game so that does happen in a subset of cases although not all not all cases have this unique uh difference but you are right to point out that there are some cases of nut carcinoma where the p63 is positive but the p40 is negative and that can be a clue to the diagnosis but that doesn't always happen so sometimes both markers are positive sometimes both can be negative so nut carcinoma can be very very uh tricky you know in terms of immunochemistry because there's so many different profiles okay let's go to p40 now p40 is also a squamous marker just like p63 and the difference is although it also is super sensitive just like p63 it is more specific meaning that it doesn't stain other things other than p63 so it stains exactly the same things that p63 does there are a few exceptions and knowing those exceptions is helpful but the important thing for this lecture is for basic immunization chemistry nuclear marker right it is a nuclear marker just like p63 right everyone you make sense yes absolutely so if you're using a marker for squamous cell carcinoma you could use either p63 or p40 and all the p40 is slightly better you they are roughly equivalent all right let's go to another one that is a very trendy marker nowadays that's called insm-1 or insulinoma associated protein 1. you can see up there in the red that i have highlighted all the things that make up the abbreviation i n s m and then one oh yes right that's how you get the name and it's called insuluma associated because it was first discovered in insulinomas oh okay and then subsequently we found out that it stains all neuroendocrine things everywhere right insulinoma like associate protein is almost the same as using synaptophysin or chromogram the the main difference is that this is a nuclear marker and those are not so this is the only nuclear marker of neuroendocrine differentiation i think this is what the second generation of neuroendocrine markers correct correct the first generation being all the other ones that we know synaptofys and chromogram and cd56 you know if you really want to use a bad marker you can also use nse which is a neuron specific enolase which also called non-specific non-specific enolase it's just it's israeli in the current day and age you shouldn't be using the nse for anything um and um adding insm-1 to your you know armamentarium of imminence to chemical stains is great because it's it's very specific just like chromogram all right let's move to another nuclear marker nuclear protein intestis or nut which we just talked about so that's why you get the name nut it comes from nuclear protein intestines and as you might expect it stains germ cells in the testis normal germ cells right in this particular photo it stains nut carcinoma which you know is a you know very aggressive carcinoma in the in the thorax and other other sites so it's a nuclear marker this particular clone c52 b1 is very very specific for nut carcinoma so it's very helpful if you have it in your lab unfortunately not very many labs have it so far but i'm anticipating as this becomes more of a well-known entity people will will start buying the antibody and get it in their lab too all right let's go to epstein-barr virus encoded rna so eber eber so eber is a stain for epstein by virus that's what we use in our lab and it's not an immunohistochemistry so be money do have you heard this term sish ever cish fish well i heard fish and fish yes but i'm not very clear i explain to you the difference yes please yeah so you know in immunohistochemistry you're looking for antigens like so all antigens are proteins in immuno in in cyto hybridization your tag is attaching not to the protein but to the rna okay so it goes straight to the genetic material not to the protein right got it so your tag attaches there and there's two ways to see that tag once it's attached one is you use the chromogen just like you would do in immunostore chemistry so if you use a chromogen it's called chromogenic so sish okay cisa like in this picture the chromogen is blue so everything that turned blue is positive for epstein-barr virus okay now what if you don't use a chromogen instead you use a fluorescent signal okay got it then we get the fish then we get fish right and the disadvantage with that is that you to look at a fluorescent signal you need a fluorescent microscope right which none of us have you might have one fluorescent microscope in your lab but everybody else is using a standard microscope right so fish is very easily readable by all every pathologist with a microscope for fish you need a special microscope and so it becomes a very specialized technique right makes sense yep yeah yeah all right so this is epstein-barr virus and of course ebv is positive in any cell that's infected with ebv and in tumors where that are driven by abv the common one you know burkitt lymphoma is the famous one but you know diffuse large b-cell lymphomas or lymphomatoid granulomatosis in the lung there are many many things that are ebv positive all of them will stain with epstein-barr virus encoded rna or which is also called ebird makes sense yep another example of a nuclear marker and the reason it stains the nucleus is that the virus is in the nucleus right it's in there right right all right so let's go now to the pax eight another one of my favorite markers and the why it's a favorite of mine is because it never stains lung cancer lung adenocarcinomas especially it never shows strong and diffuse nuclear positivity in lung adenocarcinomas so when i see this i know this is not lung it's something else and this name comes from paired box gene 8. everyone you do know what box means do you know where that comes from it's an interesting story no but i would love to do yeah there are these things called homeobox there's right called a homeobox that is important in the growth and development of drosophila which is like a some sort of a insect or bug or whatever that's used in in so biologists have been using drosophila for decades to figure out how development occurs you know they knock out a gene and the antenna doesn't form they knock out another gene and limb doesn't form so from studies on drosophila they found out a lot about how development occurs okay and how it occurs is by these genes that are called that control the homeoboxes they're called homeobox genes so there's a certain genes that control limb development certain genes that control branching morphogenesis in the respiratory tract so all these genes have some kind of an x name in them so pax 8 you know or hawks or socks so they all come from these development derived names oh yeah i know socks yes like socks 10 or pax 8 or packs 2 packs 5. that's where that thing is from so pax 8 is homeobox derived gene and paxate stains b lymphocytes as well as carcinomas of the thyroid kidney and gyn tract what it does not stain is customers of the lung especially adenocarcinoma so very helpful in you when you're trying to tell sight of origin right right this particular case it is staining a thyroid carcinoma which is papillary thyroid again this is a nuclear marker very important if it stains the cytoplasm or membrane that's not valid because that's just background staining okay it must stain the nucleus to be considered positive all right let's go to yeah exclusively same thing with gata3 which is another nuclear marker nuclear it binds the dna sequence called gata you know how you have dna sequences like that right right so gata3 gets its name because it binds the dna sequence g80a wow i didn't know that yeah i actually i think one of the coolest things about this presentation is going to be finding out how the names were derived of these things absolutely i can see that it's going to be cool so here's gata3 and it's most common famous uses are for detect for that it stains breast carcinoma and european carcinoma that's what it's famous for but it also stains steel lymphocytes it also stains some mesotheliomas and lung cancers so a lot of people have noticed that it stains so many things that it seems to be useless but that's not true you know in certain differentials it can be very helpful so if you're using it in lung adeno versus breast it can be very helpful you know if you're so you it really has to be tailored to some specific situations and then it can be very helpful in fact gata3 has replaced other breast markers as the most sensitive marker for breast carcinoma by far so i don't even use mammoglobin or gcdfb most of the time when i'm trying to tell lung from breast i just use gata3 and ttf1 and and i'm done for that right remember i can see there must be a lot of overlap between breast and lung yes there is overlap so in fact you can have for example you can have breast carcinomas that are positive for gaddafi rarely for for ttf1 i mean rarely sometimes you have lung adenocarcinomas that are positive for gaddafi so it is by no means a perfect antibody but there's almost no antibodies are perfect even ttf is not a perfect antibody the closest i can get to a perfect antibody is um uh the thing for the solitary fibrous tumor it's uh escaping my mind right stat six stat six yeah that's the closest i can get to a almost perfect immunist chemical marker that stains all examples of what it should stain and does not stain what it should not stain the rest are not perfect all right so continuing off on a theme of nuclear markers the next one is erg and the reason it gets its name is it's it's called the ets related gene erg erg and this is a marker of endothelial cells so abandon can you tell what can you see what this structure is that i'm pointing at yeah it seems to be a vessel yes artery or a vein very good yeah it's a small blood vessel kind of a capillary size vessel almost it's very small and you can see these cells that are lining it are positive right all right so these are endothelial cells so get uh erg has become a really really good marker of vascular things much better than what we had before which was ct 34 and cd31 so it is also a nuclear marker okay makes sense erg and then this so you can have a nuclear marker for a virus just with just as we did for epstein-barr virus right this one is cytomegalovirus so you have an immunostore chemical stain that will stain the nucleus that's infected with cytomegalovirus in fact it also shows small cytoplasmic dots where the cytoplasm because cmv is both in the nucleus and in the cytoplasm but in this particular picture you can't really see good cytoplasmic dots there might be some there but not great maybe at the top left right below like here uh maybe i don't know further up yeah that one that one maybe that one yeah i think mostly what we're seeing is nuclear positivity in these cells but it's a really really good immune chemical marker for cytomegalovirus again nuclear markers okay so we've seen two viruses already that you can stain with the mineralistic chemistry ebv and cm all right let's go to estrogen receptors so in breast cancers endometrial cancers estrogen receptors are in the nucleus so er stains is a nuclear marker it stains all things that normally express estrogen receptors which is normal breast normal endometrium cancers of the breast and endometrium and other you know stromal sarcomas and things that can arise from these sites also very logical right right right okay then we'll move to a very interesting name which i we've started using this marker called nkx 3.1 which is a prostate marker more sensitive than psa prostate specific antigen or psma and all that so nkx 3.1 seems like a very uh weird name to give a stain but it actually comes from two people called nirenberg and kim who were the people who are actually nobel prize winners who were involved in the discovery of this wow okay yeah that's how it comes so n is nirenberg k is kim and x is because it's a homeobox gene just like pax 8 right right so involved in development so nkx 3.1 is a great marker for prostate adenocarcinomas very very sensitive uh there is a caveat that it needs to be strongly and diffusely positive so just one of one or two cells weekly staining here and there doesn't make it but if you keep that caveat in mind it's a great great marker for prostate adenocarcinomas and in some institutions uh i think is becoming a really good adjunct to psa and many people realize that it's much more sensitive than psa for prostate cancer okay and uh dr sanjay when you say um like how much is it staining so is there an objective thing or just subjective thing subjective you know when when you're talking about strong and diffuse you're saying basically every cell in that tumor is positive okay and strong meaning that the brown is really a dark deep brown you know right right when you say focal and weak what you're saying is that most of the cells are not staining and there's an occasional cell that turns brown a little bit brown here a little bit there and it's light brown so you're almost thinking in your mind is this even really positive or not right right you know that's the kind of staining that you should ignore if it's ntx 3.1 i think i had one discussion around her to being equivocal and how that's being sent for so it was sent for fish testing and i think how they decide yes and in a stain that determines therapy like her2 or pdl1 that issue becomes very very important right it becomes super important whether it's staining 10 cells or 20 or 100 or whether it's weakly positive or strongly positive depending so it depends really on the marker how how careful you need to be about those things you know there are some markers like ttf1 even if they are focally positive it means something okay you know so you really have to know the specifics of that particular marker and how to interpret that it makes but that's a great question everyone all right let's go to the next marker which is uh sf1 steroidogenic factor one this is a marker of adrenocortical things like cortical carcinomas also sex quad stromal tumors and what we use it for is um the diagnosis of adrenal cortical carcinoma so great nuclear marker that stains those tumor cells all right and this is my favorite stain of all time stat6 yeah we just spoke about it yes the num the name comes from signal transducer and activator of transcription st80 yeah great marker for solitary fibrous tumor in the past we used to use bcl-2 and cd34 that's almost become obsolete now that we have this marker another marker which is uh some people like it some don't is tle1 transducin like enhancer of split one tl e and yp some people like it and some don't is that we don't really have a great until recently we didn't have a great marker for synovial sarcoma and a minister chemical marker and people used to use tle1 as sort of a screen to pick up those tumors but it's neither highly sensitive nor highly specific so people just gave up on that and some people said well we'll just whenever we suspect this we'll go straight to fish and just bypass this sub-optimal marker but some people still like it as a marker of synovial sarcomas it is not perfect by any means so there are many many pitfalls to this market also the nuclear market that's why i included it here right maybe in some resource constrained setting may have to use this correct exactly if you don't have fish in your lab for example you might have to use it here's another great marker is lymphoid enhancer binding factor one or left one and this has become a new marker for cll chronic lymphocytic leukemia when i was a resident we didn't have that in our lab it hadn't even come out then so it only appeared when i was already in practice and in my second job so it's a very new stain left one for clm nuclear marker and then ki-67 sorry i didn't put a picture here but everybody knows what that looks like it's just a proliferation marker that stains the nuclei it stains proliferating cells in any active phase of the cell cycle right so um you know we'll stain the nucleus always ki 67 comes from keel 67 kiel 67 that's another nice way nice thing to remember in terms of yeah there was some discussion on how to pronounce it yes i don't know maybe i'm pronouncing it wrong who knows how it's pronounced we'll have to ask somebody who comes from keel it might be actually i i think it's a city but again i could be wrong about that too maybe i've been you can find out everyone you do some research on what keel is and post it on twitter i'll try my best okay all right let's move to the next nuclear marker terminal deoxynucleotidal transferase or tdt superb marker for thymic lymphocytes okay great marker so it's always present in the normal thymus because they have lymphocytes that are tdt positive and it is also present in thymomas many thymomas or most thymomas have some amount of tdt-positive lymphocytes um unfortunately it's also positive in one other tumor that's in the differential and that's lymphoblastic lymphoma that can that it is also tdd-positive so you really have to use morphology plus ddt to figure out whether it's a lymphoblastic lymphoma or a thymoma but if your differential is let's say some other kind of carcinoma versus thymoma this is super super helpful if you have any tdt-positive lymphocytes in it that's a very uh strong sign that you're dealing with the thymoma in in an epithelial tumor so tdt and it's positive in the lymphocytes not in the epithelium cells all right and then we have a fancy marker called integrase in interactor 1 ini1 ioni1 is lost in some some a subset of tumors which are called ioni deficient neoplasms like epithelial sarcoma for example but otherwise in i1 is expressed in all cells all cells so like here there's a there's a lung here and the bronchial cells are expressing it the capillaries are expressing the epithelial cells are expressing it it's almost like a marker of you can use to show that your immune chemistry is working you know because it stains everything it should stain everything so it doesn't stain is the ionized deficient neoplasms okay that's how it's helpful it's a nuclear marker okay and then let's go to some nuclear markers that are used in lymphoma bcl six which comes from b cell lymphoma six bcl is a marker that's positive in normal germinal centers and in b cell lymphomas that are germinal center origin and then you have uh i think we're i think that's the last one i have for nuclear markers so everyone knew this is i've given you a sampling of nuclear markers that are used in immunistic chemistry okay yeah thank you so very much dr sanjay yes should we move to something else so we'll move to cytoplasmic okay yeah sure okay so the first one we'll do there is napsin a this is a lung marker also novel aspartic proteinase of the pepsin family that's how the name comes napsin first discovered by the japanese scientific community and then later on came to the us around 20 2010 and became known well known as a marker of lung adenocarcinomas so it stains alveolar pneumocytes lung adenocarcinomas and also macrophages in the air spaces so it's a little bit more dirty stain than ttf1 which only stains the pneumocytes doesn't stay in macrophages and uh you can see the difference here between knapsan and ttf1 is that knapsan is a cytoplasmic marker you see right right in fact for all cytoplasmic markers the nucleus will be left unstained so there's a little blue dot in the middle of the cell you know like an unstained part which isn't staining which is however so napstein should always stain the cytoplasm in a granular fashion and is one example of a cytoplasmic marker let's go to another cytoplasmic marker synaptophysin right and that's a neuroendocrine marker our standard neuroendocrine marker here it's staining pancreatic eyelid cells synaptophysin is one of the most sensitive neuroendocrine markers although not very specific then chromogranine so chromogram is the reverse of synaptism it's not very sensitive but it is very specific what that means is when chromogram is positive you can be almost sure that a tumor is really neuroendocrine so you can trust it however it will not stain all of the neuroendocrine tumors so it's not very sensitive got that every menu the difference between the sensitive and specific so in this example it is staining a small cell carcinoma which is a neuroendocrine carcinoma of the lung here is my favorite pan keratin stain which is a cocktail called keratin ae1 slash a3 nowadays it's become fashionable to say instead of cytokeratin you say keratin nowadays that's the fashion right that confused me in the starting yes in in my days we used to say ck7 ck20 now we say get it in seven carried into it so keratin a1 a3 is a great pan keratin marker stains everything that is epithelial normal epithelial cells carcinomas mesotheliomas anything of the epithelial origin will be positive a great pankerton stain to have and to use in your differential what so in your you know broad spectrum differential of is this carcinoma or lymphoma this is the stain to use first okay great stain to have and then a similar stain i don't have a picture here sorry it's called the cam 5.2 again i had to do some detective work to figure out where this name comes from this name comes from one of the discoverers of this stain her name was carol a mackin oh okay that's that's how cam comes from 5.2 so cam 5.2 is another great pan keratin stain that you use in immunostore chemistry all the time and it's a great complement to a1 a3 so generally i use if i'm trying to prove something is a carcinoma or not a carcinoma i try to use both keratin a1 a3 and cam 5.2 before going to the most specific keratins like 720 you know i use try to use a broad spectrum keratin okay also cytoplasmic okay where are we with the cytoplasmic markers here's kit used to be called seekit was very famous because it started the era of personalized therapy now it's just called kit and this is cd117 and cd117 stains both the cytoplasm and the membrane the name kit also has an interesting history because it comes from kitten literally like a kitten oh wow okay it was because kit was isolated from a pet cat with a with a fibro so-called fibrosarcoma okay and so the person who named it named it kit after kitten and now we just use it all the time i didn't know that that it was even named after that i had to do some detective work so the most famous use of kit is for diagnosis of that a tumor called gi stromal tumor or gist right but it's also positive and many other things like melanoma and seminoma so stains the cytoplasm and membrane okay then we have another just a marker as marker for gi stoma tumors that also has an interesting name interesting that secret comes from a kitten and dog one comes from you know sounds like a dog what's that sorry that would have been my guess yeah that's right so kit and dog you have to remember that everyone you and you'll be done with your markers for gi stromal tumors okay cats and dogs right right right okay so this was this marker was discovered on just so it's called dog dog dog all right this is also an example of a cytoplasmic marker then there's an um relatively decent stain called fumarate hydrotase fh which is lost in fumarase fumidate hydrogen deficient carcinomas like some renal cell carcinomas um just just a second dr sanjay yep um i'll just quickly go shut the door absolutely yeah yeah sure so we are talking here about cytoplasmic markers if you are joining us now in this immunostore chemistry talk we are talking about cytoplasmic markers and we have gotten as far as fumarate hydrotase and uh my discussion here is abhimanyu tushir from canada and we are having a great time just chatting about immunistic chemistry all right abhimanyu so let's move to the next cytoplasmic marker prostate specific antigen psa we already talked about this right right and we already said that nkx 3.1 is rapidly becoming more popular than this marketplace this has been the gold standard go ahead what were you saying um this is less sensitive correct right okay but has more you know we have been using this for decades as the you know as our standard marker for prostate cancer so everybody knows about psa it is a cytoplasmic marker then we have this marker that we use a lot for melanomas it's called hmb 45 human melanoma black 45 that's where the name comes from oh wow okay human melanoma black hmb45 and it's also a great marker for a group of tumors called pacomas perivascular epithelioid cell omas right and pacomas some of the things that are called pacomas are lamb lymphangiolomatosis in the lung angiomyele lipomas in the kidney these are tumors that arise in patients with tuberous sclerosis often and so that's another use of the hmb45 antibody cytoplasmic marker you see that every menu the unstained nucleus yeah the blue and you see the red chromogen right right right so not all chromosomes are brown obviously right okay let's go to another cytoplasmic stain inhibin this is a great marker for adrenocortical tumors and sex quartz stromal tumors so sf1 is the nuclear marker of these tumors and inhibition is the cytoplasmic marker of these two homes okay so you could use either one for for these and now let's you move to the last part of the talk okay are we menu so now we are going to membrane stains things that should stain the cell membrane and they may or may not stay in the cytoplasm but they should not stain the nucleus they are usually um membrane stains because they are located on the surface of the cell membrane you know like uh receptors and things like that so the most famous one nowadays is programmed death ligand one pdl one if you if you haven't heard about pdl1 as a pathologist the ground will open up and you will get sucked into into that and never heard from again i've heard about it from one of your talks okay good you must you must know about pdl one by one because it's very very important nowadays for therapy and there's so many pdl one pd1 pdl1 inhibitors so you must know about that so pdl1 is a is is a protein that is expressed on the surface of tumor cells and that's tumor cells use this to evade the immune system okay so because it's on the surface so the staining with pdl one is membrane staining got it okay so that's the important thing to know i could do like a two hour lecture on pdl one alone but i'm going to spare you the torture of that okay so let's go to another membrane marker which is called d240 and this is a marker that's positive in lymphatic things lymphatic endothelium you know lymphatic neoplasms but also in mesotheliomas that's another use for this marker so lymphatic things and mesotheliomas and this is a membrane stain another stain that we use in lung pathology is called mach 31 moc31 this is a marker that is a non-mesothelial stain so it stains uh adenocarcinomas and other tumors not mesothelioma and the name comes from monoclonal antibody against oat cell carcinoma 31. oat cell carcinoma is an old name for small cell right right so that's how it was discovered on small cell customers and then became known as a general purpose carcinoma marker so this is a membrane stain mark 31 is a membrane stain which we commonly use for the differential of mesothelioma versus adenocarcinoma in the pleura okay i use this a lot uh then all the cds everyone used so all the cds when you hear a cd there's cd that cd stands for cluster of differentiation right and all those markers are located on the membrane cell membrane so every cd marker virtually every cd marker should be positive on the cell membrane too so here you see cd45 which is the most famous broad spectrum lymphocyte marker stains all lymphocytes whether t or b and therefore stains all lymph most lymphomas whether t or b cell lymphomas there are only a few that are that are cd45 negative it stains both the cytoplasm and cell membrane similarly cd20 we started this lecture with cd20 right is a cell membrane marker it's a marker of b lymphocytes and then b cell lymphomas any b-cell lymphoma similarly cd3 is the reverse of that it's a t-cell marker so it gains t lymphocytes and any malignancies that are of t lymphocyte origin cd3 and cd20 then we have cd68 this is not now this is kind of an exception to my it should be located on the membrane rule because cd68 is actually a cytoplasmic state not a membrane state so right as i told you the rule already we have a thing that breaks that so i i guess it's not a rule then so cd68 is a cytoplasmic marker for macrophages things of macrophage origin okay cd10 is a stain that is positive in the germinal center so very similar to bcl6 that we talked about before and this is a membrane marker cd10 is used for germinal centers and for general center derived lymphomas you'll see that a lot when you go to hematopathology cd5 is like cd3 so a subset of cd3 positive cells are also positive for cd5 this is a t cell marker right cd5 is also positive in a one type of form one type of carcinoma which is carcinomas that arise in the thymus thymic carcinomas are positive for cd5 there okay then we have a cd that's positive in plasma cells and that's called cd138 and that's both positive in normal plasma cells and malignant plasma cells which is myeloma plasma cell neoplasm then we have cytokeratine stains now cytokeratin stains can be positive both in the cytoplasm and the cell membrane one of the famous ones is ck7 that stains lung epithelium for example lung epithelium and lung carcinomas and many other things the famous thing is above the diaphragm all of these things are above the diaphragm and similarly there's a below the diaphragm strain called ck20 so in this particular case it's staining pancreatic adenocarcinomas uh ck20 is also famous for staining colonic cancers so the cytokeratin stains can be both cytoplasmic and membrane so remember that in general okay then there's this uh another famous cytokeratin stain is cytokeratin five six or today we would say keratin five six right right and this is a great marker for squamous cell carcinoma not as sensitive as p63 or p40 but but pretty good and it also stains normal squamous epithelium also stains mesotheliomas so although p63 p40 don't stay in mesothelioma ck56 does stain mesotheliomas okay but those are nuclear stains and this one is cytoplasm correct very good yes so p63 and p40 are nuclear stains and this one is cytoplasmic so you've already learned a lot about about nuclear cytoplasm and you're remembering it too everyone knew so that's a great sign great sign that this is happening so okay so we'll stop it here everyone knew thank you so much for joining me it's been great to chat with you