Understanding Immunohematology and Transfusion

hello my name is wassi manani i am a medical officer with canadian blood services and i work with the national immunohematology reference laboratory you know there are going to be three sets of videos uh one for each of the tiered questions this one is for tier one and this is going to be a bit of a hybrid of a powerpoint and a chalk talk i'll be using my stylus to write on the screen and hopefully this will help enhance the education content i'm looking forward to it and let's get started we'll be starting with our first case and remember the tier 1 cases are the baseline cases that are the easiest however there are some more difficult questions thrown in so don't be too surprised when you see some hard questions so we have a 24 year old female that arrives for an outpatient surgical procedure and a type in screen is sent so for this first question let's take a look what we have we have our four typing and we have a four plus with an ata and nothing with an anti b our reverse typing shows a one plus with an a1 cell four plus with a b cell and our rh typing with two different d reagents shows four plus and four plus our dat is negative with the poly specific reagent so when we're looking at this and trying to answer the first question the first question is typing demonstrates a discrepancy in the blank so when thinking about the discrepancy we have to think about where the obviously the descriptive reaction is and most often text will look at the strength of reactivity as an indicator but the reality reality is you should really think about the forward and the reverse typing separately and think about whether or not they jive together so in this particular case the four typing presents as a group a the reverse typing presents as a group o so the question becomes which of these is the discrepant one in this particular case because the strength of this a1 is much weaker you could argue that the group o typing is the one that is discrepant versus the group a typing that's not always correct but when you have to start resolving an discrepancy you have to pick a direction you either have to pick the forward or the reverse typing in order to do your work up and you'll see with uh my workflow later on that you have to pick a direction you can't sit on the fence about it and i think that's one of the most difficult things about an discrepancy you pick one of the two and then you work it up you may be completely wrong and that's okay but in this case because the discrepancy appears to be most related to the a1 cell we would say the reverse typing is a discrepant for question two what is the first step in resolving the discrepancy you can pre-warm wash the patient cells review the antibody screen results or increase the incubation time so again let's look at this we have forward typing which we think is correct a reverse typing which we think is incorrect and with this reverse typing we have a few options right we can pre-warm it to see if it goes away washing the patient's rbcs won't do anything because that's really the the forward typing bit reviewing the antibody screen results we can do that and increase the incubation time we can do that so these three things are the three things that we can do for reverse typing if we were to pre-warm the plasma or serum the question becomes is the shotgun approach really worth it so if you pre-warm a sample you're not sure what you're pre-warming remember you can pre-warm away clinically significant antibodies and when you're doing a type in screen the whole point of doing the screen is to identify any antibodies that may interfere with your typing so in this particular case you don't pre-warm unless you know what you're pre-rolling away increasing the incubation time will likely not do much or make this one plus even worse and so that'll just make it more confusing and so the last one we have is reviewing the antibody screen results again remembering that if you have an aloe antibody or an auto antibody it can interfere but if you don't know that it's there you can't really attack it so the best way to go about this is actually just doing your antibody screen results which you would usually do at the same time as your typing if you've got multiple systems running but in this case we're kind of taking it in a stepwise format so as i promised we're going to talk about how to approach a forward and reverse typing discrepancy and this is after you've made the decision about whether the forward or reverse is discrepant remembering that you just have to pick a direction you may not be right and that's okay but you just pick a direction and you go with it so with a forward typing discrepancy we really have three main things we can think about an extra antigen being expressed a missing antigen so a loss of an antigen or mixed field agglutination that throws us off that we were not expecting with a reverse typing discrepancy we can have extra antibodies so like we have in this particular case we have one plus reactivity with an a1 cell and an individual who is group a we would not expect that or we can have missing antibodies so for example if a group a individual did not have an anti-b in their plasma we would be surprised by that so for our forward typing let's go through my thought process of how i approach it so we have a forward type and i'm sorry about my awful handwriting we can have a missing or weakened antigen correct and so from there what i usually think about is a subgroup of a or b or you can have suppressed antigen the other side of a forward typing is you can have unexpected reactivity and so you have an extra antigen you weren't expecting so with this there's a few options you can have an acquired b so this for example occurs when someone with colon cancer releases a deacetylase enzyme which then converts a group a individual to look like a group b individual by some monoclonals that's that's much less seen now nowadays than it was in the past but it can still be seen you can have this which is essentially a b which expresses a weakened form of a and therefore it looks like it's an extra antigen but the person really does express a group a it's just weakened you can have mixed field and you can have antibody coding the cell which blocks the antigen from being detected and before i forget if anyone ever wanted to know this is called b sub a so for our forward typing we can have mixed or weakened and we have subgroups that are a or b we can have a suppressed antigen and on the other side we can have unexpected antigens so we can have acquired b we can have a b sub a a mixed field or antibody coding the red cell which throws us off so one of the more common types of discrepancies with four typing is with the group a and so i'm going to focus on that for the next two slides because it's the most common thing that we run into with a subgroups so when thinking about a subgroups they give us usually four typing that's off and sometimes a reverse type that's off if they form an anti-a1 but let's focus just on the forward typing so if your four typing reagent shows a weak with an anti-a reagent and it's fully mixed field we're talking about an a3 which is the most common subtype of the a's if it's less than 10 percent mixed field then we're talking about a end if it's weak plus with an anti a comma b then we're talking about an a x and you'll recognize these most often from textbooks and the reality is even though i've learned these serologically a lot of it's done molecularly now but serologically you can get pretty darn close to what it is as long as you have a gentle hand and a good eye you can detect the difference between a full mixed field and a 10 mixed field and then the week plus with an anti-a comma b so now what happens with your a sub groups if your anti-a shows nothing well now you have to actually start going into secretor status which i'm sure many people love oops so if you're a full secretor and you've got nice strength reactivity with your a and h antigens and h being group o then you're an am if your secretor status shows weak plus for a and h now we're talking about a end and if you secrete only the h now we're talking about a l and the a l is the the elute version of a so a is expressed on such a low level you use an anti-a and it shows nothing but you can elute off the nta because it's concentrated when you run the elute and that's when you can detect it and so they only secrete h as a result of that because there's such low amounts of it so with these these of the a subgroups there's no anti-a reactivity compared to the prior slide where there's weak reactivity with anti-a reagents and again you can identify these molecularly they can be a little tough sometimes depending on the genotype the type of genotyping you run and so serologically you can get pretty close to what the answer is so now let's talk about what's going on in our case we have a reverse discrepancy so with a reverse discrepancy you can have missing or weakened antibody the other option is you can have an extra antibody up here that you weren't expecting so with the missing or weakened you have two options you can have weakened anti-a in the patient's plasma serum or weakened antibe so pretty simple with the x-ray antibody we have a few you can have an a subgroup which means you have to run the a1 lectin because you have an anti-a1 present when you don't expect it in a group a individual you can have an auto antibody cold or warm you can have rouleaux which is the stacking of coins usually due to plasma proteins and then you can also have a passive antibody so for example if someone receives a red cell unit with an alloy antibody in it it may show up or if someone receives something like ipig and it tends to bind something that it shouldn't so again to go over our reverse typing discrepancies we get a missing or weakened antibody so for example in a group b individual you might have weakened anti-a and a group a individual you might have weakened anti-b or in a group o you might have missing or weakened anti-a or anti-b for extra antibodies you can have an anti-a1 which you would identify with your a1 lectin you could have an auto antibody giving you extra reactivity rouleaux which is the stacking which is called stacking of coins when the red cells stack because of excess plasma proteins or you can have a passive antibody due to some other introduction of the antibody to the patient so the identified antibodies known to classically react at what temperature so let's take a look at our at our antibody id we have immu core cells and we have no reactivity in mts gel in the first five cells here and then the last two cells six and seven we have three and two plus reactivity so if we look across kind of making a little bit easier you can see that the texts have already kind of ruled out a lot of stuff and really if we just scan we can see that the only antigen that's present that really only has two that are reactive are is the m antigen and so we have three inch plus reactivity matching with the anti-m and we know that anti-m reacts classically at room temperature because it's a cold reacting aloe antibody so we're going to talk a little bit about the m aloe antibody and let's first discuss the serology so it's predominantly igm in cold reacting which means it reacts at a predominantly immediate spin reactivity it does show dosage so if you're m positive and positive or m positive and positive you'll get let's say one plus reactivity here and you might get three plus reactivity here it's warm reacting oh sorry if it's warm reacting it's usually an igg component of anti-m so for those cases where you've got immediate spin 37 iat and you see oneplus reactivity let's say oneplus reactivity and oneplus reactivity it's most commonly that this component is the igm this component is the igg so i j the anti-m can actually have multiple ig isotypes depending on where it reacts and they usually do not activate complement even the ones that uh react at iat phase normally don't activate complement usually um they cause hemolysis through delayed hemolysis so they'll bind to the red cell and they'll be removed from this from the from the intravascular space by the reticulo endothelial system so they don't cause intravascular hemolysis the way that for example antibodies can sorry i'm trying to get it to go next for me there we go uh we can use enhancement to help us out so let me just erase this here so if we're going to use enhancement to to bring out the anti-m you can use peg list or poly brain however if you're using these techniques you have to be really careful because if it reacts at immediate and immediate spin let's say it's two plus or three plus an immediate spin at 37 and iat if you using your peg let's say you can actually get reactivity that's like let's say one plus that kind of carries over because the peg enhances it enough that it's able to react at a warmer temperature when it normally wouldn't so just because it reacts at 37 does not mean it's clinically significant if you're going to run it in peg so if you're going to look at clinical significance of anti-m it should only be done saline because you can trick yourself into thinking it's clinically significant based on this carryover effect of using an enhancement method of like pegless or polybrain you can drop the ph down to 6.5 to enhance an anti-m and some anti-m's only react in the presence of one to two plus one or to two percent glucose solutions um in plasma and we don't really know why that is other than the fact that the glycophorin a uh protein is a glycoprotein which has sugars on it and it may be that the sugar helps enhance the antibody binding to the actual protein itself and then finally clinical implications if it reacts at greater than 37 degrees it's more likely to be clinically significant and therefore you should honor it if you look in it says that anything that does not react at 37 or above for an anti-m they pretty much ignore it and they'll just give it even if it's cross-matching compatible at a immediate spin they also recommend not doing immediate spin cross matches for these anti-m's because it essentially just uh makes more of a headache for you and then it very rarely causes hdfn it can but it very rarely does and in cardiac surgeries a lot of times we get questions about what happens if we're going to put the patient in hypo in a hypothermic state so they drop the temperature of the patient down to drop the heart rate in order to do the work and allow the heart not to be ischemic interestingly they looked at patients with anti-m's that were cold reacting only and none of them caused hemolysis or extra or that caused the delayed hemolysis so really these anti-amps don't cause any issues even when the body temperature is dropped down they're pretty much just a nuisance when they react only at immediate spin for this next question number four if a transfusion is needed and you have limited inventory would you issue cross-matching compatible units based on the antibody identified so now we have to think about what i just talked about before remembering that this antibody does not really cause hemolysis and it's only reacting really at immediate spin what would we do because it's not clinically significant you can actually especially in an emergency issue and feel pretty darn safe now if you had a larger institution or if you have a ton of time if you really want to you could cross match units to find an m negative unit remembering that about 75 percent of units are m positive one out of every four will be a negative and so if you want to play it safe you could find an m negative unit but in this particular case because it's an emergency um and we need to be able to issue units and our inventory is limited you just issue what you have on the shelf um m positive or m negative doesn't matter and it will not cause homolysis um now if you did a full serological cross match right it potentially would show up if you did immediate spin but remembering that i said it's a kind of a red hearing it tricks you because there's reactivity immediate spin it's still okay to issue it and it will show up in a full serological cross match so that should that would have been theoretically known um is the same this no this antibody causes hemolysis we know that it typically does not and this antibody causes anaphylaxis no it does not all right so now that we've identified the alloy antibody remember that you form alloy antibodies to things that you do not have on your red cells of course there's exceptions to the rule but for the most part you form alloy antibodies to things you don't have this is also called the landsteiner's law so if you phenotype a patient's red blood cells what antigens would you expect to be negative lewis a duffy a m or n and our texts were kind enough to do the phenotyping you can see the grading was negative with an m monoclonal positive control was good negative control was good so this is a valid test and we'd expect the patient would have no m antigen because they formed the alloy antibody so now back to our original question we had an discrepancy and the most likely reason why we had a discrepancy was that the red cell that we were using that was an a1 likely expressed the m antigen and the person had an anti m therefore we're getting that reactivity in this particular cell for the reverse typing so now that we know that we select a red cell reagent that is negative for our anti-m and then in an emergency you would expect the typing to be compatible in the future without typing your reagents in advance what would you use for your massive hemorrhage protocol so again we have a patient coming in they're massively bleeding let's say they're at a new hospital they don't have time to type their reagent and we know from the past slides that anti-m reacting at immediate spin does not cause clinically significant hemolysis and so you would essentially give your a positive since we know the patient's blood group and we would ignore the m in this particular case and we would give m unscreened this would be c the other thing to keep in mind is that even if the person had a clinically significant anti-m remember they don't they've very very rarely cause in vitro hemolysis or in vivo hemolysis which means that you're not getting intravascular harmonious you're not putting the blood in and it doesn't hemolyze as soon as it gets into the person's bloodstream it binds and then it gets removed later on so if you happen to transfuse a unit or two that aren't compatible they're not going to be removed right away so be able to save the person's life because the red cells are going to survive for a day or two now the clinicians can go and do other things like apheresis if they need to get rid of it or support the patient until the units are cleared but even if it was clinically significant it would not cause an issue especially in an emergency setting you need to save the person's life in the moment and that anti-m will not cause harm in the moment so our second case uh is actually nothing to do with serology per se it's reviewing the newest massive hemorrhage protocol toolkit that is available on orpcon's website so i've given you the link and we're just going to run through a few questions these will be pretty straightforward so the first question is you're preparing to update your massive hemorrhage protocol an mhp should include a multi-disciplinary team for a and you can see i've um removed this from the pro from the tool kit and you can see it should be developed by multi multi-disciplinary team including physicians from multiple hospital services including emergency trauma surgery anesthesia critical care and then obviously non-physician personnel like blood transport security communication services laboratory personnel and they also suggested someone from respiratory therapy for the second part of this applicable only for level one trauma centers that is false i thought i had it there we go um so the protocol should be tailored to each institution based on their their current resources and so therefore a one size fits all does not actually work so this one and this one are gone now and should be reviewed every five years is not true it should be reviewed every three years so that should be three the answer is a for the second question the transfusion packs should include so these are the coolers that you send out so in each cooler you've got the rbcs plasma and then plus or minus the platelet so two units of rbcs pcc which is prothrombin complex concentrate if plasma is not available only two coolers unless the clinical team calls for more and then continue no matter what the laboratory results come back all right so based on those choices let's take a look at what's in the mhp toolkit we can see for adults they all start off with four rbcs so that first one is out we can also see that um for massive hemorrhage protocols that can't provide plasma they're actually providing pccs so that one is correct if you don't have plasma you should be offering prothomben complex concentrate um and again here it shows if it's if you don't have enough to issue for plasma you can substitute it for a pcc um and then for the final question on there was whether or not you would use laboratory uh values to guide your your um your management and the answer is yes you should so we use four rbcs for the most part for the based on toolkit um you keep sending coolers until the clinical team calls you and tells you to stop sending coolers right because the communication should be two-way the entire time and you also want to stay ahead of the bleeding and then you continue on and you start to modify what you're giving based on the laboratory results so once you start getting laboratory results in they may call and start modifying um what the components are that the patient should be getting so that it's being tailored to what their uh bleeding is actually needing to in order for it to stop so the correct answer is b pcc can be used if plasma is not available so question three gets at uh the use of rh versus rh positive units and a bleeding person where you don't know the blood type so we have a mhp going on and we need to know what the appropriate pair of a person to the own egg unit is most appropriate and i pulled this image from the tool kit and you can see any woman less than 45 years of age should receive own egg so off the bat we can say that this 60 year old man doesn't need it and this 40 year old man doesn't need it and so now we're down to a 15 year old woman and a 30 year old woman they're both less than 45 however the 30 year old woman has had a hysterectomy which means she can't have a child on her own and therefore if she forms an anti-d it actually won't cause harm to a fetus because she can't carry a child and therefore she can actually receive o positive units as long as you know that she has a history of a hysterectomy and so then we're left with c being the answer a 15 year old woman uh can should be the one to receive own eggs out of these options for question four which of the following is not possible for a massively transfused patient so when you're receiving large volumes of red blood cells plasma platelets cryo and whatever else you're getting remember that many of them are colder temperatures so there's something called the deadly triad it's hypothermia hypocalcemia and dilutional coelopathy it essentially occurs because you're getting cold blood products which drops your temp and when you drop the temp you actually decrease the effectiveness of your coagulation cascade the enzymes require a certain temperature so when they drop you actually make clotting harder because there's citrate in the blood products you can actually drop the calcium levels so hypothermia hypocalcemia and then because um you may be putting in saline albumin or more plasma than red cells you can also dilute uh the you can also drop the hemoglobin content and make them diluted or you can also just by giving them plasma too many red cells um or too much albumin you can also drop the person's coag levels uh because you delete their plasma too much so either you can dilute the hemoglobin or you can dilute the plasma um most often people refer to dilution of glyculopathy as the dilution of the plasma content and therefore a drop in the collage factors from unbalanced transfusions and then finally um the inr verb of plasma once it's thought is not one it's usually above 1.3 somewhere between 1.3 and 1.7 is um the average unit so this is not possible if you're transfusing um plasma even if you replace the entire blood volume of someone with plasma that's been freshly thawed they would never be they would never be in an iron rf1 they'd be ninr somewhere between 1.3 to 1.7 so they can be hypothermic they can be hypocalcemic and they can have dilutional glyculopathy this is going to be our third and final case for tier one so let's take a look at what we have question one is a 28 year old woman presenting to the emergency department with fatigue and flu-like symptoms she's diagnosed with aml with a hemoglobin of 65. she uh the nurse informs you if days later the patient will be getting a stem cell transplant from a sibling that is b positive and in order uh for one unit is placed of red blood cells and since the patient's shorter breath before starting chemotherapy which blood group would you choose to issue for this patient so in looking at this let's just first look at the typing maybe on rh typing four typing looks good we have a four typing group a reverse typing matches with an atb they're rh negative with two monoclonal rhd reagents so therefore they are a neg so we have a person who's sorry my handwriting is not great today we have a person who's a neg going to be positive and so we and knowing that need to think about what the patient's going to be getting red cell wise now most institutions in canada in the standard practices actually you continue to transfuse based on the current blood grouping and then you switch over when the transplant happens there are some institutions less commonly and i've been a part of some of them where if you are an egg going to be paz you actually think about this part when before you ever go to transplant so in this particular case we're going to think about what most people do and which is probably what your institution does which would be this approach when the transplant happens you stop and you switch over so we're thinking about the patient who is a neg now and so as a result you would train you transfuse them with a negative units now if you're if you were happened to be at an institution where you consider the conversion blood group you'd have to think about what would be compatible with an egg and a b paws and the reason why some places do this is that if you transfuse an a neg rbc and now they start making anti-a right because they're b positive you can get hemolysis so some places as soon as they know someone's going to transplant they will take into account both blood groups and then make the decision if you're at an institution like this you'd be giving one egg or oppos depending on how strict you're going to be so however for most institutions it'll be a nag because you stick to the blood group and then when the switch happens so let's say transplant happens here you then switch over to the o negative or o positive depending on your institution but for this particular answer again it's a negative so the patient begins uh induction chemo and now you notice a discrepancy what explanation what is the explanation for the discrepancy so let's look back at our we have a four typing of group a reverse typing shows uh a b individual since they have no anti-a or atp they continue to be rh negative so they haven't fully engrafted showing any rh positive so the question is what's causing the uh the discrepancy so remember we have a forward discrepancy and we have a reverse discrepancy we have to decide which is which since we knew the historical we know that it's not the forward typing because the forward typing still types as a group a what's odd in this particular case is that our reverse typing is now different right we used to have an anti-b and it's gone now so if we take a look at this gain of a antigen on the red blood cell no we know that we haven't gained anything loss of a antigen on the red blood cell no because we can see it in the forward typing um gain of antib in the plasma serum obviously we haven't had a gain since we have no anti-b leaving us with d loss of antibes in the plasma or serum now the next question two weeks after chemotherapy and the stem cell transplant a type in screen is sent in preparation for transfusion what's the explanation for the mixed field rbc agglutination so remember this person started a pause they got transplanted so they're like in a black box so to speak and at some point they're going to come out as bpos excuse me they were an egg let me change that here they were a negative going to be positive so at some point in this black box they're going to be a negative and be positive at you know at the same time which means that you have to take that into account right so in the last question they had lost their reverse typing right and so they were just looking as group a forward but their plasma cells weren't producing the anti-b now they're in grafting more and so as a part of this a neg to b pos situation we're starting to produce both a negative and b positive cells and so now we're getting a mixed field so in the forward typing we have the patient's old red blood cells floating around giving us this anti-a forward typing and for the reverse typing we would potentially have an anti-b which we don't see anymore right because the patient's plasma cells are being destroyed as a part of the chemotherapy and re-engraftment now the new red blood cells four type is group b and um we're now starting to see anti-deform from the new red blood cells so the green is new and this blue are the old red blood cells and so you can imagine that when you're thinking about this in a test tube you've got a and b red blood cells in it which means that when you're doing your agglutination only one or the other is going to glutenate when you're using these reagents and therefore you get mixed field so as we've been describing the production of group a red blood cells no because we're going from group a to a group b production of group o we know that's not true production of group a b we know that's not true the person was is in grafting with their new group b cells which is what's giving us our new mixed phenotype now a few months after the transfusion so we would theoretically have full engraftment the person would have gone from an egg to b-paws and they would look hopefully mostly v-paws the patient presents at an outside smaller hospital with a gi bleed there's a request for two units of red blood cells with a type and screen ordered stat what blood group do you provide in an emergency so let's take a look at the forward and reverse typing forward typing looks like a b reverse typing still looks like a group o r h looks positive positive so we have an rh positive so forward reverse looks like we have a b pause the reverse makes it look like excuse me my brain is not working today a b a b so the question is what are we going to provide if we don't know what this person is and you have to think about this remember in terms of what's going to be the most compatible with both of uh the with both potential blood groups the best thing you can do in this particular case since they're both rh positive anything with rh negative is essentially thrown out as an answer right because we don't need to worry about the rh now we find out what's compatible with each of them a group a is not compatible with a group b person so not going to happen group b would be compatible however we also don't know what's going on with this group a b in the reverse we're either missing or adding an antibody and therefore we can't resolve it we don't know if the group b red cells will be attacked by an antibody that we're missing leaving us with gruvo paz because that's the safest bet to know that it will be compatible and we will have no issues so why did the patient not form that anti-a in their plasma or serum remember that the world doesn't just revolve around as much as i would love it to it does not just revolve around our red blood cells right so we have our red blood cells and they express a b a [Music] um and with this particular individual the only thing they're expressing right now is the group b and so they have group b theoretically they'd form an anti a that's however not the case because there are other organs involved right we've got the heart oh geez the heart that's my ugly liver the kidneys they all express and these organs were were predating the transplant right so all these organs express the group a so as a result the human body is tolerized to group a and won't form an anti-a now if you were to magically change the entire body over to group b you could make an anti-a but that's not the case the only thing that's producing really group b in this particular case is the anything coming out of the bone marrow which in what we're talking about are red blood cells but everywhere else in the body there's group a being expressed so the body doesn't see group as foreign as foreign so why is it that it does not group a is expressed in other parts of the body um a b antigen is not expressed only in red blood cells the a b antigen is not expressed in other parts of the body so we're not talking about the a and b antigen right the the b antigens only being expressed on the stuff coming out of the bone marrow but it's not expressed on the organs um and the a antigen is only expressed on the red cells false because they switched over to their new blood group so it should be only group b so that brings us to the end of our tier one i'm sorry for a little bit of rambling and free thought there at the end i was uh just trying to make sure i captured everything um we'll be getting to tier two and tier 3 in the next few days releasing them in a staggered format so you have time to finish them and look forward to any feedback or questions you have

hello my name is wassi manani i am a medical officer with canadian blood services and i work with the national immunohematology reference laboratory you know there are going to be three sets of videos uh one for each of the tiered questions this one is for tier one and this is going to be a bit of a hybrid of a powerpoint and a chalk talk i'll be using my stylus to write on the screen and hopefully this will help enhance the education content i'm looking forward to it and let's get started we'll be starting with our first case and remember the tier 1 cases are the baseline cases that are the easiest however there are some more difficult questions thrown in so don't be too surprised when you see some hard questions so we have a 24 year old female that arrives for an outpatient surgical procedure and a type in screen is sent so for this first question let's take a look what we have we have our four typing and we have a four plus with an ata and nothing with an anti b our reverse typing shows a one plus with an a1 cell four plus with a b cell and our rh typing with two different d reagents shows four plus and four plus our dat is negative with the poly specific reagent so when we're looking at this and trying to answer the first question the first question is typing demonstrates a discrepancy in the blank so when thinking about the discrepancy we have to think about where the obviously the descriptive reaction is and most often text will look at the strength of reactivity as an indicator but the reality reality is you should really think about the forward and the reverse typing separately and think about whether or not they jive together so in this particular case the four typing presents as a group a the reverse typing presents as a group o so the question becomes which of these is the discrepant one in this particular case because the strength of this a1 is much weaker you could argue that the group o typing is the one that is discrepant versus the group a typing that's not always correct but when you have to start resolving an  discrepancy you have to pick a direction you either have to pick the forward or the reverse typing in order to do your work up and you'll see with uh my workflow later on that you have to pick a direction you can't sit on the fence about it and i think that's one of the most difficult things about an discrepancy you pick one of the two and then you work it up you may be completely wrong and that's okay but in this case because the discrepancy appears to be most related to the a1 cell we would say the reverse typing is a discrepant for question two what is the first step in resolving the discrepancy you can pre-warm wash the patient cells review the antibody screen results or increase the incubation time so again let's look at this we have forward typing which we think is correct a reverse typing which we think is incorrect and with this reverse typing we have a few options right we can pre-warm it to see if it goes away washing the patient's rbcs won't do anything because that's really the the forward typing bit reviewing the antibody screen results we can do that and increase the incubation time we can do that so these three things are the three things that we can do for reverse typing if we were to pre-warm the plasma or serum the question becomes is the shotgun approach really worth it so if you pre-warm a sample you're not sure what you're pre-warming remember you can pre-warm away clinically significant antibodies and when you're doing a type in screen the whole point of doing the screen is to identify any antibodies that may interfere with your typing so in this particular case you don't pre-warm unless you know what you're pre-rolling away increasing the incubation time will likely not do much or make this one plus even worse and so that'll just make it more confusing and so the last one we have is reviewing the antibody screen results again remembering that if you have an aloe antibody or an auto antibody it can interfere but if you don't know that it's there you can't really attack it so the best way to go about this is actually just doing your antibody screen results which you would usually do at the same time as your typing if you've got multiple systems running but in this case we're kind of taking it in a stepwise format so as i promised we're going to talk about how to approach a forward and reverse typing discrepancy and this is after you've made the decision about whether the forward or reverse is discrepant remembering that you just have to pick a direction you may not be right and that's okay but you just pick a direction and you go with it so with a forward typing discrepancy we really have three main things we can think about an extra antigen being expressed a missing antigen so a loss of an antigen or mixed field agglutination that throws us off that we were not expecting with a reverse typing discrepancy we can have extra antibodies so like we have in this particular case we have one plus reactivity with an a1 cell and an individual who is group a we would not expect that or we can have missing antibodies so for example if a group a individual did not have an anti-b in their plasma we would be surprised by that so for our forward typing let's go through my thought process of how i approach it so we have a forward type and i'm sorry about my awful handwriting we can have a missing or weakened antigen correct and so from there what i usually think about is a subgroup of a or b or you can have suppressed antigen the other side of a forward typing is you can have unexpected reactivity and so you have an extra antigen you weren't expecting so with this there's a few options you can have an acquired b so this for example occurs when someone with colon cancer releases a deacetylase enzyme which then converts a group a individual to look like a group b individual by some monoclonals that's that's much less seen now nowadays than it was in the past but it can still be seen you can have this which is essentially a b which expresses a weakened form of a and therefore it looks like it's an extra antigen but the person really does express a group a it's just weakened you can have mixed field and you can have antibody coding the cell which blocks the antigen from being detected and before i forget if anyone ever wanted to know this is called b sub a so for our forward typing we can have mixed or weakened and we have subgroups that are a or b we can have a suppressed antigen and on the other side we can have unexpected antigens so we can have acquired b we can have a b sub a a mixed field or antibody coding the red cell which throws us off so one of the more common types of discrepancies with four typing is with the group a and so i'm going to focus on that for the next two slides because it's the most common thing that we run into with a subgroups so when thinking about a subgroups they give us usually four typing that's off and sometimes a reverse type that's off if they form an anti-a1 but let's focus just on the forward typing so if your four typing reagent shows a weak with an anti-a reagent and it's fully mixed field we're talking about an a3 which is the most common subtype of the a's if it's less than 10 percent mixed field then we're talking about a end if it's weak plus with an anti a comma b then we're talking about an a x and you'll recognize these most often from textbooks and the reality is even though i've learned these serologically a lot of it's done molecularly now but serologically you can get pretty darn close to what it is as long as you have a gentle hand and a good eye you can detect the difference between a full mixed field and a 10 mixed field and then the week plus with an anti-a comma b so now what happens with your a sub groups if your anti-a shows nothing well now you have to actually start going into secretor status which i'm sure many people love oops so if you're a full secretor and you've got nice strength reactivity with your a and h antigens and h being group o then you're an am if your secretor status shows weak plus for a and h now we're talking about a end and if you secrete only the h now we're talking about a l and the a l is the the elute version of a so a is expressed on such a low level you use an anti-a and it shows nothing but you can elute off the nta because it's concentrated when you run the elute and that's when you can detect it and so they only secrete h as a result of that because there's such low amounts of it so with these these of the a subgroups there's no anti-a reactivity compared to the prior slide where there's weak reactivity with anti-a reagents and again you can identify these molecularly they can be a little tough sometimes depending on the genotype the type of genotyping you run and so serologically you can get pretty close to what the answer is so now let's talk about what's going on in our case we have a reverse discrepancy so with a reverse discrepancy you can have missing or weakened antibody the other option is you can have an extra antibody up here that you weren't expecting so with the missing or weakened you have two options you can have weakened anti-a in the patient's plasma serum or weakened antibe so pretty simple with the x-ray antibody we have a few you can have an a subgroup which means you have to run the a1 lectin because you have an anti-a1 present when you don't expect it in a group a individual you can have an auto antibody cold or warm you can have rouleaux which is the stacking of coins usually due to plasma proteins and then you can also have a passive antibody so for example if someone receives a red cell unit with an alloy antibody in it it may show up or if someone receives something like ipig and it tends to bind something that it shouldn't so again to go over our reverse typing discrepancies we get a missing or weakened antibody so for example in a group b individual you might have weakened anti-a and a group a individual you might have weakened anti-b or in a group o you might have missing or weakened anti-a or anti-b for extra antibodies you can have an anti-a1 which you would identify with your a1 lectin you could have an auto antibody giving you extra reactivity rouleaux which is the stacking which is called stacking of coins when the red cells stack because of excess plasma proteins or you can have a passive antibody due to some other introduction of the antibody to the patient so the identified antibodies known to classically react at what temperature so let's take a look at our at our antibody id we have immu core cells and we have no reactivity in mts gel in the first five cells here and then the last two cells six and seven we have three and two plus reactivity so if we look across kind of making a little bit easier you can see that the texts have already kind of ruled out a lot of stuff and really if we just scan we can see that the only antigen that's present that really only has two that are reactive are is the m antigen and so we have three inch plus reactivity matching with the anti-m and we know that anti-m reacts classically at room temperature because it's a cold reacting aloe antibody so we're going to talk a little bit about the m aloe antibody and let's first discuss the serology so it's predominantly igm in cold reacting which means it reacts at a predominantly immediate spin reactivity it does show dosage so if you're m positive and positive or m positive and positive you'll get let's say one plus reactivity here and you might get three plus reactivity here it's warm reacting oh sorry if it's warm reacting it's usually an igg component of anti-m so for those cases where you've got immediate spin 37 iat and you see oneplus reactivity let's say oneplus reactivity and oneplus reactivity it's most commonly that this component is the igm this component is the igg so i j the anti-m can actually have multiple ig isotypes depending on where it reacts and they usually do not activate complement even the ones that uh react at iat phase normally don't activate complement usually um they cause hemolysis through delayed hemolysis so they'll bind to the red cell and they'll be removed from this from the from the intravascular space by the reticulo endothelial system so they don't cause intravascular hemolysis the way that for example antibodies can sorry i'm trying to get it to go next for me there we go uh we can use enhancement to help us out so let me just erase this here so if we're going to use enhancement to to bring out the anti-m you can use peg list or poly brain however if you're using these techniques you have to be really careful because if it reacts at immediate and immediate spin let's say it's two plus or three plus an immediate spin at 37 and iat if you using your peg let's say you can actually get reactivity that's like let's say one plus that kind of carries over because the peg enhances it enough that it's able to react at a warmer temperature when it normally wouldn't so just because it reacts at 37 does not mean it's clinically significant if you're going to run it in peg so if you're going to look at clinical significance of anti-m it should only be done saline because you can trick yourself into thinking it's clinically significant based on this carryover effect of using an enhancement method of like pegless or polybrain you can drop the ph down to 6.5 to enhance an anti-m and some anti-m's only react in the presence of one to two plus one or to two percent glucose solutions um in plasma and we don't really know why that is other than the fact that the glycophorin a uh protein is a glycoprotein which has sugars on it and it may be that the sugar helps enhance the antibody binding to the actual protein itself and then finally clinical implications if it reacts at greater than 37 degrees it's more likely to be clinically significant and therefore you should honor it if you look in it says that anything that does not react at 37 or above for an anti-m they pretty much ignore it and they'll just give it even if it's cross-matching compatible at a immediate spin they also recommend not doing immediate spin cross matches for these anti-m's because it essentially just uh makes more of a headache for you and then it very rarely causes hdfn it can but it very rarely does and in cardiac surgeries a lot of times we get questions about what happens if we're going to put the patient in hypo in a hypothermic state so they drop the temperature of the patient down to drop the heart rate in order to do the work and allow the heart not to be ischemic interestingly they looked at patients with anti-m's that were cold reacting only and none of them caused hemolysis or extra or that caused the delayed hemolysis so really these anti-amps don't cause any issues even when the body temperature is dropped down they're pretty much just a nuisance when they react only at immediate spin for this next question number four if a transfusion is needed and you have limited inventory would you issue cross-matching compatible units based on the antibody identified so now we have to think about what i just talked about before remembering that this antibody does not really cause hemolysis and it's only reacting really at immediate spin what would we do because it's not clinically significant you can actually especially in an emergency issue and feel pretty darn safe now if you had a larger institution or if you have a ton of time if you really want to you could cross match units to find an m negative unit remembering that about 75 percent of units are m positive one out of every four will be a negative and so if you want to play it safe you could find an m negative unit but in this particular case because it's an emergency um and we need to be able to issue units and our inventory is limited you just issue what you have on the shelf um m positive or m negative doesn't matter and it will not cause homolysis um now if you did a full serological cross match right it potentially would show up if you did immediate spin but remembering that i said it's a kind of a red hearing it tricks you because there's reactivity immediate spin it's still okay to issue it and it will show up in a full serological cross match so that should that would have been theoretically known um is the same this no this antibody causes hemolysis we know that it typically does not and this antibody causes anaphylaxis no it does not all right so now that we've identified the alloy antibody remember that you form alloy antibodies to things that you do not have on your red cells of course there's exceptions to the rule but for the most part you form alloy antibodies to things you don't have this is also called the landsteiner's law so if you phenotype a patient's red blood cells what antigens would you expect to be negative lewis a duffy a m or n and our texts were kind enough to do the phenotyping you can see the grading was negative with an m monoclonal positive control was good negative control was good so this is a valid test and we'd expect the patient would have no m antigen because they formed the alloy antibody so now back to our original question we had an discrepancy and the most likely reason why we had a discrepancy was that the red cell that we were using that was an a1 likely expressed the m antigen and the person had an anti m therefore we're getting that reactivity in this particular cell for the reverse typing so now that we know that we select a red cell reagent that is negative for our anti-m and then in an emergency you would expect the typing to be compatible in the future without typing your reagents in advance what would you use for your massive hemorrhage protocol so again we have a patient coming in they're massively bleeding let's say they're at a new hospital they don't have time to type their reagent and we know from the past slides that anti-m reacting at immediate spin does not cause clinically significant hemolysis and so you would essentially give your a positive since we know the patient's blood group and we would ignore the m in this particular case and we would give m unscreened this would be c the other thing to keep in mind is that even if the person had a clinically significant anti-m remember they don't they've very very rarely cause in vitro hemolysis or in vivo hemolysis which means that you're not getting intravascular harmonious you're not putting the blood in and it doesn't hemolyze as soon as it gets into the person's bloodstream it binds and then it gets removed later on so if you happen to transfuse a unit or two that aren't compatible they're not going to be removed right away so be able to save the person's life because the red cells are going to survive for a day or two now the clinicians can go and do other things like apheresis if they need to get rid of it or support the patient until the units are cleared but even if it was clinically significant it would not cause an issue especially in an emergency setting you need to save the person's life in the moment and that anti-m will not cause harm in the moment so our second case uh is actually nothing to do with serology per se it's reviewing the newest massive hemorrhage protocol toolkit that is available on orpcon's website so i've given you the link and we're just going to run through a few questions these will be pretty straightforward so the first question is you're preparing to update your massive hemorrhage protocol an mhp should include a multi-disciplinary team for a and you can see i've um removed this from the pro from the tool kit and you can see it should be developed by multi multi-disciplinary team including physicians from multiple hospital services including emergency trauma surgery anesthesia critical care and then obviously non-physician personnel like blood transport security communication services laboratory personnel and they also suggested someone from respiratory therapy for the second part of this applicable only for level one trauma centers that is false i thought i had it there we go um so the protocol should be tailored to each institution based on their their current resources and so therefore a one size fits all does not actually work so this one and this one are gone now and should be reviewed every five years is not true it should be reviewed every three years so that should be three the answer is a for the second question the transfusion packs should include so these are the coolers that you send out so in each cooler you've got the rbcs plasma and then plus or minus the platelet so two units of rbcs pcc which is prothrombin complex concentrate if plasma is not available only two coolers unless the clinical team calls for more and then continue no matter what the laboratory results come back all right so based on those choices let's take a look at what's in the mhp toolkit we can see for adults they all start off with four rbcs so that first one is out we can also see that um for massive hemorrhage protocols that can't provide plasma they're actually providing pccs so that one is correct if you don't have plasma you should be offering prothomben complex concentrate um and again here it shows if it's if you don't have enough to issue for plasma you can substitute it for a pcc um and then for the final question on there was whether or not you would use laboratory uh values to guide your your um your management and the answer is yes you should so we use four rbcs for the most part for the based on toolkit um you keep sending coolers until the clinical team calls you and tells you to stop sending coolers right because the communication should be two-way the entire time and you also want to stay ahead of the bleeding and then you continue on and you start to modify what you're giving based on the laboratory results so once you start getting laboratory results in they may call and start modifying um what the components are that the patient should be getting so that it's being tailored to what their uh bleeding is actually needing to in order for it to stop so the correct answer is b pcc can be used if plasma is not available so question three gets at uh the use of rh versus rh positive units and a bleeding person where you don't know the blood type so we have a mhp going on and we need to know what the appropriate pair of a person to the own egg unit is most appropriate and i pulled this image from the tool kit and you can see any woman less than 45 years of age should receive own egg so off the bat we can say that this 60 year old man doesn't need it and this 40 year old man doesn't need it and so now we're down to a 15 year old woman and a 30 year old woman they're both less than 45 however the 30 year old woman has had a hysterectomy which means she can't have a child on her own and therefore if she forms an anti-d it actually won't cause harm to a fetus because she can't carry a child and therefore she can actually receive o positive units as long as you know that she has a history of a hysterectomy and so then we're left with c being the answer a 15 year old woman uh can should be the one to receive own eggs out of these options for question four which of the following is not possible for a massively transfused patient so when you're receiving large volumes of red blood cells plasma platelets cryo and whatever else you're getting remember that many of them are colder temperatures so there's something called the deadly triad it's hypothermia hypocalcemia and dilutional coelopathy it essentially occurs because you're getting cold blood products which drops your temp and when you drop the temp you actually decrease the effectiveness of your coagulation cascade the enzymes require a certain temperature so when they drop you actually make clotting harder because there's citrate in the blood products you can actually drop the calcium levels so hypothermia hypocalcemia and then because um you may be putting in saline albumin or more plasma than red cells you can also dilute uh the you can also drop the hemoglobin content and make them diluted or you can also just by giving them plasma too many red cells um or too much albumin you can also drop the person's coag levels uh because you delete their plasma too much so either you can dilute the hemoglobin or you can dilute the plasma um most often people refer to dilution of glyculopathy as the dilution of the plasma content and therefore a drop in the collage factors from unbalanced transfusions and then finally um the inr verb of plasma once it's thought is not one it's usually above 1.3 somewhere between 1.3 and 1.7 is um the average unit so this is not possible if you're transfusing um plasma even if you replace the entire blood volume of someone with plasma that's been freshly thawed they would never be they would never be in an iron rf1 they'd be ninr somewhere between 1.3 to 1.7 so they can be hypothermic they can be hypocalcemic and they can have dilutional glyculopathy this is going to be our third and final case for tier one so let's take a look at what we have question one is a 28 year old woman presenting to the emergency department with fatigue and flu-like symptoms she's diagnosed with aml with a hemoglobin of 65. she uh the nurse informs you if days later the patient will be getting a stem cell transplant from a sibling that is b positive and in order uh for one unit is placed of red blood cells and since the patient's shorter breath before starting chemotherapy which blood group would you choose to issue for this patient so in looking at this let's just first look at the  typing maybe on rh typing four typing looks good we have a four typing group a reverse typing matches with an atb they're rh negative with two monoclonal rhd reagents so therefore they are a neg so we have a person who's sorry my handwriting is not great today we have a person who's a neg going to be positive and so we and knowing that need to think about what the patient's going to be getting red cell wise now most institutions in canada in the standard practices actually you continue to transfuse based on the current blood grouping and then you switch over when the transplant happens there are some institutions less commonly and i've been a part of some of them where if you are an egg going to be paz you actually think about this part when before you ever go to transplant so in this particular case we're going to think about what most people do and which is probably what your institution does which would be this approach when the transplant happens you stop and you switch over so we're thinking about the patient who is a neg now and so as a result you would train you transfuse them with a negative units now if you're if you were happened to be at an institution where you consider the conversion blood group you'd have to think about what would be compatible with an egg and a b paws and the reason why some places do this is that if you transfuse an a neg rbc and now they start making anti-a right because they're b positive you can get hemolysis so some places as soon as they know someone's going to transplant they will take into account both blood groups and then make the decision if you're at an institution like this you'd be giving one egg or oppos depending on how strict you're going to be so however for most institutions it'll be a nag because you stick to the blood group and then when the switch happens so let's say transplant happens here you then switch over to the o negative or o positive depending on your institution but for this particular answer again it's a negative so the patient begins uh induction chemo and now you notice a discrepancy what explanation what is the explanation for the discrepancy so let's look back at our we have a four typing of group a reverse typing shows uh a b individual since they have no anti-a or atp they continue to be rh negative so they haven't fully engrafted showing any rh positive so the question is what's causing the uh the discrepancy so remember we have a forward discrepancy and we have a reverse discrepancy we have to decide which is which since we knew the historical we know that it's not the forward typing because the forward typing still types as a group a what's odd in this particular case is that our reverse typing is now different right we used to have an anti-b and it's gone now so if we take a look at this gain of a antigen on the red blood cell no we know that we haven't gained anything loss of a antigen on the red blood cell no because we can see it in the forward typing um gain of antib in the plasma serum obviously we haven't had a gain since we have no anti-b leaving us with d loss of antibes in the plasma or serum now the next question two weeks after chemotherapy and the stem cell transplant a type in screen is sent in preparation for transfusion what's the explanation for the mixed field rbc agglutination so remember this person started a pause they got transplanted so they're like in a black box so to speak and at some point they're going to come out as bpos excuse me they were an egg let me change that here they were a negative going to be positive so at some point in this black box they're going to be a negative and be positive at you know at the same time which means that you have to take that into account right so in the last question they had lost their reverse typing right and so they were just looking as group a forward but their plasma cells weren't producing the anti-b now they're in grafting more and so as a part of this a neg to b pos situation we're starting to produce both a negative and b positive cells and so now we're getting a mixed field so in the forward typing we have the patient's old red blood cells floating around giving us this anti-a forward typing and for the reverse typing we would potentially have an anti-b which we don't see anymore right because the patient's plasma cells are being destroyed as a part of the chemotherapy and re-engraftment now the new red blood cells four type is group b and um we're now starting to see anti-deform from the new red blood cells so the green is new and this blue are the old red blood cells and so you can imagine that when you're thinking about this in a test tube you've got a and b red blood cells in it which means that when you're doing your agglutination only one or the other is going to glutenate when you're using these reagents and therefore you get mixed field so as we've been describing the production of group a red blood cells no because we're going from group a to a group b production of group o we know that's not true production of group a b we know that's not true the person was is in grafting with their new group b cells which is what's giving us our new mixed phenotype now a few months after the transfusion so we would theoretically have full engraftment the person would have gone from an egg to b-paws and they would look hopefully mostly v-paws the patient presents at an outside smaller hospital with a gi bleed there's a request for two units of red blood cells with a type and screen ordered stat what blood group do you provide in an emergency so let's take a look at the forward and reverse typing forward typing looks like a b reverse typing still looks like a group o r h looks positive positive so we have an rh positive so forward reverse looks like we have a b pause the reverse makes it look like excuse me my brain is not working today a b a b so the question is what are we going to provide if we don't know what this person is and you have to think about this remember in terms of what's going to be the most compatible with both of uh the with both potential blood groups the best thing you can do in this particular case since they're both rh positive anything with rh negative is essentially thrown out as an answer right because we don't need to worry about the rh now we find out what's compatible with each of them a group a is not compatible with a group b person so not going to happen group b would be compatible however we also don't know what's going on with this group a b in the reverse we're either missing or adding an antibody and therefore we can't resolve it we don't know if the group b red cells will be attacked by an antibody that we're missing leaving us with gruvo paz because that's the safest bet to know that it will be compatible and we will have no issues so why did the patient not form that anti-a in their plasma or serum remember that the world doesn't just revolve around as much as i would love it to it does not just revolve around our red blood cells right so we have our red blood cells and they express a b a [Music] um and with this particular individual the only thing they're expressing right now is the group b and so they have group b theoretically they'd form an anti a that's however not the case because there are other organs involved right we've got the heart oh geez the heart that's my ugly liver the kidneys they all express and these organs were were predating the transplant right so all these organs express the group a so as a result the human body is tolerized to group a and won't form an anti-a now if you were to magically change the entire body over to group b you could make an anti-a but that's not the case the only thing that's producing really group b in this particular case is the anything coming out of the bone marrow which in what we're talking about are red blood cells but everywhere else in the body there's group a being expressed so the body doesn't see group as foreign as foreign so why is it that it does not group a is expressed in other parts of the body um a b antigen is not expressed only in red blood cells the a b antigen is not expressed in other parts of the body so we're not talking about the a and b antigen right the the b antigens only being expressed on the stuff coming out of the bone marrow but it's not expressed on the organs um and the a antigen is only expressed on the red cells false because they switched over to their new blood group so it should be only group b so that brings us to the end of our tier one i'm sorry for a little bit of rambling and free thought there at the end i was uh just trying to make sure i captured everything um we'll be getting to tier two and tier 3 in the next few days releasing them in a staggered format so you have time to finish them and look forward to any feedback or questions you have

Transcript for:Understanding Immunohematology and Transfusion

Transcript for:
Understanding Immunohematology and Transfusion