I hope all of you had a really good break. I hope you guys were pleased with the program calendar. I do apologize for the late send out on that. But we were able to adjust the calendar to allow for an official 13 start date, so I hope you guys were pleased with that, I felt better about it myself, and I hope you guys are excited about blood bank, so blood bank is my passion, what I have the most experience in, and I'm going to turn all of you guys into blood bankers, okay when I was in this program, an internship coordinator at the Blood Bank here at UAMS told me that you have to have a healthy fear of blood bank, and so it can be intimidating, and once you guys get in the blood bank you might see that it can be a very stressful atmosphere because you'll have traumas come in, here at UAMS we are a level one trauma, and so we get to see a lot of gunshot wounds, motor vehicle accidents, motorcycle accidents, stabbing, aortic aneurysms, anything that's very trauma and will use a lot of blood products. And so the role of blood banker is to make sure that we provide safe products for transfusion into those patients. We want to make sure that we give the right ABO. So if you give somebody the wrong ABO it can be fatal. Okay, so that's one reason blood bank is sometimes so scary to students. So, again, I encourage that healthy fear. Keep that fear, but become confident in your determination of ABO Rh antibodies identification as we dig deeper into Blood Bank topics. So, just so you know that to say that I hope you guys will love it as much as I do. Okay, in this lecture, this first lecture, we're going to talk about some, we're gonna do some immunology review, and we're gonna dive into ABO group system in lecture 2. Okay, so bear with me. Probably this first lecture will be a review on how our body builds antibodies. We're really going to concentrate, and I know you guys have all had immunology. And so after this course a lot of students have told me if you guys struggled in immunology maybe you did not like it, and it is one of the courses that a lot of students say that was my least favorite course and it's just because sometimes people have a hard time grasping the antigen-antibody complex, what actually is going on, that is the main focus of blood bank. We want that antigen-antibody complex so we can see agglutination okay so we're really going to focus on making that antibody antigen complex. So students tell me after blood bank they have a better appreciation of immunology. Okay, so I'm hoping that will be the case this year as well. Okay, so what exactly is blood bank and you might hear it referred to us in different terminology. You might hear it referred to as transfusion medicine. Some hospitals call their blood bank the Department of transfusion medicine. The big term for it is immunohematology because we are combining immunology with the cells. So a combination of immunology and hematology. So what we learned last semester, we're building on it this semester. So we're going to continue to talk about those topics. And so again we're going to concentrate on the antigen and antibody complex. That's what we're studying in blood bank. That's what we want. Does the patient have the antigen? And if they lack the antigen, then guess what, they can build the corresponding antibody. And if they have the antibody, when we are thinking about giving transfusions, we want to make sure that the red cells we give them lack the antigen. So, the patient's antibody doesn't attack the product that was giving. Okay, and a good word of advice for blood bank for you guys, I'll go ahead and say it now, you have to think about blood bank forward and backwards. Okay, so if you have a reaction what does that mean? Where's my antigen? Where's my antibody? Am I testing patient plasma? That's where your antibody is. Okay in the patient plasma. Am I using patient red cells to determine what antigen is on the red cells. Your antigen is always on the red cells. Okay, so when we start mixing our antigenic antibody we're going to using commercial antisera. Right, that's your commercially manufactured antibody will react with patient cells. If you're using reagent red cells those are commercially manufactured red cells. You know the antigen is known. We're testing for the antibody in the patient plasma. Okay, so, always in the back of your mind think where's my antigen coming from? Where's my antibody coming from? Am i testing for patient antigen or patient antibody? Okay and that will help you understand this course a lot. And our ultimate goal in blood bank, of course, is to find products for transfusion that is safest for the patient. Typically in blood bank when they order products that usually if you guys are familiar with the donor process, they take off about five hundred mLs in the donor process. That unit can actually be broken down into four different components. Okay, so rather than a whole blood unit, they can do red cells. Alright, they can spin that product down. And we'll talk in depth about this process later on. They can get red cells. They can actually do your buffy coat, alright, so the white cells, if they wanted to give granulocytes only. They can be get platelets, and they can do plasma, right. And they can even, if you really want to think about it, they can break that plasma down into a step further and get cryo precipitates. So really there's five products from one whole blood unit. Back in the old days, we're gonna talk a little bit about history in just a minute, but whole blood donations used to be a thing. You went. You donated blood five hundred mLs. It had red cells, plasma, white cells, and platelets. And they would just give all of that to the patient when they needed a transfusion. That's a whole blood transfusion. However, there's a huge concern with that. You have volume overload. So, maybe the patient only needs oxygen carrying capabilities. Then they only need red cells. They don't need the plasma. They don't need the platelets. They just need the red cells. And, so, that's why we kind of break that whole blood unit into all the different components. And, so, what we're going to study is why we give those components, and how we find the safest components for the recipient. Okay, alright, so just a little bit of history on blood bank and transfusion. It's actually documented that the first transfusion occurred in 1492. Any of you guys familiar with Pope Innocent the eighth? It was thought that he was very severely sick. And it's recorded that three shepherd boys made a donation to him. Depending on one what reference you look at, they say that he drank the blood of these three boys, and ultimately, he died from that. So they tried to tie it back to the blood from the three boys being ultimately what caused his death. Who knows? It might have been his illness that caused his death. But that's the first recorded transfusion in 1492. In 1613, there was a huge discovery by William Harvey. He discovered that the blood circulates in the veins. So what this discovery allowed is they were able to enter the veins at this point, and they used needles to insert things into the circulation. Okay, and then in 1667, we had the first animal-to-human transfusion. And it was a French physician John Baptist Dennis. There was a young boy that was sick, and he transfused that young boy with blood from a lamb. That boy actually survived. And if you look at this research, the history of transfusion, you'll look up, there'll be other animal human transfusions. Some survived, some didn't. It was ultimately determined that animals have different proteins in their circulation not compatible with humans. And so that is why animal to human transfusion of course is not common practice. And then in 1824, James Blundell introduced vein-to-vein transfusion using a syringe. However, if you guys know we're going to talk about this. We use anticoagulants for our blood. And so doing a direct transfusion from donor to recipient the bloodless clotting. And that caused a huge issue. So that was the concern there And in a 1901, we have Dr. Karl Landsteiner. How many you guys have heard of him? He is the founder of the ABO blood system and agglutination. So he and his group of researchers in their lab, they drew each other's blood and they started mixing each other's red cells and plasma. And they noticed agglutination. And so they were able to identify three different ABO systems. The "A", the "B", and, at the time, they called it the "C" group. We all know now the other group is the "O." And also you have "AB." So, he was the original founder of the different ABO groups. And it is in 1921, that the volunteer blood bank services was introduced in Europe. They actually pioneered it. It was not actually until the 1930s that the volunteer blood donor services that which we know of the American Red Cross started in the United States and it became huge. They started using blood bank products in World War two for all the troops in battle. And in 1941, we had the discovery of the Rh, so the Rhesus. So if you're have the D antigen on your red cells, you're Rh positive. If you lack the D antigen in you're Rh negative. And so we'll get into the Rh antigen as well. I talked a little bit about components. Like I mentioned they used to do whole blood transfusions. Now we break though the whole blood into various components depending on what the patient needs. And as we go through this semester, we're going to talk about testing on these donor units. We do a lot of infectious disease testing on these units before they're deemed safe for transfusion. And it all goes back to AIDS because we'll talk about Ryan White who was a little eight-year-old boy who had Hemophilia A. So he had recurrent plasma transfusions from donors to replace his factor eight. And he ended up becoming HIV positive. He had AIDS from all of those transfusions. So it was actually him and his mom that led efforts and all of the infectious disease testing on our products. We'll talk about him more when we start talking about infectious disease testing of our products. Alright and like I mentioned the main point of blood transfusion, we want to have a successful transfusion. Okay we want whatever we're transfusing into the recipient whether it be red cells plasma, we want the recipient to accept it, okay so it has to be compatible, we'll talk a lot about compatibility, antigen-negative, safe for transfusion, we're going to complement that, and we want to only replace what the patient is lacking, okay so if they have been in a trauma, they've lost a lot of blood, we really they might need oxygen carrying capabilities and volume replacement, so maybe red cells and plasma are good for them, maybe even platelets, depending on how much blood they have lost, maybe your patient just has severe anemia, okay and they need oxygen carrying capabilities, we just want to give them red cells, a lot of our chemotherapy patients maybe they have pancytopenia, so they're probably going to get red cells and platelets transfusions. In their cases, you might even give them granulocytes, granulocytes, rare occasions, and again when we start talking about expiration dates of our products, granulocytes only are good for 24 hours from collection, so that requires a lot of communication between the requesting physician, the donor center, and then their receiving hospital, where it's going to be transfused okay so keep in mind, what your patient lacks we need it to be safe, if it's incompatible with that recipient, their immune system is going to destroy it, it does them no good, they're still gonna have the symptoms that they initially presented with, okay so that's our ultimate goal in blood bank, to replace the product your patient is lacking, and make it safe for them, we don't want them to have any adverse reactions from that transfusion either, ok so whether that be infectious disease, maybe that unit, they do a lot of nucleic acid testing now for like West Nile virus, HIV so maybe the unit was initially tested negative but then that donor comes back 8 weeks later and now they test positive, okay so if you can potentially expose that recipient to an infectious disease, that's some type of adverse reaction or maybe you give your recipient the wrong ABO and they have a hemolytic transfusion reaction, that's an adverse reaction, and we don't want that to occur okay so that's the role of the blood banker, to prevent those adverse reactions and when we'll talk a lot about that in this course, okay so just a little bit of immunology review, and I hope this is a review for all of you guys remember immunology is the process where the body is protected from disease and foreign invaders, so think about whether that be bacteria, virus or in the case of blood bank, maybe the patient has been transfused, they're exposed to an antigen on the donor red cells that they are negative for, then when the body recognizes, "Hey that antigen, I've never seen it before! It's foreign, I'm going to build an antibody to it." That's your body's response, and so that's oftentimes we're going to talk about different ways that patients build antibodies. Are they naturally occurring? Have they been exposed? The way that you can be exposed, for females, is if you have a previous pregnancy. If your baby is antigen positive and you are antigen negative, you can build an antibody to whatever your baby's antigen is during the feto-maternal blood exchange, okay if you've had a previous transfusion, you can be exposed to donor cells that have the antigen that you can build the antibody to, okay so we're going to talk about how the body recognizes those antigens as foreign, they must recognize from self versus foreign, all right, the body's going to say, "Hey I've never seeing this antigen before. I'm going to respond to it. I'm going to launch an immune response." Okay we're going to talk about two different types of immune responses. so what happens if your patient builds that antibody, all right, it could cause intravascular hemolysis, depending on what type of antibody it is, and you guys have all talked about the different types of antibodies IgG, IgM, IgA, IgE. Two main antibodies that we're going to talk about in blood bank, IgG and IgM, okay that's the two that we will primarily focus on, so depending on what type of antibody your patient has will determine the effect, okay, if it's an IgM antibody those are typically more severe, they're larger in size, they can result in intravascular hemolysis, okay immediate cell destruction, such as a incompatible ABO transfusion. Or is your antibody IgG? So usually those are not as severe will, attach to the red cells, your spleen will recognize that as foreign, resulting in extravascular hemolysis, so the symptoms are that are not as severe as intravascular hemolysis, and we have some tests that we'll talk about in blood bank to differentiate which type of antibody is coating the patient cells. Why is hemolysis occurring? Is it due to complement? Or is it due to some type of antibody where there's IgG, IgM? So speaking of complement, if you were to give your patient a wrong ABO transfusion, ABO antibodies are IgM. They're very large. They can activate complement that will result in intravascular hemolysis. That's going to cause immediate problems, often times that transfusion will only have lasted probably about 10 minutes. Then they've probably just started it, the patient's going to start immediately complaining of side pain at their kidneys, fever is going to spike, if that transfusion is not immediately stopped, you have intravascular hemolysis occurring, you have red cell fragmentation, those fragments are going to travel to the kidney, they can cause kidney damage Those cells can initiate DIC, okay so that is why the wrong ABO is so fatal, it's gonna cause your patient to go into DIC, okay and they're gonna have intravascular hemolysis, so they're going to be losing cells immediately okay and again so it's that intravascular hemolysis that is more severe than extravascular hemolysis okay and you guys all remember from immunology you remember the complement cascade so once complement is activated C1, C3 C4, C5, 6, 7 and then that 8, 9 complex attaches to the red cell, once that 8 and 9 complex is on the red cell membrane it's gonna puncture a hole in the cell ultimately that would cause cell lysis, okay that's what happens in the wrong abo an extravascular hemolysis typically you'll see this with IgG antibodies, it just attaches to the red cell, your spleen and your liver can recognize that as foreign, and then they will just destroy the cell, that way, extravascularly so if they've got a splenectomy, than those cells just remain in circulation for longer periods of time and the liver might help as well try to clear those cells but typically they'll just be in circulation for the length of their life, we'll concentrate a lot we have a whole section on moms and babies and so I mentioned if the baby is antigen positive for the antigen that the mom lacks she can build an antibody, if that antibody coats baby cells, then those cells will be destroyed and that can cause severe anemia in the baby, it can cause respiratory distress in the baby if it's not treated, any of you guys ever heard of an intrauterine transfusion? Sometimes if it's a very severe case they will do a transfusion into the baby while he's in utero, so that's a technique that we'll talk about in our mom and baby section okay so there's two types of immune responses and I want to use the board and really display this so we're going to talk about, this should all be review, cell-mediated versus humoral immunity, you guys remember that? I want to talk just review that really quick and then how that applies to blood bank. okay so let's say we're inside the body right, we're going to use bacteria because it's easy, you got a bacteria, here's your bacteria, it has all of these specific antigen sites on it these are all of your antigen sites on it, the body's going to launch two types of immune responses, you have cellular, cell-mediated involving the T cells, okay you have a phagocytic cell here, here is your bacteria, your phagocytic cell can engulf it, right and then this phagocytic cells becomes an antigen-presenting cell, so your bacteria is engulfed, all these antigen sites that were on the bacteria are now on this cell, so here's your antigen-presenting cell, now has all of the antigens being exposed on it, so it will present those antigens to what? So your T cells, so here's your T cells, put in the types of T cells, right, you have your CD4 helper cells, you have your CD8 suppressor cells, Your CD4 cells, once they're activated, they can activate your natural killer cells, they're also going to release cytokines and then what's hopefully going to happen in your T cells now express those antigens that were on the bacteria those antigens or receptor sites, I should say, combine directly to the bacteria, they can directly kill the bacteria, okay same things would apply if this is a virus or donor cells, where this antigen has been introduced into the patient's body, so that's cellular, now we have humoral, so then your suppressor cells are going to become cytotoxic "cyto" meaning cells, and "toxic" meaning physical agent resulting in cell death humoral, so you have your bacteria, think about your bacteria remember I mention those specific antigens sites, along those specific antigens on that bacteria have specific epitopes, that should be a triangle specific epitope, so on your B cells, and one thing that you can you notice, that your CD4 helper cells, they have the antigens, on them now right, they can actually present to your b-cells as well and they can activate these b-cells, what do b cells become when they're activated, plasma cells, what do we know about our plasma cells? they produce our antibodies, so under B cells, here's your antigen from the bacteria, there's receptor sites on your b-cells that combine to that bacteria right because they've been stimulated by the T cells, all right so now they combined that bacteria, activating the B cells, b cells are going to transform plasma cells and then the plasma cells are going to build clones of cells that recognize this bacteria and then they will build antibodies to that bacteria, okay this is not effective at immediately fighting this infection, this is to recognize future exposures alright So you'll have your memory b-cells producing the antibody so these are proteins. I remember from immunology that the B cell has to have a certain antigen for it to become a memory cell. yeah. so once it's exposed, let's say that this bacteria (the memory Bcell) now recognizes it, and it's going to build antibodies these antibodies will help fight off any type of future exposures, does that make sense? all right so then you have these antibodies that can attack the bacteria attack your donor cells. What's going to happen? Phagocytosis? Well it depends on what type of antibody it is. So what's gonna happen is these antibodies will attack when there's bacterial cells you'll have the antibodies attached like that and then this kind of lives in circulation until it gets into the spleen and then the spleen will destroy this, resulting in extravascular hemolysis, okay this is what we typically see when these antibodies are built and then on re-exposure it will attach to whatever the object is formed to, on red cells, bacteria, virus, and then the spleen can take it out, that make sense to everybody so the cellular is intravascular and humoral is extravascular? I don't want to make that distinction because it depends on what type of antibody is being produced, you could also have these cells producing because your B cells produce a specific clone of antibodies, so if you have an amnestic response, the secondary immune response, if you've already been exposed to it, typically it's going to be a IgG related which would be extravascular hemolysis, if you have an IgM antibody such as the wrong ABO or some of our cold reactive antibodies that could result in intravascular and extravascular, as well so it depends, I don't want to make that specific connection, it just depend on what type of antibody is being involved, I will post this chart and if it's not already there, I'll post it in the lecture one section in blackboard but from that what I want you guys to remember, humoral immunity involves your t cells, your CD4, CD8, cytotoxic, your natural killer cells, humoral involves your b cells transforming through plasma cells, producing the antibodies to destroy whatever foreign object can be introduced into the body Compliment, if you are dealing with the IgM antibody, most often times all of your IgM antibody can activate complement, once complement is activated like we mentioned, it will attach to that cell membrane ultimately resulting in cell destruction, I mean that's intravascular, we have a whole lecture dedicated to the complement cascade what happens, how we detect that, and blood bank, one thing is, if you guys think about the complement cascade, you think about C3 and C3B and C3D, when complement has been activated, and the cascade has completely gone through its process, C3B and C3D remain on the cell membrane, so we are able to detect that in our blood bank testing, when we are testing, when we do our DAT, and we are looking at what is coating patient red cells that is how we detect complement, okay and that's how we can tell that complement has been activated, and again we'd have the whole lecture coming up on that, and this is just to demonstrate what's going on when complement has been activated, notice that when C1 is activated, we have C4 activated, which C2a will cleave into C3B so we have C3b, which is more involved the phagocytosis process; C3A is going to be involved in inflammation it's going to cause your vessels to constrict and so as complement cascade, continues you'll have your C5, C6, C7, C8, C9 complex attached to the cell, ultimately resulting in cell lysis we'll be talking more indepth about compliment in a future lecture okay so what exactly are antigens? Think about antigens as being substances capable of inducing a immune response in your patients body okay whether that antigen is on a bacteria, whether that the antigen is on a virus, whether that antigen is from donor unit cells okay whether the red cell antigen on the cells, they're usually composed of protein carbohydrate or lipid, again they may be free such as on the bacterial membrane or they're attached to your cells, whether that be your patient cells or your donor cells, if you take it back to hematology, and think about your red cell membrane remember we talked about how the structure of the red cell has proteins in it and then you have some glycoproteins, that are within that, and it's got carbohydrates, they extend from those proteins so this is your carbohydrate molecule right here, this is where your ABO antigens would be they are consisting of an oligosaccharide, so several sugars mixed together we're going to talk about that in the next lecture, and then on some of them will have Rh antigens, your Duffys it is thought that if your antigen sites protrude from the cell that they are very antigenic, okay so that makes sense, our ABO antigens very strong, very antigenic, as well as our Rh, they're very strong too and that is because they extend from the cell rather than being directly on the cell membrane like some of our other antigens that we are going to talk about and then our antigens, we'll talk about the epitope, the epitope is the portion of the antigen where the antibody combines, and we will talk about the process of goodness of fit when we are trying to detect if the patient has the antibody or if we are trying to identify the antigen on the patient cells we want a goodness of fit, we want the antibody to fit the specific epitope of the antigen, think about it like a lock and key we want the antibody to attach that antigen, once that occurs that gives us agglutination, we are able to visualize that in blood bank, that is what we are looking for that let's us know the patient has a corresponding anybody or antigen that's your goodness of fit if for any reason the epitope changes or anything like, that affects the binding of that antibody to the antigen and that will affect how well we can detect it and part of blood bank is encouraging this antigen-antibody complex to occur, we want this to occur, so we have some enhancement media, we have some ways that we were trying to induce this agglutination, and everything in blood bank is for a reason so when we centrifuge our specimens we're encouraging the antigen-antibody complex we're pushing them closer together, when we incubate at 37 degrees some antibodies have a desired temperature of reaction, so we want to mimic what's going on inside the body to allow the antibody to react, okay and so everything is for a reason, and if you're dealing with an IgG molecule which we'll talk about in just a minute, think about how small it is you guys remember from immunology it has just two antigen binding sites versus IgM has ten, IgM is very large and usually it does not need any encouragement to allow for agglutination, okay we can pretty much visual and that it's going to be pretty strong reaction, IgG is so small it has a harder time breaking down that zeta potential and bringing the cells together so we can see agglutination okay how can you build (antibodies to) antigens, usually you can through anything you're exposed, to let's say ingestion, inhalation, it's in the air you breathe, like spores, your ABO, you have the antigens on your red cells right, how do we build the corresponding antibody's to the ABO, so your ABO antibodies are naturally occurring, they're IgM, and so remember if you lack the antigen you can build the antibody, so if your type "A" you have the "A" antigen on your red cells but meaning that you lack the "B" antigen, so you have anti-B in your plasma, where did that anti-B come from, it's naturally occurring, it's in the air you breathe usually about six months of age you'll start building those antibodies, okay so it's in the food you eat, being exposed to things, the air you breathe, those are naturally occurring, and we talked about our antibodies, they are proteins, immunoglobulins produced by the plasma cells, usually they're produced by direct exposure to an antigen, or if that macrophage becomes exposed to it and becomes the antigen presenting cell and then again here's your plasma cells and then your antigen presenting cells two types of antibodies you guys need to be aware of, we have allo-antibody and and that is an antibody to a foreign substance, okay that's an alloantibody something that you are lacking, you are antigen negative and you have built an antibody to it, that's an allo-antibody versus an auto-antibody, auto-antibody is when you build an antibody to your self, okay auto-antibodies are blood bank how do I word it we hate Auto antibodies, right because the patient's going to react with himself, everything going to be positive, and it's really hard to rule out any underlying alloantibodies okay and you guys as we start talking about auto-antibodies you'll see what I mean by that, all right still in immunology review, the immunoglobulin structure, you guys all remember your immunoglobulin has two heavy chains and two light chains, the heavy chains are constant, and for IgG that heavy chain is gamma, for IgM that heavy chain is Mu, that's why we call them IgG versus IgM and then you have two light chains, those can be variable, it can be either lambda or Kappa, okay so whatever your heavy chain is determines what type of antibody you have, is it IgG or it's an IgM? In blood bank those are the two that we are most concerned with and here's your immunoglobulin structure, I'm hoping you guys have all seen this before, you know what it looks like but just as a review your heavy chains are on the outside and here's your light chain, and then you have your FAB region this is where your antigen binding site is For an IgG molecule you have two and here's your FC region, your crystallizable region this is your complement binding region also there are some receptor sites on here, neutrophils and other things combined to these immunoglobulins as well, if you notice it's kind of like a mirror image if you were to divide this down, you have a mirror image, notice that you have a cage right here down in the middle, it's just composed of disulfide five bonds, we have some processes in blood bank that we will talk about what we will break this disulfide bond up, and we will open up the IgG molecules to allow for further binding, and so just remember that cage region allows for flexibility in that molecule as well kinda lets it move around better to meet the antigen to allow it for binding, we also have some enzymes processes that we can do, like I mentioned that will break those bindings and allow the IgG molecules to open up and fold on itself and so this just differentiates the two differences between IgG and IgM, remember IgGs are very small in size, they have two binding sites IgM molecule is very large, it is joined by the J protein in the middle and it has 10 binding sites, so when you think about agglutination, and you guys start doing your ABO interpretations that we're going to do tomorrow and in lab, those are all IgM, and so they can react after initial spin one concern with IgG is they're so small they have a hard time breaking down that zeta potential, allowing all the cells to come together, we usually have to incubate them, maybe even add an additional reagent to be able to see the agglutination reaction, we're going to talk about the process of agglutination, it's actually two stages, we have what we call sensitization, where the antibody attaches to the red cells, but maybe we haven't seen agglutination yet in blood bank Let's say you're dealing with an IgG antibody, it's going to attach to the red cell, but it's so small in size, maybe it cant attach to another red cell so if you guys think about agglutination, when we see agglutination, that means the antibody is bringing multiple red cells together, kind of like how a cluster of grapes and that is what we look for in blood bank, that is agglutination, IgG has a very hard time of bringing those red cells together because of its size, okay we have some tests in blood bank to help IgG, so we do our ABO determinations tomorrow we will be dealing with IgM antibodies, there is a disclaimer with that I will get to in lecture two, this is all review for you guys right now IgG vs. IgM okay so let's just talk about some of our on IgG and IgM characteristics, I mentioned that IgG is your gamma, it only has one monomer, so two antigen binding sites Think about the size of IgG compared to the size of IgM Remember I mentioned IgG is small, it has the capability to cross the placenta keep that in mind, that is very important blood bank when we start serving moms and babies, okay because if mom has an IgG antibody, guess what it can cross the placenta, it can attach to baby red cells, it can cause respiratory distress in the baby, it can cause anemia, and that will ultimately end up in early delivery or intrauterine transfusion, depending on how severe the case is we have a whole section that talks about hemolytic disease of the newborn and that is why, because mom's IgG antibody can cross the placenta, versus IgM, they're so large in size, it's not going to get across that placenta, okay so some of our IgM antibodies that we'll talk about such as Lewis A, Lewis B, those cannot cross the placenta so we will start talking about clinically significant (antibodies) other than our ABO antibodies, other IgM antibodies not considered clinically significant remember our IgM antibodies can definitely activate complement, there's only a couple of IgG antibodies that have the capability to activate complement, and when we start talking our IgG antibody we'll identify those, and remember naturally-occurring IgM antibodies and typically naturally occurring IgG antibodies are typically only built after exposure, okay so for females if you've had a pregnancy and you could build an IgG antibody, not a problem for your current pregnancy, it's a problem for any future pregnancy, if Mom's Rh negative, the dad is Rh positive (and) the baby gets that D-antigen, the mom is Rh negative, baby is Rh positive, then mom is exposed to the D antigen, she could build anti-D D is the most antigenic antigens that we have. Have you guys ever heard of RhoGAM? RhoGAM is given to pregnant females who are Rh negative to prevent, kind of like a prophylactic treatment, to prevent the production of anti-D, it's the only antibody that we have that capability for if any of you guys ever invent something for Kell, Kell is the next most antigenic antigen Why we don't have something for Kell, I don't know yet, but if you guys ever get that you can be rich, alright so if you have ever heard of RhoGAM you know somebody that has received RhoGAM, that's the reason why, they're Rh negative If they don't receive RhoGAM, they could potentially build in anti-D which could coat future pregnancy, future baby cells, and that could result in you hemolytic disease of the newborn, early delivery, intrauterine transfusion, or ultimately fetal death if it goes untreated That's how severe it is. And like I mentioned, we have some enhancers but we don't need to use it for IgM, that molecule is so large, we will be able to visually see agglutination Versus IgG, we have to encourage it, we have to help it (by adding enhancers) Location of your antigen and antibody, I've already said and I want to say it, again if you can wrap your head around this I think will blood bank will be very easy for you, your antigen will always be on the red cells, whether you're testing patient red cells whether that be a donor or recipient, or you're phenotyping in donor cells we are identifying antigens on donor cells, or you're using reagents red cells, your reagent red cells provide the known antigen your antibody is in the plasma, if you're using patient plasma mixing with the reagent red cells, we're identifying the antibody in the patient, and remember the rule, "Landsteiner's rule," if you lack the antigen on your red cells, you can build the antibody in your plasma. Where are your antigens? On your red cells. Where is your antibody? In the plasma. So you have to think about blood bank forward and backwards. So if you're doing patient testing and you're doing your forward testing, we know you're looking for the antigen that's on your patient red cells, if you're doing your reverse testing, you're using patient plasma, your testing for the antibody So whatever it is you're trying to identify, blood banking can be complicated, forward is easy but there's always discrepancies, there's always problems, and that's where blood bankers become really good problem solvers. You're going to have to ask yourself these questions. Where's my discrepancy? Is it an antigen problem or an antibody problem? So if people have autoantibodies, would it be an antibody problem? So, people with autoantibodies, they have an antigen on their red cells that they had built an antibody to. Sometimes we will see that after an infection with infectious mononucleosis or Mycoplasma pneumoniae, that virus causes the patient to build that antibody If they have I antigen on their red cells, anti-I, which is an known autoantibody, we will see it quite frequently in blood bank, they have I antigen on their red cells, and if they have the I antibody in their plasma it's a self antibody, it's an auto-antibody, which will cause reactions we aren't there yet, but we do have auto control where you will make patient cells with patient plasma, your autocontrol is going to be positive of course because the antibody is attacking the patient's own cells Anytime you have a positive autocontrol that indicates an auto-antibody And drugs can cause a positive autocontrol, we will get all into that So we talked a lot about the antigen-antibody complex, we want that in blood bank, I think I've made that clear sometimes we have to create that reaction in the laboratory, okay we're looking for that antigen-antibody complex to occur we want that agglutination, and that antigen-antibody complex is dependent upon the structure of the molecule if you think about IgG versus IgM, the epitope of your antigen, it's referred to as the specificity so sometimes like when we add our reagent antisera, let's say you're doing the D testing, so we're testing for the D antigen on our patient red cells, our antisera is anti-D so that D antigen has a specific epitope, so the specificity of the anti-D is to that epitope of the D antigen Does that make sense? And then here's your good fit that I mentioned earlier and depending upon your good fit, what type of binding, is it hydrogen? Is ionic? Is it electrostatic? And then in blood bank we can encourage them to fit by incubation temperatures, time pH of saline that we use, any enhancement media that we add it is all to ensure the good fit think about it as the lock and key. Antigen and antibody complex occurs So the antigen-antibody reaction occurs in two stages the first stage is going to be sensitization, that means that the antibody has attached to the antigen on the cell, okay in certain cases when you might not be able to see that, if it's an IgG reaction, IgGs are so small the antibody might have bound to the cell, but we are unable to visualize that because it's so small, okay versus the second stage that is true agglutination, that's where the antibody has bound to multiple cells and then we have the clustered grapes, we have agglutination so sensitization means that the antibody has attached to the antigen but we are unable to visualize that, if you think about this you have the number of red cells, you have some plasma added, for energy anybody complex to occur it's really just by chance, all right especially when we're taking patient plasma fixing with screening cells, this is your antibody screen, the antibody screening cells have a known antigenic profile, so we know what antigens are on those cells what antibody is present on patient plasma, that's the unknown, that's what we're testing, so if you have a reaction, if you have sensitization and agglutination really just by chance, okay so we're adding it, we're seeing what happens we don't know in the screening phase, so we try to increase probability of that collision, so if you think about antigens being on the surface of your red cells, antibodies being in the plasma we mix them together, you have a lot of forces that are in place to prevent that agglutination, you have the zeta potential, the negative charge around the cells that naturally repulses them from each other, keeps them from coming together, alright you have that working against us, and so that's one reason some of the things we do in blood banks, such as centrifugation, bring the cells together, break down that zeta potential, so we can view that agglutination, okay and so another thing that we will do is will increase the antibodies, so oftentimes when we start talking about our ABO determination and we add plasma to our test tubes, we add two drops of patient plasma, one drop of cells, and that's only to increase the amount of antibody, to increase the concentration of the antibody, that should allow more chance that the antigen-antibody complex will occur okay I mean that's really just to refer to as the serum to cell ratio, so increasing serum or plasma, will increase the chance of that collision, all right increase that likelihood of that sensitization occurring, increasing the red cells will not, okay so if any time you're testing, you don't see any agglutination, but you suspect it's there, we might try to increase the amount of plasma, then some environmental factors that we try to control, temperature number one, incubation, time incubation, and remember IgG antibodies typically react at 37 degrees, they're warm reacting, IgM are what we call cold reacting or room temperature reacting, okay so sometimes after initial spin, that means incubation at room temp, we immediately spin and read for agglutination, our IgM antibodies will react, our IgG antibodies will not, we have to carry it through incubation phase, and usually we will add a reagent to it to enhance it pH is important too we have our blood bank saline, normal saline, and it's usually about pH of seven and then our ionic strength, and we're going to talk about this, I believe it's in lecture three or four, we'll go into depth about all of these, the incubation times, your temperatures, centrifugation speed, everything like that, we'll go into depth okay so after sensitization, after that antibody has attached to the red cells, we want to develop what we call lattice, and so what's gonna happen is the antibody is not only going to bind to one red cell, it's actually going to bind to multiple red cells, that's what gives us our agglutination okay that's what we can see and blood bank, that's what our desired reaction is I wish I had a better whiteboard, so here's what's going on, to really visualize agglutination, so here's your test tube, okay let's say you have patient plasma and you have a reagent red cells, we are doing a back type maybe so here's your red cells with the antigen, they're commercial red cells okay the antigen is known, we add our patient plasma with the antibodies, what's going to happen is the antibody sensitization will (occur) but we can't see that yet, all right we're unable to see that yet, this is sensitization, in agglutination, after we centrifuge, after we incubate at 37° then what's gonna happen is that antibody that's already bound is going to bring red cells together, we can visualize that, that is agglutination when you have a cluster of red cells together, that's what we look for with agglutination that make sense to everybody? Now how we get there, sometimes we have to encourage whether we add a some enhancement media, we increase our incubation, there's a reagent that we'll use, it's called AHG, that you guys are learn about, we add it to further enhance the lattice formation of the IgG antibodies until I mentioned this, agglutination is influenced by the distance between the red cells, so we have that zeta potential working against us okay often especially with IgG, IgG is going to have a hard time because that zeta potential, the negative charge surrounding those cells, is going to naturally repulse those cells, so they're going to want to be away from each other we want them to come together, okay and so there's some enhancement media that will add to break down that zeta potential, that's one reason we centrifuge to try to force them closer together, so we can visualize that agglutination, and then we want to make sure that we have the zone of equivalence, okay we want to make sure that we have equal amounts of antigen and antibody to allow for agglutination to occur, okay that's called the zone of equivalence and we use normal saline, so we'll use a 0.85% normal saline so if you have a negative charge around the cells, the cells are happy in that normal saline, and it's kind of supposed to help aid in breaking down that zeta potential, and you guys remember did we talk about zeta potential a little bit? You guys remember that the cells are surrounded by the negative charge, that charge makes the cells repel each other, okay so that's what we're fighting in blood bank, we'll talk about that a lot, and like I mentioned, IgM, typically (you) don't have a problem getting the agglutination with IgM, it's IgG that we have to encourage the visualization of agglutination because they are just so small, they have a hard time breaking down that zeta potential, and so here's an example Here you have your negative charge surrounding the cells, notice that IgM with its multiple binding sites is better able to bring those cells together So not only is it larger in size, which can break down that the zeta potential easier, but it has more binding sites and so more cells are able to come together, so we are able to easier visualize agglutination with the IgM molecules, this is what agglutination looks like and this is an IgM antibody so that's what agglutination is, and I mentioned that zone of equivalence, all right so we want equal amounts of antibody, equal amounts of antigen and we want to avoid, did you guys talk about pro-zone and post-zone in the immunology? we want to avoid excess antibody, and we want to avoid excess antigen, both of these will cause false negative reactions, okay if you have too much antibody, too much antigen if there's too much antigen and all the antigen sites and are bound up, if there's too much antibody and all the antigen sites are bound up, that could cause a false negative, if there's not enough antibody present that could also cause a false negative, prozone (is) excess antibody, postzone (is) excess antigen This is what it should look like in the medium, where you have an equal amount antibody and antigen allows for equal agglutination to occur, notice if you have prozone, too much antibody, all the antigen sites are bound up, we will not be able to see that visualization of agglutination, that's false negative, if you have too much antigen, that's also a false negative, we won't be able to visualize agglutination, so this is where we want to be, we want to being able to see that agglutination occur, the antigen-antibody complex occurring timing, speed of centrifugation (is) very important as well, again centrifugation forces those cells close together, allowing for that that agglutination, so we're going to talk a lot about our reactions in blood bank, we'll grade our reactions, there's some subjectivity there, but whenever you think about a positive reaction versus a negative reaction, there's another reaction that you always have to keep in mind, and that's if after you're doing your testing, you're reading your reactions and you know there's any type of hemolysis so if you're patient plasma was yellow prior to adding to the tube, and after you speed down, now your plasma has a red tinge to it and your cell button is really small, that means hemolysis occurred, cell destruction, antigen-antibody complex occurred, and it resulted in immediate cell lysis okay so hemolysis has always considered a positive reaction in blood bank Why? Because complement can immediately hemolyze the cells, okay so if you ever have hemolysis in your test tube reaction, it's probably an antibody that can activate complement, and so just to further differentiate primary immune response from secondary immune response, this should also still be review from immunology, remember upon initial exposure you'll have your B cells, you'll have your plasma cells being activated, producing the antibody, typically in initial immune response those are IgM related, so with primary immune response to your antibodies being produced is typically IgM, okay after exposure those titers of that antibody are going to drop down, if that patient were to ever be exposed again to the same antigen, and then upon secondary exposure, secondary immune response or amnestic response then it's typically going to be an IgG antibody So IgM is associated with primary, IgG is associated with secondary or amnestic response so you have this kind of lag thing in the middle here, that's where your titer drops down low, when we do our blood bank testing, we'll talk about some of the antibodies that are known to do this, like your Kidd families that are notorious for this they're titer drops down really low, in our blood bank testing we are unable to detect antibodies when the titer is so low, so what's going to happen? If your patient has previously had JKa antibody that was identified the titer is so low right now that it is undetectable, (and if) that patient gets transfused with a JK+ unit, (then) it going to cause a secondary, amnestic immune response of IgG antibodies, that patient is going to present with a delay in hemolytic transfusion reaction, everything is going to be okay until about two to three days later, two to three days later that patient is going to present with symptoms of anemia, jaundice, feel real tired, maybe even longer than two to three days maybe seven days later, it just depends on how quickly that titered anybody increases, so that's the difference between primary and secondary, and we'll talk about which antibodies are notorious for this, JKa is the number one okay so that completes lecture one. Anybody have any questions on blood bank so far? okay