Immunohematology. Done by : Zaid Al-Ghnaneem

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1 Immunohematology Done by : Zaid Al-Ghnaneem

2 Hello everyone, in this sheet we will talk mainly about immunohematology which is the reactions between our immune system with Antigens found mainly within blood and how our immune system deals with them, it is also called blood banking. In the beginning please note that doctor put the reference as follow : References: -Blood Groups and Red Cell Antigens (Laura Dean) -Cellular and molecular immunology, 8th edition Slides are in Bold text. Introduction to replace blood lost by hemorrhage or to correct defects caused by inadequate production of blood cells The major problem is the immune response of the recipient ABO alloantigens are the most important not only on RBCs If the individual lacks blood group antigen, he will produce naturally IgM to that antigen if given blood with that antigen: complement activation & transfusion reaction There is many diseases that occur to blood and they lead to decrease it abnormally, such as anemia or thalassemia and many others, mainly we have immune responses in these diseases, now this immune response can occur to any part of blood such as RBC, WBC, or Platelets. Doctor said that student asked him about blood transfusion and WBC : doctor said that when the physicians make blood transfusion they make HLA typing before transfer, or it is preferred to remove WBC before transport WBC Depleted transfusion because : 1- it may transport infections. 2- the body may not respond well to them. Now, as we know everyone has blood type differ from other, for example if the blood group is A that means the Antibody in the same person is Antibody B, the antibodies are IgM type. Blood type and cross match Before a blood transfusion, two blood tests known as a "type andcross match" are done the recipient's blood type is determined, i.e., their ABO type and Rh D status donor blood may still be incompatible because it contains other antigens that are not routinely typed but may still cause a problem if the recipient's serum contains antibodies that will target them therefore, a "cross match" is done to ensure that the donor RBCs actually do match against the recipient's serum see next slide Before we do blood transport, we have to do 2 tests : Typing, and Cross matching

3 In the typing mainly we determine ABO and Rh but there is a lot of antigens on RBC not only ABO or Rh, we have to detect them! how? by cross matching, because the blood of the recipient may make immune response to these other antigens, so TYPING determine ABO and Rh is not Enough! How we do cross matching? We take serum from recipient, mix it with RBCs from donor, if antibodies from serum binds with Antigens from RBCs, that means there is Agglutination. Cross match Small amount of the recipient's serum is mixed with a small amount of the donor RBCs the mixture is then examined under a microscope if incompatible, the donor RBCs are agglutinated by antibodies in the recipient's serum Immune-mediated transfusion reactions Destruction of incompatible RBCs = hemolytic transfusion reaction acute or after days (delayed) can be life-threatening Destruction of incompatible donor white blood cells febrile non-hemolytic transfusion reaction (FNHTR) mild and resolve by itself Destruction of incompatible donor platelets post-transfusion purpura When RBCs destroyed, that means Hemolysis we call it hemolytic transfusion reaction, note that hemolysis due to ABO is very danger and may lead to death, also hemolysis may occur due to other antigens ( remember we say not only ABO and Rh, there is a lot of Antigens ) this hemolysis may be Acute or Delayed and acute is more serious. If the destroyed cells was WBC This will lead to febrile non-hemolytic transfusion reaction (FNHTR) and it is less serious than RBC hemolysis. If the Platelets are destroyed we call it post-transfusion purpura and it will lead to thrombocytopenia and the patient will have bleeding. Now all these destruction (RBC, WBC, Platelets ) their patients suffered from chills, fever, shaking, and aching It is better to use a specific blood component rather than whole blood. Also, all WBCs are now removed from donated blood to reduce infections and WBC incompatibility. Acute type of hemolytic transfusion reaction: within 24 hours, often during the transfusion

4 Acute is 2 types : Intravascular hemolysis, and Extravascular hemolysis. Intravascular hemolysis: -Most severe -Strong activation of complement may cause shock and high fever due to TNF, IL-1, etc. -Hemoglobin released into plasma and excreted in urine (hemoglobinuria) acute tubular necrosis (AKI) -Jaundice (bilirubin in blood) -Large amounts of tissue factor from destroyed RBCs DIC -Mainly against ABO antigens few other antigens (of the Kidd & P blood group systems ) When hemoglobin is passed within kidney, it will injury the tubules and lead to Acute renal failure (ARF) or what we call it Acute tubular necrosis (AKI) Extravascular hemolysis: -RBCs are removed from the body by macrophages of liver & spleen -especially due to IgG Fc bound to their receptors on macrophages the Rh blood group mediate this type of RBC removal -may also: Abs bound to C3b then bind C3b receptors on macrophages such antibodies include those directed against antigens of the ABO, Duffy, and Kidd blood groups *slower & more controlledthan intravascular hemolysis less Hb or bilirubin in blood and less jaundice mainly chills & fever Here in Extravascular it is mainly due to response to Rh antigens not ABO and it is IgG not IgM, So.. Intravascular due to ABO antigen mainly IgM Extravascular due to Rh antigen mainly IgG Now we have to know that ABO antibodies are Preformed that means they are developed before exposure to antigen, from birth! On the other hand the Rh antibodies are formed when it exposure to Antigen, so firstly exposure to antigen then the antibodies will develop, for example the pregnant lady when she is negative Rh and her fetus is positive Rh, when delivery some of the antigens will pass from fetus to mother and it will lead to antibody formation ( before delivery the mother was not having Rh antibodies ) so if the mother became pregnant again with positive fetus, Antibodies will pass from her to the fetus and it will lead to death of the fetus.

5 Question from student : who determines if its intravascular or extravascular? answer is Antigen, as we said when it is ABO and there is a lot of IgM it is intravascular, when it is Rh and a lot of IgG it is extravascular. Delayed type of hemolytic transfusion reactions: after 1-14 days the hemolysis is "delayed" because the antibodies are only present in low amounts initially -Much less severe than acute reactions -Especially against Kidd & Rh antigens although the recipient is also previously sensitized by the time a cross match is done, the level of antibody in the recipient's plasma is too low to cause agglutination, making this type of reaction difficult to prevent with time extravascular hemolysis Here we are talking about Delayed which doesn t occur immediately, it occurs after 1-14 days and it is less serious than acute, Mainly it comes with Rh and Kidd antigens, why delayed? because the Antibodies when the body expose to these antigens was little in quantity. Transfusion associated lung injury (TRALI) rare and occasionally fatal transfusion characterized by a sudden onset of shortness of breath antibodies from donor s plasma attack WBCs of recipient these WBCs will accumulate in lung vasculature and secrete inflammatory mediators with resulting pulmonary There is something called TRALI, and it means : it is condition due to transfusion, but! here the cause is NOT antibody from recipient, but Antibody from the donors plasma that attack the vessels of lung of the recipient and lead to condition resembles ARDS Acute respiratory distress syndrome ABO Blood Group Antigens Carbohydrates attached to membrane proteins or lipids synthesized by polymorphic glycosyltransferase enzymes a common core glycan on chromosome 9 (3 allelic variants) H antigen (= fucosylated glycan)

We know that ABO are antigens, and these antigens found on the surface of RBCs, not all the people have the same antigens because there is polymorphisms in the enzyme " glycosyltransferase" that adds

6 We know that ABO are antigens, and these antigens found on the surface of RBCs, not all the people have the same antigens because there is polymorphisms in the enzyme " glycosyltransferase" that adds the outer ring, for example in person it will add N-acetyl-galactosamine, in other person it will add Galactose, an so on.. The addition is on the OUTER side, the person with blood type O the enzyme is not working ( non functional ) so it will not add group to the OUTER surface of the CORE. The CORE is called H antigen or Fucosylated glycan. The CORE is formed by an enzyme called fucosyl transferase glycosyltransferase adds these fucosyl transferase adds this Ok, now just think of it, a person doesn t have the fucosyl transferase enzyme, so it will not have the H antigen ( Core) (Fucosylated glycan), this person will NOT accept blood transfusion from Group A or B or O, ONLY it will accept from one person, a person like him doesn t have the H antigen (Core) (Fucosylated glycan) So to make thing better can donate blood Person without H antigen Person without H antigen + ABO blood cannot donate blood Person with H antigen ( group A or B or O ) Person without H antigen We call the person that doesn t have H antigen Bombay blood group or ABH (H instead of O) OO individuals are said to be blood type O AA and AO individuals are blood type A BB and BO individuals are blood type B AB individuals are blood type AB What is Bombay blood group??? We answer it above go read it From wikipedia : For this reason people who have Bombay phenotype can donate red blood cells to any member of the ABO blood group system (unless some other blood factor gene, such as Rhesus, is incompatible), but they cannot receive blood from any member of the ABO blood group system (which always contains one or more of A and B and H antigens), but only from other people who have Bombay phenotype.

Universal recipients Universal donors ABO typing and solid organ transplants These antigens are also expressed on endothelial cells hyperacute rejection may occur ABO is important in organ

7 Universal recipients Universal donors ABO typing and solid organ transplants These antigens are also expressed on endothelial cells hyperacute rejection may occur ABO is important in organ transplantation, we have to know that ABH antigens are not only on RBC! they are also found on Epithelial tissue and Endothelial tissue, and there is some cancers that express Antigen A more than B for example. ABO incompatibility between mother and fetus..generally does not cause problems Why? Because we said that ABO is mainly IgM, right? but also there is small amount of IgG and remember IgG can pass the placenta not IgM, so since IgM cannot pass placenta there is no incompatibility between mother and fetus Other blood group antigens, Rhesus (Rh) Antigen Non-glycosylated, hydrophobic cell surface proteins Rh proteins are encoded by 2 genes one of which is important here RhD 15% of the population have deleted or alterated RhD allele these people are not tolerant to RhD antigen will make antibodies if exposed We have to know that Rh antigen is protein not carbohydrate, made of 2 genes each gene will lead to polypeptide, so we will have 2 polypeptides, the most important one is RhD ( so Rh sometimes called RhD because D is the most important) Remember Rh antiboies are made by Sensitization and not Preformed.

8 Other Rh antigens Polypeptides of Rh antigen are 2 as we said, one of them is D and it is the important one, and the other could be : C,D,E,c,e The polypeptide D the first one again it is the most important one, so when we say somebody is Rh positive we mean he/she has D polypeptide and vice versa. Rhesus (Rh) Antigen, clinical significance If Rh-negative mother carries Rh-positive fetus she will be sensitized by his RBCs usually during birth IgM will be formed and will be class-switched to IgG next pregnancy: IgG will cross the placenta into the Rhpositive fetus hemolytic disease of the newborn (HDN) will result = erythroblastosis fetalis *Prevention: by administration of anti-rhd antibodies to the mother within 72 hours of birth of the first Rh-positive baby Pregnant lady ( negative Rh ) after she delivered first baby (positive Rh) we give her within 72 hours of the birth of first baby an Anti-Rhd antibodies(passive immunity) to stop and prevent Sensitization. Question from student : what is the difference that I give her Anti-RhD Antibody or formation of this Antibody naturally? answer is when you give her ready antibody it will not lead to activation of lymphocytes and killing mechanisms and so on. -other Rh antigens, such as c, C, E, and e, can also cause problems -HDN can also be caused by incompatibility of the ABO blood group usually less severe than Rh-caused

9 As mentioned above, HDN ( Hemolytic disease of the newborn ) due to Rh problems mainly, but also ABO can produce this disease. Coombs test = antiglobulin test or AGT we use anti-human globulin (anti-ig, Coombs reagent ) in the final step and check if agglutination of RBCs occurs (visual detection) *Direct for diagnosing HDN or Indirect for preventing HDN *Can be also used for diagnosing autoimmune hemolytic anemia Now we can prevent the occurrence of disease in the baby if we know the mother has Antibodies for Rh or NOT.. we have 2 tests, Direct and Indirect In direct : we take RBCs from the fetus and expose them to Antiglobulin or Coombs reagent (exogenous) من عندي ( in Lab to know if there is agglutination or not, if yes, that means the baby has the (يع ن disease HDN, so we DIAGNOSED him. In Indirect : Serum from mother + RBCs that are Rh+ from the lab + anti-ig from the lab, and then see if there is agglutination or not, if yes that means the child maybe will get the disease so I can treat the mother to stop and desensitization these Antibodies so I PREVENT HDN from occur. Usually these 2 tests done together (Direct and Indirect) We can use same tests for autoimmune hemolytic anemia also. Other blood group antigens Kell antigens Duffy antigens Kidd antigens MNS antigens Other blood group antigens Lewis Antigens fucosyltransferases Le (dominant, functional) & le (recessive, nonfunctional) Se (dominant, functional) & se (recessive, nonfunctional) Le-a will be produced if: Le + se Le-b will be produced if: Le + Se Le a-, Le b- (= Lewis negative people) if homozygous for le (even if they have Se because it works on Le-a and converts it to Le-b) Lewis antigens, cont d not only on RBCs

10 important as ligands for E-selectins & P-selectins not important in transfusion reactions Le(b) and H antigens are receptors for H.pylori Doctor doesn t explain these 2 slides and said he didn t know why he puts these 2 slides Lewis antigens and said that he forgave us I couldn t find this topic lewis antigens in lippincott book, if you find it Share it. Good luck all and hope you get benefit from all immunity lectures

Immunohematology (Introduction) References: -Blood Groups and Red Cell Antigens (Laura Dean) -Cellular and molecular immunology, 8 th edition

Immunohematology (Introduction) References: -Blood Groups and Red Cell Antigens (Laura Dean) -Cellular and molecular immunology, 8 th edition Introduction to replace blood lost by hemorrhage or to correct