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1 De La Salle Health Sciences Institute DEPARTMENT OF PATHOLOGY IMMUNOHEMATOLOGY The study of immunologic reactions involving all components of blood

od Deals with antigens, antibodies and antigen-antibody reactions

Application transfusion of blood & its components diagnosis, prevention & management of immunization associated with pregnancy leukocyte testing for organ transplantation laboratory resolution of parentage problems DEFINITION OF TERMS ANTIGEN (Ag) Any substance that stimulates the production of antibodies AGGLUTINOGEN Antigen on the surface of RBCs ANTIBODY (Ab) Proteins produced by the Reticuloendothelial system in response to antigen stimulation AGGLUTININ Antibody that attacks RBC Antigens and manifests this activity by clumping of the RBC HEMOLYSIN Abs that attack RBC Antigens and manifests this activity by lysis of the RBCs AGGLUTINATION Clumping of red blood cells as a result of antibodies binding to antigenic sites of adjacent red cells NATURAL ANTIBODIES Abs that appear without antigenic stimulation, during childhood, decreases with age ACQUIRED/ IMMUNE ANTIBODIES Appear upon introduction of Ag by disease, transfusion, pregnancy, and substances chemically related to RBC Antigens COMPLETE ANTIBODIES Bivalent Antibodies will directly agglutinate appropriate RBCs INCOMPLETE ANTIBODIES Univalent Abs coats RBC surface but cannot directly agglutinate them ISOANTIBODIES / ALLOANTIBODIES Abs produced against Ags from genetically different individuals of the same species AUTOANTIBODIES Abs produced against ones own tissues WARM AND COLD ANTIBODIES WARM ANTIBODIES Abs which react best in vitro at body temperature (37C) Usually IgG Require exposure to foreign Ag before they are produced, hence acquired Most are clinically significant (EXCEPT ABO blood group which

2 are IgM) Can coat red cells at body temp and lead to removal by macrophages (extravascular hemolysis) Maternal IgG Abs can cross the placenta and attack fetal red cells

COLD ANTIBODIES Abs which react best at 4-10C Usually IgM Exist in humans regardless of whether they have been pregnant or transfused, therefore natural Most are not clinically significant, with the major exception being the ABO IgM antibodies When significant, IgM antibodies are very efficient at fixing complement and causes intravascular hemolysis Maternal IgM are NOT able to cross the placenta

BLOOD GROUP SYSTEMS 1. ABO BLOOD GROUP 2. Rh BLOOD GROUP SYSTEM 3. OTHERS A. MNS B. I/i C. DUFFY D. KELL E. KIDD F. P G. LUTHERAN H. LEWIS ABO BLOOD GROUP First blood groups discovered Most significant for transfusion practice ABO compatibility is essential before other pretransfusion test is performed ABO antigens are the only antigens for which reciprocal antibodies consistently and predictably exist in serum of normal individuals

BIOCHEMISTRY: Paragloboside: core backbone of all of the antigens in the ABO blood group RBC] Gl GalNAGGal Gl: glucose Gal: Galactose NAG: N-acetylgalactosamine H antigen: precursor to the A and B antigens made by adding a fucose (a 5-carbon sugar) to paragloboside After (and only after) the H antigen is made, A or B antigens can be added A and B antigens functionally mask the H antigen The more A or B that is made, the less H is present Type O cells have no A or B and express the most H antigen A antigen Formed by the addition of N-acetyl galactosamine to the H

3 antigen B antigen Formed by the addition of galactose to the H antigen

GROUP A Express A antigen on RBC surface Genotypes AA or AO Have naturally occurring, clinically significant, predominantly IgM (with a small amount of IgG) antibodies against type B (anti-B) Subgroups o A1 (80%) o A2 (20%) o Significance: some with A2 have antibodies against the A1 subgroup (anti-A1) GROUP B Express B Ag on RBC surface Genotypes BB or BO Have naturally occurring clinically significant, predominantly IgM (with a small amount of IgG) antibodies against type A cells GROUP O Have neither A nor B antigens on their RBC Genotype OO (universal donors) Have naturally occurring, clinically significant, very high titer, anti-A, anti-B and anti-A,B antibodies Maternal anti-A,B can cross the placenta to cause hemolytic disease of the newborn Group O cells have the most H antigen GROUP AB Express A and B Ag on RBC surface Genotypes A1B or A2B have no ABO antibodies (universal recipients)

Blood Group A B AB O

RBC Antigen A B A&B none

Serum Ab Anti-B Anti-A None Anti-A Anti-B Anti-A,B

Blood Group/ Phenotype A B AB O

Genotype A1O / A1 A1 / A1 A2 A2O / A2A 2 BB / BO A1B / A2B OO

4 ABO BLOOD GROUP TESTING Forward Grouping / Forward Typing Also called cell typing Testing for antigens on red cells Utilizes commercial sera containing known antibodies (anti-A and antiB) versus patients red cells Reverse grouping /typing Also called serum or back typing Uses patients serum versus commercial A1 and B cells Analyzes patients serum for the presence of anti-A and anti-B antibodies Forward Typing Anti-A Anti-B (+) (--) (--) (+) (+) (+) (--) (--) (+) = Agglutination (--) = No agglutination Common Causes of ABO Discrepancies 1. Abnormal Antigens Person has A2 blood group with an anti-A1 formation Forward Typing Reverse Typing Anti-A Anti-B A1 cells B cells (+) (--) (+) (+) Acquired B phenotype Seen in group A patients with exposure to gram-negative bacteria by way of intestinal obstruction, gram-negative sepsis or colon cancer Forward Typing Reverse Typing Anti-A Anti-B A1 cells B cells (++) (+) (--) (+) Reverse Typing A1 cells B cells (--) (+) (+) (--) (--) (--) (+) (+) Blood Group A B AB O

2. Abnormal Antibodies or lack of appropriate antibodies Weak or missing antibodies : newborn, elderly, immunosuppression and other conditions that yield hypo / agammaglobulinemia Forward Typing Reverse Typing Anti-A Anti-B A1 cells B cells (+) (--) (--) (--) Non- ABO antibodies (eg polyagglutinins, multiple myeloma) present that cause agglutination of test red cells Forward Typing Reverse Typing Anti-A Anti-B A1 cells B cells (+) (+) (+) (+)

5 BOMBAY (Oh)PHENOTYPE Patients lack the H gene and therefore cannot make H antigen, A or B antigen on their red cells Forward typing: O Discrepancies on reverse typing: Serum agglutinates A1, B, and O cells Confirmatory testing is done by using an anti-H reagent made from Ulex europaeus plant (red cells + anti-H = NO agglutination) Have very strong anti-A, anti-B, and anti-H and can only receive cells from a Bombay donor

Rh BLOOD GROUP Complex blood group with >50 described antigens Nomenclature systems Fisher-Race (English) antigens: D, C, E, c and e Wiener (American) antigens: Rho, rh, rh, hr, hr Antigens Lack corresponding naturally occurring Abs (when present they are the result of sensitization caused by receipt of D-positive RBCs from another person Fisher-Race D C E c e Wiener Rho rh rh hr hr

Antibodies Individuals who lack the antigen may be exposed to it through transfusion or pregnancy Anti-D (Rho), anti-C (rh), anti-E(rh), anti-c(hr) and anti-e(hr) have all been known to cause hemolytic transfusion reaction and hemolytic disease of the newborn Warm-reacting, exposure-requiring IgG antibodies that are clinically significant Infants < 4 months usually do not form new antibodies against any incompatible red cell antigens

Rh BLOOD GROUP TESTING Testing for D(Rho) is the most common Rh test performed Rh-positive simply means D-positive D antigens are potent immunogens. Of D-negative patients, 80% will develop an anti-D when transfused with a single unit of D-positive blood. WEAK D PHENOTYPE (Du) Weakly expressed D antigens that require more sensitive testing to detect Quantitative difference in D antigen Reduced # of D antigen expressed on red cells All apparently D-negative blood donors must have a weak-D test to avoid false classification

6 PARTIAL D / D MOSAIC / D VARIANT PHENOTYPE Qualitative difference in D antigen Number of D antigen on the red cell is not reduced but protein structure is altered If alloimmunized, may produce anti-D antibodies If donating blood should be labeled as D-positive if receiving blood, they should be labeled as D-negative and receive D-negative units.

TRANSFUSION-RELATED TESTING Identify clinically significant antigens on red cells Identify plasma antibodies to red cells Detect antibodies and complement bound to red cells IgM and IgG antibodies comprise 80% of the circulating Abs and are the most significant Abs for transfusion-related testing To detect Ab (particularly IgG) or complement bound to RBC, antihuman globulin reagent (Coombs reagent) can be added Agglutination (clumping) of RBCs is the end result of testing indicating a positive reaction COMMON TESTS USED IN IMMUNOHEMATOLOGY I. ANTIGLOBULIN (COOMBS) TEST Remains the most important single test in Ab detection Principle: Red blood cells sensitized by IgG or complement can be made to agglutinate by adding antihuman globulin DIRECT COOMBS TEST (DAT) Detects RBCs that have already been sensitized with IgG Demonstrates that in vivo coating of RBC by Ab has occurred but does NOT identify the antibody INDIRECT COOMBS TEST (IAT) Detects antibodies to RBC antigens present in the patients serum Detects in vitro red cell sensitization if red cells contain antigen corresponding to serum antibody Procedure: STEP 1: patients serum (with unknown Ab) + RBC (with known Ag) STEP 2: product of step 1 + Coombs reagent Indirect Antiglobulin Test Patients serum Detects in vitro red cell sensitization if red cells contain antigen corresponding to serum antibody Useful in: 1. Detection and identification of unexpected antibodies 2. Compatibility testing (crossmatching) 3. Red cell antigen phenotyping 4. Investigation of transfusion

Direct Antiglobulin Test Patients red cells Detects in vivo antibody coating (sensitization) of red cells

Useful in: 1. Detection of hemolytic disease of the newborn (employing infants red cells) 2. Investigation of transfusion reactions 3. Detection of autoimmune hemolytic anemia (AIHA)

7 4. Detection of red cell sensitization by drugs (penicillin, cephalothin, alphamethyldopa) II. RED CELL TYPING Forward Typing Reverse Typing Rh Typing III. CROSSMATCHING Absence of agglutination or hemolysis is essential to the safety of blood transfusions Agglutination or hemolysis in any phase of the transfusion (ie incompatibility) = presence of Ab and its corresponding Ag Uses To detect antibodies in the donor or recipient To detect ABO typing discrepancies Major crossmatch tests patient (recipient) serum + donor red cells Detects Abs in the patients serum that may destroy transfused donor RBCs Primarily functions to determine the ABO compatibility of the donor cells Minor crossmatch uses recipients red cells + donors serum Detects Abs in the donor serum which may react with Ag in the recipient No longer required as part of the cross match procedure Tested in 3 phases (major crossmatch): Immediate spin in saline at room temperature Incubation at 37C with enhancement medium Antiglobulin phase after washing incubated cells with saline Effects of temperature on antibodies IgM antibodies are usually cold antibodies and are often detected in the immediate spin phase of testing (performed at room temperature) IgG antibodies usually react optimally at 37C, are referred to as warm antibodies, and are often detected in the antihuman globulin phase of testing Antibodies that do not react at body temp (37C) are usually not clinically significant reactions 5. Detecting Du antigen (weak D)

IV. ANTIBODY SCREEN Use: to demonstrate unexpected antibodies in the serum of the recipient that may destroy donor RBCs that were thought to be compatible on the basis of the Rh and ABO typing Has replaced minor crossmatching Procedure: Recipients serum + type O reagent red cells (with known antigens Will NOT detect errors in ABO typing since reagent RBCs are all group O

V. PRE-TRANSFUSION TESTING / COMPATIBILITY TESTING 1. Review of recipients blood bank history 2. ABO and Rh typing of recipient & donor 3. Antibody screening of recipient & donor serum 4. Major crossmatching

HEMOLYTIC DISEASE OF THE NEWBORN Also referred to as erythroblastosis fetalis Occurs when the mother is alloimmunized to antigen(s) found on the RBC of the fetus Destruction of fetal RBCs by mothers IgG antibodies

HDN Due to Rh Incompatibility Set-up: Rh(-) mother + Rh(+) baby Rh (-) person exposed to Rh(+) blood will develop reaction after 2 4 weeks Mother develops antibody against the Rh(+) blood coming from the baby First baby is not affected; HDN occurs during subsequent pregnancies HEMOLYSIS



Extramedullary hematopoiesis Hepatosplenomegaly

Hypoxic injury to the liver & heart Hydrops

be Jaundice

May Kernicterus

prevented by giving anti-Rh to Rh(-) mother in the ante-natal (28 weeks) & immediate postnatal period (within 72 hours after delivery) Factors which affect maternal response to Rh(+) fetal RBCs Concurrent ABO incompatibility Dose of immunizing antigen Isotype of antibody (IgM vs IgG) Due to ABO Incompatibility Set-up: O mother + A or B baby ABO incompatibility occurs in 20%-25% of pregnancies Hemolytic disease occurs only in 10% Treatment warranted in 1 out of 200 cases Not as severe as HDN due to Rh incompatibility because Most Anti-A & Anti-B are IgM (do not cross the placenta) A & B antigens are poorly expressed in neonatal red cells Many cells other than red cells express blood group A and B antigens