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Transcript of Basic Concept of Immunohematology.ppt.pptx
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Basic Concept of Immunohematology
Leni Lismayanti, dr., SpPK
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Lecture Overview
1. Basic immunohematology concept.2. Erythrocyte antigens and
antibodies (ABO blood group system).
3. Immunohematology tests.
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Basic Immunohematology Concept
Immunohematology:Serologic, genetic, biochemical, and molecular study of antigens associated with membrane structures on the cellular constituents of the blood, and immunologic properties & reactions, of all blood components and constituents.
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Immunohematologist
• perform variety of serologic laboratory examinations,
• evaluate & interprete the reactions observed, …
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Immunohematologist
• provide selected advanced investigations to aid in the study of: pathogenesis, diagnosis, prevention, management of immunization associated with transfusion, pregnancy and organ transplantation.
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Blood Group Antigens• Genetically encoded erythrocyte antigen
systems.• Immunologic diversity expressed by other
blood constituents (leukocyte, platelet, plasma).
• Produced by alleles at a single gene locus or by a group closely linked loci, constitute a blood group antigen system.
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• Blood group genes located on autosomal chromosomes inherited following Mendelian rules useful genetic markers.
• Codominance (+) genetic heterozygotes at a particular locus will express both gene products.
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• Membrane associated structure of blood cells and constituents of plasma:– Antigens (capable to
react with a complementary antibody or cell receptor)
– Immunogens (able to elicit an antibody- mediated immunologic response if introduce as a foreign substance into a responsive host).
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• Antigen has a variety of epitopes (specific antigenic determinants).
• Epitopes:– Discrete– Immunologically active regions of the
antigen– Molecular configuration confers:
• The ability to interact with specific lymphocyte membrane receptor, or
• Secreted complimentary antibody
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Immunogenicity
• Ability of antigen to elicit immune response.
• Determined by:– Certain innate characteristics of the antigen– Host’s genetically determined immune
responsiveness
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Characteristics of antigen
• Determined immunogenicity:– Degree of foreignness– Molecular size & configuration (affected
by temperature,pH,ionic environment)– Antigenic complexity (number of
available epitopes).
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Number of epitopes in RBCAntigen Phenotype Number of epitopes
A A1 adult 810-1170 X 103 D
A A1 newborn 250-370 X 103 D
B B adult 750 X 103 D
D D -- 110-202 X 103 D
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• Blood groups antigen vary greatly in their ability to elicit an immune response.
• A,B and D (Rh0) most immunogenic (blood transfused must be matched for these antigens)
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Chemical Characteristics
• Most potent immunogens: complex macro-molecular glycoproteins & lipoproteins.
• RBC antigen: glycoproteins, lipoproteins, glycolipids.
• Immunogenicity of antigen relates to the total complex molecular structure (area where antigen combines with specific antibody).
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• This structure:– usually limited to one
or a few simple structure
– Exposed on the exterior, mobile surface of the molecule.
– Also called immunodominant structure (determine the specificity and optimal binding energy of antigen-antibody interactions.
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Antigen Density• The number of antigenic sites on a foreign
substance• Contribute to:
– strength and end results of an immunologic response
– efficiency of antibody binding – extent of complement activation determining
likelihood of RBC hemolysis• Identification techniques: RIA, ELISA,
Electron microscope with ferritin-labelled anti-immunoglobulin, flowcytometry.
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Blood Groups Antibodies
• Immunoglobulins and antigen binding
• Blood group alloantibodies and autoantibodies
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Immunoglobulin and Antigen Binding• Immunoglobulin (Ig): protein molecules
that are produced in response to antigenic stimulation demonstrate specific antibody activity.
• Antibody specificity determined by hypervariable/complementary-determining regions of Ig molecules.
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Immunoglobulin and Antigen Binding
• Amino acid sequence heterogeneity in hypervariable region, allows for variation in the configuration of the peptide chains in the variable loops, determines the combining specificity of each antibody.
• The combining site of an antibody, where it is in physical contact with an epitope, is called the paratope.
• Binding involves formation of multiple noncovalent bonds, between antigen and amino acids of paratope.
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• The attractive forces between antigen and antibody, become significant when the distance between the interacting groups is small.
• The acttractive forces:– Electrostatis and van der Wall’s forces– Hydrogen bonds– Hydrophobic interactions
Ig and Antigen Binding
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The better the physical fit between epitope and paratope higher overall binding energy greater affinity of the resulting reaction between antigen &
antibody.
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Blood Group Alloantibodies and Autoantibodies
• Majority of clinically significant blood group antibodies: IgG, IgM, IgA*
• Blood group antibodies:– Alloantibodies: reacts with foreign antigen/not
present on the patient’s own RBC– Autoantibodies: reacts with an antigen on the
patient’s own cell.• Alloantibodies:
– Natural antibody – Immune antibody
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• Natural antibody– Naturally occuring alloantibodies:– Unknown stimulus.– Appear regularly in the serum of persons who
lack the corresponding antigens.– May produced in a small subset of individuals.
• Immune antibody– Result of immunization to foreign RBC antigen– Exposure through:
• Blood transfusion• Pregnancy (delivery)
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The Complement System & Blood Banking
• Complement involve in:– Sensitization & destruction of transfused
RBC by alloantibodies.– Destruction of autologous RBC by
autoantibodies• Complement important in
immunohematology testing
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Role of Complement in RBC Destruction
• Antibody binding to RBC antigen activation of complement (by classical pathway).
• Mode of destruction & extent of hemolysis depends on:– Class of Ig involve– Activity of individual’s RES– RBC destruction hemolysis:
• Intravascular• extravascular
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Intravascular Hemolysis
• Binding of antibodies directed against antigen
• Activation of complement (terminal membrane-attack complex polymerized to form pores in the RBC membrane ECF enters cell swelling burst by osmotic lysis; IgM, IgG).
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Extravascular Hemolysis
• Mainly by IgG• Removescomplement coated RBC
(mechanism unclear).
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Erythrocyte Antigens & Antibodies
• > 700 antigens organized into 29 blood group systems by the International Society of Blood Transfusion (ISBT).
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ABO Antigens
• Also express in many tissues, body fluids, platelet and endothel).
• Most important blood group system in transfusion and organ transplantation.
• 3 antigens: A, B, H (biosynthetic precursor of A & B antigens).
• 4 phenotypes: group A, B, AB, O• A & B: autosomal codominant antigens
expressed on group A, B & AB RBC
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Group O• Autosomal recessive reflecting the
absence of a functional ABO gene• Express H antigen• Most frequent
ABO antigen expression usually accompanied by the presence of naturally occuring antibodies against the missing
antithetical antigens.
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Null and Weak Phenotypes• ABO antigens can:
– Weakened weak A, weak B phenotypes
– Anomalous:• Inherited: cis-AB• Acquired:
– Absence (Null phenotype):• Classic Bombay completely absence of
all ABH antigens on RBC surface• Para-Bombay shows little/no antigen in
RBC but normal in secretion/body fluids.
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ABO antigen Biochemistry
• Carbohydrate• ABH antigens
expressed on RBC glycoproteins & glycosphingolipid (type 2,3,4 chain) RBC origin.
Type 1 chain are synthesized by gastrointestinal mucosa secreted into plasma passively adsorbed onto RBC
membrane
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Molecular Biology• The expression of ABO antigen is
controlled by 3 separate gene loci:– ABO located in chromosome 9– FUT1(H gene) in chromosome 19– FUT2(Se gene) --> in chromosome 19
• Each gene codes for a different enzyme (glycosyltransferase) which attaches specific monosaccharides onto precursor dissacharide chain.
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Molecular Biology
• 4 type of dissacharide chains:– Type 1: found in plasma & secretion
substrate for FUT2 gene.– Type 2,3,4: only in RBC substrate for
FUT1 gene.
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ABO Antibodies
• Weak or absent in newborn 3-6 mo• 5-10 yo adult level• Advancing age slight decrease• Detected at room temperature, saline agglutinins
with optimal reactivity at 40C.• Mostly IgM.• IgG (reactive at 370C) can occur after
transfusion/pregnancy; higher titer; less readily neutralized by soluble blood group substances.
• Can fix complement hemolysis in vivo/vitro • Can cause: hemolytic transfusion reaction &
hemolytic disease of the new born.
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Less common ABO antibodies
• Anti-A1
• Anti-H
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Immunohematology Tests
• Hemagglutination• Antihuman Globulin Test• Compatibility Testing
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Reference book
• Henry’s Clinical Diagnosis and Management by Laboratory Methods. 21st ed. McPherson RA, Pincus MR. Saunders Elsevier. 2007; pp: 617-24; 628-32; 647-68.
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