Basic Concept of Immunohematology.ppt.pptx
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Transcript of Basic Concept of Immunohematology.ppt.pptx
Basic Concept of Immunohematology
Leni Lismayanti, dr., SpPK
Lecture Overview
1. Basic immunohematology concept.2. Erythrocyte antigens and
antibodies (ABO blood group system).
3. Immunohematology tests.
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.
Immunohematologist
• perform variety of serologic laboratory examinations,
• evaluate & interprete the reactions observed, …
Immunohematologist
• provide selected advanced investigations to aid in the study of: pathogenesis, diagnosis, prevention, management of immunization associated with transfusion, pregnancy and organ transplantation.
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.
• 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.
• 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).
• 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
Immunogenicity
• Ability of antigen to elicit immune response.
• Determined by:– Certain innate characteristics of the antigen– Host’s genetically determined immune
responsiveness
Characteristics of antigen
• Determined immunogenicity:– Degree of foreignness– Molecular size & configuration (affected
by temperature,pH,ionic environment)– Antigenic complexity (number of
available epitopes).
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
• 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)
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).
• 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.
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.
Blood Groups Antibodies
• Immunoglobulins and antigen binding
• Blood group alloantibodies and autoantibodies
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.
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.
• 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
The better the physical fit between epitope and paratope higher overall binding energy greater affinity of the resulting reaction between antigen &
antibody.
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
• 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)
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
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
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).
Extravascular Hemolysis
• Mainly by IgG• Removescomplement coated RBC
(mechanism unclear).
Erythrocyte Antigens & Antibodies
• > 700 antigens organized into 29 blood group systems by the International Society of Blood Transfusion (ISBT).
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
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.
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.
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
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.
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.
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.
Less common ABO antibodies
• Anti-A1
• Anti-H
Immunohematology Tests
• Hemagglutination• Antihuman Globulin Test• Compatibility Testing
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.