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.