Red Cell Antigens and Antibodies

Introduction

There are over 450 possible blood group antigens distributed on the red cell membrane in a mosaic pattern.
Each is recognized by a specific antibody.
These antigens are genetically determined by a pair of allelic genes. Thus one may be homozygous or heterozygous for any antigen. Genes for antigens that are linked together constitute blood groups. See definition bellow.

Blood Group Systems

Definition: A blood group system includes those antigens which are produced by alleles at a single gene locus or by alleles at other loci so closely linked on the chromosome as never, or very rarely, to cross over.

Five of the Nine Classical Systems that are Clinically important (and one extra):

(1) ABO - Chromosome 9 * Ü This one you need to know

(2) Rh - Chromosome 1p * D antigen is the important one Ü This one you need to know

(3) Kell - Chromosome 7 *

(4) Duffy - Chromosome 1q *

(5) Kidd - Chromosome 18 *

(6) Lewis ( these antigens are plasma Ag that are absorbed to the membrane).

The number of genes and antigens per system varies widely (e.g. the # of alleles at a single site is at least 20 in the Lutheran system and 17 in the Kell system.

Each RBC holds many antigens: e.g. 10⁶ molecules of A antigen/RBC; 7.5X10⁵ B antigen/RBC; 50k of D/RBC

Other Blood Group Systems: Xga, Diego, Cartwright etc. Their antigens or antibodies are rarely clinically significant

Genetic Basis of Blood Group Antigens (Simplistically!)

Some Basic Tenets

Codominance is the general rule, which means the product of each of a pair of alleles is recognized serologically.
There may be more than two alleles at each locus.
Amorphic genes exist = there is no recognizable antigen product, e.g. Group O. or Rh (-) for the lack of D Ag.

ABO Genetics: 4 possible alleles of genes (A₁, A₂ B, O)

Alleles code for transferase enzymes that move an immuno-dominant sugar to the terminal position on sugars reaching out from the cerimide in the membrane.
A₁ and A₂ transfer the same sugar GalNAC but A₁ is more efficient and produces a slightly different configuration
B gene product that transfers a D-Gal to the same terminal position on the chain of sugars
O is a truncated gene so it is an amorph, which means it has no tranferase activity and is not detectable.
AB individuals buts GalNAc on some chains and D-Gal on others and both are immuno-detectable.

MNSs Genetics

Simplest possible. Two alleles of importance M and N (Ss not as important). Can either have Heterozygous MN or Homozygous MM or NN.
A single locus on Chr. 4 has either M or N

Rh Genetics: multiple pairs of allelic genes so closely linked that they do not cross over and are inherited together.

One loci codes for presence or absence of D. Htrz or Hmoz for D is Rh (+) (85% of Pop), Hmoz without D = Rh(-) (15%)
There are linked loci for additional genes Cc and Ee. They are inherited w the D genotype. Not real important now.

Kell system: 4 closely linked antigens, all of which depend on gene for the precursor substance.

There are 17 known alleles at the Kell loci. This locus is linked to 3 other sets of alleles of minor importance.

Antigens have variable Structure, which affects their reactivity serologically

Some are transporters, some are surface enzymes, some are adhesion molecules, some are part of RBC membrane, some are receptors, etc.)

Glycosphingolipids –Ex: ABO, P, I; these are CHO so are indirect gene products of enzymes (tranferases)

Series of sugars bound to ceramide and extending from membrane surface

Ceramide: A long base, sphingosine, plus a fatty acid.
Sugar chains, 4 at least; may be branched

For ABO group, terminal sugar determines the antigen. Precursor gene product H puts the fucose sugar on the end of the sugar chain. Then the genes for the following groups add terminal sugars as in III B above.

Antigenic determinates not an integral part of membrane but extend through it to the surface.
Lewis is a plasma Ag absorbed on surface of RBC, therefore it is genetically determined =direct gene product = protein

Glycophorins Carry the Ag MNSs Group, MN carried on glycophorin A (a SGP) and Ss on glycophorin B (d SGP)

Long chain glycosylated amino acids bound by actin to spectrin extending through the membrane with Ag waving on the surface with blood groups in the terminal positions.
Glycophorin A - Carries Ag’s M and N which differ only in amino acids 1 and 5 at N-terminus
Glycophorin B - Carries Ag’s S and s which have identical amino acids through first 28. Ss are linked to U Ag
Antigens are not integral part of membrane

Glycooproteins: embedded in membrane and do not significantly extend beyond surface; Proteins are therefore direct gene products.

Rh and Kell (or at least Kell precursor substance).

Integral membrane components. Lack of precursors for Kell Þ abnormal RBC membraneÞ Acanthocyte. No Rh Þ stomatocyte. Lack of either Þ shortened survival - hemolytic state

Rh antigens - complex protein. Phospholipids, hydrophobic; Major portion embedded in lipid bilayer.

Kell antigens - a glycosylated protein possibly bound to actin.

Duffy antigen - membrane bound protein; cytokine receptor for IL-8 and P. vivax

Blood Group Antibodies

This is how the Ag have been defined; Can be:

(1) heterophile =against different species
(2) allo = against same species, e.g. ABO
(3) auto antibodies = against one’s own cells)

Defs:

(1) Complete: Agglutination of cells suspended in saline
(2) Incomplete: No agglutination of saline suspended cells

But Agglutination in macromolecular media - albumin, dextran, PVP (polyvinylpyrrolidone)
But Agglutination of enzyme treated cells: papain, trypsin, bromelin, ficin
But Agglutination by antiglobulin serum

(3) Naturally occurring antibodies: Developing without prior exposure to red blood cells.
(4) Acquired antibodies: develop after red cell exposure by transfusion or pregnancy.

IgM Antibodies - Example - Anti A and Anti B

Virtually always agglutinate cells in saline suspension.

Frequently "naturally" occurring - no prior exposure to red blood cells required because exposure is from dusts, pollens, etc. during infancy so patient who is A Group will have antibody to B. Patient with O group will have anti A and B

Large molecule (MW = 900,000), potentially 10 combining sites, span – 300 angstroms, so can bridge the minimum gap between RBC due to electrostatic repulsion of negative surface charge in saline Þ agglutination

Due to multiple binding sites: Rapidly reacting, usually at room temp, usually activating complement because there is a high probability of 2 combining sites hitting adjacent antigens.

Produces hemolysis in vitro (more likely agglutination) and intravascularly in vivo Þ hemogobinemia/hemoglobinuria or more likely extravascularly Þ Ý bilirubin.

Not transported across placenta.

Historically discovered by Lansteiner-ABO system 1900. - ABO System

Pt who are Group A will make Anti B but can also make anti A₁.

IgG Antibodies - Usually "Incomplete"

Result of prior red cell exposure - transfusion or pregnancy. Very, very rarely "naturally" occurring.

Do not agglutinate cells in saline suspension.

Small MW 160,000 - 2 Ab combining sites (2Fab portions).

Span at maximum - 140 Å so can not span RBCs in saline

React more slowly and usually best at 37^o

May or may not activate complement

Actively transported across the placenta. Hemolysis in vivo, rarely in vitro

Agglutinate cells suspended in albumin which ß charge; protease treated cells and react by Coombs' anti Ig test.

Example: Anti-Rh (Anti-D) (almost never activates compliment)

Hemolytic disease of the newborn
Landsteiner-Weiner - similar antibody from rabbits immunized with cells of Rhesus monkey, thus Rh.
Ag highly immunogenic. 1 ml of blood may alloimmunize a human. Rh (-) Patient should only get Rh(-) blood, especially premenopausal women (except urgent emergency) to prevent Hemolytic Disease of newborn.

Antiglobulin Reaction: Coombs' Test (this is how many Groups were discovered: Rh, Kell, Duffy, Kidd and Xga)

Definition - procedure for detecting incomplete antibodies, either on RBCs or in serum, using anti-human Ig serum.

Antiglobulin serum prepared by injecting another species: rabbit, goat, etc. with human Ig, complement components, etc.

Animal produces antibody in its serum: Anti-human globulin against g Ig or complement.

Originally a mix of antibodies to human Ig. Now monospecific: Anti-IgG, anti-C3b, anti-C3d or anti-IgM
Polyspecific serum: A blend of anti-IgG and C3b and/or C3d

Serum reacts with either IgG and/or C3 components bound to RBC membrane resulting in agglutination.

Antiglobulin Direct Test: A test performed on cells

Test for in vivo reaction of antibody with RBC Ag to determine if the RBC is coated with IgG or complement?
Method: RBCs washed in saline 3-4 X. Antiglobulin serum added and tube centrifuged. Agglutination = + test
Used to ID:

(1) Hemolytic disease of the newborn
(2) transfusion reactions (donor cells coated with recipient's Ab)
(3) Autoimmune Hemolytic Anemia
(4) Drug induced immune hemolytic anemia

Antiglobulin Indirect Test - A serum test to determine if an "incomplete" antibody is present in the serum.

Method: Incubate test cells in serum mins at 37^o . Wash cells 3-4 X. Add anti-Ig serum, read for agglutination
Used to ID:

(1) Compatibility testing - unknown serum and cells
(2) Blood grouping - known serum - unknown cell
(3) Ab screening - unknown serum, selected cells

(4) Ab ID - unknown serum, Panel of Group O cells with all major Ag identified (previously in lab).
(5) Ab titration - serum known, selected known cells

Mechanisms of red cell destruction by antibody in vivo

Intravascular Hemolysis - Hemoglobinemia, Hemoglinuria (IgM, some IgG) Complement-MAC C5-9, rapid, massive.

Extravascular Hemolysis Þ unconjugated Bili. Macrophages of RE destroy RBCs with IgG+C3b> IgG>IgG₁>C3b and bi>IgG_2or4 .