RED CELL ANTIGENS

 Consist of complex molecules of different types that coat the surface of the

erythrocyte.

 Some are glycolipids identified with the lipid bilayer

 Others are glycoproteins and complex carbohydrates associated with the

integral glycophorins

 Most of them terminate in SIALIC ACIC molecule which gives the red cells

their strong net negative charge that allows the cells to repel each other.

 The antigens are capable of stimulating an immune response in animals that

lack them.

 300 antigens are known to date and are grouped into 15 blood group

systems.

 Studies on the red cell antigens and their corresponding antibodies constitute

the science of Immunohaematology.

 Application of this knowledge is the basis of blood transfusion science and

blood banking.

 Most blood group antigens are believed to be integral components of the cell

membrane. Absence of some may result in a disease state characterized by

cell lysis.

1
 Construction of these antigens on the red cell is influenced indirectly (if they

are carbohydrate based) by gene-specified enzymes or direct synthesis (if

they are protein antigens). Eg; construction of ABO antigens involves genes

on chromosome 9.

 These genes code for enzymes known as glycosyl transferases which add

specific sugars onto the glycolipids:

1) H-gene codes for L-fucosyl transferase.

2) A-gene codes for N-acetylgalactosaminyl transferase.

3) B-gene codes for D-galactosaminyl transferase.

4) O-gene is an amorphic gene and does not code for

detectable enzymes. Individuals that inherit the O-gene only

produce the H substance (antigen).

 Antigens of other blood groups are produced from precursor substances. Eg,

A, B, Lewis, ect. are produced from H substances

 Systems such are ABO, P, Lewis and Ii share the same precursor.

 Some antigens are strictly protein in nature, e.g Rhesus, M, N, Duffy, S, s

and U. These are integral components of the red cell membrane.

 Deletion of genes responsible these antigens may result in shortened red cell

survival (haemolysis).

 Some blood group antigens are found in body fluid such as saliva and urine.

2
Structurally related blood group systems (ABO, H, P, Ii and Lewis)

 A blood group system consists of antigens produced directly or indirectly by

allelic genes.

 Inheritance of these genes results in the expression of the antigens on the

surface of the red cell membrane or body fluids.

 Antigen expression can also be influenced by genes inherited independently

at different loci. Eg,:

A and B antigens are affected by I gene or alleles at H gene locus.

 ABO, H, P, I or Lewis antigens are constructed on structurally related

carbohydrate molecule known as Lactoceramide.

 The activities of some of the genes of these antigens may affect the

expression of the other antigens.

 Antigens appear when specific sugars are added by transferases to the end of

oligosaccharides.

 Soluble forms are found in plasma and other body fluids.

3
 1. ABO BLOOD GROUP SYSTEM

- The most important blood group system, which lead to the development of

routine grouping system.

- Discovered by Landsteiner in 1900 through a series of experiments on

samples from colleagues.

- Serum + red cells = agglutination or haemolysis in some mixtures but

not in other

- Landsteiner classified blood samples into one of the three groups A, B and O.

The fourth group AB was discovered by De castello and Sturli.

- Serum of each person contained antibody/ antibodies directed against

antigens absent from the person's red cells.

- ABO remains the most important blood group to date.

- Antibodies are consistently absent from the persons who have the antigens.

- ABO incompatibility between the recipient and donor is the foundation on

which all other pretransfusion testing rest.

4
ROUTINE ABO GROUPING

REACTION OF CELLS REACTION of SERUM GROUP FREQUENCY

WITH- WITH- W B I O

Anti-A Anti-B Anti-AB A cells B cells O cells

0 0 0 + + 0 O 45 49 79 40

+ 0 + 0 + 0 A 40 27 16 28

0 + + + 0 0 B 11 20 4 27

+ + + 0 0 0 AB 4 4 <1 5

Antigens of the ABO system

- Glycosphingolipids carrying A and B oligosaccharides are integral parts of the

red cell membrane, epithelial and endothelial cells.

- present in soluble forms in plasma.

- Glycoproteins for A and B antigens are found in secretions such as saliva.

- Genes at three separate loci ( ABO, Hh, Sese) control the expression of A

and B antigens.

- A, B, O genes are located at ABO locus on chromosome 9.

- A and B genes encode for glycosiyltransferases that produce A and B

antigens.

5
- O gene is amorphic and no detectable antigen is found due to its gene action.

- O persons lack A and B antigens but carry lots of H antigen.

- H antigen is a precursor of both A and B antigens.

- Hh and Sese genes are closely linked.

- H gene produces enzyme, L-fucosyltransferase to construct H antigen on

which A and B are produced

- T he h gene is very rare. No antigen is produced in association with h.

- If the hh gene is inherited, the individual will not have A, B and H antigens.

These are referred to as Bombay persons. They are capable of producing

anti-A, anti-B and anti-H.

- Se is directly responsible for H antigenic expression (indirectly for A and B on

the glycoproteins in epithilial secretions such as saliva.

- 80% of the population are secretors because they inherited the secretor gene

(Se) and can produce the H antigen that is convertible to A or B.

- The se gene has no demonstrable product, hence it is amorphic.

- Infant oligosaccharides are linear in structure, hence the have

immunodominant sugar added. Their reaction with appropriate antibody are

generally weak.

- Adult oligosaccharides are branched, hence have many immunodominant

sugars resulting strong reactions with appropriate antiserum.

- Blood group-active glycoproteins on cell surface or body fluids are either type

1 or type 2.

6
- Glycolipids produced by the red cell membrane are predominantly type 2

chains.

- The chain is carried on the glycosphingolipids called paraglobosides.

- A and B antigens are constructed at the expense of H antigens.

- There are alleles at ABO locus that encode for weak forms of A and B

antigens which are known as subproups of A or B.

- Secretions of Sese contain type 1 and type 2 chains without A, B or H activity

(Oh phenotype) on the red cells. H and Se each code for different

fucosyltransferase.

- H gene codes for an enzyme that produces H antigen on type 2 chains on red

cells while that produced by Se gene prefers type 1 chains in body fluids/

secretory glands.

- O gene produces a protein, which can be detected immunologically but has

no transferase activity.

- Red cells of O persons carry readily detectable unconverted H antigen.

- H, A and B are found in secretions of genetically hh persons who are Se.

- Therefore, it means that H gene and Se gene code for different L-

fucosyltransferases.

- A and B are detected in direct agglutination with anti-A and Anti-B,

respectively. ABO reagents sometimes produce weak reactions with red cells

of newborns than with adult red blood cells.

- A and B red cells are not fully developed at birth until 2-4years of age.

7
- The antigenic expression remains constant throughout adult life.

Subgroups of A

- These are phenotypes that differ from others of the same ABO group with

respect to the amount of A antigen carried on red cells and saliva.

- The two principle subgroups of A are A1 and A2 . They are distinguished by

use of anti-A1 from human serum or lectin dolicho biflorus seed.

- A1 A2

- Anti-A1 + 0

- Anti-A + +

- Red cells from 80% of A and AB persons are agglutinated by anti-A1 and are

known as A1 and A1B.

- Red cells from 20% of A and AB that are not agglutinated by anti-A1, but by

anti-A are known as A2 and A2B.

- Anti-A1 is found in serum of 1% to 8% of A2 persons and 22% to 35% of A2B

persons.

- Antii-A1 may cause discrepancies in ABO testing and incompatibilities in

crossmatching.

- Anti-A1 is considered clinically significant if it reacts at 37co.

- It is not necessary to test for red cells to confirm their subgroup unless when

working with samples from people who are known to have serum that

contains anti-A1 .

8
- Subgroups weaker than A2 are very rare and are characterized by decreased

number of A antigenic sites and increased number of H antigenic sites.

- They are classified on the basis of:

- 1. Degree of red cell agglutination by anti-A and anti-A1 .

- 2. Degree of red cell agglutination by anti-AB

- 3. Presence or absence of anti-A1 in serum.

- 4. Degree of H activity on the red cell.

- 5. Presence of A and H substances in saliva secretions.

- Red cells of AX, Ael, Aint or A3 are very rarely found in transfusion practice.

- Ax red cells are agglutinated by anti-AB, and not by anti-A. They react with

monoclonal anti-A reagent.

- Ael fails to react with anti-A or anti-AB of any origin. Absorption studies are

done to determine whether the red cells carry the antigen at all.

- Aint can be identified only by Anti- A with which they react more weakly than

A1 cells and react more strongly with anti-H than A2 cells.

- A3 cells can produce a characteristic mixed-field agglutination reaction

with anti-A and anti-AB.

- Am cells can be identified by their ability absorb anti-A. Saliva studies,

absorption / elution techniques are useful.

- Weak subgroups of B are even less common than those of A, but the reaction

patterns are the same.

9
 SEROLOGICAL REACTIONS OF A AND B PHENOTYPES

- Phenotype anti-A anti-B anti-AB anti-H anti-A1 Saliva

- A1 4+ 0 4+ 0 4+ A, H

- Aint 4+ 0 4+ 3+ 2+ A, H

- A2 4+ 0 4+ 2+ 0 A, H

- A3 2+ mf 0 2+mf 3+ 0 A, H

- Am +/0 0 +/0 4+ 0 A, H

- Ael 0 0 0 4+ 0 H

- Ax +/0 0 +/2+ 4+ 0 H

- B 0 4+ 4+ 0 0 B, H

- B3 0 2+ mf 2+ mf 4+ 0 B, H

- Bm 0 0 +/0 4+ 0 B, H

- Bx 0 +/0 2+/0 4+ 0 H

mf = Mixed field agglutination

Antibodies to A and B antigens

 People possess antibodies to A and B antigens that are absent from their red

cells

 Serum testing is made possible in addition to red cell testing.

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 Presence of antibodies without known stimulation is sometimes called "

natural occurrence''

 Presence of antibodies as a result of known stimuli, e.g pregnancy and

transfusion is sometimes called ''immune occurrence''.

 Naturally occurring antibodies are thought to be due to exposure to bacteria

in the environment and make people produce antibodies to A or B-like

structures on the bacterial cell wall.

 Anti-A occurs in B and O persons. Anti-B is found in A and O persons.

 Group AB persons have got both A and B. So they do not produce antibodies

against these antigens.

Times of appearance:

 Anti-A and anti-B are produced after the first few months (3-6 months ) of

life. But, some infants are capable of producing antibodies at birth.

 Production remains constant throughout adult life.

 Detection of the antibodies in infants may not be valid as some would have

come from the mother.

Reactivity of anti-A and anti-B:

 The anti-A and anti-B from B and A persons, respectively, are predominantly

of the IgM type.

 Anti-AB from the O persons is mostly of the IgG type which crosses the

placenta.

 Antibodies to A and B react best at Room temperature or below.

11
 They are efficient activators of complement at 37co resulting in complement

mediated cell lysis.

 Donors and recipients can be found whose sera cause haemolysis of ABO

incompatible red cells at temperatures below 37c0.

 Haemolysis should be suspected if a pink to red discoloration appears in the

supernatant or when the cell button is reduced in size.

 HAEMOLYSIS must be interpreted as a positive reaction result.

 Haemolyis does not occur in EDTA blood sample.

Anti-AB ( group O serum)

 Reacts with A and B cells

 It is mostly of the IgG type

 Capable of causing HDN

 Eluate prepared from A group cells that have been used to adsorb group O

serum contain anti-A that reacts with A and B cells

 Similar findings are obtained after using B cells to adsorb O serum.

 Therefore, anti-AB in O persons is inseparable.

 Saliva from A and B secretors can inhibit anti-AB activity with either A or B

cells.

Anti-A1

 B group serum contains both anti-A1 and anti-A.

 B serum agglutinates both A1 and A2 cells.

 Following adsorption with A2 cells group B serum will agglutinate only A1 cells.

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 Before adsorption:

 A1 cells A2 cells

 B serum + +

 After adsorption:

 B serum + 0

 A1 cells have quantitatively more A antigens than A2 cells

 Anti-A1 is a weakened form of anti-A.

 Anti-A1 reacts with A1 cells because A1 has more antigenic sites than A2

 Sera of certain weak subgroups of A may contain anti-A1.

 A1 and A2 cells are differentiated by use of adsorbed group B serum or lectin

anti-A1.

 Lectin anti-A1 will reacts with both A1 and A2 before dilution , but with only A1

after dilution.

THE H SYSTEM

 H H, Hh and hh genotypes are responsible for the H and Oh (Bombay)

phenotypes.

 H gene produces H antigen which is a precursor of other antigens.

 The amount of H antigen diminishes in this order:

13
 O > A2 > B> A2B > A1 > A1B

 Potent anti-H is found in Oh phenotype who lack H on their cells

 Anti-H may be in A1, A1B or B persons due very little unconvertible H

antigens, but it usually very weak and react at room temperature .

 Anti-H in Oh phenotypes reacts very well over a wide thermal temperature

(4-37c0) with all cells except Oh.

Oh phenotype ( Bombay)

 these are red cells without H, A and B antigens

 first discovered in Bombay, India

 front typing gives an apparent O blood group

 back typing shows presence of anti-A, ant-B and ant-H

 Lectin anti-H fails to agglutinate Oh red cells.

Para- Bombay phenotype, Ah and Bh

 Ah and Bh lack serologically detectable H antigen but carry small amounts of

A or B.

 Weak reactions with anti-A or anti-B

 Non-reactive with lectin anti-H or anti-H serum of Oh persons

 Produced by variant H gene that codes for minute amounts of H antigens.

 Serum of Ah and Bh persons contain anti-H - Ah has anti-H and antii-B; Bh

has anti-H and anti-A

14
 2. THE LEWIS BLOOD GROUP SYSTEM

 Comes from the same precursor substance with ABO antigens

 Common antigens are Lea and Leb

 Not indigenous to the red cell

 Carried on plasma glycolipids that are later absorbed from plasma

to the red cell membrane.

 Presence depend on inheritance of one Le two le genes.

 Addition of fucose by Le gene to type 1 oligosaccharides produce

Lea antigen.

 Interaction of Le, Se, H genes give rise to Leb

 More Leb is adsorbed to the red cell membrane readily than Le a

 Type 1-------> H substance------------> Leb

Se/ H transferase

 le is an amorphic gene

15
phenotypes and frequencies in Lewis

REACTIONS WTH: ADULT FREQUENCIES

Anti-Lea Anti-Leb Phenotype W B

very very
0 0 Le (a- b-) rare rare

+ 0 Le (a+ b-) 22 23

0 + Le (a- b+) 72 55

+ + Le (a+ b+) 6 22

Antibodies to Lewis System

 occur almost exclusively in Le (a- b-) persons

 they are naturally occurring.

 Le (a- b+) individuals do not make anti-Lea because there are

small amounts of Lea in plasma and saliva.

 Anti-Lea and anti-Leb may occur together in serum.

 They are of the IgM type.

 There is no HDN because of their being IgM and antigens are

poorly developed at birth.

16
 May cause in vitro haemolysis with enzyme treatment of red cells.

 Most agglutinate saline suspended red cells.

 A few may cause positive Coombs reactions.

 Anti-Leb sera are of two types:

a) that which react best with O and A2 cells ( most common)

and has been designated anti-LebH

b) that which is equally reactive with Leb of all ABO phenotypes

and has been designated LebL

 Saliva from O Le(a-b-) persons neutralizes anti-LebH if they have H

substances.

 Anti-Lec is a cold agglutinin which agglutinate Le (a- b-) cells from

people who are sese (non secretors).

 Anti-Led agglutinates Le (a- b-) cells ( secretor). They react more

strongly with type 1 oligosaccharides and are referred to as anti-

type 1H.

A. Lewis in pregnancy

 Pregnant females sometimes test Le (a- b-).

 May produce antibodies against Lewis antigen

17
B. Lewis in transfusion practice

 Antigens readily adsorb to and elute from the red cell membrane.

 Transfused red cells quickly assume the Lewis phenotype of the

recipient.

 Lewis antibodies in the recipient are easily neutralized by Lewis

blood group substances in the donor plasma. NO HAEMOLYSIS OF
TRANSFUSED CELLS.
C. Lewis antigens in children
 Infants type Lewis negative Le(a- b-)
 Some can show small amounts of Lewis when tested with
monoclonal antibodies.
 Reliable Lewis grouping in infants not possible until after six years.
 In children incidences of Lea are higher than Leb. Le (a+ b+) may
be observed as a transient phase:

Leb Le(a+b-)--------> Le(a+b+) ------> Le(a- b+) in adults(80%)

Lea Le(a+b-) is 80% in infants and diminishes to 22% in adults

 Cord cells are agglutinated by antibodies that agglutinate Le(a+b-)
and Le(a-b+) but not Le(a-b-) adult red cells.
 The antigen in cord cells is called Lex.

18
 3. THE Ii BLOOD GROUP SYSTEM
 Antigens are found on type 2 chain
 Also found on membrane associated glycoproteins and
glycosphingolipids
 The structures are eventually convertible to H, A or B
 I specificity appears when the branched structure of type 2 chain
appears.
 When disaccharide units beta-GlcNac and beta-Gal are linked to an
unbranched chain the i specificity results.
 Foetal red cells are rich in i antigen. Their membrane carry minute
amounts of branched oligosaccharides, hence, poorly developed I
antigens.
 I antigens develop during the first two years of life and i is lost in
the process.
 Adult red cells react strongly with anti-I than with anti-i
 Rare adults without I antigen are known to exist and they are
capable of producing anti-I alloantibodies.

19
Amounts of Ii antigens on different human red cells
Phenotype antigen_________
I i
I adult much trace
I cord little much
I adult (I-) trace much___

Antibodies to I and i antigens

 Anti-I is usually identified by its failure to react with cord cells.
 An autoanti-I exists that react best at 4c0 and is often associated
with infection (mycoplasma gondii).
 An autoanti-i also exists in association with infection
mononucleosis (cold agglutinin).
 A naturally occurring anti-I is found in I negative persons:
- IgM type
- Reacts best in with saline suspended red cells (room
temperature)
- May cause haemolytic transfusion reaction (HTR).
- No HDN
- Binds complement

20
Serological behavior of antibodies to I/i blood group system
Anti-I Anti-i
4c0 I adult 4+ 0-1+
i cord 0-2+ 3+
i adult 0-1+ 4+

22c0 I adult 2+ 0
i cord 0 2-3+
i adult 0 3+

complex reactivity
 Anti-IH is common in sera of A1 and react strongly with H+ and I+
cells
 O cells react positively but not A1 cells
 Red cells of A1, I+ persons react poorly with anti-IH due to weak
H.
 Anti-IH fails or reacts poorly with cord red cells or i adult cells

21
4. P BLOOD GROUP SYSTEM

 Immunisation studies by Landsteiner led to the discovery of the P

blood group system in 1927. M and N blood groups were also
discovered at the same time.
 It was originally called P, but name was changed to P1
 P was assigned to antigens present on all human red cells
 Red cells without P1 are called P2.
 P1 is present in 80% of whites and 94% of blacks
 Another antigen of the P system is Pk
 P antigens are built through the addition of sugars to the
glycosphingolipid.
 The process is analogous to the development of H, A and B
antigens.
 Pk is developed through the addition of Gal in alpha (1-4) linkage
to the terminal Gal residue of lactosylceramide (ceramide
hexoside or CDH).
 CDH can be converted to P antigen (globoside) by addition of
GalNAc.
 Addition of Gal to paragloboside will result in P1
 Paragloboside is the precursor of A, B, H, Ii antigens found on red
cells.
 Several phenotypes of P blood group exist.
 Pk is found when Pk has not been converted to P antigen.

22
 There are some very rare people who lack P1, P and Pk. These are
referred to as p phenotype.
Antibodies to the P blood group system
 Persons who are Pk have a very potent alloanti-P that reacts with
P1 phenotype (P1 + P2) and P2 phenotype (PI- P+).
 Biphasic autohaemolysin anti-P exists in persons with Paroxymal
Cold Haemoglobinuria ( PCH):
- Associated with STDs, especially syphilitic conditions
- It is sometimes known as Donath-Landsteiner antibody
- It is of the IgG type
- It binds complement under low temperature and causes
haemolysis under warm condition (37c0).
 p persons produce anti-P1+ P + Pk (anti-Tja).
- It is a potent haemolytic IgM.
- it causes both HTR and HDN
- It has been curiously associated with abortion in early pregnancies
involving p women.
 Anti-P1 is almost always found in P2 persons
- it reacts optimally at 4c0 but may occasionally react at 37c0
- it is neutralized by fluids from hydatid cyst
- It is almost always of the IgM type.
- Has not been associated with HTR and HDN

23
Phenotypes and frequencies in P blood group system________
Reactions with: phenotype frequ.
Anti-P1 Anti-P anti-Pk -PP1Pk Ph'type white black
+ + 0 + P1 79% 94%
0 + 0 + P2 21 06
0 0 0 0 p v. rare
+ 0 + + P1 k v. rare
0 0 + + P2 k v. rare_________

5. THE RHESUS BLOOD AND LW GROUP SYSTEM

 The Rhesus blood group system is very complex
 The first human anti-D was discovered by Levine and Stetson in
1939 in serum of mother who a fetus with severe and fatal HDN
 Landsteiner and Wiener, in 1940, raised antibodies in guinea pigs
and rabbits that reacted with 85% of human red cells during their
immunization studies. This was called Rhesus factor.
 Levine and Katzin found several postpartum women with similar
antibodies to those raised in animals.
 Wiener and Peters observed examples of anti-Rh in Rhesus
negative patients who had received ABO compatible Rhesus
positive blood units.

24
 Antigens detected by anti-Rhesus and human anti-D are not
identical.
 The Rhesus antigens are protein in nature and are found only on
the red cell membrane.

Rhesus positive and Rhesus negative

 Rhesus positive means presence of D antigens and Rhesus
negative means absence of D antigen.
 Rho is the earlier name for D antigen but is no longer used.
 D antigen is genetically determined and the genes are autosomal
dominant.
 Rhesus genes are found on chromosome 1.

Other important Rhesus antigens

 More studies have revealed more Rhesus antigens. By mid 1940s
four additional antigens were found-:
-C
-E
-c
-e
 Subsequent studies have revealed more than 40 antigens
belonging to the Rhesus system.

25
 The five principle antigens make up >99% of clinical problems due
to Rhesus.
 Immunological activity of Rhesus arises from surface material with
several antigenic determinant areas.
 Composition of these determinants is genetically determined.
 Many variations and combinations have been recognized.
 The five principle antigens make the backbone of the Rhesus
system.
 The CDE nomenclature is used for most applications in Blood
transfusion purposes.
 A combination of CDE and Rh-Hr terminology is used.

Clinical Significance of Rhesus

 D, after A and B, is an important antigen in transfusion practice.
 Rhesus negative persons do not regularly have anti-D in their
serum.
 Formation of anti-D always follows immunization through
pregnancy or transfusion.
 A high proportion of Rhesus negative persons exposed to D
produce anti-D
 Immunogenicity of D antigen is by far the greatest.

26
 80% of D - (Rhesus negative) persons who received just one unit
of D+ (Rhesus positive) blood produced anti-D.
 Therefore, blood for all recipients and donors is routinely typed for
D status.
 D - (Rhesus negative) recipients can be identified and be given D-
(Rhesus negative) blood

Inheritance and Nomenclature of Rhesus antigens

 Genes are inherited inseparably or as haplotypes. They
codominant.
 Three systems of nomenclature have been used to express genetic
and serological information about Rhesus system.

A. System Notation
1. Rh-Hr was devised by Wiener who believed that the immediate
gene product is the agglutinogen. Each agglutinogen is made up
of several serological specificities called epitopes or factors.

2. CDE was devised by Fisher and Race who believed that there are
closely linked loci forming the trigene complex each of which
codes for an antigen.

27
3. Rosefield who based her theory on simple serological
observations postulated numerical designation. Symbols were
then used to facilitate the recording of data.

Equivalent Notations of the Rhesus system____________________

Numerical
Designation CDE Rh-Hr___
Rh1 D Rho
Rhw1 DU Kh0
Rh2 C rh'
Rh3 E rh''
Rh4 c hr'
Rh5 e hr''
Rh6 ce(f) hr
Rh7 Ce rhi
Rh8 Cw rhw1
B. Phenotype Notation
 In Wiener terminology single italic letters designated genes. Eg.
R for genes that do include Rho (D) and r for genes that do not
include Rho(D) with various superscript symbols (R1, R2, R0,
RZ, r', r'', r and ry ) to denote different alleles.
 Gene products were designated by Roman type Rh and rh with
various subscripts to indicate the haplotype of origin. Eg, R was
used for products including Rho (D):
- Rh0 = D
- Rh' = C
- Rh'' = E

28
- hr' = c
- hr'' = e
 shorthand notation phenotype employs single letters R and r for
genes producing or not producing D, respectively.
-R1 for C, D and e
-R2 for c, D and E
-r for c and e
-R0 for c, D and e
Determination of Rhesus Phenotypes from Five Principle Antiserums
Reactions with Anti-
Phenotypes
-D -C -E -c -e Rh-Hr CDE
+ + 0 + + R1r CcDe
+ + 0 0 + R1 CDe
+ + + + + R1R2 CcDEe
+ 0 0 + + R0 cDe
+ 0 + + + R2r cDEe
+ 0 + + 0 R2 cDE
+ + + 0 + RZR1 CDEe
+ + + 0 0 RZ CDE
0 0 0 + + r ce
0 + 0 + + r'r Cce
0 0 + + + r"r cEe
0 + + + + r'r'' CcEe

29
Phenotypes and Genotypes

 only five reagent antiserums are routinely available:

-anti-D
-anti-C
-anti-E
-anti-c
-anti-e

 routine pretransfusion testing include only D testing

 other antiserums are used only when resolving discrepancies
caused by antibodies or in family studies.
 assortment of Rhesus antigens on the person's red cells
constitutes his Rhesus phenotype
 based on the assortment of antigens, the probable genotype may
be constructed. Therefore, knowledge of gene frequencies in
single gene complex is very important.
 Knowledge of genotypes is important in:

1. population studies
2. investigation of disputed parentage

30
Unique problems of the D antigen

 To determine whether a person has one or two genes that

produce C and c or E and e is easy.
 Positive reactions for C and c or E and e mean the individual has
both genes.
 Positive reaction for D may mean the genotype could be Cde/cDE
or CDe/ cDE because d has not been serologically determined.
 Typing for D simply determines the presence or absence of the D
antigen.
 If D is absent, it can be assumed, correctly, that the person carries
two genes that do not code for D production. For example,
Cde/cde
 If D is present it not simple serologically, to test and establish
whether two genes are present that code for D antigen:
E.g. CDe/ CDe
Or
Cde/CDe

31
Position effect

 Cis effect involves the interaction of genes on the same

chromosome; e.g. weak E antigen is produced by cDE gene
compared to strong expression of E by cdE gene.
 Trans effect involves interaction of genes on the opposite
chromosome. Eg. Weak expression of C and E in a person who
has the genotype- CDe/cDE (R1R2) than when genotypes are
CDe/cde (R1r) or cDE/cde (R2r).

Homo-/heterozygous D expression
 Most D- (Rhesus negative) persons are homozygous for r gene
(rr)
 A gene that does not produce D but produces C or E is known as r'
and r'', respectively.
 A gene that produces both C and E but not D (ry) is very rare.
 It is difficult to establish the zygosity of D+ (Rhesus positive)
persons.
 Genotypes can be assigned by making references to frequencies
of individual gene complexes in populations.

Effects of race

 Most people with phenotype CDe are likely to have genotype

CDe/CDe (R1R1).

32
 r' is most infrequent in all population groups. Therefore, CDe
phenotype will be most unlikely to have the genotype CDe/Cde
(R1r1).
 Racial origin should be used in order to influence deduction on
genotypes. Eg:
a) A white person with cDe phenotype would most likely
be cDe/cde (R0r) because r is more common in whites
than R0 .
b) A black person with cDe phenotype would most
probably be cDe/cDe (R0R0) because R0 is common in
blacks.

Weak expressions D antigens (Du)

 Not all D+ cells react equally with anti-D blood grouping reagent.
 Most cells show clear-cut macroscopic agglutination and these can
be reliably classified as D+ (Rhesus positive).
 Some are not readily agglutinated by anti-D and cannot be
classified as D- (Rhesus negative). They may not be directly
agglutinated by anti-D.
 Additional tests are needed to demonstrate the presence of weak
D antigen.
 Weak reactivity with anti-D is usually designated DU.
1. Genetically transmissible DU

33
 Some Rhesus genes may code for weakly reactive D antigen.
 This characteristic is quantitative.
 These are common in blacks
 They are usually products of R0 (cDe) in blacks
 In whites it occurs as a product of R1(CDe) or R2 (cDE) genes
 This form of weak D is sometimes called " Low-grade DU'' which
gives a weak or negative agglutination with the majority of anti-D
reagents.
2. Position effect DU
 Weakened D antigen by ''C'' in trans position. Eg. weak D in
CDe/Cde (R1r') compared to strong D in CDe / cde (R1r).
 This is sometimes known as ''High grade DU'' or ''gene interaction
DU''.
3. D Mosaics
 Due to D antigens that lacks part or parts of its structure. It
qualitatively differs from normal D.
 D is made up of 4 subunits.:
RhA RhB
RhC RhD
 If one or more subunits is missing, the D antigen will be weak.
 If the Dmosaic person is given blood , he/she will produce antibodies
(anti-D) against the missing subunit on transfusued red cells. This
antibody will not react with the person's own D antigen.
 Targett (Rh40) is a good manifestation of D mosaicism.

34
Significance of DU in Donors and Recipients

1. DU donor blood
 DU donor blood is potentially capable of immunizing D negative
recipient :
- But 68 units transfused into 45 D- recipients did not cause anti-D
production. This is because DU is a very weak immunogen than
''normal'' D.

 In practice DU is labeled as Rhesus positive.

 There is accelerated destruction of DU donor cells in D- person
with anti-D already in his/her plasma.
 HDN has been reported in DU infants.

2. DU Recipients
 Most DU recipients are recorded as D- and will be safely given
Rhesus negative blood. This is considered wasteful.
 Some workers prefer to give D+ blood to recipients who are DU .
This may lead to careless and incorrect interpretation of tests for
D antigen.

35
Other Rhesus antigens
 The number of Rhesus antigens now exceeds 40.
 Cis product antigens or compound antigens are formed by
the interaction of genes on the same chromosome and are usually
inseparable.
 They do not involve the D antigen. E.g. R1 (CDe) = Ce, a cis
product that almost always accompanies C and e.
- RZr (CDE/cde) do not have Ce, but ce (f) and CE as compound
antigens.
 Antibodies to cis product antigens are useful in determining the
exact genotype.

Deletions
 Rare genes do exist that code for Rhesus material that lack
activities at Ee, Cc sites resulting in _De, cD_, _D_ or _ _ _
 Absence of Rhesus antigens other than D result in strong D
activity.
 Strong D is identified in the course of studying unexpected
antibodies . eg. anti-C, anti-c and anti-e.

G antigen

36
 It is present in all red cells possessing C or D antigens.
 Anti-G appears to be anti-C+D
 Anti-G can not be separated into anti-C and anti-D
 G appears to be an entity of C and D:
1. D- immunized to C- D+ cells may produce anti-C
and anti-D
2. D- persons exposed to C+D- cells may produce
antibodies that appear to contain anti-D component.
 G cells may lack D but show weak C expression (- - eG)
 D- G+ cells is found in blacks.
 C and c, and E and e are known as antithetical antigens,
respectively. Antithetical antigens are always all present or
one of them. There is never a situation when all are
absent.

LW ANTIGENS

 The antigen identified by animal antiserum of Landsteiner Wiener

was not the same or identical to that defined by the human
antiserum reported by Levine and Stetson.
 D+ cells gave a strong reaction with animal antiserum than D-
cells.

37
 Antigens identified on Rhesus monkey is present on all human red
cells although D- cells from adults give, comparably. weak
reactions.
 Cord cells in both D+ and D- persons react strongly.
 As the Rhesus terminology was strongly established LW was
suggested by Levine for the antigen found in monkeys and
characterized by animal antiserum in honor of Landsteiner and
Wiener.
 LW genes are found in chromosome 19.

LW phenotypes
 Rare persons exist who lack LW antigens although they have
normal activity of Rh with or without D antigen.
 These can form alloanti-LW.
 The notation LW1 and LW2 were initially adopted to describe,
respectively, strong reaction of anti-LW with D+ and weak
reactions with D- of adult cells. This was later found to be
incorrect.
 LW3 was adopted for those cells, which lacked LW altogether and
produced anti-LW.
 LW4 was adopted for LW negative proposita ( Mrs Big ) with anti-
LW that reacted with LW1, LW2 and LW3 :
-new born infant had weakly positive DAT
-Her serum contained anti-LW with a titer of 1/32000
against D+ cells and 1/400 against D- cells.

38
 Anti-LW has sometimes been identified in LW+ persons due to
transient LW- during the time of antibody formation. Normal LW+
returns as the antibody disappears.
Antithetical LW antigens
 In 1981 Nea was reported as a new blood group antigen found in
5% of Finish persons.
 Anti-Nea shows variations similar to those of anti-LW and relation
between Nea and LW was established.
 Anti-Nea had a strong reaction in D+ than D- red cells.
 Nea cord cells showed strong reaction with anti-Nea despite the D
status.
 11 unrelated LW3 persons showed that they were all Ne (a+).
 Studies have shown that LW and Ne are allelic gene products
 Tippet and Sistonen suggested to change the names of LW
antigens as follows:
1. LW (1-3)------------------------------------->LWa

2. Nea ------------------------------------------>LWb

3. Mrs Big's LW4 ----------------------------->LW (a- b-). Her antibodies

were thought to be anti-LWab

4. Red cells from transient LW- ---------->LW (a- b-)

39
 Rh null Syndrome and Rhmod
 43 persons in 14 families were found to be without Rhesus
antigens at all.
 These persons are referred to as Rhnull:

1). For the expression of Rhesus antigens regulator gene X1r is

required. Persons with X0r gene do not have Rhesus expression
X1r/X1r = strong Rhesus expression
X1r/X0r = weak Rhesus expression
X0r /X0r = no Rhesus expression

2). Homozygosity for an amorphic gene r results in Rhnull

phenotype characterized by absence of detectable products with Rh
antiserums.

 Red cells without Rhesus antigens tend to exhibit an abnormal

membrane, resulting in severe haemolysis. This condition is known
as hereditary stomatocytosis. Altered activities of S, s and U
may also lead to this disorder.

40
Rhmod
 A less complete suppression of Rhesus antigens may result in
Rhmod phenotype.
 This is due to lack of modifier gene XQ. The red cells do not
completely lack the Rhesus and LW antigens, but the activity is
reduced. Haemolysis is also present.

Antibodies to Rhesus antigens

 Most antibodies are due to immunization. But some, such as anti-
E and anti-CW may be naturally occurring.
 Anti-D is the most powerful antibody followed by anti-c and anti-
E
 Few examples of antibodies to Rhesus antigens give strong
reactions with saline suspended red cells.
 Most react best in high-protein antihuman globulin reagent (AHG
or DAT) or enzyme technique.
 Enzyme technique is important in the detection of weak or
developing antibodies to Rh ntigens.
 Detectable antibodies persist for many years.
 When levels fall, subsequent exposure may result in rapid
secondary immune response.
 Antibodies to Rhesus antigens are mostly of the IgG type.
 They cross the placenta and cause HDN.
 They may also cause HTR.
 They react best at 37 0C.

41
 OTHER BLOOD GROUPS
 In addition to ABO and Rhesus antigens, over 300 other antigens
have been detected on the human red cell surface. Some are
found on other body tissues.
 Antigens found on red cells of almost all persons are known as
public or high frequency antigens.
 Antigens in a few persons' red cells are known as private or low
frequency antigens.
 Some antigens were found by identification of corresponding
antibodies in the serum of patients.

42
1. THE MN and SsU BLOOD GROUP SYSTEM
M and N
 Discovered by Landsteiner and Levine in 1927 during
immunization studies by injecting human red cells into a rabbit.
 M and N behave as products of allelic genes.
-Red cells type as: M+ N-
M- N+
M+ N+
 The above phenotypes represent the homozygosity of M and N
respectively, and the heterozygosity for both.
 M and N antigens are carried on the sialoglycoproteins (SGPs),
Alpha SGP is glycophorin A that carries the two antigens.
 There are variant antigens of M which may cause discrepant M
and N typing: eg, Mg is a product of a rare gene at MN locus. It
does not react with anti-M or anti-N.
 Persons with genotype MgN will give reactions like M- N+. The
apparent genotype will be NN (not true).
 Persons with genotype MgM will give reactions such as M+ N- .
The apparent genotype will be MM ( false again)
 Anti-Mg occurs as a saline agglutinin. But Mg antigen is so rare
that the incidence of anti-Mg production is only 1-2%.

S,s and U antigens

43
 The S and s antigens are products of the allelic genes found at
locus that is closely related to MN locus. The gene complex that
produces N and s is more commonly found than that producing N
and S.
 A small proportion of black people are S-s- The S-s- cells are also
negative for U antigen which is usually a high frequency antigen.
 Only 16% of S-s- cells are U+.
 S, s and U antigens are found on the delta SGP with a small
segment that duplicates the alphaN SGP sequence causing the
presence of N-like antigen , "N" in almost all red cells regardless of
MN type.
 U negative persons lack delta SGP altogether.

Phenotypes and frequencies in MNSsU system

Reactions with anti-:

Ph'type Fre'ncy(%)

W B

M N S s U

+ 0 M+N- 28 26

+ + M+N+ 50 44

0 + M-N+ 22 30

44
 + 0 + S+s-U+ 11 3

+ + + S+s+U+ 44 28

0 + + S-s+U+ 45 69

0 0 0 S- s- U- 0 <1%

0 0 + S-s- U+ very rare

Antibodies to MNSsU system

a) Anti-M and Anti-N

 Anti-M is detected quite frequently in sera as saline agglutinin and

reacts best at room temperature.

 Most antibodies of this type are naturally occurring.

 Anti-M is predominantly of the IgM type but there are

examples that are wholly or partially of the IgG type

 Agglutination at room temperature with IgG is possible due

high density of M antigen on the red cells.

45
  Some anti-M may show strong reactivity at pH of 6.5

 Anti-M is rarely clinically significant because it reacts best at

room temperature.

 It is not detected using enzyme-treated red cells.

 IgG fraction of anti-M may cause HDN as well as HTR

 Anti-N is comparatively rare than anti-M.

 It is invariably of the IgM type

 May behave as a weakly reactive cold agglutinin.


Few persons of rare phenotype M+N-S-s-U- and M+N-S-s-

N+W have been found to have a powerful anti-N of the IgG

type.


Anti-N-like antibody has been found in haemodialyis patients


Anti-N has been associated with kidney- graft rejection.


Anti-N is not detected using enzyme treated red cells.


It is naturally occurring


Reacts best at room temperature.

b) Anti-S, -s and -U

46

Unlike anti-M and anti-N these antibodies are almost always

immune type.

They are usually of the IgG type

They are capable of causing HDN and HTR

They react best at 37 0C

Detected by using AHG reagent

Anti-S occurs about as infrequently as anti-N.

Anti-s is found less frequently than anti-S because:

a) s negative phenotype is less frequent than S-

b) s antigen is less immunogenic than S

 Anti-U is found less frequently than the other two because U is a

high infrequency antigen


Anti-U must be considered when serum from a previously

transfused or pregnant black patient contains an antibody to high

frequency antigen. Possible U- can be suspected if S-s- results are

obtained.


Antibodies to S, s and U are not detected using enzyme treated

red cells.

47
2. LUTHERAN BLOOD GROUP SYSTEM

First example of anti-Lua was found in 1945.

The main phenotypes of the Lutheran antigens are:

Lu ( a+ b- )

Lu ( a- b+ )

Lu ( a+ b+ )

Lu ( a- b- )

Lu ( a- b- ) is very rare that ; 1) may result from an amorphic

gene (lu) inherited from each parent ; 2) may arise from a

dominant trait inherited as an inhibitor gene ( In(lu)) which

prevents normal expression of Lutheran and other blood group

antigens such as Jka, Jkb, P1, i, and Augustine (Aua) and 3) due to

X-borne suppressor gene.

Associated Antigens

 High incidence antigens such as Lu4, LU5, Lu6, Lu7, Lu8, Lu11, Lu12

and Lu13 have been assigned to the Lutheran blood group system

because their antibodies do not react with Lu (a- b-) phenotype.

48
 Low incidence antigens such as Lu9 and Lu14 have been assigned

to the Lutheran blood group system because of their antithetical

relationship with high frequent antigens, Lu6 and Lu8, respectively.

 Lutheran genes are found on chromosome 19.

Antibodies

 They are rarely encountered in routine practice

 Anti-Lua and anti-Lub are mostly immune type. But there are also

naturally occurring fractions.

 They are mostly of the IgG type

 Anti-Lua does not cause HDN because the antigens are poorly

developed at birth.

 Anti-Lua has not been associated with HTRs.

 Anti-Lub has been associated with diminished survival of

transfused red cells (HTR) and HDN.

 Both are known to cause mixed-field agglutination

 Both are not detected by red cells treated with trypsin,

chymotrypsin and pronase .

 Some anti-Lutheran antibodies are of the IgA type. If the IgA is of

the monomer type it can potentially cause HDN.

49
 The antibodies react best in AHG and 37 0C .

Phenotypes and frequencies of the Lutheran blood group

system.
Reactions with Anti:

Phenotype Frequency

-Lua -Lub

+ 0 Lu (a+ b-) 0.15%

+ + Lu (a+ b+) 7.5%

0 + Lu (a- b+) 92 %

0 0 Lu (a- b- ) very rare

3. KELL BLOOD GROUPSYSTEM

 The Kell antigen was first demonstrated in 1946, through an

antibody that caused HDN.

 The gene (K) responsible for Kell antigens is present in 9% of

whites and 2% of blacks.

 An antithetical antigen k was confirmed to be present by

demonstrations using anti-Cellano which reacted with 99.9 % of

human red cells.

50
 The gene (k) is responsible for Cellano and is allelic to K gene

Other Antigens

 Other antithetical antigens of the Kell blood group system are:

-Kpa

-Kpb

-Jsa

-Jsb

 Kpa is antithetical to Kpb, while Jsa is antithetical to Jsb

 These anigens, along with K and k are produced by genes closely

linked at loci - like those of the Rhesus system.

 Ko phenotype represents absence of principle Kell antigens on the

red cell surface.

 Several high frequency antigens have been assigned to the Kell

blood group system because the identifying antibodies have been

found to not react with Ko red cells.

 K11 and K17 are antithetical antigens, with K11 being of high

frequency and K17 low frequency.

 A numerical nomenclature was proposed:

51
 Numerical Kell

K1 K

K2 k

K3 Kpa

K4 Kpb

K6 Jsa

K7 Jsb

 K5 was designated Ku antigen as identified by antibodies

produced by Ko persons.

 K antigen is extremely immunogenic, hence anti-K is frequently

found as a very strong antibody in transfused persons who lack

this antigen.

Phenotypes and frequencies in Kell blood group system

Reactions with anti-:

Frequency(%)

-K -k -Kpa -Kpb -Jsa -Jsb phe'type White Black

+ 0 K+ k- 0.2 rare

+ + K+ k+ 8.8 2

52
0 + K- k+ 91 98

+ 0 Kp(a+b-) rare 0

+ + Kp(a+b+) 2 rare

0 + Kp(a-b+) 98 100

+ 0 Js(a+b-) 0 1

+ + Js(a+b+) rare 19

0 + Js(a-b+) 100 80

0 0 0 0 0 0 K0 v. rare v. rare

___________________________________________________

The Kx antigen

 Kx seems to be the precursor of other Kell system antigens.

 It is determined by gene on the X chromosome known as X 1k

 Kx is normally present on granulocytes and fibroblasts

 Except on K0, Kx is found in trace amounts on red cells.

 Kx antigen is curiously associated with cellular function

 In the absence of X1k the granulocytes lack the Kx, resulting in

them failing to perform normal phagocytic functions. This defect is

known as Chronic Granulomatous Disease (CGD).

53
The McLeod phenotype

 Red cells without Kx have a shortened survival (haemolysis),

decreased H2O permeability, acanthocytic morphology and marked

depression of Kell antigens. These cell abnormalities are known as

McLeod phenotypes.

 Persons with McLeod phenotypes have poorly defined abnormal

neuromuscular system characterized by elevated creatine

phosphokinase (CPK) levels in serum. There is disordered

muscular functions in the elderly.

Sex-linked genes influencing the Kx expression

Gene Kx/GRAN Kx/RBC K/RBC CPK CGD

X 1k yes trace normal No No

X 2k No No d'ed in'ed Yes(II)

X 3k No trace normal No Yes(I)

X 4k Yes No d'ed in'ed No

___________________________________________________

54
Antibodies to Kell blood group system

a) Anti-K and Anti-k

1. Anti-K is frequently found in patients after transfusion.

 Has appeared as saline agglutinin in persons never exposed to K+

cells . This very rare.

 Most examples are immune type

 React best in AHG and 37 0C

 Some can bind complement

 Has caused HTR on numerous occasions- both delayed and

immediate.

 It may cause HDN since it is of the IgG type.

 Since 90% of donors are K- it is easy to find compatible donors for

patients with anti-K.

2. Anti-k has serological characteristics similar to those of anti-K

although it occurs much less frequently that anti-K because 1/500

(.2%) people lack k antigen.

 Since 99.98% are k+ it will be difficult to get compatible donrs.

b) Other antibodies to Kell blood group system

55
Anti-Kpa, -Kpb, -Jsa and -Jsb are much less common than anti-K

 They show similar serological behavior to anti-K

 They are considered clinically significant

 They are rare because the antigens are less immunogenic

 Generally patients with antibodies to high frequent antigens may

transfusion problems due to lack of compatible donors; we

depend on the use of RARE DONOR FILES.

 Anti-Ku in K0 persons may be a mixture of antibodies to other Kell

antigens. It reacts with red cells from all Kell phenotypes.

 Its behavior is similar to that of anti-Rh29 of Rhesus (total

Rhesus).

Rhnull vs Ko

4.DUFFY BLOOD GROUP SYSTEM

 A Pair of antigens, Fya and Fyb, are encoded by a pair of

codominant allelic genes.

 The third gene, Fy at the same locus has high frequency among

the blacks and produces no Fya or Fyb antigens

56
 blacks who are Fy (a- b-) are considered to have the FyFy

genotype.

 A fourth allele FyX encodes for a weakened Fyb:

- Fyb goes undetected in FyaFyX, FyFyX OR FyXFyX

- It is detected by using a powerful anti-Fyb OR use of absorption/

elution techniques.

 Duffy antigens are good receptors for some malaria parasites.

Hence, Fy(a- b-) persons are less susceptible to some malaria

infections ( plasmodium vivax).

 Genes for Duffy are found on chromosome 1

 Fya and Fyb antigens are carried on minor membrane glycoprotein.

 They are destroyed by enzyme treatment.

Antibodies to Duffy blood group system

 Anti-Fya is commonly encountered

 Anti-Fyb is less common

 Anti-Fya and anti-Fyb are of the IgG type.

 They can cause HDN and HTR

 Both react best in IAHG and at 370C

 Both are immune type

57
 Both are not detected using enzyme treated red cells

 Weak anti-Fya and anti-Fyb can give convincing reactions with

homozygous Fya and Fyb.

Rare antibodies

i) Anti-Fy3

 A Fy(a- b-) white person produces an anti-Fy3 which acts as a

combination of anti-Fya and anti-Fyb which is not separated by

absorption/elution technique.

 Reacts well with enzyme treated Fya+ and Fyb+ red cells.

 Fy3 is considered to be an enzyme resistant antigen found in Fy

positive , but absent in Fynegative red cells.

ii) Anti-Fy4

 Reacts strongly with red cells of Fy(a- b-) persons as well as

Fy(a+ b-) and some Fy(a- b+) from blacks, but not with Fy(a+

b+)

 Reacts with papain-treated red cells

58
 Fy4 is considered to be a product of Fy gene, which, in a

homozygous state gives rise to Fy(a- b-) in blacks.

iii) Anti-Fy5

 Is similar to anti-Fy3 except that it give negative reaction with

Rhnull cells as well as those of Fy(a- b-) in blacks.

 Reacts with Fy(a- b-) of the whites.

 Fy5 is thought to be a product of the interaction of the Rhesus and

Duffy genes.

Phenotypes and Frequencies of Duffy Blood Group System

Reactions with anti-:

Phenotype Frequency___

-Fya -Fyb W B

+ 0 Fy(a+ b-) 17 9

+ + Fy(a+ b+) 49 1

59
0 + Fy(a- b+) 34 22

0 0 Fy(a- b-) v. rare 68

_____________________________________________________

5.THE KIDD BLOOD GROUP SYSTEM

 It is the simplest blood group system so far that consist of two

antigens Jka and Jkb which are antithetical antigens

 Four phenotypes are defined by anti-Jka and anti-Jkb .

 Phenotype Jk(a- b-) is very rare and is found in people of Pacific

Islands.

 It results from silent gene Jk OR presence of an inhibitor gene

In(Lu).

Antibodies to Kidd antigens

 First recognized in 1951 as an anti-Jka associated with HDN

60
 Anti-Jkb was first recognized in 1953 in association with delayed

type HTR

 Both are immune type

 Both are of the IgG type

 Both react best in IAT and at 37 0C

 They are weak on first detection. May become undetectable after

storage due to Complement dependence.

 They show a dosage effect where they react very well with red

cells encoded by homozygous genes ( double dose of antigens).

 Anti-Jkb is notorious for causing delayed haemolytic transfusion

reaction (de-HTR)

 Jk(a- b-) have sera that react with Jka and Jkb cells, but not with

Jk(a- b-).

 Jk(a- b-) cells are resistant to lysis by 2M urea, where as Jk(a+ b-

), Jk(a- b+) and Jk(a+ b+) cells are rapidly destroyed.

 Locus for Jk genes is on chromosome 18.

Phenotypes and Frequencies of Kidd Blood Group System

Reactions with anti-:

P'type Frequency

61
-Jka -Jkb white black

+ 0 Jk(a+ b-) 28 57

+ + Jk(a+ b+) 49 34

0 + Jk(a- b+) 23 9

0 0 Jk(a- b-) exceedingly rare

______________________________________________________

6. SEX-LINKED BLOOD GROUP ANTIGEN: Xga

 In 1962, an antibody was discovered that reacted with red cell

antigens in women more than in men

 X- borne characteristics = Xga

 The antigen is destroyed by enzyme treatment of red cells.

 Anti-Xga reacts best in IAT and at 370C

 The antibody is of the IgG type

 Mostly of the immune type

 May cause HDN and HTR

 Some may agglutinate saline suspended red cells

 Anti-Xga may be used in tracing transmission of genetic triats.

Phenotype males females

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Xg (a+) 65.6% 88.7%

Xg (a-) 34.4 11.3 %

7.Ch and Rg

 Chido and Rogers antigens are part of the C4 molecule of the

human Complement proteins.

 It is not intrinsic to the red cell membrane.

 It behaves as if it resides on the C4d fragment

 Anti-Ch and anti-Rg are easily neutralized by serum and plasma

from person positive for the antigen.

Examples of High and Low Frequency Antigens Unrelated to

Principle Blood Group Systems

High frequency Low frequency

Augustine (Auta) 99.9% Bishop

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Gerbich (Ge) 99.9% Box

Gregory (Gya) 99.9% Hunt

Chido and Rogers 95.9% Wra

Sda

8. The Sda antigen

 A fairly high frequency antigen that is widely distributed in body

fluids and mammalian tissues.

 It may disappear during pregnancy

 It has a strong expression in Cad individuals

 Anti-Sda may cause mixed field agglutination

Additional Pairs of Antithetical Antigens

SYSTEM REACTIONS

With anti-: P'TYPE Fre'ncy(%)

64
Diego -Dia -Dib

+ 0 Di(a+ b-) O+

+ + Di(a+ b+) very rare

0 + Di(a- b+) 100

Cartright -Yta -Ytb

+ 0 Yt(a+ b-) 92

+ + Yt(a+ b+) 8

0 + Yt(a- b+) very rare

Dombrook -Doa -Dob

+ 0 Do(a+ b-) 17.2

+ + Do(a+ B+) 49.5

0 + Do(a- b+) 33.3

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Colton -Coa -Cob

+ 0 Co(a+ b-) 89.3

+ + Co(a+ b+) 10.4

0 + Co(a- b+) .3

Sciana -Sc1 -Sc2

+ 0 Sc:1, -2 99.7

+ + Sc: 1, 2 .3

0 + Sc: -1, 2 very rare

O+ stands for avery high frequency in Oriental populations and

American Indians. There are insufficient data to calculate

frequencies in Blacks.

Platelet antigens

 They are carried on a variety of structures on the platelet.

 Testing platelet antigens may be of little benefit, but :

66
 -may help as an additional test in HLA matching

-plt specific antibodies have been found in Post-

Transfusion Purpura (PTP) and Neonatal

Thrombocytopaenic Purpura (NATP)

platelet antigens-: Glycoprotein

Pi IIIa

Bak IIb alpha

Pen IIIa

Ko ?

Br Ia-IIa

PIE Ib alpha

PIT V

_________________________________________________

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 HLA TYPING

 HLA (human leukocyte antigens) antigens are gene products

found on the surfaces of all nucleated cells.

 They are glycoprotein in structure

 Cells with these antigens are : -lymphocytes

-granulocytes

-monocytes

-platelets

-nucleated red cells

 Mature non-nucleated red cells lack HLA antigens.

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 HLA system has been variously designated as; 1)

histocompatibility locus antigens, 2) human leukocyte antigens, 3)

transplantation antigens and 4) tissue antigens.

 HLA antigens are the second after ABO in importance in

influencing the survival of organ transplants.

 Immunological recognition of the differences in HLA antigens is

probably the first step in the rejection of transplanted tissue.

 HLA antigens are determined by genes present on the major

histocompatibility complex (MHC) of chromosome 6.

-HLA-A, -B, and C antigens are termed Class I

-HLA-D antigens (DP, DQ and DR) are termed class

II

The Human Leukocyte Antigens______________________________

Class I Class II

Antigens: HLA- A(27) HLA- DR(22)

- B(58) -DP(6 )

-C(10) -DQ(9)

________________________________________________________

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Distribution : plts, nucleated cells, B-lymphocytes,

Macrophages, T-

cells,

Macrophages,

monocytes

_______________________________________________________

Structure: large polypeptides, two peptide,

chains (MHC code) and alpha and beta,

alpha and beta2 micro- (both MHC code)

globulin.

________________________________________________________

Interaction with: CD 8 Lymphocytes CD4 , mixed

Lymphocyte

reaction

________________________________________________________

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 In 1950 several investigations revealed presence of

leukoagglutinins in transfused patients.

 It became necessary to do HLA typing before tissue

Transplantation

 Methods for typing HLA antigens are:

1) Serological tests; - for HLA-A, HLA-B and HLA-C

2) Two stage lymphocytotoxicity; -for HLA-A, HLA-B and HLA-C

3) Mixed lymphocyte culture(MLC);- for HLA-DR, HLA-DQ and HLA-

DP.

 These are used to assess compatibility of donor and recipient.

Typng Methods

1) STANDARD HLA CROSSMATCHING

Principle- based on the reaction of recipient serum that has

preformed leukoagglutinins with donor lymphocytes in the presence

of complement:

i. Lymphocytes + ab --co--> Cell lysis = INCOMPATIBLE

ii. Lymphocytes + ab --co--> No lysis = COMPATIBLE

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2) HLA CLASS I(ABC) ANTIGEN TYPING

Principle - The phenotyping of HLA Class I antigens is based upon

reacting lymphocytes isolated from the recipient and the antisera of

known specificity in the presence of complement. The endpoint is

lymphocytotoxicity ( cell lysis) which is detected by phase

microscopy:

i. T-lymphocyte + antiserum --co--> cell lysis = INCOMPATIBLE

OR

ii. T-lymphocytes + antiserum --co--> no lysis = COMPATIBLE

3) HLA CLASS II (DR, DQ, DP) ANTIGEN TYPING

Principle- detection of Class II antigens using purified forms of B-cell

preparations is based on antibody- specific complement dependent

disruption of cell membrane of the lymphocyte and the

demonstration of cell death by uptake of Trypan Blue dye.

i) B-lymphocyte + ab----co---> cell death

72
 Dead cells + dye ------->in'sed dye uptake = INCOMPATIBLE

OR

ii) B-lymphocytes + ab---co---> no cell death

Viable cells + dye ----------> no dye uptake = COMPATIBLE

3. MIXED LYMPHOCYTE CULTURE

Principle - cells (lymphocytes) from two genetically dissimilar

individuals are mixed. Recognition of disparate "D" antigen stimulates

DNA synthesis. The uptake of 3H-Thymidine is then quantified and

compared with that of three unrelated controls. The lower the

response, the better the correlation with graft survival.

APPLICATION OF HLA ANTIGEN TYPING

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1. For the selection of donor-recipient pairs for renal and BM

transplantation. BM transplantation requires a rigid HLA

matching to avoid acute graft rejection and graft vs host

disease.

2. To avoid febrile haemolytic transfusion reaction. If immunized

with leukocyte and platelet HLA antigens future platelet

concentrates should be HLA identical.

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