3. REVIEW OF LITERATURE -...

Review of Literature

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3. REVIEW OF LITERATURE

3.1 HUMAN BLOOD GROUPS INTRODUCTION, TERMINOLOGY AND FUNCTION

What is the definition of a blood group? Taken literally any variation or polymorphism detected in the blood could be considered a

blood group. However, the term blood group is usually restricted to blood cell surface

antigens and generally to red cell surface antigens.10

Definition of blood group: A blood group could be defined as ‘An inherited character of the red cell surface, detected

by a specific alloantibody’.11

Inherited variations in human red cell membrane proteins, glycoprotein and glycolipids,

are detected by alloantibody. Alloantibody is an antibody produced in one individual

against the red blood cell antigens of another individual of the some species.12

These alloantibodies occur either ‘naturally’ as a result of immunization by ubiquitous

antigens present in the environment or as a result of alloimmunization by human red cells,

usually introduced by blood transfusion or pregnancy. Although it is possible to defect

polymorphism in red cell surface proteins by other methods such as DNA sequence

analysis, such variations cannot be called blood groups unless they are defined by an

antibody.13


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Table 1: The blood group systems14

Discovery of the ABO blood groups first made blood transfusion feasible and disclosure

of the Rh antigens led to the understanding and subsequent prevention of hemolytic

disease of the newborn. Although A B O and Rh are the most important systems in

transfusion medicine, many other blood group antibodies are capable of causing

Hemolytic Transfusion Reactions (HTR) or Hemolytic Disease of New born (HDN).15

Thus blood group systems other than ABO and Rh systems are definitely important and

need our attention while practicing transfusion medicine.

Blood group terminology:16 The problem of providing a logical and universally agreed nomenclature has dogged blood

group serologists almost since the discovery of the ABO system.


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It is important to understand how blood groups are named and how they are categorized in

to system, collections and series.

The International Society of Blood Transfusion (ISBT) working party on terminology for

Red cell surface antigens was set up in 1980 to establish a uniform nomenclature that is

‘both eye and machine readable’. Part of the brief of the Working Party was to produce a

nomenclature in keeping with the genetic basic of blood groups’ and so a terminology

based primarily around the blood group systems was devised. First the systems and the

antigens they contained were numbered, then the high and low frequency antigens

received numbers and then in 1988, collections were introduced. Numbers are never

recycled, antigens that become part of a system or collection are given a new number and

their original number becomes obsolete.

Blood group antigens are categorized into 29 systems, five collections and two series.

The working party produced a monograph in 1995 to describe the terminology which has

been updated and has a website (http://www.iccbba.com/page 25. html). The ISBT

terminology provides a uniform nomenclature for blood groups that can be continuously

updated and is suitable for storage of information on computer databases.

Every authenticated blood group antigen is given a six digit identification number. The

first three digits represent the system (001-029), collection (205-210) or series (700 for

low frequency, 901 for high frequency), the second three digits identify the antigen. For

example, The Lutheran system is system 005 and Lua, the first antigen in that system has

the number 005001. Each system also has an alphabetical symbol; that for Lutheran is LU.

So Lua is also LU 001 or, because redundant sinistral zeros may be discarded, LUI.

For phenotypes, the system is followed by a colon and then by a list of antigens present

each separated by a comma. If an antigen is known to be absent, its number is preceded by

a minus sign. For example, Lu (a-b+) becomes LU : -1,2.

Genes have the system symbol followed by a space or asterisk followed in turn by the

antigen number representing the gens. For example, Lutheran gene Lua become LU1 or

LU*1.

http://www.iccbba.com/page%2025


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Genotypes have the symbol followed by a space or asterisk followed by the two alleles or

haplotypes separated by a stroke. For example Lua / Lub becomes LU ½ or LU* ½ and Lua

Lu6/Lub Lu9 would be Lu1, 6/2,9 or Lu*1,6/2,9. Genes and genotypes are always italicized

or underlined.

Blood Group Systems:16 A blood group system consists of one or more antigens. These are governed by a single

gene because or by a complex of two or more very closely linked homologous genes with

virtually no recombination occurring between them. Each system is genetically discrete

from every other blood group system. Any two systems may be shown to be different

either by demonstrating that the genes segregate at meiosis through the analysis of families

or by the gene loci being allocated to different chromosomes or to clearly distinct parts of

the same chromosome. New antigens should only be assigned to a system when it is

proven that the antigen is controlled by a gene at the blood group system locus.

In some system, the gene directly encodes the blood group determinant, whereas in others,

where the antigen is carbohydrate in nature, the gene encodes a transfer are enzyme that

catalyses biosynthesis of the antigen. A, B and H antigens, for example, may all be located

on the same macromolecule, yet H-glycosyl-transferase is produced by a gene on

chromosome 19 while A and B – transferase, which require H antigen as an acceptor

substrate are products of a gene on chromosome 9. Hence H belongs to a separate blood

group system from A and B. Regulator genes may affect expression of antigens from more

than one system: In(Lu) down- regulates expression of antigens from both Lutheran and P

systems, mutations in RHAG are responsible for Rhnull Phenotype, but may also cause

absence of U (MNS5) and Fy5 antigens. So absence of an antigen from cells of a null-

phonotype is never sufficient evidence for allocation to a system. Four systems consists of

more than one gene locus: MNS comprises three loci, Xg and Chido / Rodgers have two.

Collections:16 Collections were introduced into the terminology in 1988 to bring together genetically,

biochemically or serologically related set of antigens that could not at that time, achieve


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system status. For example, before setting up the Cromer collection the allelic antigens

TCa, TCb and TCc had the numbers 900020, 700035 and 700036, respectively, and WESa

and WESb had the numbers 700042 and 900033; yet all five reside on the same

macromolecule. Together with five other biochemically and serologically related antigens,

these antigens became the Cromer collection. Initially they could not become the Cromer

system as they had not been shown to be genetically distinct from all existing systems; that

required another couple of years.

Eleven collections have been created, six of which have subsequently been declared

obsolete : the Gerbich (201), Cromer (202) and Indian (203) Collections have become

systems; Auberger (204), Gregory (206) and Wright (211) have been incorporated into the

Lutheran, Dombrock and Diego systems respectively.

Table 2: Blood group collections.16

Low frequency antigens, the 700 series Red cell antigens that do not fit into any system or collection and have incidence of less

than 1% in most populations tested are given a 700 number.


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Table 3 Low frequency antigens: the 700 series.17

The 700 series currently consists of 22 antigens. Thirty two 700 numbers are now obsolete

as the corresponding antigens have found homes in systems, or can no longer be defined

because of a lack of reagents.


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Table 4: Frequencies of low frequency antigens.17

High frequency antigens, the 901 series.

Originally, antigens with a frequency greater than 99% were placed in a holding file called

the 900 series, equivalent to the 700 series for low frequency antigens. With the

establishment of the collections, so many of these 900 numbers became obsolete that the

whole series was abandoned and the remaining high frequency antigens were relocated in

a new series, the 901 series which now comprises 11 antigens.


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Table 5 High frequency antigens: the 901 series.18

Obsolete : 901004 Joa is now DO5;901006 Oka is OK1; 901007 JMH is JMH1; 901010 Wrb is DI4; 901011 is RAPH1.

Table 6 Frequencies of high frequency antigens in various populations.18


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Structures and Functions of Blood Group antigens:19 For the half century following Landsteiner’s discovery, human blood groups were

understood predominantly as patterns of inherited serological reactions. From the 1950s

some structural information was obtained through biochemical analyses, first of the

carbohydrate antigens and then of the proteins. In 1986, GYPA, the gene encoding the MN

antigens was cloned and this led into the molecular era of blood groups. A great deal is

now known about the structures of many blood group antigens, yet remarkably little is

known about their functions and most of what we know has been deduced from their

structures.

1. Membrane Transporters:

Membrane transporters facilitate the transfer of biologically important molecules in an out

of the cell. In the red cell they are polytopic, crossing the membrane several times, with

cytoplasmic N and C termini and are N-glycosylated on one of the external loops.

Band 3, the Diego blood group antigen is an anion transporter, the Kidd glycoprotein is a

urea transporter, and the Colton glycoprotein is a water channel. The Rh proteins and the

Rh-associated glycoprotein (RhAG) have structure characteristic of membrane transporter

(except the Rh proteins are not glycosylated). There is some evidence to suggest that the

Rh complex could be involved in ammonium transport.

2. Receptors and adhesion molecules:

The Duffy glycoprotein is polytopic, but has an extracellular N-terminus. It is a member of

the G protein coupled super family of receptors and might function as a receptor for

chemokines. The Lutheran glycoproteins, LW glycoprotein and CD 147, the OK

glycoprotein are members of the immunoglobulin super family (IgSF). The IgSF is a large

family of receptors and adhesion molecules with extra cellular domains containing

different numbers of repeating domains with sequence homology to immunoglobulin

domains. CD47 and CD 58 are also red cell IgSF glycoproteins, but do not express blood

group activity. The functions of these structures on red cells are not known, but their

primary functional activity may occur during erythropoiesis. Some other red cell surface

antigens with structures that suggest they could function as receptors and adhesion


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molecules are CD44, the Indian antigen, the Xg and CD99 glycoproteins, CDW/08, the

JmH blood antigen and CDW 75.

3. Complement regulatory glycoproteins: Red cell have at least three glycoproteins that exist, at least in part, to protect the cell from

destruction by autologous complement. Two, decay-accelerating factor (DAF, CD55), the

Cromer glycoprotein, complement receptor-1 (CR1, CD35), the Knops glycoprotein,

belong to the complement control protein super family. CD59, the most important of these

for protecting against autologous complement is not polymorphic and does not have blood

group activity. The major function of red cell CR1 is to bind and process C3b/C4b-coated

immune complexes and to transport to the liver and spleen for removal from the

circulation.

4. Enzymes: Two blood group glycoproteins have enzymatic activity. The Yt glycoprotein is

acetylcholinesterase, a vital enzyme in neurotransmission. The Kell glycoprotein is an

endopeptidase that can cleave a biologically inactive peptide to produce the active

vasoconstrictor, endothelin. The red cell function for both of these enzymes is unknown.

The structure of the Dombrock glycoprotein suggests that it belongs to a family of ADP-

ribosyltransferases.

5. Structural Components: The shape and integrity of the red cell is maintained by the membrane skeleton a network

of glycoproteins beneath the plasma membrane. At least two red cell membrane

glycoproteins have an extended cytoplasmic domain, which functions to link the

membrane with its skeleton. These proteins are band 3, the Diego antigen and glycophorin

C and its isoform glycophorin D, the Gerbich blood group antigens. Mutations in the

genes encoding these proteins can result in abnormally shaped red cells.

6. Components of the glycocalyx:

Band 3 and glycophorin A, the MN antigen are the two most abundant glycoproteins of

the red cell surface. The N-glycans of band 3, together with those of the glucose


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transporter, provide the majority of the red cell ABH antigens, which are also expressed

on some other glycoproteins and on glycolipids. The extra cellular domains of glycophorin

A and other glycophorin molecules are heavily O-glycosylated. Carbohydrate at the red

cell surface constitutes the glycocalyx, or cell coat, an extra cellular matrix of

carbohydrate that protects the cell from mechanical damage and microbial attack.


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3.2 BLOOD GROUP SYSTEMS (MAJOR & MINOR)

The ABO Blood Groups : (ISBT No. 001)

Introduction:20 The science of blood group serology materialized in 1900 with the discovery of the ABO

blood groups by Landsteiner. Together with the development of anticoagulants, it was this

discovery that made the practice of blood transfusion possible. Landsteiner mixed serum

and red cells from different individuals and found that in some tests the cells were

agglutinated (clumped) and in others they were not, demonstrating individual variation.

The mixing of serum, or at least antibodies with red cells followed by observation of the

presence of absence of agglutination is the basis for most methods for determining blood

group phonotype in use today. By 1910, the ABO blood groups had been shown to be

inherited characters, in the 1950s they were shown to represent oligosaccharide chains on

glycoproteins and glycolipids and in 1990 the gene encoding the enzymes responsible for

synthesis of the ABO antigens was cloned.

ABO is considered a blood group system because it was discovered on red cells and its

antigens are readily detected by haemagglutination techniques, on red cell. However, they

are also present in many different tissues and organs and in soluble form in secretions and

so are often referred to as histo-blood group antigens.

Because they are widely expressed, ABO antigens are a major consideration in solid organ

and bone marrow transplantation.21

ABO antigens, antibodies and inheritance:22

At its simplest, the ABO system consists of two antigens, A and B. the indirect products of

the A and B alleles of the ABO gene. A third allele, O produces no antigen and is recessive

to A and B. There are four phenotypes : A, B, AB and O.


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Table 7 ABO antigens, antibodies, and genotypes.22

Table 8 Routine ABO Grouping results and Phenotype frequencies.23

The A phenotype results from the genotypes A/A or A/O, B phenotype from B/B or B/O,

AB from A/B and O from O/O. Although many variations of the ABO phenotypes exist,

almost all are basically quantitative modifications of the A and B antigens.

Landsteiner’s rule states that individuals lacking A or B antigen from their red cells have

the corresponding antibody in their plasma. In this respect, ABO is unique among blood

group polymorphisms. Violations of Landsteiner’s rule in adults are rare.

A1 & A2 :

The A phenotype can be subdivided into A1 and A2. A1 is the more common phenotype in

all populations. A1 & A1, B red cells have a stronger expression of A antigen than A2 and

A2 B respectively. With most anti-A reagents, A1 red cells agglutinate faster, give a

stronger agglutinate and are agglutinated by higher dilutions of Anti A, than A2 cells.

Estimated numbers of antigens site per red cell can be summarized as follows:24

A1: 8-12 x 105, A2:1-4 x 105 ,A1B:5-9 x 105 ,A2B 1 x 105


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Table 9 Differentiating Characteristics of the A1 & A2 subgroups. 25

In addition to this quantitative dichotomy there is also a quantitative difference between

A1 & A2. About 2% of A2 and 25% of A2B individuals produce an antibody called anti A1,

that reacts with A1 & A1B cells, but not with A2 or A2B cells. The usual serological

interpretation of this is that both A1 & A2 cells have A antigen, but A1 cells have an

additional antigen called A1, absent fromA2 cells. Some lectins, which are sugar binding

proteins of non-immune and non-human origin, agglutinate red cells. Appropriately

diluted lection from the seeds of Dolichos biflorus, an Indian legume is a very effective

anti A1 reagent, agglutinating A1 & A1B cells, but not A2 or A2B cells.

Table 10 Serologic differentiation of the ABO Groups.25

Antigen, Phenotype and gene frequencies:26 The four phenotypes – A,B,O and AB- are present in most populations, but their

frequencies differ substantially throughout the world. Populations with a group O

phenotype frequency greater than 60% are found in native people of the Americas and in


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parts of Africa and Australia, but not in most of Europe or Asia. Some native people of

South and Central America are virtually all group O and probably were entirely so before

the arrival of Europeans. The frequency of A is quite high (40-60%) in Europe, especially

in Scandinavia and parts of Central Europe. Relatively high A frequency is also found in

the Aborigines of South Australia (upto 77%) and in certain Native American tribes where

the frequency reaches 50%. A2 is found mainly in Europe, the near East, and Africa, but is

either very rare or absent from indigenous populations throughout the rest of the world.

High frequencies of B are found in Central Asia (about 40%). In Europe, B frequency

varies from between 8% and 12%.

ABO antibodies: Anti A and Anti B are almost invariably present when the corresponding antigen is absent.

With the exception of new born infants, deviations from this rule are rare. Missing

antibodies may indicate a weak subgroup of A or B, chimerism,

hypogammaglobulinaemia, leukemia and lymphoma or occasionally old age. ABO

antibodies detected in the sera of neonates are usually IgG and maternal in origin, but are

rarely IgM and produced by the fetus. Generally, ABO agglutinins are first detected at an

age of about 3 months and continue to increase in titer, reaching adult levels between 5

and 10 years. Although ABO antibodies are often referred to as ‘naturally occurring’, they

probable appear in children as a result of immunization by A and B substances present in

the environment.

Changes in the characteristics of anti A or B occur as a result of further immunization by

pregnancy or by artificial means, such as incompatible transfusion of red cells or other

blood products. Typical serologically detectable changes are increase in titer and avidity of

the agglutinin, increase in haemolytic activity and greater activity at 370 C.

Anti –A and –B molecules may be IgM, IgG or IgA; some sera may contain all three

classes. Anti –A and –B of non-stimulated individuals are predominantly IgM, although

IgG and IgA may be present. IgG2 and IgG1 anti –A and –B are predominant, with IgG3

and IgG4 playing minor role.26 It is interesting that ABO titers have progressively

depressed over the past two decades with increasing consumption of pasteurized,

commercially packaged foods that are relatively sterile. This trend may be reversed with


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increasing use of probiotic nutritional supplements that contain live bacteria. The latter

have shown to stimulate ABO antibodies, with marked increases in ABO titers within

several months.27

The importance of the ABO system to transfusion and transplantation

medicine:28

ABO is the most important blood group system in transfusion medicine, because

transfusion of ABO incompatible red cells will almost always result in symptoms of a

hemolytic transfusion reaction (HTR) and may cause disseminated intravascular

coagulation, renal failure and death.

Two types of ABO incompatibility can be distinguished:

• Major incompatibility, where antibodies in the recipient will destroy transfused red

cells (eg. A to O, B to O, A to B, B to A).

• Minor incompatibility, where antibodies in the donated blood will destroy the

recipient’s red cells (eg. O to A or O to B).

Major incompatibility in blood transfusion must be avoided. Although minor

incompatibility can usually be disregarded when the donor does not have exceptionally

high levels of ABO antibodies, whenever possible donor blood do the same ABO group of

that of the patient should be used for transfusion. Signs of red cell destruction may be

apparent following transfusion of group O whole blood or in exceptional circumstances,

packed red cells to recipients of other ABO groups. This is the result of destruction of the

patient’s red cells by transfused ABO antibodies. Anti –A1 is rarely clinically significant

and most examples are not active above 250C, though there are few reports of HTRs

caused by anti-A1.

IgG anti-A, -B and –A,B are all capable of causing hemolytic disease of the fetus new

born (HDFN), though HDFN caused by ABO antibodies is uncommon and almost only

occurs in A1, B, or A1B babies of group O mothers. Despite the presence of IgG ABO


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antibodies in the serum of most group O women, severe ABO HDFN is rare for two main

reasons;

1) Fetal red cells have a relatively low density of A and B antigens.

2) Soluble A and B substances are present in fetal plasma and other body fluids and can

neutralize maternal antibodies. The complement deficiency of fetal plasma may also

play a part in the rarity of ABO HDFN.

ABO antibodies cause rejection of incompatible kidney, liver and heart transplants, but

can usually be disregarded for tissue transplants, including cornea, skin and bone.

Haemopoietic stem cells do not express ABO antigens, so ABO is often disregarded when

selecting a stem cell donor. However, major ABO incompatibility may lead to haemolysis

of infused red cells with a bone morrow transplant and can give rise to pure red cell

aplasia and delayed red cell chimerism in non- myeloablative stem cell transplants.

The appearance of apparent auto anti-A or –B following transplantation of minor

incompatible solid organs (eg. O organ to A recipient) results from the presence of

lymphoreticular tissue transplanted with the organ. Typically these antibodies are IgG,

appear 7-10 days after transplantation and last for about one month. They are often

responsible for haemolysis and have caused acute renal failure and even death.

Haemolysis induced by antibodies of graft origin may also be a complication of minor

ABO incompatibility in stem cell transplantation, especially in patients treated with

cyclosporine for prophylaxis against graft – versus – host disease.

The biochemical nature of the ABO antigens:28 A and B antigens are oligosaccharides. The most abundant structures on red cell carrying

ABO activity are the N-linked oligosaccharides of red cell surface glycoprotein,

predominantly the red cell anion exchanges (AE1, the Diego blood group antigen or band

3) and the glucose transporter (GLUT1), although some other glycoprotein are also

involved. ABO-active oligosaccharides are also present on glycolipids.

Oligosaccharides are chains of monosaccharide sugars : D-Glucose (Glc); D-galactose

(Gal); D-mannose (Man); N-acetyl-D-glucosamine (GlcNAc); N-acetyl-D-galactosamine

(GalNAc); L-fucose (Fuc).


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Fig 1 Diagrammatic representation of A- and B-active oligosaccharides, plus the H-active oligosaccharide, the precursor of A and B. R: remainder of molecule.

An oligosaccharides is A-active when the terminal monosaccharide is GalNAc, in α13

linkage to a Gal residue that also has Fuc in α12 linkage, whereas an oligosaccharides is

B-active when the terminal monosaccharide is Gal, in α13 linkage to the α,1,2-

fucosylated Gal residue. GalNAc and Gal are the immunodominant sugar of A and B

antigens, respectively. Group O red cells lock both GalNAc and Gal from the α1,2 –

fucosylated Gal residue, so express neither A nor B. The A and B trisaccharides may be

attached to several different core oligosaccharides chains, but in red cells the fucosylated

Gal residue is usually in α14 linkage to GlcNAc. This is called a type 2 core structure,

less abundant core structures, called type 3 and 4, are only present on glycolipids and may

also be involved in A & B activity. Type 3 and type 4 structures express A antigen on A1

phenotype red cells, but not on A2 cells, which may account for the qualitative differences

between A1 and A2. ABH antigens expressed on RBC glycoprotein and most

glycosphingolipids (type 2,3 and 4 chains) are of RBC origin. In contrast, type 1 chain

ABO antigens are synthesized by gastrointestinal mucosa, secreted into plasma, and

passively adsorbed onto red cell membrane. Synthesis of type 1 chain ABO antigens is

linked to the Lewis blood group system.29


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Biosynthesis of the ABO antigens and ABO molecular genetics: Oligosaccharides are built up by the stepwise addition of monosaccharide. The addition of

each monosaccharide requires a specific transferase an enzymes that catalyses the transfer

of the monosaccharide from its donor substrate, a nucleotide molecule carrying the

relevant monosaccharide, to its acceptor substrate, the non-reducing end of the growing

oligosaccharide chain.

The first step in the synthesis of ABH antigens is the synthesis of the H or group O

antigens, the immediate biosynthetic precursor of both A and B antigens. The H antigen is

formed by the addition of fucose (FUC), in an α1-2 linkage, to a terminal galactose. This

reaction is catalyzed by two different enzymes, depending on whether the fucose is being

added to a type 1 or type 2 chain oligosaccharide acceptor. Fucosyl transferase type 1

(FUT1), the product of the H or FUT1 gene, catalyzes the formation of type 2 chain H

antigen. In contrast, fucosyltransferase type 2 (FUT2), the product of the secretor gene,

catalyzes the transfer of fucose to type 1 chain precursors to form type 1 chain H or Led

antigen. Inactivating mutations in FUT1 are responsible for the Bombay and Para Bombay

phenotypes. Bombay and Para Bombay nonsecretors also have inactivating mutations in

FUT2.

Once H antigen is formed, it can serve as a substrate for A gene and B gene

glycosyltransferase.29

Fig 2 Biosynthetic pathways for production of A and B antigens from their precursor (H).


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A-transferase, the product of the A allele, is a GalNAc-transferase, which catalyses the

transfer of GalNAc from UDP-GalNAc(donor) to the fucosylated Gal residue(acceptor).

B-transferase, the product of the B-allele, is a Gal-transferase, which catalyses the transfer

of Gal from UDP-Gal to the fucosylated Gal residue of the acceptor. The O allele

produces no active enzyme, and so the fucosylated Gal residue remains unsubstituted(and

express H antigen).

Biochemically the A and B antigens are very similar, differing only by the presence of an

N-acetyl group. It is fascinating that such a minor chemical modification should have such

profound immunologic consequences. Removal of the N-acetyl group on A antigen by

circulating deacatylase enzymes is responsible for the acquired B phenotype.29

The genetic basis for oligosaccharide blood groups is fundamentally different from that of

the protein blood groups. Protein antigens are encoded directly by the blood genes, but the

genes governing carbohydrate polymorphism encode the transferase enzymes that catalyze

the biosynthesis of the blood group antigens. FUT1 (H gene) and FUT2 (Se gene) are

located on chromosome 19q13.3 and reflect a gene duplication (Lowe 1994). FUT1 is a

365 amino acid, type II transmembrane glycoprotein, composed of a large, 240 amino

acid, carboxy – terminal catalytic domain, which is anchored within the Golgi lumen by a

short transmembrane and a cytosolic domain. More than 20 mutant FUT1 alleles have

been described.29

The ABO gene locus is located on chromosome 9q34 and encodes the A and B

glycosyltransferases. The gene is large spanning 18 Kb, and contains seven exons,

although exons 6 and 7 encode the majority of the active enzyme. The product of the ABO

gene is a 41-kD, 353 amino acid type II transmembrane glycoprotein, Comparison of A

and B enzymes shows nearly 98% identity, differing by four key amino acids at residues

176, 235, 266 and 268. Amino acid 268 is absolutely critical in determining the activity

and substrate specifically (UDP-GalNAc vs UDP-Gal) of the enzyme. Substrate

specifically is also influenced by amino acid residues 235 and 266. The polymorphism at

residue 176 is not biologically significant. To date, more than 200 ABO alleles have been

identified.30


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Table 11 Key Amino Acids in distinguishing A,B, and Hybrid Glycosyltransferase.30

The cloning and sequencing of the ABO gene locus have also uncovered the molecular

basis of group O and ABO subtypes. Two deletion mutants (O1, O1 var) are the most

common and the ancestral genes for most other group O alleles.

O1 has same sequence as A1, apart from a deletion of a single nucleotide in the part of the

gene encoding the stem of the enzyme. This disrupts the three nucleotide amino acid code

and also introduces a code for termination of translation of the mRNA. Consequently the

O1 allele encodes a truncated protein, which could have no enzyme activity.

O2 has a single amino acid change at position 268, which appears to disrupt the catalytic

site and results in either no enzyme activity or possibly trace A-transferase activity.

It is interesting to note that the O2 allele (O03), found in many Europeans, contains a single

missense mutation at amino acid residue 268. O03 and a related allele (Aw08) have been

linked to ABO typing discrepancies by an absence of anti-A and/or anti-B in these

individuals. It has been suggested that the absence of anti-A or anti-B is the result of weak

residual enzyme activity; however this has not been confirmed.


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Fig 3 Model of glycosyltransferase products of five alleles of ABO, showing the positions of the amino

acid substitutions that are most important in determining the specificity of the enzymes.

Several ABO subtypes are also the result of mutations at the ABO gene locus, including

single – nucleotide polymorphisms (SNPs) and nonsense, frame shift and translation-

initiator mutations. For example, the A2 and Ael phenotype are associated with a single

nucleotide deletion (nucleotide 1060) and frameshift, resulting in the loss of a stop codon

and synthesis of a longer A enzyme with decreased activity. Single point mutations appear

to be responsible for the decreased enzyme activity of A3, AX, Afinn, Aend, B3, BX and BV

alleles. In contrast, the cis –AB and B(A) alleles, which can synthesize both A and B

antigens are molecular chimeras with characteristics of both A1 and B gene consensus

alleles.

H, the precursor of A and B.31 An antigen called H is present on the red cells of almost everybody, but is expressed much

more strongly on group O and A2 cells than on A1 and B cells. H is the biosynthetic

precursor of A and B; the fucosylated Gal structure that is converted to the A-active

trisaccharide by addition of GalNAc or to the B-active trisaccharide by addition of Gal. In

group O individuals that H antigen remains unconverted and is expressed strongly. In A1

and B individuals most of the H antigens are converted to A or B structures, but in A2

individuals in which the A- transferase is less efficient than in A1, many H-active

structures remain.


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The ultimate enzyme in the biosynthesis of H on red cells is a fucosyltransferase (FucT1),

which catalyses the fucosylation of the Gal residue of the H precursor.

Fig 4 Biosynthetic pathway for the production of H, the precursor of A and B.

FUT1, the gene encoding this fucosyltransferase is genetically independent of ABO;

FUT1 is on chromosome 19, ABO is on chromosome 9. ABO and H are therefore,

separate blood group systems. Very rare phenotypes exist in which homozygosity for

mutations in FUT1 results in no H being present on the red cells and consequently no A or

B antigens are expressed on the red cells regardless of ABO genotype.

ABH secretion:14 In addition to their presence on red cells A, B and H antigens are widely distributed in the

body and in most people are present as soluble glycoprotein in body secretions. A genetic

polymorphism determines whether H antigen and consequently A and B antigens are

present in secretions. About 80% of Caucasians are ABH secretors; their secretions

contain A plus a little H if they are group A, B plus some H if they are group B and an

abundance of H if they are group O. Secretions of ABH non-secretors, who represent

about 20% of the population, contain no H and consequently neither A nor B.


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Secretor phenotypes are usually determined by detecting ABH substances in saliva by

haemagglutination inhibition techniques.

Table 12 Haemagglutination-inhibition test to determine ABH secretor status from

saliva.31

Boiled saliva is mixed with anti-A, -B and –H reagent and group A, B and O red cells,

respectively are added to the mixtures. Lectin from the seeds of common gorse, Ulex

europaeus, is generally used as an anti-H reagent. In secretors, the soluble blood group

substance will bind to this antibody or lectin and block agglutination of the appropriate

indicator cell. In non-secretors, the antibodies are not blocked and agglutination occurs.

The enzyme responsible for synthesis of H antigen on red cells is a fucosyltransferase

produced by the FUT1 gene. This gene is active in mesodermally derived tissues, which

includes the haemopoietic tissue responsible for production of red cells. FUT1 is not

active in endodermally derived tissues, which are responsible for body secretions such as

saliva. Another gene FUT2, which produces a fucosyltransferase (FucT2) very similar to

that produced by FUT1, is active in endodermally derived tissues, so FUT2 controls

secretion of H. Homozygosity for inactivating mutations in FUT2 is responsible for the

non-secretor phenotype. Such inactivating mutations are common in FUT2. The most

common Caucasian FUT2 inactivating mutation converts the codon for tryptophan – 143

to a translation stop codon. In group A and B non-secretors, the A and B genes are active

in the endodermal tissues and produce active transferases in the secretions, but these

enzymes are unable to catalyze the synthesis of A and B antigens in the secretions because

their acceptor substrate, the H antigen, is absent.


28

H-deficient red cells:31 Phenotypes with H-deficient red cells are rare. Homozygosity for inactivating mutations in

FUT1, the gene encoding the fucosyltransferase responsible for biosynthesis of H on red

cells, results in red cells with no H antigen. As H-deficient red cells look the precursor of

A and B, they are always group O. If A or B genes are present, active A- or B- transferases

will be present, but unable to produce A or B antigens in the absence of their acceptor

substrate, the H antigen.

Red cell H-deficient individuals may be ABH secretors or non-secretors. Red cell H-

efficient non-secretors (the Bombay phenotype) produce anti-H, plus anti-A and –B. Anti

H is clinically significant and has the potential to cause severe HTRs and HDFN.

Consequently, anti –H can cause a serious transfusion problem because H-deficient

phenotypes are rare and compatible blood is very difficult to find. Secretors with H-

deficient red cells have H antigen in their secretions, but not on their red cells. They do not

produce anti-H, but may make a related antibody called anti-HI, which is not usually

active at 370C and not generally considered clinically significant. Anti-HI is also quite a

common antibody in individuals with A1 phenotype.

Further Complexities:31 Many rare subgroups of A and B exist, in which there are different degrees of weakness of

the A or B antigens. In some cases there is unexpected secretion of A or B substance. The

most commonly used names of A subgroup phenotypes are A3, Aend, AX, Am, Ay and Ael.

They are defined by characteristic serological patterns and for most of the A subgroups

one or more mutations in the ABO gene has been identified, but there is not always a

straight forward correlation between genotype and phenotype.

Subgroups of B-B3, Bx, Bm and Bel- are serologically analogous to the subgroups of A.

They are extremely rare.

On very rare occasions, the ABO groups appear to break the rules of Mendelian

inheritance, with for example, group O parent of a child with a weak A phenotype (Ax). In


29

one such family the father had the genotype A/O1, with an A allele producing so little A

antigen that it is not detected by standard serological methods, but the child had the

genotype A/O2 in which the expression of the same A allele appears to have been

enhanced by the O2 allele in trans.

The phenotype called cisAB is very rare, but particularly interesting. Red cells with the

cisAB phenotype are group AB, although both A and B are usually expressed somewhat

weakly. The interesting point is that in cisAB both A and B are inherited from the same

parent and can be passed on to the same child. The reason is that cisAB represents a single

allele at the ABO locus that encodes a single transferase enzyme with dual A- and B-

transferase activity. The Cis AB- transferase has leucine at position 266, typical of A-

transferase, but alanine at position 268, typical of B- transferase.

Acquired Changes:31 On rare occasions group A people may acquire B antigen and become group AB, although

the B antigen is generally weak and there is some weakening of the A antigen. In most

cases this phenomenon occurs in patients with diseases of the digestive treat, usually colon

carcinoma. The usual explanation for acquired B is that the bacterial enzymes in the blood

remove the acetyl group foam GalNAc, the immunodominant sugar of A antigen, to

produce galactosomine, which is similar enough in structure to Gal, the immunodominant

sugar of B antigen, to cross react with some anti - B.

Weakening of A antigen is common in group A patients with acute myeloid leukemia

(AML). In some cases all red cells show weakness of the A antigen, whereas in others two

populations of A and O red cells are apparent. Leukemia associated changes in B and H

antigens are less common. Between 17% and 37% of patients with leukemia have a

significantly lower A, B or H antigenic expression compared with healthy controls.

Occasionally modifications of ABH antigens are manifested before diagnosis of

malignancy and therefore indicate preleukemia states.

A or B antigens on red cells can be abolished, in vitro, by converting them back to H

antigen by removal of the inmunodominant sugar with an appropriate enzyme, an

exoglycosidase : α – N- acetylgalactosaminidase (A-zyme) or α - galactosidase (B-zyme).


30

Such enzymes are obtained from a variety of sources such as Chryseobacterium (A-zyme)

and green coffee beans (B-zyme). The long term aim of this research is to produce

universal group O donor red cells from blood of all ABO groups.

Biological Role:32 The biological role of ABH antigens is still not known. Multiple studies have linked

specific ABO types with a higher incidence of many disease, including autoimmune,

neoplastic and infectious disorders. Depression of A and B antigen expression can occur in

malignancy and is often associated with increased metastatic potential. Malaria has been

shown to bind A and B antigens with rosette formation, a possible risk factor in cerebral

malaria. Genotyping studies suggest a “parent of origin effect” between severe malaria and

inheritance of a functional maternal ABO allele. A and B antigen expression may also

stabilize the clustering and spatial organization of sialoglycoproteins, which also serve as

malaria receptors.

Numerous other associations between ABO and diseases have been reported, mostly based

on observed ABO phenotype frequency discrepancies between patients with the disease

and the healthy population. For example, group A people appear to be more susceptible

than those of other ABO groups to carcinoma of the stomach and colon, group O people

appear to be more susceptible to gastric and duodenal users and group B are more likely to

have Streptococcus pneumoniae and Escherichia coli infections.

Almost nothing is known about the functions of ABO antigens, on red cells or elsewhere

in the body. ABH antigens are very abundant on red cells. The ABH antigens contribute to

the glycocalyx or cell coat, an extra cellular matrix of carbohydrate that protects the cell

from mechanical damage and attack by pathogenic micro-organisms.

It is probable that the A/B polymorphism is at least 13 million years old and has almost

certainly been maintained by selection. The nature of the selection pressures involved,

however, remains a mystery.33


31

MNS (ISBT No.002) BLOOD GROUP SYSTEM

Following the discovery of the ABO blood group system, Landsteiner and Levine began

immunizing rabbits with human RBCs, hoping to find new antigen specificities. Among

the antibodies recovered from these rabbit sera were anti-M and anti-N both of which were

reported in1927. Data from family studies suggested that M and N were antithetical

antigens. In 1947, Walsh and Montgomery discovered S, a distinct antigen that appeared

to be genetically linked to M and N. Its antithetical partner s, was discovered in 1951.

Family studies (and later, molecular of genetics) demonstrated the close linkage between

the genes controlling M, N, S and s antigens.34

The frequencies of the common M N and S s phenotypes are listed in following Table 13.

Table - 13 Phenotypes of the MNSs System.35

There is a disequilibrium in the expression of S and s with M and N. In whites, the

common haplotypes were calculated to appear in the following order of relative frequency:

Ns > Ms > MS > NS. In 1953, an antibody to a high incidence antigen U, was reported by

Weiner. The observation by Greenwalt et al that all U-RBCs were also S-s- resulted in the

inclusion of U into the system. Forty-three antigens have been included in the MNS

system, making it almost equal to Rh in size and complexity. Most of these antigens are of

low incidence and were discovered in cases of HDN or incompatible cross match. Others

are high incidence antigens. Antibodies to these low and high incidence antigens are not

commonly encountered in the blood bank.36


32

Table 14 Summary of MNS antigens.36


33

Basic concepts:36

M and N Antigens

The M and N antigens are found on a well characterized glycoprotein called glycophorin

A (GPA), the major RBC sialic acid-rich glycoprotein (sialoglycoprotein, SGP). The

antigens are defined by the first and fifth amino acids on this structure, but antibody

reactivity may also be dependent on adjacent carbohydrate chains, which are rich in sialic

acid.

Fig. 5 Structure of Glycophorin A(GYPA) and B(GYPB)

There are about 106 copies of GPA per RBC. The antigens are well developed at birth.

Because M and N are located at the outer end of GPA, they are easily destroyed or

removed by the routine blood bank enzymes, ficin, papain and bromelin and by the less

common enzymes trypsin and pronase. The antigens are also destroyed by ZZAP, a

solution of dithiothreitol (DTT) and papain or ficin, but they are not affected by DTT

alone, AET,α - chymotrypsin, chloroquine or glycine - acid - EDTA treatment. Treating

RBCS with neuraminidase, which cleaves sialic acid (also known as neuraminic acid or

NeuNAC), abolishes reactivity with only some examples of antibody. M and N antibodies


34

are heterogeneous; some may recognize only specific amino acids, but others recognize

both amino acid and carbohydrate chains.

M and N have not been detected on lymphocytes, monocytes or granulocytes by

immunofluorence flow cytometry, nor have they been detected on platelets. GPA and M

and N have been detected on renal capillary endothelium and epithelium.

S and s Antigens:

S and s antigens are located on a smaller glycoprotein called GPB that is very similar to

GPA. S and s are differentiated by the amino acids at position 29 on GPB. S has

methionine, whereas s has threonine. The epitope may also include the amino acid

residues at position 34 and 35 and the carbohydrate chain attached to threonine at position

25. There are fewer copies (about 1.7 - 2.4 x 105) of GPB than GPA per RBC. In addition,

there is about 1.5 times more GPB produced by the S gene than by the s gene. S and s also

are well developed at birth.

S and s antigens are less easily degraded by enzymes because the antigens are located

farther down the glycoprotein and enzyme - sensitive sites are less accessible. Ficin,

papain, bromelin, pronase and chymotrypsin can destroy S and s activity, but the amount

of degradation may depend on the strength of the enzyme solution, length of treatment,

and enzyme to call ratio. Trypsin does not destroy the S and s antigens; neither does DTT,

AET, Chloroquine nor glycine-acid-EDTA treatment. Like M and N, S and s are

considered RBC antigens; they are not found on platelets, lymphocytes, monocytes and

granulocytes.

Anti -M :

Many examples of anti-M are naturally occurring saline agglutinins that react blow 37oC.

50 to 80 percent of anti-M are IgG or have on IgG component. They usually do not bind

complement, regardless of their immunoglobulin class, and they do not react with enzyme

treated RBCs. The frequency of saline reactive anti-M in routine blood donors is 1 in 2500

to 5000. It appears to be more common in children than in adults and is particularly


35

common in patients with burns. Because of the antigen dosage, many examples of anti-M

may react better with M+N - RBCs (genotype MM) than with M+N+ RBCs, (genotype

MN). Very weak anti - M may not react with M+N+ RBCs at all, making antibody

identification difficult. Antibody reactivity can be enhanced by increasing the serum - to -

cell ratio or incubation time, or both, by decreasing incubation temperature or by adding a

potentiating medium such as albumin, low ionic strength solution (LISS) or poly ethylene

glycol (PEG).

Some examples of anti - M are pH - dependent, reacting best at pH 6.5. These antibodies

may be detected in plasma, which is slightly acidic from the anticoagulant but not in

unacidified serum. Other example of anti - M react only with RBCs exposed to glucose

solutions. Such antibodies react with M+ reagent RBCs or donor RBCs stored in

preservative solutions containing glucose but do not react with freshly collected M+

RBCs. The significance of both pH dependent and glucose - dependent antibodies in

transfusion is questionable.

As long as anti - M does not react at 37oC, it is not clinically significant in transfusion. It is

sufficient to provide units that are cross match compatible at 37oC and the antiglobulin

phase without typing for M antigen. Sometimes compatible units carry the M antigen; for

example, M+N+ RBCs, which do not react with weak anti-M. Only rarely do such units

stimulate a change in the antibody’s thermal range. Anti-M rarely causes hemolytic

transfusion reactions, decreased cell survival, or HDN. However, when 37oC - reactive

IgG anti - M is found in pregnant woman, the physician should be forewarned, some HDN

cases have been severe.

Anti N:

The serologic characteristics as of the common anti - N (made by individuals whose RBCs

type M+N - and S + or s+) are similar to those of anti-M; a cold reactive IgM or IgG saline

agglutinin that does not bind complement or react with enzyme-treated RBCs. Anti N can

demonstrate dosage, reacting, better with M-N+ (NN) RBCs than with M+N+ (MN)

RCCs. Rare examples are pH or glucose - dependent. Also like anti- M, anti- N is not

clinically significant unless it reacts at 37oC. It has been implicated only with rare cases of

mild HDN.


36

Anti - N is rarer than anti - M because the terminal end of GPB carries the same amino

acid sequence and sugars as GPA when N is exposed (GPAN). This N-like structure called

'N', may prevent N - individuals from recognizing N as a foreign antigen. The most potent

antibodies are found in rare individuals who type M+N-S-s - and lack N and 'N'.

Anti N is also seen in renal patients regardless of their MN type, who are dialyzed on

equipment sterilized with formaldehyde. Dialysis - associated anti - N reacts with any N+

or N- RBC treated with formaldehyde and is called anti-Nf. Formaldehyde may alter the M

and N antigens so not they are recognized as foreign. The antibody titer decreases when

dialysis treatment and exposure to formaldehyde stop. Because anti - Nf does not react at

37oC, it is clinically insignificant in transfusion. However, it has been associated with the

rejection of a chilled transplanted kidney.

Anti - S and Anti - s :

Most examples of anti - S and anti - s are IgG, reactive at 37oC and the antiglobulin test

phase. A few express optimal reactivity between 10oC and 22oC by saline indirect

antiglobulin test. It anti - S or anti - s specificity is suspected, incubating tests at room

temperature and performing the antiglobulin test without incubating at 37oC may help its

identification. Dosage effect can be exhibited by many examples of anti - S and anti -s,

although it may not be as dramatic as seen with anti M and anti - N

The antibodies may or may not react with enzyme - treated RBCs, depending on the extent

of treatment. Treated RBCs should be tested for S and s antigen expression with known

antisera before enzyme reactions are interpreted. Although seen less often than anti - M,

anti - S and anti - s are more likely to be clinically significant. They may bind

complement, and they have been implicated in severe hemolytic transfusion reactions with

hemoglobinuria. They have also caused HDN. Units selected for transfusion must be

antigen - negative and cross match Compatible. Antibodies to low - incidence antigens are

commonly found in reagent anti - S, and these can cause discrepant antigen typing results.

Biochemistry:

GPA, the structure carrying the M and N antigens has a molecular weight of 36 KD and

consists of 131 amino acids. The hydrophilic NH2 terminal end, which lies outside the

RBC membrane, has 72 amino acid residues, 15 O - glycosidically linked oligosaccharide


37

chains (GalNAc - serine / threonine) and 1 N - glycosidic chain (sugar - asparagine). The

portion that traverses the membrane is hydrophobic and contains 23 amino acids. The

hydrophilic COOH end, which contains 36 amino acids and no carbohydrates, lies inside

the membrane and interacts weakly with the membrane cytoskeleton. M and N antigens

differ in their amino acid residues at positions 1 and 5. M has serine and glycine at these

positions, where has N has leucine and glutamic acid.

GPB, the structure carrying S, s and U antigens, has a molecular weight of 20KD and

contains 72 amino acids and 11 O - linked oligosaccharide chains and no N - glycans. It

has an outer glycosylated portion of 44 amino acids, a hydrophobic portion of 20 amino

acids that traverses the RBC membrane, and a short cytoplasmic ‘tail’ of 8 amino acids.

The first 26 amino acids on GPB are identical to the first 26 amino acids on N Form of

GPA (GPAN). This N activity of GPB is denoted as 'N' (N-quotes) to distinguish it from

the N activity of GPAN. Anti - N reagents do not recognize the 'N' structure. The U

antigen, expressed when normal GPB is present is located very close to the RBC

membrane.

Most O - linked carbohydrate structures on GPA and GPB are branched tetrasacharides

containing one GalNAc, one Gal, and two NeuNAc (sialic acid). Heterogeneity does occur

within these chains (they can lack a sugar or have sugar substitutions) but their significant

feature is NeuNAc, which helps give the RBC its negative charge. About 70 percent of the

RBC NeuNAc is carried by GPA, and about 16 Percent is carried by GPB.

GPA associates with protein band 3, which affects the expression of the antigen Wrb of the

Diego blood group system (located on protein band 3). GPB appears to be associated with

the Rh protein and Rh-associated glycoprotein complex as evidenced by the greatly

reduced S and s expression on Rhnull RBCs.

Other antigens within the MNS system have been evaluated biochemically and at the

molecular level. Some are associated with altered GPA because of amino acid

substitutions and / or changes in carbohydrate chains. Others are expressed on variants of

GPA or GPB. Still others result from a genetic event that encodes a hybrid glycophorin

that has parts of both GPA and GPB. This altered glycophorins are associated with

changes in glycosylation, changes in molecular weight, loss of high - incidence antigens or


38

the appearance of novel low - incidence antigens and / or alterations in the expression of

MNS antigens.

Genetics:36

The genes GYPA and GYPB, which code for GPA and GPB, respectively and located on

chromosome 4 at 4928 - q31. The known alleles for GYPA (M/N) and GYPB (S/s) are

codominant. Because of their many similarities and primate antigen studies, some suggest

that GYPB arose from a duplication of an ancestral GYPA gene and that the other alleles

arose by further mutations. GYPA is organized into seven exons: exon A1 encodes a leader

protein that helps insert the structure into the membrane during the formation; exon A2

encodes the first 26 amino acids; exon A3 has inverted repeat sequences known to be sites

for DNA recombination; exon A4 encodes the remaining extracellular portion; exon A5,

the transmembrane protein; and exons A6 and A7, the cytoplasmic portion. Exon A7 also

contains the stop codon.

GYPB has a size and arrangement similar to those of GYPA but only five exons; exons B1

and B2, which are nearly identical to exons A1 and A2, encode a leader protein and amino

acids 1 through 26; exon B3 is analogous to exonA4, encoding the portion of the molecule

that carries S and s; exon B4 similar to A5, encodes a larger transmembrane portion

because of a mutation that affects an mRNA splice site; and exon B5 encodes the

cytoplasmic portion and final stop codon. There is no counterpart to exon A3 because of

another splice site mutation, nor is there an exon A6 or A7 counterpart.

A third highly homologous gene, GYPE, does not appear to make a glycoprotein that has

been definitively recognized on the RBC surface.

Misalignment of GYPA and GYPB during meiosis followed by an unequal crossing over

appears to provide an explanation for some of the variant glycophorin observed in the

MNS system. The resulting now reciprocal GYP(A-B) and GYP(B-A) genes encode GP (A-

B) and GP (B-A) hybrid glycophorins, respectively.


39

Fig. 6 Misalignment during meiosis leading to crossover and reciprocal GYP(B-A) and GYP(A-B) hybrids For more complex variant glycophorins, a gene conversion event probably occurs. Gene

conversion is not completely understood; it involves a nonreciprocal exchange of genetic

material from gene to another homologous gene. The point of fusion between the GPA and

GPB part in the hybrid glycophorin can give rise to novel antigens, e.g. antigens of low

incidence. Also, expected antigens may be missing if the coding exons are replaced by the

inserted genetic material.

Fig. 7 Gene conversion event, transferring one strand of DNA to the misaligned homologous gene


40

M and N Lectins:36

A number of plant lectins have proved useful in studying GPA biochemistry, some with

practical application. These having N reactivity include Bauhinia variegata. Bauhinia

purpura, and Vicia graminea. When diluted, V. graminea lectin behaves as anti-N and so

makes an appropriate typing reagent.

GPA and GPB Deficient phenotypes:

RBCs of three rare phenotypes lack either GPA or GPB or both GPA and GPB and,

consequently, all MNS antigens that are normally expressed on those structures.

U- phenotype :- The U (for universal) antigen is located on GPB very close to the RBC

membrane between amino acids 33 and 39. This high - incidence antigen is found on

RBCs of all individuals except 1 % of American blacks (and 1 to 35% of African blacks)

who lack GPB because of a partial or a complete deletion of GYPB. They usually type S- s

- U - and make anti - U in response to transfusion or pregnancy. Anti - U is typically IgG

and has been reported to cause hemolytic transfusion reactions and HDN.

The U antigen is resistant to enzyme treatment; thus, most examples of anti - U react

equally well with untreated and enzyme-treated RBCs; there are rare examples of broadly

reactive anti - U, however, that do not react with papain - treated RBCs.

Some examples of anti-U react with apparent U-RBCs, although weakly by adsorption and

elution. Such RBCs are said to be U variant (Uvar). Uvar RBCs have an altered GPB that

does not express S or s. There is a strong correlation between the low - incidence antigen

He (MNS6), found in 3 percent of African- Americans and Uvar expression.

Because examples of anti-U are heterogeneous, U - units selected for transfusion must be

cross matched to determine compatibility. Some patients may tolerate Uvar units, others

may not. If the patient is U - and N-, the antibody may actually be a potent and anti - N

plus anti - U, making the search for compatible blood even more difficult.


41

En(a-) phenotype:36

In 1996 Darnborough et all and Furuhjelm et al described an antibody to the same high -

incidence antigen, called Ena (for envelope), which reacted with all RBCs except those of

the propositi. Both En(a-) individuals appeared to be M-N- with reduced NeuNAc on their

RBCs. The RBCs of the two individuals are mutually compatible.

Most En(a-) individuals produce anti -Ena, which is an umbrella term for reactivity against

various portions of GPA unrelated to M or N, but not all antibodies detects the same

portion. Anti- Ena Ts recognizes a trypsin - Sensitive (TS) area on GPA between amino

acids 20 and 39. Anti - Ena Fs reacts with a ficin sensitive (FS) area between amino acids

46 and 56, and anti - Ena FR reacts with a ficin resistant (FR) area around amino acids 62

to 72. Although the gene responsible for this phenotype has been termed En, it is now

known that En (a-) phenotype has more than one origin. Typically, the En (a-) phenotype

results from homozygosity for a gene deletion at the GYPA locus; consequently, no GPA

is produced but GPB is not affected. This type of En (a-) inheritance is called En(a-) Fin.

The En (a-) phenotype in the English report, however probably represents heterzygosity

for a hybrid gene along with the rare Mk gene; this type is often called En (a-) UK.

Mk phenotype:36

The rare silent gene Mk was identified in 1964 by Metaxas and Metaxas - Buhler when

they studied and M - N + (NMk) mother who had an M + N - (MMk) child. The RBCs of

these rare individuals expressed half the amount of normal MN and Ss on SDS - PAGE

analysis.

In 1979 Tokunaga et al reported finding two related MkMk blood donors in Japan. The

RBCs of these individuals typed M- N-S-s-U-, but they had a normal hematologic picture.

More individuals have since been identified, and it is now known that the Mk gene

represents a single, near complete deletion of both GYPA and GYPB, thus Mk is the null

phenotype in the MNS system. The MkMk genotype is associated with the decreased RBC

sialic acid content but increased glycosylation of RBC membrane bands 3 and 4.1


42

The Miltenberger series:36

The Miltenberger series of low - incidence antigens was developed to bring order to a

group of specificities that appear to be related to one another. This relationship, however,

is purely serologic. The Miltenberger classification still appears in the literature, although

it is no longer feasible to continue expansion of the original series.

After the first five antigens were defined, Cleghorn performed a series of cross tests with

their respective antibodies and separated the RBCs into five phenotype: MiI ; MiII, MiIII,

MiIV and MiV. These phenotypes have been redefined and expanded to 11 with the

discovery of additional antigens.

Table 15 The Miltenberger Series. 36

Biochemical and molecular studies have simplified understanding of the Miltenberger

series. Each phenotype represents a distinct variant GP or hybrid molecule. Tippett et al

proposed that the Miltenberger terminology be dropped in favor of one that recognized the

glycoprotein variant and the name of the first propositus.

Autoantibodies:

Autoantibodies to M and N have been reported. Not all examples of anti - M in M +

individuals or anti -N in N+ individuals are autoantibodies. Many fail to react with the

patient's own RBCs. It may be that these individuals have altered GPA and that their

antibody is specific for a portion of the common antigen they lack.


43

Autoantibodies to U and Ena are more common and may be associated with warm - type

autoimmune hemolytic anemia.

Disease Associations:

GPAM may serve as the receptor by which certain pyelonephritogenic strains of

Escherichia coli gain entry to the Urinary treat.

The malaria parasite Plasmodium falciparum appears to use alternative receptors including

GPA, GPB and GPC for cell invasion; some of these receptors also involve NeuNAc. In an

attempt to identify the receptor, the invasion rate into cells with normal and rare

phenotypes was studied. Reduced invasion is seen with En(a-), U -, MkMk, Tn and cad

RBCs (which have altered oligosaccharides on glycophorins), Ge -, and normal RBCs

treated with neuraminidase and trypsin.


44

THE P BLOOD GROUP (ISBT No. 003) AND GLOBOSIDE

(ISBT No. 028) BLOOD GROUP SYSTEMS AND RELATED

ANTIGENS

Introduction:37

Traditionally, the P Blood group comprised the P, P1 and Pk antigens and later, Luke. The

biochemistry and molecular genetics although not completely understood as yet, make it

clear that at least two biosynthetic pathways and genes at different loci are involved in the

development and expression of these antigens. Consequently, these antigens cannot be

considered a single blood group system.

Currently, in ISBT nomendature P1 is assigned to the P blood group system (003), P to the

new Globoside blood group system (028) and Pk and LKE are assigned to the Globoside

collection (209).

The P blood group was introduced in 1927 by Landsteiner and Levine. In their search for

new antigens, they injected rabbits with human RBCs, and produced an antibody, initially

called anti-P, that divided human RBCS into two groups : P+ and P-.

In 1951 Levine et al described anti - Tja (now known as anti - PP1Pk) an antibody to a high

- incidence antigen that Sanger later showed was related to the P blood group. Because

anti - Tja defined an antigen common to P + and P - cells and was made by an apparent P

null individual, the original antigen and phenotypes were renamed. Anti P become anti -

P1; the P+ phenotype became P1; the P - phenotype become P2, and the rare P null

individual become p.

The P blood group become more complex in 1959 when Matson et al described a new

antigen, Pk. Pk is expressed on all RBCs except those of the very rare p phenotype, but it is

not readily detected unless P is absent, i.e. in the P1k and P2

k phenotypes.

There are two, common phenotypes: P1 and P2, and three rare phenotypes: p, P1k and P2

k.

The P1, phenotype describes RBCs that react with anti – P1, and anti – P; the P2 phenotype

describes RBCs that do not react with anti - P1 but do not react with anti - P. When RBCs

are tested only with anti P1, and not with anti P, the phenotype should be written as P1+ or


45

P1-. RBCs of the p phenotype do not react with anti -P1, anti P or anti - Pk. RBCs of the

P1k phenotype react with anti – P1 anti -Pk but not with anti – P. RBCs of the P2k

phenotype react with anti -Pk but not with anti -P1 or anti -P.

Individuals with the P phenotype (P null) are very rare: 5.8 in a million. P nulls are slightly

more common in Japan, North Sweden and in an Amish group in Ohio.

Table 16 P Blood group system.38

Basic Concepts:39

The P blood group antigens, like the ABH antigens, are synthesized by sequential action

of glycosyltransferares, which add sugars to precursor substances. The precursor of P1 can

also be glycosylated to type 2H chains, which carry ABH antigens. P1, P or Pk may be

found on RBCs, lymphocytes, granulocytes and monocytes. P can be found on platelets,

epithelial cells and fibroblasts. P and Pk have also been found in plasma as

glycosphingolipids and as glycoproteins in hydatid cyst fluid. The antigens have not been

identified in secretions. RBCs carry approximately 14 x 106copies of globoside, the P

structure, per adult RBC and about 5 x 105 copies of P1.

The P1 Antigen :

The expression of P1 changes during fetal development. The antigen is found on fetal

RBCs as early as 12 weeks, but it weakens with gestational age. Ikin et al found that

young fetuses were more frequently and more strongly P1+ than older fetuses. The antigen

is poorly expressed at birth and may take upto 7 yrs to be fully expressed.


46

Antigen strength in adults varies from one individual to another, a fact first noted by

Landsteiner and Levine, who found that some P1+ people were P1 strong (P1+S) and others

were P1 weak (P1+ w). These differences appear to be quantitative, not qualitative, and may

either be controlled genetically or represent homozygous versus heterozygous inheritance

of the gene coding for P1. The strength of P1 can also vary with race. Blacks have a

stronger expression of P1 than whites. The rare dominant gene In (Lu), inhibits the

expression of P1 so that P1 individuals who inherit this modifier gene may type

serologically as P1.

The P1 antigen deteriorates rapidly on storage. When old cells are typed or used as

controls for typing reagents or when older cells are used to detect anti - P1 in serum, false-

negative reactions may result.

Anti - P1:

Anti - P1 is a common, naturally occurring IgM antibody in the sera of P1 - individuals.

Anti P1 is typically a weak, cold reactive saline agglutinin optiomally reactive at 4oC and

not seen in routine testing. Stronger examples react at room temperature, and are rare

examples react at 37oC and bind complement, which is detected in the antiglobulin test

when polyspecific reagents are used. Antibody activity can be neutralized or inhibited with

soluble P1 substance or bypassed using prewarm test methods.

Examples of anti- P1 that react only at temperatures below 37oC can be considered

clinically insignificant. For the examples of anti - P1, which are stronger at room

temperature, setting up tests using a prewarm technique may be necessary to determine if

the antibody is reactive at 37oC.

Because P1 antigen expression on RBCs varies and deteriorates during storage, antibodies

may react only with RBCs having the strongest expression and give inconclusive patterns

of reactivity when antibody identification is performed. Incubating tests at room

temperature or lower or pretreating test cells with enzymes can enhance reactions to

confirm specificity.


47

Providing units that are crossmatch-compatible at 37oC and the antiglobulin phase,

without typing for P1, is an acceptable approach to transfusion. Giving P1+ units under

these circumstances does not cause a rise in antibody titer or a change in its thermal range

of reactivity.

Rare examples of anti-P1 that react at 37oC can cause in vivo RBC destruction; both

immediate and delayed hemolytic transfusion reactions have been reported. These rare

antibodies react well in the antiglobulin phase, bind complement, and may lyse test cells,

especially if they are enzyme - treated.

HDN is not associated with anti-P1, presumably because the antibody is usually IgM and

the antigen is so poorly developed on fetal cells.

Advanced concepts:39 Biochemistry: The RBC antigens of the P blood group exist as glycosphingolipids. As with ABH, the

antigens result from the sugars added sequentially to precursor structures. Biochemical

analyses have shown that the precursor substance for P1 is also a precursor for type 2H

chains that carry ABH antigens. However, the genes responsible for the formation of the

P1 and ABH antigens are independent. As with ABH, P blood group antigens are resistant

to enzyme, DTT, chloroquine or glycine - acid - EDTA degradation.

Fig. 8 Synthesis of P blood group antigens


48

Genetics:

The gene encoding the enzyme responsible for the synthesis of Pk from lactosylceramide,

4 - α-galactosyltransferase (Gb3 or Pk synthase) was cloned independently by three

research groups in 2000. The gene encoding the 3 - β - N - acetylgalactosaminyl -

transferase (Gb4 synthese) that is responsible for converting Pk to P was cloned in 2000.

Several mutations in both genes have been identified that result in the P and Pk

phenotypes.

The genetic relationship of P1 to P and Pk is not yet understood. RBCs of the p phenotype

are P1 - in addition to P - and Pk - (the P1 - status of p RBCs cannot be explained). It is

still unclear if another gene is involved in the synthesis of P1 or if another mechanism

exists. The P1 gene (located on chromosome 22) and the P gene (located on chromosome

3) are inherited independently.

The gene for the synthesis of LKE has not yet been cloned.

Other sources of P1 Antigen and Antibody:

The discovery of strong anti -P1 in two P1 - individuals infected with Echinococcus

granulosus tapeworms led to the identification of P1 and Pk substance in hydatid cyst fluid.

This fluid was subsequently used in many of the studies that identified the biochemical

structures of the P blood group.

A P1 like antigen has also been found on RBC, in plasma, in droppings of pigeons and

turtledoves, and in the egg white of turtledoves. Exposure to these birds may place P1 -

bird handlers at risk of making strong, clinically significant anti P1. The P1 antigen in bird

droppings may be attributed to certain gram - negative avian bacteria rather than the birds

themselves.

Strong antibodies to P1 have also been associated with fascioliasis (bovine liver fluke

disease), Clonorchis sinensis, and Opisthorchis viverrini infections. P1 substance has been

identified in extracts of Lumbricoides terrestris (the common earthworm) and Ascaris

suum.

Soluble P1 substances have potential use in the blood bank and are commercially

available. When it is necessary to confirm antibody specificity or to identify underlying


49

antibodies, these substances can be used to neutralize anti -P1 if pre warmed methods do

not eliminate reactivity.

Anti - PP1Pk:

Originally called anti - Tja, anti - PP1Pk was first described in the serum of Mrs. Jay, a p

individual with adenocarcinoma of the stomach. Her tumor cells carried P system

antigens, and the antibody was credited as having cytotoxic properties that may have

helped prevent metastatic growth postsurgery (the T in the Tja refers to tumor).

Anti - PP1Pk is produced by all p individuals early in the without RBC sensitization and

reacts with all RBCs except those of the p phenotype. Unlike antibodies made by other

blood group null phenotypes, the anti - P, anti - P1 and anti - Pk components of anti - PP1Pk

are separable through adsorption. Components of anti - PP1Pk have been shown to be IgM

and IgG. They react over a wide thermal range and efficiently bind complement, which

makes them potent hemolysins. Anti - PP1,Pk has the potential to cause severe hemolytic

transfusion reactions and HDN.

The antibody is also associated with an increased incidence of spontaneous abortions in

early pregnancy. Although the reason for this is not fully known, it has been suggested that

having an IgG anti - P component is an important factor. Women with anti - P and anti -

PP1Pk and a history of multiple abortions have successfully delivered infants after multiple

plasmapheresis to reduce their antibody level during pregnancy.

Allo anti - P:

In addition to being a component of the anti - PP1Pk in p individuals, anti - P is found as a

naturally occurring alloantibody in the sera of all Pk individuals. Its reactivity is similar to

that of anti - PP1Pk. In that it is usually a potent hemolysin reacting with all cells except

the auto control and those with the p phenotype. However, it differs from anti - PP1Pk in

that does not react with cells having the extremely rare Pk phenotype and the individual

making the antibody may type P1+. Alloanti - P is rarely seen, but because it is hemolytic

with a wide thermal range of reactivity it is very significant in transfusion. IgG class anti-P

may occur and has been associated with habitual early abortion.


50

Autoanti - P associated with Paroxysmal Cold Hemoglobinuria:

Anti - P specificity is also associated with the cold - reactive IgG autoantibody in patients

with paroxysmal cold hemoglobinuria (PCH). Historically, this rare autoimmune disorder

was seen in patients with tertiary syphilis, it is now more commonly presents as a transient

acute condition secondary to viral infection, especially in young children. The IgG

autoantibody in PCH is described as a biphasic hemolysin; in vitro the antibody binds to

RBCs in the cold and, via complement activation, the coated RBCs lyse as they are

warmed to 37oC. The autoantibody typically does not react in routine test systems but is

demonstrable only by the Donath-Landsteiner test.

Anti - Pk :

Anti - Pk has been isolated form some examples of anti PP1Pk by selective adsorption with

P1 cells. Autoanti - Pk has been reported in the serum of P1 individuals with biliary

cirrhosis and autoimmune hemolytic anemia. Anti - Pk activity can be inhibited with

hydatid cyst fluid.

Disease Associations:

Several pathologic conditions associated with the P blood group antigens have been

described; parasitic infections are associated with anti – P1, early abortions with anti -

PP1Pk or anti - P, and PCH with autoanti - P.

The P system antigens may also be associated with urinary tract infections. Some

pyelonephritogenic strains of E.coli ascend the urinary tract in ladder like fashion by

adhering to P1 and/or Pk glycolipids on uroepithelial cells. The fimbriae or pili of such

organisms have receptor sites for structures involving Gal (α1-4) Gal (B1-4), the terminal

sugars for P1 and Pk. Other globoside associations have been identified with infection.

Streptococcus suis, which occasionally causes meningitis and septicemia in humans, binds

exclusively to Pk antigen. A class of toxins secreted by Shigella dysenteriae, Vibrio

cholerae, Vibrio parahemolyticus, and some pathogenic strains of E.coli also have binding

specificity for a Gal (α1-4) Gal (β1-4) moiety. In addition, globoside is the receptor of

human parvovirus B19.


51

THE Rh AND RhAG BLOOD GROUP SYSTEMS :

(ISBT No. 004 and O30)

Introduction : 40

The Rh blood group system was discovered in New York in 1939, with an antibody in the

serum of a woman who had given birth to a stillborn baby and then suffered a hemolytic

reaction as the result of transfusion of blood form her husband. Levine and Stetson found

that the antibody agglutinated the red cells of her husband and those of 80% of ABO

compatible blood donors. Regrettably, Levine and Stetson did not name the antibody. In

1940, Landsteiner and Wiener made antibodies by injecting rhesus monkey red cells into

rabbits. These antibodies not only agglutinated rhesus monkey red cells, but also red cells

from 85% of white New Yorkers and appeared to be the same as Levine and Stetsons'

antibody and other human antibodies identified later. By 1962, however it was clear that

rabbit and guinea pig anti-rhesus reacted with a determinant that was genetically

independent of that determined by the human antibodies, despite being serologically

related. In consequence, the antirhesus antibodies were renamed anti-LW, after

Landsteiner and Wiener, and the human antibodies remained as anti-D of the Rh (not

rhesus) blood group system.

As early as 1943, Rh started to become complex. From their work with four antisera, anti-

C, -c, -E and -e, detecting two pairs of antithetical antigens, Fisher and Race postulated

three closely linked loci producing D/d, C/c and E/e. Anti-d has never been found and

does not exist. Wiener, in New york worked with antibodies of the same specificities, but

came up with a different genetical theory involving only one gene locus. In 1986, Tippett

provided another alternative theory: two loci, one Producing D or no D, the other

producing C/c and E/e. Shortly after, Tippett's Theory was validated by molecular genetic

studies.


52

Haplotypes, genotypes, and phenotypes:40

Although there are only two Rh gene loci, RHD and RHCE, the Fisher - Race theory of

three loci D/d, C/c and E/e, is still appropriate for interpreting serological data because C/c

and E/e represent different mutation sites within RHCE. No conclusive evidence of

recombination between D/d and C/c, C/c and E/e or D/d and E/e has been found.

The three pairs of alleles can comprise eight possible haplotypes. All have been identified

and are shown in following table.

Table 17 Eight Rh haplotypes and their frequencies in English, Nigerian and Hong

Kong Chinese populations. 40

These eight haplotypes can be paired to form 36 different genotypes. However, form these

36 genotypes, only 18 different phenotypes can be recognized by serological tests with

anti - D, -C, -c, -E and -e. This is because there is no anti-d, so homozygosity (D/D) and

heterozygosity (D/d) for D cannot be distinguished serologically. For example, DCe/dce

cannot be distinguished serologically from DCe/Dce. Furthermore, in D/d and C/c

heterozygotes, it is not possible to determine whether D is in cis (on the same

Chromosome) with C or with c, so, for example DCe/dce cannot be distinguished from

Dce/dCe. The same applies to C/c and E/e so DCe/ DcE cannot be distinguished from

DCE/Dce by usual serological techniques.

The most common Rh phenotype in a Caucasian population is D + C+ c+ E-e +. This is

often written as DCe/dce (R1r)


53

Although this written in the format of a genotype, it is not a true genotype, but a probable

genotype. It is not a true genotype because D+ C+ c+ E -e+ could also result from DCe/

Dce (R1R0) or Dce/dCe (Ror’). It is a probable genotype because, in a Caucasian

population, DCe/dce is 15 times more common than DCe/Dce and 650 times more

common than Dce/dCe.

In an African population however, the haplotype Dce is more common than dce, so the

probable genotype D+C+c+E -e+ would be DCe/Dce. The probable genotype for

D+C+c+E+e+ is DCe/DcE (R1R2) but DCe/dcE (R1r”), DcE/dCe (R2r’), DCE/dce (Rzr),

Dce / DCE (R0Rz) and Dce/dcE (R0r’’) are all alternatives of lower incidence. It is

important to remember that probable genotypes and true genotypes are not always the

same.

An alternative notation, with no genetical implications, is the numerical terminology of the

International Society of Blood Transfusion (ISBT) : D is RH1; C,RH2; E,RH3; c,RH4;

and e,RH5. The common Rh phenotype D+ C+ c+E-e+ is RH: 1, 2,-3, 4, 5.

Table 18 Common Rh types by three nomenclatures.41

Biochemistry and molecular genetics:41

Rh phenotypes are controlled by two genes : RHD, which encodes the D antigen, and

RHCE, which encodes the Cc and Ee antigens. Both genes have 10 exons and share about

94% sequence identity throughout all introns and exons. They are extremely unusual for

closely linked homologous genes in that they are in opposite orientation on the


54

chromosome; that is, in tail-to-tail configuration (5'RHD3' - 3'RHCE5'), the coding strand

of RHD becoming the non-coding strand of RHCE, and vice versa.

Another gene, SMP1 encoding, a small membrane protein, is located between the Rh

genes. RHD is flanked by two 9kb regions of 98.6% identity, the Rh boxes.

Fig. 9 Diagram representing the 10 exons of the RHD and RHCE genes in opposite orientation, the Rh boxes (regions of identity), and the SMP1 gene.

RHD and RHCE encode proteins of 417 amino acids, although the N- terminal methionine

is cleaved from the mature proteins. The RhD and RhCcEe proteins differ by between 31

and 35 amino acids, according to RHCE allele.


55

Fig. 10 RH Proteins ( RHD and RHCE Proteins)

Interpretation of the amino acid sequences predicts that the Rh proteins cross the

membrane 12 times, providing six extracellular loops, the potential sites for expression of

Rh antigens. N- and C- termini are inside the cytosol. It is partly become the Rh proteins

have this sort of polytopic structure that the Rh system is so complex. Both proteins Rh D

and RhCE possess two three molecules of palmitate (C16 fatty acid) covalently linked to

transmembrane cysteine residues. Palmitoylation of Rh proteins may help to maintain the

phospholipid asymmetry of the RBC membrane.

Rh antigens are very dependent on the shape of the molecule and may also involve

interactions between more than one of the extracellular loops, Minor changes in the amino

acid sequence, such as a single amino acid change, even within a membrane spanning

domain, can cause conformational changes that create new antigens and affect the

expression of existing ones. Unlike most cell membrane proteins, the Rh proteins are not

glycosylated.

Within the red cell membrane, the Rh proteins are associated with a glycoprotein called

the Rh-associated glycoprotein (RhAG). RhAG shares 33% identity with the Rh proteins

and has a very similar arrangement in the membrane. Unlike the Rh proteins, RhAG is


56

glycosylated, with a single N-linked sugar on the first extracellular loop. It is produced by

the RHAG gene on chromosome 6. Although RhAG does not carry any Rh antigens, its

presence is essential for expression of Rh antigens and in its absence no Rh antigens are

expressed.

D antigen (RH1):

In the field of transfusion medicine, D is the most important Rh antigen, and the most

important blood group antigen after A and B. Anti-D can cause severe and fatal hemolytic

transfusion reactions (HTRs) and hemolytic disease of the fetus and newborn (HDFN). At

least 30% of D- recipients of transfused D+ red cells make anti-D.

D+ and D- phenotypes are often referred to as Rh+ and Rh-. Between 82% and 88% of

Europeans and North American Caucasians are D+, around 95% of Black Africans are D+.

D is a high - frequency antigen in the Far East, reaching 100% in some populations.

D antigen expression varies quantitatively. Even among the common phenotypes there is

readily detectable quantitative variation of D, with less D expressed in the presence of C.

Current evidence suggests that D is a highly complex antigen, depending on both specific

amino acids and the tertiary structure of the RHD protein itself. At least nine "D- specific"

amino acids (Me+169, Met 170, Ile 72, Phe 223, Ala 226, Glu 233, Asp 350, Ala 353, and

Gly 354) have been identified as functional D epitopes. The nine amino acids lie along the

third, fourth and sixth external loops of the RHD protein, creating six distinct D - epitope

clusters or footprints. 43

Molecular basis of the D polymorphism:

The D- phenotype results from absence of the Rh D protein. This explains why no antigen

antithetical to D was found. Consequently, the symbol d simply represents in absence of

D. In Caucasians the D- phenotype almost always results from homozygosity for a

deletion of RHD. This deletion appears to have occurred between a 1463 bp region of

identity in each of the Rh boxes. D+ people can be homozygous or hemizygous for the

presence of Rh D.

Sixty-six percent of D- Black Africans have an intact RHD, but this gene is inactive owing

to a nonsense mutation in exon 6 ( the codon for Tyr 269 converted to a translation


57

termination codon) and a 37 bp insertion in exon 4 that might introduce another premature

termination codon. This inactive RHD, called RHDψ, produces no D protein and no D

antigen.

Table 19 Molecular basis for RH and RHAG antigens.43


58

D variants:

Fig. 11 The Rh gene cluster

Numerous variants of D exist, mostly caused by mutations within the RHD gene. D

variants have been ranked into two main classes:44

1. Weak D (formerly Du) in which the whole D antigen is expressed, but expressed

weakly. Because all D epitopes are present, individuals with weak D cannot make

anti-D when immunized by a normal, complete D antigen. Weak D is usually

associated with amino acid substitutions in the membrane - spanning or cytosolic

domains of the Rh D protein, which are not exposed to the outside of the

membrane.

Because of their location, it is hypothesized that these mutations may interfere with

the assembly or efficient insertion of the Rh D protein. Furthermore, because all

the mutations are intramembranous or intracellular, it is assumed they do not

significantly alter the presentation of D epitopes on the extracellular loops. This

may explain why most weak D individuals do not make allo anti-D when


59

transfused with D-positive blood. In these individuals, the number of Rh d

molecules is decreased 40-100 fold ranging from 66-5200 molecules per red cell.

2. Partial D, in which part of the D antigen is missing. That is, only some D epitopes

are expressed, and these may be expressed normally or weakly. Because some or

most of the D epitopes are missing, individuals with partial D can make an

antibody to those epitopes they lack, following immunization with complete D

antigen, and this antibody behaves as anti D in tests with red cells of common D

Phenotypes. Partial D is usually associated with amino acid changes in the exposed

extra cellular loops of the Rh D protein.

This dichotomy, however, is no longer valid. Some D variants have been classified as

weak D (e.g. weak types 4.2 and 15), yet individuals with these variants have subsequently

been found who have made anti-D. Some partial D antigens, such as DVI, have distinctly

weakened expression of those epitopes they have. Consequently, the terminology is

misleading and a new terminology is required.

Table 20 Some symbols given to D variants.44

Another type of D Variant is DEL, in which the D is expressed so weakly it cannot be

detected by conventional serological methods and requires specialist techniques, in

particular adsorption and elution. Despite the very low level of D expression on DEL red

cells, they have still immunised D- patients to make anti D following transfusion.

Consequently it is possible that all D variants have the potential to immunise D-

transfusion recipients.


60

Numerous monoclonal antibodies to the D antigen have been produced. By definition,

each monoclonal antibody detects only one epitope. Analyses of tests with many such

antibodies against red calls expressing different D variant antigens has led to the definition

of 30 reaction patterns, interpreted as 30 epitopes of D (ep D).

Same D variant proteins express an antigen that is specific for that partial D antigen. For

example, red cells with DIVa variant D always express Goa (RH30), those with DV

variant express DW (RH 23) and those with DFR express FPTT (RH 50).

Many D variants arise from missense mutations in RHD, which result in one or several

amino acid substitutions in the Rh D protein. In some cases, however, the mutation

represents an exchange of genetic material between RHD and RHCE, so that sections of

RHD are replaced by the equivalent section of RHCE.

Fig. 12 Diagram of the 10 exons of nine examples of RHD-CE-D and RHCE-D-CE(DHAR) genes responsible for D variant phenotypes. Red boxes = RHD exons;blue boxes = RHCE exons.

For example, in the most common type of DVI, exons 4, 5 and 6 of RHD are replaced by

exons 4, 5 and 6 of RHCE, resulting in a hybrid protein in which part of the third

extracellular loop and the fourth and fifth loops have the sequence of a CcEe protein.


61

Rh - Negative Phenotype:45

Rh - Negative (D-) occurs in approximately 15% of white donors, almost always in

association with ce/ce or rr phenotype. In most Caucasian people, D - reflects a deletion of

the entire RHD gene. In blacks, D- can result from gene deletion or from inheritance of an

RHD pseudogene. Nearly 60% of D - negative black people inherit a mutant RHD allele

(RHψ/D) containing a 37 - base pair (bp) internal duplication, frame shift, and premature

stop codon. RHD genes containing nonsense mutations and nucleotide deletions have also

been reported in some D- negative Japanese and Caucasian donors.

Clinical Significance of anti-D:46

Anti- D is clinically the most important red cell antibody in transfusion medicine after anti

A and anti-B. It has the potential to cause severe HTRs and D+ blood must never be given

to a patient with anti D. As atleast 30% of D-recipients of transferred D+ red cells make

anti-D, D-positive red cells are not routinely transfused to D- patients.

Anti - D can cause severe HDFN. This occurs when IgG anti - D in an immunised mother

crosses the placenta and facilitates destruction of D+ fetal red cells. The effects of HDFN

caused by anti -D at its most severe are fetal death at about the 17th week of pregnancy. If

the infant is born alive, the disease can result in hydrops and jaundice. If the jaundice leads

to kernicterus, this usually results in infant death or permanent cerebral damage. In most

cases of HDFN, the mother was immunised to produce anti-D by fetal D+ red cells during

a previous pregnancy. These D+ red cells leak into maternal circulation via a

transplacental haemorrhage, which generally occurs during delivery, but sometimes

happens earlier in the pregnancy. Anti-D immunisation can be prevented, in most cases,

by administration of a dose of anti-D immunoglobulin to the D- mother immediately after

delivery of a D+ve baby. It is still not absolutely clear how the anti-D immunoglobulin

prevents immunization, but it is probably due to rapid removal of the D +fetal cells from

the maternal circulation. In order to prevent the less common occurrence of the mother

being immunized during the course of pregnancy, anti-D immunoglobulin may be

administered to D- pregnant women antenatally, and this has become the usual practice in

many countries.


62

It is imperative that D+ red cells are never transfused to D- girls or premenopausal

women. If a D- young woman is given D+ blood products, then anti -D immunoglobulin

should be given. Anti-D in women with a D variant antigen can cause severe HDFN in a

fetus with a complete D antigen. If a woman known to have a D variant antigen gives birth

to a D+ baby; she should be given anti- D immunoglobulin.

D testing :46

According to the UK guidelines patient samples are tested in duplicate by direct

agglutination with IgM monoclonal anti-D selected to give a negative result with category

DVI cells. An antiglobulin test for detecting weak D variants should not be carried out.

Patients with very weak D variants or DVI will be found to be D- and will receive D-

blood. This will do no harm and will prevent the DVI patients from making anti-D and

ensure that they receive anti-D immunoglobulin following a D + pregnancy. For typing

donors, however at least one of the antibodies should detect DVI and weak forms of D.

C,c,E and e antigens (RH2, RH4, RH3, RH5):46

C and c antigens are the products of alleles of RHCE. C and c have frequencies of 68%

and 81% respectively in English blood donors. In Black Africans the frequency of c is

much higher and the frequency of C is much lower, where as in Eastern Asia the opposite

is the case: C is higher and c is lower. E and e represent another pair of RHCE alleles. In

all populations e is significantly more common that E.

Table 21 Frequencies of C, c, E and e antigens in three populations.46

In contrast to the complexity of the D antigen epitope, the C/c and E/e antigens are single

amino acid polymorphisms on the RhCE protein. Three additional amino acid


63

polymorphisms (Cys16Trp, Ile60Leu and Ser68Asn) may help stabilize the C and c

antigens. Arg 229 appears critical to the e and f expression. Because C/c and E/e epitopes

are present on the same protein, alloantibodies dependent on the expression of both C/c

and E/e antigens can occur. Four alloantibodies with “Compound" Rh specificity have

been described; anti-ce (f for RH6), anti-Ce (RH7), anti- cE (RH 27) and anti CE (RH 22).

These antibodies react only with RBCs carrying the appropriately paired antigens, in cis,

on the same RhCE protein. The RHCE protein is home to several additional high and low-

incidence antigens. 47

C/c primarily represents a Ser103Pro substitution in the second extracellular loop, encoded

by RHCE exon 2, although the situation is more complex than that. Ser103 is essential, but

not sufficient, for C Specificity; for full expression of C, the proteins must have ser103,

cys16, and some other downstream amino acids characteristic of the RhCcEe protein.

Cys16 is not, however, a requirement for all epitopes of C as some rare RH variants have

Ser103 and Trp16, yet express a weak, abnormal C. The c antigen is determined almost

entirely by the presence of Pro103. The E/e polymorphism is basically dependent on a

Pro226 Ala substitution, in the fourth extracellular loop, encoded by RHCE exon 5,

though changes to other residues do have some effect on e expression.48

Clinical significance of Cc Ee antibodies:48

All Rh antibodies should be considered to have the potential to cause HTRS and HDFN.

For transfusion to a patient with an Rh antibody, antigen-negative blood should be

provided wherever possible. Anti -c is clinically the most important Rh antigen after anti-

D and often causes severe HDFN, whereas anti - C, -E and -e rarely causes HDFN and

when they do the disease is generally (but not always) mild.

Compound antigens : ce, CE, cE (RH6, RH7, RH22, RH27) ant G (RH12).48

Antibodies to compound antigens detect red cells when they have specific C/c and E/e

antigens encoded by the same gene. For example, anti-ce (also known as anti - f) only

reacts with cells of individuals who have a dce or Dce complex that is, with c and e in cis.

Consequently, red cells with the phenotype D+ C+ c+ E + e + will react with anti-ce, but

not anti-Ce, if the genotype is Dce/dce, but will react with anti-Ce but not anti-ce, if it is

DCe/DcE. Anti-ce is a common component of anti-c and the anti-e sera, but is


64

occasionally found as a single specificity. Most anti-C and anti- C + D sera contain some

anti -Ce. Both anti -CE and anti -cE are rare antibodies.

The G antigen (RH12) is a high frequency antigen present on virtually all D- Positive and

C - Positive RBCs. G has been identified as Ser103, a C-type antigen on RHD and RHCE

proteins. It is not surprising that anti -G alloantibodies have both anti - C and anti - D

specificity (anti - C + D) and are frequently accompanied by anti - C (anti-G + anti -C).49

Anti-G reacts with red cells that have D or C, that is D+C+, D+C- and D-C+ cells. The

primary defining amino acid of C is Ser103 in the RhCcEe protein. The Rh D protein also

has serine at this position. Consequently, anti-G recognizes the presence of Ser103,

whether it is in the context of a D protein or a CcEe protein, in contrast to anti - C, which

is more conformationally dependent and only recognizes the presence of Ser103 in the

context of CcEe protein. Anti-G is often present in sera containing anti - D plus anti - C,

and can confuse serological investigations of HDFN.

Cw, Cx, MAR (RH8, RH9, RH51):50

Cw is a relatively low frequency antigen in all populations, although its incidence is quite

variable. In an English population Cw has a frequency of 2.6% and similar frequencies are

found in most Caucasian populations. Cx is a rare antigen, with an incidence of between

0.1% and 0.3% in Caucasian population. Cw and Cx are usually produced by DCe

complexes that produce a weakened form of C. Cw is associated with a GluA1Arg

substitution and Cx with an A1a36Thr substitution in a CcEe protein, with resultant

conformational changes in the molecule responsible for the weakness of C.

The high-frequency antigen MAR is abolished by either the Cw or the Cx substitution, so it

appears that the presence of both Ala37 and Glu41 are required for MAR expression.

VS, V (RH20, RH10).50

VS has a frequency of about 30-40% in Black African populations, but is rare in other

ethnic groups. VS is represented by a Leu245val substitution in a CcEe protein and is

associated with weak e. VS+ red cells are usually also V+, although about 20% of VS+ red

cells lack V and, in addition to the Leu245val substitution, have a Gly336Cys substitution.

The haplotype containing the altered RHCE gene associated with a VS+V- phenotype


65

contains no RHD, but does not have an RHD -CE - D hybrid gene, which produces no D,

but does produce an abnormal C.

RHAG Alloantigens :51

RHAG is a new blood group system with three antigens; O1a, Duclos and DSLK. All three

are the result of an amino acid polymorphism in the RhAg protein. Both Duclos and

DSLK are associated with weak U expression, suggesting an interaction between RhAg

and GYPB.

Rh-deficient phenotypes - Rhnull and Rhmod. Rhnull erythrocytes lake all Rh antigens as a

result of an apparent absence of RhD and RhCE proteins. In addition, Rhnull erythrocytes

lack the high frequency antigens Fy5 and LW and may have markedly decreased

expression of S/s and U antigens. The absence of these non-Rh antigens reflects the

complex topologic association of Duffy, LW and GYPB proteins with Rh proteins on

RBC membranes.

Extremely rare (/1 in 6 Million), Rhnull cells have abnormalities in RBC morphology

(spherocytes, stomatocytes), water content, cell volume, cation fluxes, carbon dioxide

permeability and phospholipid asymmetry. Rhnull cells show increased osmotic fragility

and a shortened circulating half-life, often accompanied by a mild hemolytic anemia (Rh

deficiency syndrome). Because Rhnull individuals can become sensitized to multiple Rh

antigens, including high-frequency antigens, transfusion support can be quite difficult.

Some alloimmunized Rhnull individuals can produce anti-RH29, which reacts with all

RBCs except Rhnull.

Rhnull has two types of inheritance:52

1. Homozygosity for inactive Rh hoplotypes. These individuals have no RHD (like

most D - people) and are homozygous for RHCE containing inactivating

mutations, so that neither Rh protein can be produced.

2. Normal active RHD and RHCE genes, but homozygosity for inactivating mutations

in RHAG, an independent gene encoding the RhAG. RhAG does not express Rh

antigens, but is closely associated with the Rh protein in the membrane. In the

absence of RhAG, Rh antigens are not expressed. Some mutations in RHAG permit


66

reduced quantities of RhAG to be produced, which results in low level expression

of all Rh antigens; the Rhmod phenotype.

Rh antibodies:53

Antibodies against Rh antigens are routinely encountered in the blood bank. D is the most

immunogenic Rh antigen, followed by c, E, C and e. In general, antibodies against Rh

antigens are the result of immune stimulation by transfusion or pregnancy. Exceptions

include some examples of anti- Cw and anti-E with can be naturally occurring. Most

antibodies against Rh antigens are of IgG isotype (IgG1, and IgG3), although rare

examples of IgM and IgA are known. Anti-Rh antibodies reactive at 37oC and are usually

detected in the AHG phase of testing. The reactivity of anti-Rh antibodies can be enhanced

with enzyme-treated RBCS.

Clinically, antibodies against Rh are associated with hemolytic transfusion reactions.

However, because Rh antibodies do not fix complement, incompatible RBCs are almost

always cleared through extravascular destruction. To prevent sensitization to the D

antigen, Rh negative patients should be transfused with Rh negative RBCs. This is

particularly true for young girls and women of child bearing age. For transfusion in

alloimmunized patients, RBC units should be negative for the Rh antigen of interest and

cross match compatible with the recipients’ serum through the AHG phase of testing. One

possible exception may be R1R1 (DCe / DCe) patients who have developed anti-E

alloantibodies. Because these patients are at increased risk of delayed hemolytic

transfusion reactions because of the subsequent development of anti - c, many blood

bankers advocate transfusing only R1 R1 units to R1R1 patients.

Antibodies against Rh antigens are also a major cause of HDFN. All Rh-negative women

should receive Rh immune globulin (IgG anti - D) prophylactically in midpregnancy,

following an invasive procedure (i.e. amniocentesis) and immediately after delivery to

prevent alloimmunization. Rh immune globulin prophylaxis is also recommended in

women with partial D phenotypes because these women can be at risk for D

alloimmunization. Rh immune globulin may also be given following transfusion of Rh D+

platelet concentrates or after accidental transfusion of Rh D + RBCs. In the latter, Rh

immune globulin is given after two - volume RBC exchange with Rh D - negative RBCs.

Administration of one vial of Rh immune globulin is recommended for every 30 ml whole


67

blood or 15 ml packed RBCs transfused. Rh immune globulin should be given within 72

hrs of exposure to prevent active immunization. Rh immune globulin is not given to Rh

negative women who are already immunized to D antigen. (i.e. have anti - D).

It is also recommended to give Rh immune globulin to Rh-negative women with anti-G

alloantibodies. Anti-G behaves as an anti C+ anti D because of recognition of a Ser103 or

C - type antigen on both Rh D and RhCE proteins. In general, HDFN secondary to anti -G

or anti - C + anti-G is mild when compared with HDFN due to anti D. However, because

these women may still become immunized to D-specific epitopes on the Rh D protein,

many blood bankers advocate giving Rh immune globulin to Rh - negative women with

anti - G antibodies. Separation of anti -G from a true anti-C + anti -D is very laborious,

requiring sequential adsorption and elution. One clue suggesting the presence of anti G is

an anti - C titer at least fourfold higher than anti-D. It is not necessary however, to separate

anti-G from anti-C + anti-D for routine transfusion. With very rare exceptions, RBCs

negative for D and C antigens are also negative for G antigen.

Putative function of the Rh proteins and RhAG.54

The Rh proteins and RhAG are homologous structures, with identical conformation in the

membrane and 33% sequence identity. Their multiple membrane- spanning conformation

with both termini in the cytosol is characteristic of membrane transporters. The Rh

proteins and RhAG show a degree of amino acid sequence homology with ammonium

transporters in lower animals and plants. Yeast cells lacking ammonium transporters fail to

grow in low ammonium medium but will grow successfully in that medium following

transfection with RHAG. Furthermore, transport of an ammonium analogue into Xenopus

oocytes was enhanced 8-10 fold following transfection of the cells with RHAG, and

transient expression of RhAG in a human cell line (HeLa) enhanced the permeability for

ammonium ions and for ammonia. It is feasible, but strictly speculative, that RhAG and

the Rh protein Complex in red cells is involved in ammonium transport, possibly so that

red cells can function to carry ammonium away from the brain to the liver or kidney for

metabolism or excretion.

An alternative suggestion, is that the macrocomplex involving Rh and band 3 functions as

an oxygen / carbon dioxide gas exchange channel. This would make sense, as the primary

purposes of the red cell are transport of oxygen and conversion of carbon dioxide to


68

bicarbonate, by carbonic anhydrase II (CAII) in the red cell cytoplasm. The macrocomplex

is ideally located to channel CO2 to and from CAII and O2 to and from hemoglobin.

Fig. 13 Model of the Rh macrocomplex in the red cell membrane


69

LUTHERAN BLOOD GROUP SYSTEM (ISBT No. 005)

INTRODUCTION:55

Lutheran antigens have been recognised since 1945, when the first example of anti - Lua

was discovered in the serum of a patient with lupus erythematosus diffusus, following the

transfusion of a unit of blood carrying the corresponding low-incidence antigen. The new

antibody was named Lutheran, a misinterpretation of the donor's name, Luteran. In 1956

Cutbush and Chanarin described anti - Lub, which defined the antithetical partner to Lua.

The blood group system appeared complete until 1961, when Crawford et all described the

first Lu(a-b-) phenotype. Unlike most null phenotypes at the time, this one demonstrated

dominant inheritance. In 1963 Darnborough et al found a more traditional Lu (a-b-)

phenotype inherited as a recessive silent allele.

Using rare Lu (a-b-) RBCs to test antibodies to unknown high - incidence antigens, some

sera showed a phenotypic relationship to Lutheran. That is, they reacted with all RBCs

tested except those with the Lu (a-b-) phenotype, even though the antibody producers

appeared to have normal Lutheran antigens. Those specificities were not identical to one

another, and they were given the numeric designations Lu4, Lu5, Lu6 and so on, to

represent their association to the system. Within this group three pairs of antithetic

antigens (Lu6 and Lu9, Lu8 and Lu14, and Lu 18 and Lu19) have been shown to be

inherited at the Lutheran locus; several antigens have been shown to be located on the

Lutheran glycoprotein, and three antigens (Lu11, Lu16, Lu17) often referred to as para-

Lutheran, have limited evidence that they belong to the Lutheran system.


70

Table 22 Summary of the Lutheran Antigens:55

Basic concepts:

Lua and Lub Antigens:

Lua and Lub are antigens produced by allelic codominant genes. Most individuals are

Lu(b+); only a few are Lu (a+).

Table 23 Phenotypes of the Lutheran system.56

Lutheran antigen expression is variable from one individual to another; antigen expression

on one individual’s RBCs can also vary. The number of Lub sites per RBC was estimated

to be from 1640 to 4070 on Lu(a-b+) RBCs and 850 to 1820 on Lu (a+b+) RBCs.

Although the antigens have been detected on fetal RBCs as early as 10 to 12 weeks

of gestation, they are poorly developed at birth and do not reach adult levels until age 15

years. Lutheran antigens have not been detected on platelets, lymphocytes, monocytes, or

granulocytes by means of sensitive radioimmunoassay or inmunofluorescent techniques.


71

Lutheran glycoprotein, however, is widely distributed in tissues; brain, lung pancreas,

placenta, skeletal muscle, and hepatocytes (especially fetal hepatic epithelial cells).57

Anti - Lua :57

Most examples of anti Lua are IgM naturally occurring saline agglutinins that react better

at room temperature than at 37oC. A few react at 37oC by indirect antiglobulin test. Some

are capable of binding complement, but invitro hemolysis has not been reported. Lutheran

antibodies are unusual in that may be IgA as well as IgM and IgG.

Anti - Lua often goes undetected in routine testing because most reagent cells are Lu (a-).

Anti Lua is more likely encountered as an incompatible crossmatch or during an antibody

workup for another specificity. Experienced technologists recognize Lutheran antibodies

by their characteristic loose, mixed - field reactivity in a test tube. Anti - Lua is not

profoundly altered with the common blood bank enzymes ficin and papain, but it can be

destroyed with trypsin, chymotrypsin, pronase, AET and DTT. Most Lua antibodies are

clinically insignificant in transfusion. There are no documented cases of immediate and

only rare and mild delayed transfusion reactions due to anti - Lua.

Because Lutheran antigens are poorly expressed on cord RBCs, cases of HDN associated

with anti-Lua are mild. Infants may exhibit weakly positive or negative direct antiglobulin

tests and mild to moderate elevations in bilirubin. Many require no treatment; others

respond to simple photo therapy.

Anti-Lub :58

Although, the first example of anti - Lub was a room temperature agglutinin; and IgM and

IgA antibodies have been noted, most anti - Lub is IgG and reactive at 37oC at the

antiglobulin phase. It is made in response to pregnancy or transfusion.

Alloanti - Lub reacts with all cells tested except the autocontrol, and reactions are often

weaker with Lu (a+b+) RBCs and cord RBCs. Ficin or papain does significantly alter

reactivity. AET or DTT should destroy Lub antigen through disruption of the disulfide

bond of the glycoprotein but this may require optimal conditions. Autologous RBCs will

test Lu (a+) if typing sera are available.


72

Anti - Lub has been implicated with shortened survival of transfused cells and post -

transfusion jaundice, but severe or acute hemolysis has not been reported. Chromium

survival studies demonstrate a rapid initial clearance of some Lu (b+) RBCs but much

slower removal of those remaining. Anti Lub may be regarded as clinically significant, but

blood should not be withheld in emergency situations, just because compatible units

cannot be found. Like anti-Lua, anti-Lub is associated with only mild cases of HDN.

Biochemistry :58

Fig. 14 Lutheran glycoprotein

Using immunoblot methods and a monoclonal antibody (BRIC 108), which initially

appeared to have Lub – like activity, Parsons et al identified two proteins with molecular

weights of 85 and 78 kd. These two glycoproteins, now known as the Lutheran

glycoproteins, contain both N-and O- linked oligosaccharides and intrachain disulfide

bonds. Subsequent immunoblotting with human antibodies to Lutheran antigens has

demonstrated that Lua, Lub, Lu3, Lu4, Lu6, Lu8, Lu12, Aua(Lu18) and Aub(Lu19) are

located on the Lutheran glycoprotein. The predicted 85 KD protein contains 597 amino

acids with five potential N – glycosylation sites. It traverses the cell membrane just once

and has a cytoplasmic domain of 59 amino acids. A smaller isoform (78KD) lacks part of

the cytoplasmic domain. The external portion consists of five disulfide – bonded domains


73

(three are constant, and two are variable). The Lutheran glycoproteins belong to the

lmmunoglobulin super family of proteins. Although its biologic rote is still uncertain, the

Lutheran protein most probably plays some role in adhesion or intracellular signaling.

The molecular basis for the four pairs of antithetical antigens and several of the high

incidence antigens has been determined through the creation of Lutheran glycoprotein

mutants.

Genetics :58

The Lu gene is located on chromosome 19 at position 19q13.2 – q13.3, along with genes

that govern expression of severed blood group antigens (H, Se, Le, Lw, Oka) and genes for

C3, apolipoprotein C-II (APO), and myotonic dystrophy. A linkage between Lu & the Se

gene (FUT2) was the first example of autosomal linkage described in humans.

Lu (a-b-) phenotypes :59

Three genetic explanations for the Lu (a-b-) phenotype have been described.

Table 24 Summary of Lu (a-b-) phenotypes.59

Dominant In(Lu) type:59

The first Lu (a-b-) family study was reported by the propositus herself. Because the

phenotype was seen in successive generations in 50 percent of members in her family and

others, and because null individuals passed normal Lutheran genes to their offspring, the

expression of Lutheran was thought to be suppressed by a rare dominant regulator gene

later called In(Lu) for “Inhibitor of Lutheran”. In(Lu) segregates independently form

Lutheran. Blood donor screenings have shown the frequency of this type Lutheran null to

be 1:3000 to 1:5000.


74

Dominant type Lu (a-b-) RBCs carry trace amounts of Lutheran antigens as shown by

adsorption elution studies. For example, a person who inherits two normal Lub genes plus

In(Lu) will type Lu (a-b-) with routine methods but will adsorb and elute anti-Lub.

Individuals with the In(Lu) type of Lu (a-b-) RBCs do not make anti-Lu3.

Inheriting just one In(Lu) gene prevents normal expression of all Lutheran antigens, as

well as P1, i and An wj, which are genetically independent. The I antigen appears

unaffected. The expression of CR1, the structure that carries the Knops system antigens,

does not appear to be regulated by In(Lu).

In(Lu) may also affect RBC shape and metabolism. The osmotic fragility of In(Lu) RBCs

is normal, but these cells significantly resist lysis when incubated in plasma at 37oC.

In(Lu) RBCs appear to lose more K+ than they gain Na+ under these conditions.

Recessive LuLu type :59

In some families, the Lu(a-b-) phenotype demonstrates recessive inheritance, the result of

having two rare silent alleles LuLu at the Lutheran locus. The parents and offspring of

these nulls may type Lu(a-b-), but dosage studies and titers show to carry a single dose of

Lub.

Unlike the In(Lu) type, recessive Lu(a-b-) people truly lack all Lutheran antigens and can

make an inseparable anti Luab called anti-Lu3. They also have normal antigen expression

of P1, i, and the many other antigens that In(Lu) affects. This distinction emphasizes the

importance of testing an antibody against recessive Lu (a-b-) RBCs before calling its

antigen phenotypically related to Lutheran.

Recessive x-liked Inhibitor type:59

In 1986 Norman et al described an Lu (a-b-) phenotype in a large Australian family that fit

neither an In(Lu) nor a LuLu pattern. All Lu(a-b-) family members were male and carried

trace amounts of Lub detected by adsorption-elution. Although P1 expression was weak, i

was well expressed, but I was depressed. The pattern of inheritance suggested an X-borne

inhibitor to Lutheran. The researchers proposed calling the locus XS, XS1 being the

common allele and XS2 the rare inhibitor that suppresses in a hemizygous state. There

have been no other families reported with this rare X-linked Lu(a-b-) phenotype.


75

Lu (w) phenotype :59

Several Lu (a-b+w) and Lu (a+w b+w) individuals with weakened Lutheran antigens have

been described. Although not proved, this phenotype also known as Lu(w), may result

from In(Lu) with a lesser degree of penetrance or an allele to In(Lu) that causes less

suppression.

Anti – Lu3:59

Anti – Lu3 is a rare antibody that reacts with all RBCs except those testing Lu (a-b-). The

antibody looks like inseparable anti-Luab and recognizes a common antigen, Lu3, that is

present whenever Lua or Lub is present. Anti Lu3 is usually antiglobulin reactive.

This antibody is made only by LuLu individuals i.e. the recessive type of Lu(a-b-). RBCs

from dominant and X-linked type Lu(a-b-) individuals can be safely transfused to patients

with anti-Lu3.

Lu6/Lu9 and Lu8/Lu14:59

In 1972, Lu6 and Lu8 designations were given to two nonidentical antibodies directed

against high-incidence antigens related to the Lutheran system. The antibodies reacted

with all RBCs except autologous and Lu(a-b-) cells, but they were made by Lu(a-b+)

individuals.

In 1973, Molthan et al described anti-Lu9, an antibody that reacted with 2 percent of

random donors and that gave very strong reactions with Lu6 RBCs. In 1977 Judd et al

described anti-Lu14, another antibody to a low-incidence antigen that was strongly

expressed on Lu:8 RBCs.

Aua (Lu18) and Aub (Lu19):59

Aua (Auberger) was described in 1961 by salmon et al as an antigen found in 80 percent of

whites. In 1989 its antithetical antigen, Aub was reported by Frandson et al. Because the

antigens were suppressed by In(Lu) and were destroyed by trypsin, chymotrypsin, and

pronase, they were closely associated with the Lutheran system except that, in one family


76

study, they were inherited independently. Serologists considered then another set of

antigens suppressed by Lutheran inhibitors.

Aua and Aub were subsequently shown to be expressed on the Lutheran glycoprotein and

the Au(a-) family members associated with the earlier genetic exclusion were retested and

found to test Au(a+). Family linkage studies also demonstrated that the Auberger and

Lutheran antigens were controlled by the same gene.

Other Lutheran Antigens:59

Lu4, Lu5, Lu7, Lu12, Lu13 and Lu20 are antigens of very high incidence that are absent

from Lu (a-b-) RBCs, but they have not been shown to be inherited at the Lu locus. All

have been shown to be located on the Lutheran glycoprotein, and all but Lu20 have been

shown to be inherited. Their antibodies parallel the characteristics of anti Lu6 and anti

Lu8; they do not react with Lu(a-b-) RBCs or autologous cells, which carry otherwise

normal Lutheran antigens.

The high – incidence antigens Lu11, Lu16, and Lu17 are phenotypically related to

Lutheran. These antigens are absent from Lu(a-b-) RBCs of the recessive and In(Lu)

types, but they have not been shown to be located on the Lutheran glycoprotein nor have

they been shown to be inherited; evidence that these antigens belong to the Lutheran

system is thus very limited.

The An/Wj antigen was once associated with the Lutheran system because it is not

expressed or only weakly expressed on In (Lu) Lu(a-b-) RBCs and was given the

designation Lu15, this became obsolete when An/wj was found on LuLu RBCs.

Lu10 is another designation no longer used. It was reserved for singleton, an antigen

thought to be the allele of Lu5; the antibody reacted strongly with Lu:-5 RBCs. However,

five other examples of Lu:-5 cells were later tested and found to be negative with the

singleton serum.


77

LEWIS SYSTEM: (ISBT No. 006) Introduction:60

The Lewis blood group system is unique because Lewis antigens are not synthesized by

the RBCs and incorporated into the RBC membrane structure. Instead, these antigens are

manufactured by tissue cells and secreted into body fluids. These antigens, therefore, have

been referred to as a system rather than as a blood group because they are found primarily

in the secretions and the plasma. These antigens are then absorbed onto the RBC

membrane from plasma, but they are not really an integral part of the membrane structure.

Because Lewis soluble antigens are manufactured by tissue cells, antigen production

depends not only on the inheritance of Lewis genes but also on the inheritance of the

secretor gene. Genetic interaction also exists between the Lewis and ABO genes because

the amount of Lewis antigen detectable on the RBC is influenced by the ABO genes.

Inheritance:60

Lewis genes (Le) do not code for the production of Lewis antigens but rather produce a

specific glycosyltransferase, L- fucosyltransferase. This enzyme adds L- fucose to the

basic precursor substance. The Le gene is located on the shortarm of chromosome 19 and

is distantly linked to the Se and H genes, which are located on the same chromosome. This

gene codes for the specific glycosyltransferase, α-4-L-fucosyltransferase, which transfers

L-fucose to type 1 chain oligosaccharide on glycoprotein or glycolipid structures. The

structure formed is known as Lea-soluble antigen. Type 1 chain refers to the beta linkage

of the number 1 carbon of galactose to the number 3 carbon of N-acetylglucosamine

(GlcNAc) residue of the precursor substance.

Fig.15 Structure of type-1and type-2 chains


78

The Lea – antigen is then secreted and adsorbed onto the RBCs, lymphocytes and platelet

membranes from plasma. Lewis antigen is also found on other tissues; such as the

pancreas, stomach, intestine, skeletal muscle, renal cortex and adrenal glands.

In the addition of L-fucose to type 1 chain catalyzed by Lewis enzyme, the number 1

carbon of L-fucose is attached to the number 4 carbon of GlcNAc. This transfer reaction

can occur only in type 1 precursor structures. Addition of L-fucose cannot occur with type

2 precursor structures because, in type 2 structures, the number 4 carbon of GlcNAc is

already linked to galactose.

The Lewis (Le) Phenotypes:60

The Lewis (a+b-) phenotype : Nonsecretors.

Lea substance is secreted regardless of the secretor status. Therefore, an individual can be

a nonsecretor (sese) of ABH and still secrete Lea in the body fluids, producing the

phenotype Lewis a- positive b-negative on the RBCs – Le (a+b-). All Le (a+b-)

individuals are nonsecretors of ABH substances. Lewis enzyme has been detected in

saliva, milk, submaxillary glands, gastric mucosa and kidney and cyst fluids. Lewis

fucosyltransferase has not been detected in plasma or in RBC stroma. Lewis antigens

produced in saliva and other secretions are glycoproteins, but Lewis cell-bound antigens

absorbed from plasma onto the RBC membranes are glycolipids.

The phenotype Lewis a results from the transfer of fucose to a type 1 chain by the action

of enzyme L-fucosyl transferase.


79

Fig. 16 Action of Le gene transferase enezyme.

Fig. 17 Formation of Lewis antigens (Lea, Leb, Aleb, BLeb) adsorbed from plasma.


80

The Lewis (a-b+) phenotype, secretors:

The phenotype Le (a-b+) is the result of the genetic interaction of Lele and Sese genes. Leb

antigen represents the product of genetic interaction between Le(FUT3) and Se(FUT2)

genes. Lewis and secretor genes are two independent genes that code for different but

related ∝-2-L-fucosyltranferases. The inheritance of the Se gene codes for the enzyme ∝-

2-L-fucosyltranferases, which adds L-fucose to type 1 precursor substance and thus forms

type 1 H. The inheritance of the Le gene codes for the addition of another L-fucose that is

added to the sub terminal N-acetyl glucosamine and thus forms Leb antigen.

Some precursor chains are not acted on by the Se fucosyltransferase but may accept L-

fucose from the Lewis fucosyltransferase forming Lea. Therefore, both Lea- soluble and

Leb – soluble antigens can be found in the secretions. However, only Leb adsorbs onto the

RBC from plasma. This is probably because higher concentrations of Leb in plasma allow

Leb – soluble antigen to compete more successfully for sites of adsorption onto the RBC

membrane. As a result, the RBCs of these individuals always phenotype as Le (a-b+), even

though both Lea and Leb soluble antigens are present in the secretions and plasma. In

secretors, Le and Se fucosyltransferases compete for the type 1 chain precursor. Se

glycosyltransferase catalyzes the synthesis of H on type 1 chain precursor structures. The

respective amount of Lea and type 1 H substances formed in secretions are determined by

the ratio of these two fucosyl transferases.

In nonsecretors (sese), only Lea antigens are formed in secretion, as no Se enzyme is

present in secretory cells. As a result, all type 1 chain precursor glycoproteinss are

available for the Le enzyme ∝-4-L-fucosyltransferase. Therefore, only Lea antigen is

secreted by the tissue cells and subsequently adsorbed onto the erythrocyte from plasma,

yielding the phenotype Le (a+b-).

The Lewis (a-b-) Phenotypes : Secretors or Nonsecretors

The lack of the Lewis antigens on the RBCs of this group is not caused by the absence of

Lewis gene (FUT3) but rather by specific point mutations in the Le gene. These mutations

give rise to a nonfunctional or partially active Lewis transferase (Lew) causing the negative


81

expression of the Lewis antigen on the RBCs. According to this finding, the Lewis antigen

is absent only on the RCBs tested serologically with the Lewis antisera; however, the

Lewis antigen is present in the tissues and secretions of the Le (a-b-) secretors.

The main substances found in the secretions of Le (a-b-) individuals depend on the

secretor status and the ABO genotype of the person. The Le (a-b-) nonsecretors express

type 1 precursor, whereas the Le (a-b-) secretors express H type 1 substances plus ABH

antigens associated with the related ABO genes inherited.

The lele genotype is much more common in blacks than in whites. All Le(a-b+)

individuals are ABH secretors and also secrete Lea and Leb. All Le(a+b-) individuals are

ABH nonsecretors, yet all secrete Lea.

In terms of Le (a-b-) individuals, 80 percent are ABH secretors and 20 percent are ABH

nonsecretors. The antigen expressed by Le (a-b-) secretors is Type 1 H (Led) substance,

whereas the antigen expressed by Le (a-b-) nonsecretors is type 1 precursor substance

(Lec).

Biochemistry of Lewis Antigens :60

The Lewis antigens or substances found in the secretions are glycoproteins. In plasma,

Lewis antigens are glycolipids ( glycosphingolipids). These antigens are carried by

lipoproteins present in plasma that adhere to RBC membranes, forming

glycosylceramides. The exact site for the synthesis of Lewis glycolipids in the plasma is

not known; however, it has been postulated that they may originate mainly from the

intestinal tract epithelial cells. Other exocrine organs such as the liver, kidney and

pancreas also contribute to the plasma glycolipids.

About one-third of the total Lewis glycolipids in blood are bound to the RBCs and the rest

are in plasma. Using plasma as a source of Lewis glycosphingolipids, Le (a-b-) RBCs

incubated with Lea- positive or Leb- positive plasma can be converted to Le (a+b-) or Le

(a-b+) depending on the substance present in the plasma. With saliva as a source of Lewis

substances, Le (a-b-) RBCs cannot be converted into Lewis positive phenotypes because


82

Lewis substances in saliva, being glycoproteins are not adsorbed onto the RBC

membranes.

The substances present in secretions and antigens present on RBCs, depending on Lele,

Sese and ABO genes inherited are listed in the following table.

Table 25 Substances Present in Secretions and Antigens Present on Red Cells, Depending on the Lewis, Hh, Se, and ABH Genes Inherited.60

Lewis gene-specified fucosyltransferase competes with A and B gene-specified enzymes

for the same type 1 H substrate. As a result, individuals who inherit, Le and Se genes have

more Lewis and fewer A or B plasma glycolipids than Se, lele individuals. Lea and Leb

antigens, once formed in secretions, can no longer be used as substrates for H and A or B

enzymes, owing to chain termination signaled by the Lewis transferase. As a result,

presence or absence of the Le gene affects the concentration of H, A and B type 1 chain

substances found in secretions. Le gene does not affect synthesis of type 2 chain H, A and

B soluble antigens found in secretions.

Development and changes of Lewis Antigens after Birth :60

In plasma there are no detectable Lewis glycosphingolipids at birth. Therefore, cord blood

and RBCs from newborn infants phenotypes as Le (a-b-). It is the low level of plasma

Lewis antigens that account for this fact, because the plasma of newborn infants is

incapable of transforming Le (a-b-) adult cells into Lewis-positive phenotype. When the

genotypes Le and sese are inherited, Lewis antigens (Lea and Leb) are not detectable on

cord RBCs, but these infants do secrete Lea substance in their saliva. Lewis

glycosphingolipids become detectable in plasma after approximately 10 days of life. In

individuals who inherit Le and Se genes, a transformation can be followed from the Le (a-

b-) phenotype at birth to Le (a+b-) after 10 days, to Le (a+b+) and finally to Le (a-b+), the


83

true Lewis phenotype after 6 to 7 years. In contrast, individuals who inherit Le and Sese

genes phenotype as Le (a-b-) at birth and transform to Le (a+b-) after 10 dyas. The Le

(a+b-) phenotype persists throughout life. Individuals with lele genes phenotype as Le (a-

b-) at birth and for the rest of their lives.

Changes in Lewis Phenotype :60

A decrease in expression of Lewis antigens has been demonstrated on RBCs from many

pregnant women, resulting in Le (a-b-) phenotypes during gestation. It has been suggested

that the physiologic changes in the composition of blood that affect the distribution of

Lewis glycolipid between plasma and RBCs are responsible for this phenomenon. The

large increase in the ratio of plasma lipoproteins to RBC mass that occurs during

pregnancy may also be responsible for Le (a-b-) phenotypes. In this instance, a greater

amount of Leb glycolipids would be bound to plasma lipoproteins instead of adsorbing

onto the RBCs. Lack of expression of Lewis antigens (Lea and Leb) has been demonstrated

on the RBCs of patients with cancer, alcoholic cirrhosis and viral and parasitic infections.

This transformation on Lewis-positive phenotypes to Lewis-negative phenotypes is caused

by abnormal lipid metabolism, changes in triglycerides and high density lipoproteins and /

or other neoplastic changes occurring in cancer patients. Other factors involved in the

expression of the Lewis phenotypes may be genetic, such as a single-point mutation

caused by Leucine to arginine substitution in the Lewis fucosyltransferase. This mutation

causes a decrease in adsorption of Lewis antigen onto RBCs from plasma, resulting in Le

(a-b-) phenotype.

Lewis antibodies :60

Antibodies to Lewis blood group antigens (anti-Lea and anti Leb) are frequently detected

in antibody screening procedures. Lewis antibodies are generally produced by Le (a-b-)

persons. Lewis antibodies are considered naturally occurring because they are present

without previous exposure to the antigen-positive RBCs. They are generally IgM in nature

and do not cross the placenta to cause hemolytic disease of the newborn (HDN). Because

they are IgM antibodies, these hemolysins can activate complement and therefore can

occasionally cause in vivo and in vitro hemolysis. Lewis antibodies are more reactive with

enzyme – treated cells than with untreated cells. Anti Lea and anti Leb may occur together

and can be neutralized by the Lewis substances present in plasma or saliva.


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Anti Lea :

Anti Lea is the most commonly encountered antibody of the Lewis system produced in 20

percent of individuals with Le (a-b-) phenotypes. The antibody is often of the IgM class;

however, some may have IgG components or may be entirely IgG. The IgG form of anti-

Lea does not bind to the RBCs as efficiently as does the IgM form and thereby is not

generally detected in routine blood bank procedures. The IgM form of Lea antibody binds

complement and therefore can cause in vivo and in vitro hemolysis. The antibody

reactivity is enhanced by enzyme-treated RBCs. The Lea antibody is frequently detected

with saline-suspended cells at room temperature. However, it sometimes reacts at 370 C

and Coombs phase and therefore can cause hemolytic transfusion reactions. Anti Lea is

easily neutralized with plasma or saliva that contains Lea substance. Persons who are Le

(a-b+) do not make anti Lea because the Lea antigen structure is contained within Leb

antigen epitope and Le (a-b+) persons have Lea substance present in their plasma and

saliva.

Anti Leb :

Anti Leb is not as common or generally as strong as anti-Lea. Although it is usually an IgM

agglutinin, it does not fix complement as readily as anti-Lea. Anti-Leb is usually produced

by a Le (a-b-) individual; only occasionally will an Le (a+b-) individual produce an anti-

Leb. Like anti-Lea, anti-Leb is neutralized by plasma or saliva containing Leb substance.

Anti-Leb can be classified into two categories anti-LebH and anti-LebL. Anti-LebH reacts

best when both the Leb and the H antigens are present on the RBC, such as group O and

A2 cells. Anti-LebH is probable an antibody to a compound antigen. In phenotyping RBCs,

especially A1 and A1B cells, the antiserum being used is not anti-LebH. Anti-LebL is the Leb

antibody that recognizes any Leb antigen regardless of the ABO type. Anti LebH can be

neutralized by either H or combined H and Leb substance. Anti-LebL is the antibody of

choice for phenotyping RBCs.

Neither anti-LebH nor anti-LebL is frequently implicated in hemolytic transfusion reactions,

and anti-Leb in general has no clinical significance.


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Anti Lex :

Anti Lex agglutinates all Le (a+b-) and Le (a-b+) RBC and is formed by all individuals

who are phenotyped as Le (a-b-). Anti Lex also agglutinates approximately 90% of all cord

blood initially phenotyping as Le (a-b-). The binding site of Lex antibodies is the smaller

disaccharide structure of fucose - ∝ (14) GlcNAc-R. The reactivity of the Lex

determinant is inhibited by the Lea glycolipid similar to the inhibition of normal anti Lea.

This occurs because the specifications of both antibodies are determined by a fucose -

∝(14) GlcNAc linkage, which is present in both the Lex determinant and Lea antigen as

a product of the Lewis ∝-4-L- fucosyltransferase.

Le (a-b-x+) cord cells do not react with anti-Lea or anti –Leb. This may be a result of the

hidden nature of the Lex determinant which is covered by the addition of many more

carbohydrates to the disaccharide chain. The fact that the reactivity of anti-Lex cannot be

separated using Le (a+b-) or cord RBCs indicates that this antibody is detecting a Lewis

precursor antigen present in the biochemical structure of Lea and Leb.

Other Lewis Antigens :60

The Lewis (a+b+) phenotype: Partial secretors.

It is postulated that a weak variant of the secretory gene (Se) referred to as Sew is

responsible for the expression of Le (a+b+) phenotype. The Le (a+b+) phenotype is

referred to as partial secretory phenotype and is frequently found in Polynesians,

Australians and Asians. Individuals with the Sew gene have a reduced amount of ABH

substances in their secretions because of the competition of Le and Se transferase for type

1 precursors. In non-group O individuals, there is even more competition for H type 1 by

A and B glycosyltransferase and therefore less Leb is formed. The Lewis (a+b+)

phenotype has been reported more in group O than in non-group O individuals. Detection

of the Le (a+b+) phenotype is also dependent on the potency and type of the antisera used.

Weak expression of secretor transferase (Sew) is caused by point mutations in the coding

region of FUT2.


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Lec and Led (Type 1 precursor and Type 1 H structure):

Lec represents Type 1 precursor structure and Led is type 1 H structure. There are two

types of Lec substances referred to as single Lec and branched Lec.

Lec present in Saliva, Plasma, and on RBC is a combination of a branched structure and

unbranched. The branched structures are the main glycolipids on RBCs in Le (a-b-) non

secretors and cause agglutination using polyclonal antibodies. The branched structures are

either absent or poorly detected on Le (a-b-) cord cells because of the lack of specificity of

anti-Lec antibodies for the cord cells. Lec in its unbranched single form

(lactotetraosylceramide) does not cause agglutination on the RBCs with polyclonal

antibodies. Single Lec is the precursor to Lea and Led (type 1 H). Led is formed by the

action of the Se transferase on type 1 precursor substance (Lec).

Lec is analogous to Lea and secreted only by ABH nonsecretors (sese). Lec is adsorbed

onto the RBC only after exposure to the plasma or secretions containing the soluble

antigen. This close similarly between Lea and Lec accounts for the observation that the Le

(a-b-) individuals who produced anti Lea are all ABH secretors with an Le (a-b-c-d+)

phenotype. The presence of Lec in Le (a-b-) ABH nonsecretors probably prevents the

formation of anti- Lea, in as much as Lec and Lea are biochemically very similar. Saliva

from these Le (a-b-c+d-) ABH nonsecretors has been found to contain Lec soluble

antigens.

Led antigens is analogous to the Leb antigen and is found only in Le (a-b-) secretors; its

correct phenotype is Le (a-b-c-d+). Led is also thought to be biochemically similar to Leb.

All Oh (Bombay phenotype) individuals have a phenotype of either Le (a+b-), if the Lewis

gene (Le) is inherited, or Le (a-b-), if the Lewis genotype is lele.

Interaction among ABO, H, Se and Le genes results not only in the formation of Lea and

Leb substances but also in the compound antigen products ALeb and BLeb.


87

This has been confirmed because anti A1Leb antibody has been described in the Lewis

system. The compound antibody reacts only with RBCs that possess both the A1 and the

Leb antigenic determinants and is belived to be the result of genetic interaction among the

A1, Le, H and Se genes.

Lex and Ley :

Lex(X) is referred to as a type 2 isomer of Lea which is formed by the addition of fucose -

∝ (13) GlcNAc linkage on the type 2 chain. This Lex is the same antigenic structure

called stage- specific embryonic antigen-1 (SSEA-1).

Ley (Y) is defined as a type 2 isomer of Leb and is made by addition of fucose - ∝ (13)

GlcNAc linkage on type 2 H substance.

Other Lewis-related type 2 structures are ALex (AX), ALey (AY) and BLey (BY). ALey

and BLey are important markers of cell membrane.

Lewis and secretor fucosyltransferases can use Type 1 and type 2 precursor substances to

form type 1 and type 2 antigens; however type 2 antigens are primarily made by other ∝-

3- fucosyltransferases, such as FUT4, FUT5, FUT6 and FUT7.

Clinical Significance of Lewis Antibodies:60

Although some cases of hemolytic transfusion reactions caused by anti- Lea have been

reported and there have been causes of in vivo RBC destruction due to anti-Leb, Lewis

antibodies are generally considered insignificant in blood transfusion practices.

This is because :

1) Lewis antibodies can be neutralized by the Lewis substance present in the plasma and

can be thereby be decreased in quantity.


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2) The Lewis antigens dissociate from the RBCs as readily as they bind to the RBCs. The

Lewis-positive donor RBCs can become Lewis-negative RBCs following transfusion

into an individual with a Lewis-negative phenotype. These antigens released into the

plasma can further neutralize any Lewis antibodies present in the recipient plasma.

3) Lewis antibodies are generally IgM and therefore cannot cross the placenta and cause

HDN. In addition, Lewis antigens are not fully developed at birth.

For these reasons, the presence of Lewis antibodies in a patient’s serum, does not require

transfusions of Lewis negative RBCs, as long as pretransfusion tests performed at 370C

and Coombs phase are compatible and there is no evidence of in vitro hemolysis. The

Lewis antibodies reactive at 370C and antihuman globulin phase, however should not be

ignored because these antibodies can cause in vivo RBC destruction. In the presence of

multiple antibodies, Lewis antibodies often complicate antibody identification, but they

can be easily inhibited with Saliva from secretors or with commercially available Lewis

substance.

Biological Significance of the Lewis System :60

Although the Lewis system is not considered a significant system in transfusion medicine

it has significance at the tissue level for the establishment of a biologic relationship

between blood group antigens and disease.

Lewis system is associated with factors causing certain disease, such as peptic ulcers,

ischemic heart disease, cancer and kidney transplant rejection. In addition, Lewis antigens

have receptors to interact with micro organisms expressing a particular lectin, for eg. Leb

has receptors for Helicobacter pylori.

Altered expression of Lewis antigens such as sialylated Lewis a and sialylated Lewis x

have been associated with different types of carcinomas and their related prognoses. Sialyl

Lex and Lea antigens are found to serve as ligands mediating adhesion of cancer cells to

the endothelium of blood and lymphatic vessels, which contributes to the tumor metastatic

process.


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THE KELL (ISBT No. 006) AND Kx (ISBT No. 019)

BLOOD GROUP SYSTEM :

Introduction:61

The Kell blood group system consists of 46 high incidence and low incidence antigens; it

was the first blood group system discovered after the introduction of antiglobulin testing.

Anti-K (originally called Kell) was identified in 1946 in the serum of Mrs. Kellaher. The

antibody reacted with the RBCs of her newborn infant, her older daughter, her husband

and about 7 percent of the random population. In 1949 Levine et al described anti-

k(Cellano), the high-incidence antithetical partner to K. Kell remained a two antigen

system until Allen and Lewis and Allen et al described the antithetical antigens Kpa and

Kpb in 1957 and 1958 respectively. Likewise, JSa described in 1958 by Giblett and JSb

described by Walker et al in 1963 were found to be antithetical and related to the Kell

system.

The discovery of the null phenotype in 1957, designated K0, helped associate many other

antigens with the Kell system. Antibodies that reacted with all RBCs except those with the

K0 phenotype recognized high-incidence antigens that were phenotypically related. More

discoveries followed, including the description of the McLeod phenotype by Allen et al in

1961. This phenotype, with its weakened expression of Kell antigens and pathologic

syndrome, brought into focus another antigen, Kx made by a gene on the X chromosome.


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Table 26 Kell and XK Blood group antigens. 62

Basic Concepts :63

For many years it was believed that Kell blood group antigens are found only on RBCs.

They have not been found on platelets or on lymphocytes, granulocytes or monocytes by

means of using immunofluorescent flow cytometry. There is recent evidence, however,

that Kell glycoprotein is abundantly present in testes, and to a lesser extent, in other

tissues.

The K antigen can be detected on fetal RBCs as early as 10 weeks (k at 7 weeks) and is

well developed at birth. The total number of K antigen sites per RBC is quite low:

Hughes-Jones and Gardner found about 6000 sites on K+ k- RBCs and only 3500 sites on

K+ k+ RBCs, although others have found up to 18000 sites per RBC. Despite its lower

quantity, K is very immunogenic.


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The antigens are not denatured by routine blood bank enzymes ficin and papain but are

destroyed by trypsin and chymotrypsin when used in combination. Thiol-reducing agents,

such as 100-200 mM DTT, 2- mercaptoethanol, AET and ZZAP (which contains DTT in

addition to enzyme), destroy Kell antigens but not Kx. Glycine acid – EDTA (an IgG

removal agent) also destroys Kell antigens.

Table 27 Phenotype frequencies in the Kell system.64

K and k Antigens:65

Excluding ABO, K is rated second only to D in immunogenicity. When K- people are

transfused with a unit of K+ blood, the probability of their developing anti K may be as

high as 10 percent. Fortunately, the incidence of K antigen is low and the chance of

receiving a K+ unit is small. If anti – K develops, compatible units are easy to find.

Antibodies to K antigens are seldom encountered. Only 2 in 1000 individuals lack k and

are capable of developing the antibody. The likelihood that these few individuals receive

transfusions and become immunized is even less.


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Kpa, Kpb and Kpc Antigens :65

Alleles Kpa and Kpb are low – incidence mutations of their high-incidence partner Kpb. the

Kpa antigen is found in about 2 percent of whites. The Kpa gene is associated with,

suppression of other Kell antigens on the same molecule, including k and JSb. The effect

appears to result from a reduced amount of the Kell glycoprotein (produced by the Kpa

allele) inserted in the RBC membrane.

The Kpc antigen is even more rare. In 1979 Yamaguchi et al discovered several siblings

from consanguineous married couple in Japan who typed Kp (a-b-) but otherwise had

normal Kell antigens. The researchers concluded that both parents carried a new allele,

Kpc, for which the children were homozygous. Gavin et al then showed that Kpc and

Levay, the first low-incidence antigen ever described, were identical.

Jsa and Jsb Antigens:65

The Jsa antigen, antithetical to the high-incidence antigen Jsb, is found in about 20 percent

of blacks but in fewer than 0.1 percent of whites. The incidence of Jsa in blacks is almost

10 times greater than the incidence of the K antigen in blacks. Jsa and Jsb were linked to

the Kell system when it was discovered that K0 RBCs were Js (a-b-).

Anti – K :65

Outside the ABO and Rh antibodies, anti-K is the most common antibody seen in the

blood bank. Anti-K is usually IgG and reactive in the antiglobulin phase, but some

examples agglutinate saline-suspended RBCs. About 20 percent bind complement, but

they are seldom lytic. The antibody is usually made in response to antigen exposure

through pregnancy and transfusion and can persist for many years.

Naturally occurring IgM examples of anti-K are rare and have been associated with

bacterial infections.


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Some examples of anti-K react poorly in methods incorporating low-ionic media, such as

LISS and in some automated systems. The most reliable method of detection is the indirect

antioglobulin test. Routine blood bank albumin or enzyme methods do not affect antibody

reactivity, but DTT, ZZAP, 2ME, AET and glycine-acid-EDTA treatments destroy Kell

antigens. The potentiating medium, PEG, may increase reactivity.

Anti-K has been implicated in severe hemolytic transfusion reactions. Although some

examples of anti-K bind complement, in-vivo RBC destruction is usually extravascular via

the macrophages in the spleen. Anti-K is also associated with severe HDN. The antibody

titer does not always accurately predict the severity of disease; stillbirth has been seen

with anti-K titers as low as 64. Fetal anemia in anti-K HDN may be associated with

suppression of erythropoiesis due to destruction of erythroid precursor cells rather than

destruction of circulating antigen positive RBCs; Kell glycoprotein is expressed early on

fetal RBCs.

Antibodies to Kpa, Jsa and Other Low-incidence Kell antigens :65

Antibodies to the low incidence Kell antigens are rare because so few people are exposed

to these antigens. Because routine antibody detection RBCs do not carry low-incidence

antigens, the antibodies are most often detected through unexpected incompatible cross

matches.

The serologic characteristics and clinical significance of these antibodies parallel anti-K.

The original anti-Kpa ,was naturally occurring, but most antibodies are “immune”.

Antibodies to K, Kpb, Jsb and other High-incidence Kell antigens :65

Antibodies to high-incidence Kell system antigens are rare because so few people lack

these antigens. They also parallel anti-K in serologic characteristics and clinical

significance.


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The high-incidence antibodies are easy to detect but difficult to work with because most

blood banks do not have the antigen-negative panel cells needed to rule out other

alloantibodies nor do they have typing reagents to phenotype the patient’s RBCs.

Testing an unidentified high-incidence antibody against DTT- or AET- treated RBCs is a

helpful technique; reactivity that is abolished with DTT or AET treatment suggests that the

antibody may be related to the Kell system and enables the technologist to exclude

common alloantibodies. Caution is needed before assigning Kell system specificity until

antigen-negative RBCs are tested because DTT and AET also denature JMH and high-

incidence antigens in the LW, Lutheran, Dombrock, Cromer and Knops systems. Finding

compatible units for transfusion can be difficult, siblings and rare donor inventories are the

most likely sources. Patients with antibodies to high-incidence antigens should be

encouraged to donate autologous units and if possible, to participate in a rare donor

programme.

Biochemistry :65

The Kell antigens are located on 93-KD RBC membrane glycoprotein that consists of 731

amino acids and spans the membrane once. The N- terminal domain is intracellular, and

the large external C-terminal domain is highly folded by disulfide linkages. The Kell

glycoprotein is covalently linked with another protein, called Kx, by a single disulfide

bond. The Kx protein (440 amino acids and 37kD) is predicated to span the RBC

membrane ten times.

The Kell glycoprotein is a member of the M13 (neprilysin) family of zinc endopeptidases

and has been shown to cleave big endothelins, but how this relates to the physiologic role

of the Kell glycoprotein remains unclear.


95

Fig. 18 Diagram of KELL and XK protein.


96

Genetics :65

The KEL gens located on chromosome 7 (at 7q33) is organized into 19 exons of coding

sequence. Single base mutations encoding amino acid substitutions are responsible for the

different Kell antigens. A rare silent allele at the KEL locus has been designated K0.

No KEL haplotype has been shown to code for more than one low-incidence antigen.

People who test positive for two low-incidence Kell antigens have always been found to

carry the encoding alleles on opposite chromosomes. For example, someone who types

Kp(a+) and JS(a+) is genetically kKpa JSb on one chromosome and kKpb JSa on other.

The XK gene, which encodes the KX antigen, is independent of KEL and is located on the

X chromosome at position Xp21.1.

The Kx Antigen :65

Kx is present on all RBCs except those of the rare McLeod phenotype. When Kell

antigens are denatured with AET or DTT, the expression of Kx increases. Because of this

inverse relationship, it was initially suggested that Kx might be a precursor or backbone

for Kell, and Kx was given the designation K15. However, it is now known the Kx is

carried on a separate protein that is encoded by a gene on the X chromosome. Kx has been

placed in its own blood group system called XK, and the number K15 is obsolete.

The Ko phenotype and Anti-Ku (K5):65

K0 is a silent Kell allele. Inheriting two K0 genes results in the recessive null phenotype.

Ko RBCs lack expression of all Kell antigens. Several mutations responsible for the Ko

phenotype have been identified in unrelated propositi. Ko cells have no membrane

abnormality and survive normally in circulation. The phenotype is rare; data suggest a

frequency of 1:25,000 in whites.

Immunized individuals with the Ko phenotype typically make an antibody called anti-Ku

(K5) that recognizes the “Universal” Kell antigen (Ku) present on all cells except Ko.

Anti-Ku appears to be a single specificity and cannot be separated into components. Anti-

Ku has caused both HDN and hemolytic transfusion reactions.


97

Because Ko RBCs are negative for k, Kpb, JSb and so forth, they are very useful in

investigating complex antibody problems. They can help confirm a Kell system specificity

or rule out other underlying specificities. When Ko cells are not available, they can be

made artificially by treating normal cells with DTT, AET or glycine-acid-EDTA.

Other Kell Antigens :65

K 11 and K 17 :

In 1971 Guevin et al described anti-Cote (now called anti-K11), which reacted with all

RBCs tested except those of the propositus, two of her eight siblings and Ko RBCs. The

antigen appeared to be phenotypically related to the Kell system. Three years later,

Strange et al noticed through family studies of K+Wk (a+) individuals that the low-

incidence antigen Wka was always inherited with k and not with K. WKa and K11 were

subsequently confirmed to be alleles by Sabo et al. Wka was given the name K17 to show

its placement into the Kell system.

K14 and K24 :

K14 is a high-incidence antigen phenotypically related to the Kell system. In 1985 its

antithetical low-incidence antigen, K24 was described.

Low – incidence Antigens with no known Antithetical partners: K10 (Ula), K23 and

K25 (VLAN).

K10 (Ula) is found in about 3 parents of random Finns, 0.46 percent of Japanese and 1 of

12 Chinese. A high incidence antithetical antigen has not yet been described.

K23 was detected in a maternal serum associated with a positive DAT on the RBCs of the

woman’s third child. The antibody was no longer reactive when the paternal RBCs were

treated with AET. Immunoprecipitation studies demonstrated the antigen was carried on

the Kell glycoprotein. Although the antibody in this case caused a strong positive DAT, it

did not cause clinical HDN.

K25 (VLAN) was initially identified through an incompatible cross match. Because the

antibody-antigen reactivity was abolished after the RBCs were treated with AET, a new


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Kell system antibody was suspected. VLAN was shown to be part of the Kell system

through the monoclonal antibody-specific immobilization of erythrocyte antigens

(MAIEA) assay.

High-incidence Antigens with No known Antithetical Partners : K12, K13, K18, K19,

K22, K26 and K27.

These discrete, very high-incidence antigens have all been shown to be governed by the

KEL locus and / or expressed on the Kell glycoprotein. None of the antigens are found on

K0 RBCs, and they are weakly expressed on McLeod phenotype RBCs. K:-18 is the only

Kell antigen that has not been shown to be inherited.

Miscellaneous Kell Antigen Assignments :

The antigen designations K8 (also called Kw), K9 (KL) and K15 (KX) are obsolete. The

specificities KL, Kx and Km (K20) are related. Kx and Km are high-incidence antigens

present on all RBCs with two exceptions : RBCs with the McLeod phenotype lack Kx and

Km; K0 RBCs lack Km but strongly express Kx. K16 is a high-incidence k-like antigen

seen on k+ RBCs but not expressed on McLeod RBCs; it is unclear if this effect is

qualitative or quantitative.

Table 28 Summary of Kell Antigens on RBCs having Common, K0, and McLeod Phenotypes.65

Altered Expression of Kell Antigens :65

Weaker than- normal kell antigen expression is associated with the McLeod phenotype

and the suppression by the Kpa gene (cis-modified effect) on Kell antigens (most obvious

when there is a K0 gene in trans). Weak expression of Kell antigens on RBCs of K: -13

individuals may be due to the cis effect, similar to Kpa, but the antithetical low incidence

partner has not yet been identified.


99

Depressed Kell antigens are also seen on RBCs with the rare Gerbich – negative

phenotypes Ge : -2, -3 and Ge : -2, -3, -4. The phenotypic relationship between Gerbich

and Kell is not understood.

Marsh and Redman proposed the umbrella term Kmod to describe other phenotypes with

very weak Kell expression, often requiring adsorption-elution tests for detection. As a

group, these RBCs have a reduced amount of Kell glycoprotein and enhanced Kx

expression. Some Kmod individuals make and antibody that resembles anti-Ku, but that

does not react with other Kmod RBCs (Unlike anti Ku made by Ko individual).

Patients with autoimmune hemolytic anemia, in which the autoantibody is directed against

a Kell antigen, may have depressed expression of that antigen. Antigen strength returns to

normal when the anemia resolves and the DAT becomes negative. This phenomenon

appears to be more common in the Kell system than in others.

RBCs may appear to acquire Kell antigens. McGinnis et al described a K – patient who

acquired a K-like antigen during a Streptococcus faecium infection. Cultures containing

the disrupted organism converted K- Cells to K+ but bacteria –free filtrates do not.

Autoantibodies :65

Marsh et al reported that 1 in 250 autoanibodies do not react with Ko RBCs and therefore

related to the Kell system. The actual frequency of these antibodies could be much higher,

because the study detected autoantibody with pure Kell specificity, not mixtures of Kell

with other autoantibodies. They may be benign or hemolytic.

Most Kell autoantibodies are directed against undefined high-incidence Kell antigens, but

identifiable autoantibodies to K, Kpb and K13 have been reported. Mimicking specificities

have been reported, such as when an apparent anti-K is eluted from DAT + K- RBCs and

the anti-K in the eluate can be adsorbed onto K-RBCs.


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THE DUFFY (008) BLOOD GROUP SYSTEM

Introduction:66 The Duffy blood group system was named for Mr. Duffy, a multiply transfused

hemophiliac who in 1950 was found to have the first example of anti Fya . One year later

the antibody defining its antithetical antigen, Fyb, was described by Ikin et al in the serum

of a woman who had three pregnancies.

In 1955 Sanger et al reported that the majority of African Americans Tested were

Fy (a-b-). The gene responsible for this null phenotype was called Fy. FyFy appeared to be

a common genotype in blacks, especially in Africa; the gene is exceedingly rare in whites.

In 1975 Miller et al made the observation that Fy (a-b-) RBCs resist infection in vitro by

the monkey malaria organism Plasmodium knowlesi. It was later shown that Fy (a-b-)

RBCs also resist infection by P. vivax.

Antibodies to the four additional antigens assigned to the Duffy blood group system, Fy3,

Fy4, Fy5 and Fy6 are rarely encountered. RBCs that are Fy (a-b-) are also Fy : -3, -5, -6.

Fy5 is also not present on Rh null RBCs, regardless of the Fya or Fyb status of those RBCs.

Basic Concepts :66

Fya and Fyb Antigens :

The Duffy antigens most important in routine blood bank serology are Fya and Fyb. They

can be identified on fetal RBCs as early as 6 weeks gestational age and are well developed

at birth. There are about 13,000 to 14,000 Fya or Fyb sites on Fy (a+b-) and Fy (a-b+)

RBCs, respectively; there are half that number of Fya sites on Fy (a+b+) RBCs. The

antigens have not been found on platelets, lymphocytes, monocytes or granulocytes, but

they have been identified in other body tissues, including brain, colon, endothelium, lung,

spleen, thyroid, thymus and kidney cells.


101

Table 29 Phenotype frequencies in the Duffy system.67

Fya and Fyb antigens do not store well in saline suspension and tend to elute from RBCs

stored in a medium with low pH or low ionic strength. This can lead to inhibitory

substances in the supernatant fluid, which can weaken the reactivity of an anti Fya or anti-

Fyb. These changes are not seen in RBCs stored in licensed anticoagulants or the reagent

solutions used by commercial manufacturers for reagent RBCs.

Fya and Fyb antigens are destroyed by common proteolytic enzymes, such as ficin, papain,

bromelin and chymotrypsin and by ZZAP; they are not affected by AET or glycine-acid-

EDTA treatment. Neuraminidase may reduce the molecular weight of Fya and Fyb, but it

does not destroy antigenic activity; neither does purified trypsin.

Anti Fya and Fyb :68

Anti Fya is a common antibody and is found as a single specificity or in a mixture of

antibodies. Anti Fya occurs three times less frequently than anti-K. Anti - Fyb is 20 times

less common than anti - Fya and often occurs in combination with other antibodies. The

Fya antigen appears to be more immunogenic in Fy (a-b+) whites than in Fy (a-b-) blacks.

The antibodies are usually IgG and react best at the antiglobulin phase. Some examples of

anti- Fya and anti-Fyb bind complement. A few examples are saline agglutinins. Antibody

activity is enhanced in a low ionic strength medium. Because anti- Fya and anti Fyb do not

react with enzyme treated RBCs, this is a helpful technique when multiple antibodies are

present. Some examples of anti- Fya and anti- Fyb show dosage, which may be more

obvious with examples of saline agglutinins. This is significant because some reagent

RBCs that appear to be from homozygote (and have a double dose of either Fya or Fyb)

may actually be from heterozygote if they are from black donors; a silent allele, Fy is

commonly found in blacks. For example, Fy (a+b-) RBCs will have a double dose of Fya if

they have from a white Fya Fya donor but will have a single dose of Fya if they are from a


102

black donor who is genetically Fya Fy. Additional phenotypic markers commonly found in

black donors can give a clue to the possible presence of the silent Fy allele : Ro, S-s-,

V+VS+, Js (a+), Le (a-b-).

Anti - Fya and anti- Fyb have been associated with acute and delayed hemolytic transfusion

reactions. Once the antibody is identified, Fy (a-) or Fy (b-) blood must be given; finding

such units in a random population is not difficult.

Anti Fya and anti Fyb are associated with HDN ranging from mild to severe.

Rare antibodies with mimicking Fya and Fyb specificity have been reported e.g. anti- Fya

that can be adsorbed onto and eluted from Fy (a+b-) RBCs. Issit and Anstee suggest that

these may represent alloantibodies with “sloppy” specificity made early in an immune

response.

Biochemistry :68

Duffy antigens reside on a glycoprotein of 336 amino acids that has a relative mass of 36

kD and two N- glycosylation sites. The glycoprotein is predicted to traverse the cell

membrane seven times and has tow predicted disulfide bridges.

The amino acid at position 42 on the Duffy glycoprotein defines the Fya and Fyb

polymorphism : Fya has glycine and Fyb has aspartic acid. The Fy3 epitope, as defined by

monoclonal antibody is on the third extra cellular loop and Fy6 appears to involve amino

acids 19 through 25.

The Duffy glycoprotein is a member of the super family of chemokine receptors and is

known as the Duffy antigen receptor for chemokines.


103

Fig. 19 Duffy glycoprotein or Duffy antigen receptor for chemokine molecule

(DARC).

Genetics :68

In 1968 the Duffy gene was linked to a visible inherited abnormality of chromosome 1,

thus becoming the first human gene to be assigned to a specific chromosome. The gene is

located near the centromere on the long arm of chromosome at position 1q22-23. The Fy

locus is syntenic to Rh, that is, they are on the same chromosome, but they are far enough

apart that linkage cannot be demonstrated and serologically they appear to segregate

independently.

There are three common alleles at the Fy locus : Fya and Fyb that encode the antithetical

antigens. Fya and Fyb, respectively and a silent allele Fy, that is the major allele in blacks.

The Fy gene in Fy (a-b-) blacks has been found to be an Fyb variant with a change in the

promoter region on the gene, which disrupts the binding site for mRNA transcription in

the RBC. Consequently, Fy (a-b-) blacks do not express Fyb on their RBCs but express Fyb

in other tissue. The presence of Fyb in tissues presumably precludes the recognition of Fyb


104

as foreign; thus, no anti-Fyb is made by these individuals. A molecular analysis of

Fy (a-b-) whites revealed a 14 base pair deletion in the Fy gene, resulting in a reading

frame shift and the introduction of a translation stop codon. These individuals carry no

Duffy protein on their RBCs or on other tissues and thus can form anti Fyb and anti Fy3 .

Typing for Duffy antigens has been performed on the RBCs of chimpanzees, gorillas and

old and new world monkeys. The results suggest that Fy3 developed first, then Fyb , and

that Fya arose during human evolution.

Fyx :- 68

Fyx was described in 1965 by Chown et al as a new allele at the Fy locus. It does not

produce a distinct antigen but, rather, an inherited weak form of Fyb that reacts with some

but not all examples of anti-Fyb. Fyx has been described in white populations. Individuals

with Fyx may type Fy (b-), but their RBCs adsorb and elute anti-Fyb. They also have

depressed expression of their Fy3 and Fy5 antigens. There is no anti-Fyx.

The decreased expression of Fyb due to Fyx appears to be related to a reduced amount of

Duffy glycoprotein on the surface of RBCs.

Fy3 Antigen and Antibody : 68

In 1971 Albrey et al reported finding anti- Fy3 in the serum of an Fy (a-b-) white

Australian female. It reacted with all RBCs tested except those of the Fy (a-b-) phenotype.

Because it was an inseparable anti- Fya Fyb, it was thought to react with an antigenic

determinant or precursor common to both Fya and Fyb and was called Fy3. Unlike Fya and

Fyb, the Fy3 antigen is not destroyed by enzymes.

Anti-Fy3 is a rare antibody made by Fy(a-b-) individuals who lack the Duffy glycoprotein.

The Fy(a-b-) phenotype has been found in white, black, and Cree Indian families. Blacks

with the Fy (a-b-) phenotype rarely make anti- Fy3. Examples of anti Fy3 produced by

non-blacks appear to react with all Duffy positive cells equally well. Those made by

blacks are similar, but they react weakly or not at all with Duffy positive cord RBCs.

There may be a subtle difference in the Duffy glycoprotein expressed on tissue that is

recognized as foreign. Some patients who make anti Fy3 initially make anti- Fya.


105

Fy4 Antigen and Antibody :68

In 1973 Behzad et al described anti-Fy4 in the serum of a young Fy (a+b+) black female

with sickle cell anemia. The antibody reacted with RBCs from all Fy (a-b-) blacks, many

Fy (a+b-) and Fy (a-b+) blacks, but not usually with Fy (a+b+) blacks ,and not with whites

of any Duffy type. It was concluded that most Fy (a-b-) blacks carry an Fy4 antigen,

perhaps in place of Fya, Fyband Fy3 and are genetically Fy4 Fy4. The Fy4 antigen, like Fy3,

is not destroyed by enzymes. No other example of anti-Fy4 has been reported, it is now

thought unlikely that Fy4 is located on the Duffy glycoprotein.

Fy5 antigen and Antibody :68

Anti Fy5 was discovered by Colledge et al in 1973 in the serum of an Fy (a-b-) black child

who later died of Leukemia. Initially it was thought to be a second example of anti- Fy3

because it reacted with all Fy (a+) or Fy (b+) RBCs but not with Fy (a-b-) cells. The

antibody differed in that it reacted with the cells from an [ Fy (a-b-) Fy:-3 white female ],

but it did not react with Fy (a+) or Fy (b+) Rhnull RBCs and reacted only weakly with Fy

(a+) or Fy (b+) D - - RBCs.

Sometimes, sera containing anti-Fy5 also contain anti- Fya. Several examples of anti- Fy5

have been reported in multiple transfused Fy (a-b-) sickle cell patients with a mixture of

other antibodies.

The molecular structure of Fy5 is not known, but it appears to be the result of interaction

between the Rh complex and the Duffy glycoprotein. People who are Fy (a-b-) and / or

Rhnull do not make Fy5 antigen and are at risk of making the antibody; although few do.

Like Fy3, Fy5 is not destroyed by enzymes.

Fy6 Antigen and Antibody :68

In 1987 Nichols et al described a murine monoclonal antibody that reacted much like anti-

Fy3, except that its reactivity was destroyed by ficin, papain and chymotrypsin. Trypsin

enhanced its reactivity. The antibody appeared to define the Duffy receptor used by

P.vivax to penetrate RBCs. It is now known that Fy6 involves amino acids 19 to 25 on the

extra cellular domain of the Duffy glycoprotein. No human examples of anti-Fy6 have

been reported to date.


106

The Duffy – Malaria Association :68

A correlation between the Duffy antigens and malaria infection has long been suspected.

Since 1955 it has been known that Africans and black Americans were resistant to

infection by P.vivax and that these some populations are Fy (a-b-).

Data from human and old and new world monkey RBCs and their susceptibility to

invasion by P.vivax and P.knowlesi indicate that Fy6 is important for invasion for P.vivax.

The monoclonal anti-Fy6 has been shown to block invasion of RBCs by P.vivax.


107

THE KIDD (009) BLOOD GROUP SYSTEM

Introduction:69

In 1951 Allen et al reported finding an antibody in the serum of a Mrs. Kidd, whose infant

had HDN. The antibody, named anti-Jka, reacted with 77 percent of Bostonians. Its

antithetical partner Jkb, was found 2 years later by Plaut et al. The null phenotype Jk(a-b-)

was described by Pinkerton et al in 1959. The propositus made on antibody to a high-

incidence antigen called Jk3, which is present on any RBC positive for Jka or Jkb.

No other antigens associated with the Kidd system have been descried.

Basic Concepts :

Table 30 Phenotypes of the Kidd System.70

There are notable racial differences in antigen frequency; 91 percent of blacks and 77

percent of whites are Jk (a+); 57 percent of blacks and only 28 percent of whites are

Jk (b-).

Jka and Jkb antigens are well developed on the RBCs of neonates. Jka has been detected on

fetal RBCs as early as 11 weeks; Jkb has been detected at 7 weeks. Although this early

development of Kidd antigens contributes to the potential for HDN, anti Jka and anti Jkb

are only rarely responsible for severe HDN. Jk (a+b-) RBCs carry 14,000 antigen sites per

cell. The Kidd antigens are not very immunogenic.


108

Kidd antigens are not denatured by papain or ficin; treatment of RBCs with enzymes

generally enhances reactivity with Kidd antibodies. Kidd antigens are also not affected by

chloroquine diphosphate, AET, DTT or glycine-acid-EDTA.

The antigens are not found on platelets, lymphocytes, monocytes or granuocytes by means

of sensitive radio immunoassay or immunofluorescent techniques.71

Anti-Jka and Anti-Jkb :71

Kidd antibodies demonstrate dosage, are often weak, and are found in combination with

other antibodies, all of which make them difficult to detect.

Anti Jka is more frequently encountered than anti Jkb, but neither antibody is common. The

antibodies are usually IgG (antiglobulin reactive) but may also be partly IgM and are made

in response to pregnancy or transfusion.

Many anti Jka and anti Jkb react more strongly with RBCs that carry a double dose of the

respective antigen and may not react with Jk (a+b+) RBCs. An anti Jka that reacts only

with Jk (a+b-) RBCs can give inconclusive panel results and appear compatible with Jk

(a+b+) cells. Anti Jka and anti Jkb should be ruled out only with Jk (a+b-) and Jk (a-b+)

panel cells respectively. To ensure that stored antisera can indeed detect weak expressions

of the antigen, Jk (a+b+) RBCs should be tested in parallel as the positive control.

Antibody reactivity can also be enhanced by using LISS or PEG (to promote IgG

attachment), by using four drops of serum instead of two (to increase the antibody /

antigen ratio), or by using enzymes such as ficin or papain. In-vitro hemolysis can

sometimes be observed with enzyme-treated RBCs; antigen dose may influence this

hemolytic activity.

Many examples of the Kidd antibodies bind complement. Rare examples are detected only

by the complement they bind (i.e. they are nonreactive in antiglobulin tests using anti-IgG

reagents). Using polyspecific reagents with both anti-IgG and anti complement can be

helpful in these situations.


109

Kidd antibodies that are complement-dependent do not store well. When anti Jka or anti

Jkb specificity cannot be confirmed in a stored serum, fresh normal serum ( as a source of

complement) can be added and tested with polyspecific antiglobulin reagent.

The titer of anti Jka or anti Jkb quickly declines in vivo. A strong antibody identified

following a transfusion reaction may be undetectable in a few weeks or months. This

confirms the need to check blood bank records for previously identified antibodies before

a patient is transfused.

The decline in antibody reactivity and the difficulty in detecting Kidd antibodies are

reasons why they are a common cause of hemolytic transfusion reactions, especially of the

delayed type. Although intravascular hemolysis has been noted in severe reactions, coated

RBCs more often are removed extravascularly. The rate of clearance of incompatible

RBCs can vary but is usually rapid.

Contrary to their hemolytic reputation in transfusion, most Kidd antibodies are only rarely

associated with severe cases of HDN.

Biochemistry :71

Heaton and McLoughlin reported in 1982 that Jk (a-b-) RBCs resist lysis in 2M urea. With

Jk (a+) or Jk (b+) RBCs, lysis in 2M urea occurs within 1 minute; with Jk (a-b-) cells,

lysis is delayed 30 minutes.

Sinor et al identified the Jka protein as a single band with a relative mass of 45 kD that was

not affected by reduction and alkylation and appeared not to be glycosylated. A cDNA

clone was subsequently isolated that produces the Kidd glycoprotein and RBC urea

transporter. The predicted glycoprotein has 389 amino acids with 10 membrane-spanning

domains and two N-glycosylation sites, one of which is extracellular.


110

Fig. 20 The Kidd/urea transporter glycoprotein. The site of Jka/Jkb polymorphism at residue 280 is indicated by a solid line. The N-glycosylation site is indicated by a branched structure on the third extracellular loop. The 10 transmembrane domains are represented by amber cylinders.

Genetics :71

Jka and Jkb are inherited as codominant alleles. The Jk locus is on chromosome 18 at

position 18q11-q12. The gene, a member of the urea transporter gene family, is organized

into 11 exons. The Jka/Jkb polymorphism is associated with an amino acid substitution at

position 280, predicted to be located on the fourth extracellular loop of the glycoprotein.

Molecular studies have demonstrated the silent Jk allele can arise from mutations in both

the Jka and Jkb alleles.

Jk (a-b-) phenotype and the Recessive Allele, Jk :

People with the null phenotype lack Jka, Jkb and the common antigen Jk3. Although very

rare, the Jk (a-b-) phenotype is most abundant among polynesians and it has also been

identified in Filipinos, Indonesians, Chinese and Japanese. The null phenotype has also

been reported in several European families (Finnish, French, Swiss and English) and in the

Mato Grosso Indians of Brazil. The delayed lysis of Jk (a-b-) RBCs in 2M urea has proved

an easy way to screen families and/ or populations for this rare phenotype.

No clinical abnormalities have been associated with the Jk (a-b-) phenotype to date.

Family studies show that most Jk (a-b-) nulls are homozygous for the rare “silent” allele

Jk. Parents of JkJk off spring and children of JkJk parents type Jk (a+b-) or Jk (a-b+) but


111

never Jk (a+b+); because they are genetically JkaJk or JkbJk. Their RBCs also demonstrate

a single dose of Jka or Jkb antigen in titration studies.

Jk (a-b-) phenotype and the Dominant In(Jk) Allele:

Okubo et al discovered a dominant pattern of inheritance within a Japanese family and

proposed the existence of a dominant inhibitor to the Kidd system, In(Jk). Dominant type

Jk (a-b-) RBCs adsorb and elute anti – Jk3 and anti Jka and/or anti Jkb (depending on

which genes were inherited), indicating that the antigens are expressed but only very

weakly. Individuals with the In (Jk) Jk (a-b-) phenotype do not make anti-Jk3. Family

studies show that the In(Jk) gene does not reside at the Jk locus.

Anti Jk3 :

Allo anti-Jk3 is an IgG antiglobulin-reactive antibody that looks like an inseparable anti

JkaJkb. Because panel cells are Jk (a+) or Jk (b+), anti Jk3 reacts with all RBCs tested

except the auto-control. Most blood banks do not have the rare cells needed to confirm

anti-Jk3; however, they can easily determine its most probable specificity by means of

antigen typing. The individual making the antibody will type Jk (a-b-). Like other Kidd

antibodies, anti Jk3 reacts optimally by an antiglobulin test, and the reactivity is enhanced

with enzyme pretreatment of the RBCs.

Anti-Jk3 has been associated with severe immediate and delayed hemolytic transfusion

reactions and with mild HDN. Compatible units are best found by typing siblings or

searching the rare donor files.

Auto antibodies :71

Auto antibodies with Kidd specificity (anti- Jka, anti- Jkb and anti Jk-3) are rare, but they

have been associated with autoimmune hemolytic anemia. Some examples are drug

related; one was found in a patient taking alpha-methyldopa (Aldomet); another was

Chlorpropamide dependent.

Examples of benign auto anti- Jka have been associated with butyl, ethyl, methyl and

propyl esters of parahydroxybenzoate or paraben. These chemicals are used in some


112

commercially prepared LISS, cosmetics, food preservatives and pharmaceuticals. The

antibodies are seen when paraben containing LISS is used in antibody detection tests with

Jk (a+) cells.

As with other blood groups, Kidd auto antibodies may have mimicking specificity or be

associated with depressed antigen expression.

Disease Associations :71

Although Jka and Jkb are thought to be human RBC antigens, three organisms have been

associated with Jkb-like specificity. Two Enterococcus faecium and Micrococcus, were

able to convert Jk (b-) cells to Jk (b+) and one Proteus mirabilis, may have been the

stimulus for an autoanti-Jkb.


113

DIEGO BLOOD GROUP SYSTEM (ISBT 010)

Introduction:72

The Diego blood group was first described in 1955 in a case of HDFN and named after

Mrs. Diego, the person who made the antibody to the low-incidence antigen Dia. The

antithetical antigen Dib was described in 1967. In 1953, a low-incidence antigen named

Wra was found, but the association with the Diego blood system was not apparent. The

Diego system has implications in population genetics because the Dia antigen is found in

people of Mongolian descent.

Biochemistry and Genetics :

The SLC4A gene, located on chromosome 17, contains 20 exons. The Diego protein is a

transmembrane glycoprotein spanning the membrane 14 times. The protein accounts for

25% of total red cell protein.

Fig.21 Structure of anion exchange protein AE1 (Band 3).

The Diego antigens are carried on the erythroid band 3 protein(anion exchange 1 or AE1).

The long N- terminus is in the cytoplasmic region and functions as an anchor point for the

membrane skeleton through interaction with peripheral membrane proteins. It also serves

as a binding site for hemoglobin and glycolytic enzymes. The C-terminus is cytoplasmic

and binds carbonic anhydrate II (CA II). Carbon dioxide is hydrated to bicarbonate by CA

II. Band 3 acts as ion exchanger that permits bicarbonate ions to cross the membrane in


114

exchange for chloride ions. Band 3 deficiency results in membrane surface area loss and

the generation of spherocytic red cells. The genetic back ground to the polymorphisms is a

single – nucleotide change in the band 3 gene that gives rise to an amino acid substitution.

In addition, for at least the Wrb antigen, an interaction of the band 3 protein and GPA is

required for expression.

Dia and Dib :

There are 21 antigens associated with the system. The first antigens described were the

low-incidence antigen, Dia and the antithetical high-incidence antigen, Dib. The Dia

antigen is very rare except in Chinese, Japanese, and native peoples of North and South

America where it can approach 54% frequency. The Dia and Dib antigens are located on

the seventh extracellular loop of band 3. The single amino acid substitution changes from

leucine for Dia at position 854 to proline for Dib.

Table 31 Diego Blood Group Antigens. 73

Table 32 Dia / Dib Phenotype Frequencies.74

Phenotypes Caucasians / Blacks Asians / South Americans Indian

Di (a-b+) >99.9% 64-90%

Di (a+b+) Rare 10-36%

Di (a+b-) Very rare rare


115

Wra and Wrb :74

A new antibody responsible for a fatal case of HDFN was first described by Holman in

1953. The low incidence antigen Wra, was named after the antibody maker, Mrs. Wright.

Further examples of anti- Wra causing severe transfusion reactions appeared after. The

high-incidence antibody defining Wrb was discovered in 1971 in the serum of a woman

who has positive for the low-incidence Wra antigen. Her antibody was provisionally called

anti- Wrb. In 1988, the antigens were proven to be antithetical to each other. Early

serologic studies initially showed an association with GPA, the glycoprotein that carries

the MNS system antigens. Wrb expression is dependent on the presence of GPA. En (a-)

cell, which lack GPA, type as Wr (a-b-). Other GPA variants are also associated with the

lack of expression of Wrb. It now appears that the Wrb antigenic structure is formed by

both GPA and band 3 protein sequences.

Low-incidence Diego Blood Group Antigens :74

As of 1995, there were 37 low-incidence antigens listed in the ISBT 700 series of antigens

not assigned to a particular blood group system. Researchers suspected that more antigens

could be linked to the Diego blood group system because of the size and amount of band 3

proteins on red cells. They began looking at various antigens in the 700 series to see if

there was a connection. Seventeen antigens have now been assigned to the Diego system.


116

Table 33 Other low frequency antigens of the Diego system and their frequencies in various populations.75

The location of the antigens on the molecule has been determined. There are seven loops

in the extracellular area of the band 3 protein. ELO is located on loop 1 and Fra is on

loop 2. Eleven antigens are located on loop 3 with six more antigens on loop 4. Dia and

Dib are on the seventh loop. It is thought that the closeness of the antigens on the loops

may account for some of the characteristics of the antigens and antibodies that define

them.


117

Diego Antibodies :76

Nineteen of the 21 Diego antigens are of low incidence. The likelihood of being transfused

with a unit positive for one of the antigens is rare and the antibodies are rarely

encountered. With the exception of ELO, they have not been implicated with HDFN. The

first report of anti-ELO causing HDFN occurred in 1992 with a second report in 1993

when the same patient had another baby. It has been noted that some individuals have

antibodies to several low-incidence antigens. BOW and NFLD each arise from amino acid

substitutions at position 561. The substitution for Jna and KREP is found at position 556.

The assignment of so many low-frequency antigens to the band 3 protein helps explain

some observations among reference laboratory workers. In addition, some of the

specifications are Rh- and GPA related further indicating the relationship between band 3,

GPA and Rh proteins.

In routine pre-transfusion testing, the antigens are not likely to be carried on the screening

cells. In addition, if immediate spin or computer crossmatches are performed, the

individual with an antibody to one of the low-incidence antigens will generally not be

detected. Some of the antibodies are naturally occurring and are of no clinical significance

for transfusion.

Anti Dia and anti-Wra have caused severe and fetal HDFN and have been involved in

immediate and delayed transfusion reactions. Anti-Dia is rare in most populations, but

ethnicity should be considered during an investigation of an antibody to a low- incidence

antigen. In contrast, anti-Wra is commonly found as an apparent, naturally occurring

agglutinin or as an immune IgG antibody with other antibodies to low incidence antigens

and associated with autoimmune hemolytic anemia. In the IgG form, anti-Wra is

associated with HDFN and HTR.

Antibodies to the high-incidence Dib antigen cause problems in locating compatible blood

because of the rarity of the negative phenotype. The antibody has caused mild HDN,

moderate immediate and delayed transfusion reactions. Not much is known about anti-Wrb

as few individuals have been described with the antibody, but it is suggested that it could

cause accelerated destruction of antigen-positive red cells.


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THE CARTWRIGHT BLOOD GROUP SYSTEM YT (ISBT 011)

Introduction:77

The Yt blood group system has historically been referred to as the Cartwright system. It

was first described in 1956 when anti-Yta was first described. It consists of two antithetical

antigens, Yta and Ytb. The carrier molecule for the antigens is an enzyme, GPI-linked

glycoprotein called acetylcholinesterase (AChE).

The genetic locus for Yt is on chromosome 7. A single nucleotide mutation causes an

amino acid change from histidine for Yta to asparagine for Ytb at position 353. The

mutation does not have any effect on the overall structure of AChE nor on its functions in

nervous tissues.

The Yta antigen is a high-frequency antigen found in 99.8% of populations. Ytb is

infrequent, except in Israeli Jew, Israeli Arabs and Israeli Druse where the incidence can

be as high as 26%. A transient null phenotype has been described in a patient that

developed a probable antibody to AChE. The loss of AChE coincided with the loss of Yta

antigen. As AChE levels returned to normal, the antigen was detected.

Table 34 Yt system Phenotype Frequencies.77

Phenotype Frequencies (%)

Yt (a+b-) 91.9

Yt (a+b+) 7.8

Yt (a-b+) 0.3

Yt (a-b-) Transient – extremely rare

GPI – linked molecules and PNH :

AChE is an example of a carrier molecule for blood group antigens that is linked to the red

cell membrane by a glycosyl phosphatidylinositol (GPI) anchor. The acquired

hematopoietic stem cell disorder paroxysmal nocturnal hemoglobinuria (PNH) results

from the absence or deficiency in expression of GPI-linked proteins. All red cell antigens

that are carried on a GPI-linked carrier molecule will not be expressed in PNH.


119

Even though the Yt (a-b+) phenotype is relatively low, many examples of anti-Yta have

been described. Ytb appears to be a poor immunogen and is rarely seen except in

individuals who make other blood group antibodies. Both antibodies are the result of

transfusion or pregnancy and are not known to be naturally occurring. They are generally

not implicated in transfusion reaction or HDFN. In fact, there are several reports of

woman with anti-Yta bearing a Yt (a+) child and no indication of HDFN. The same holds

true for a woman with anti-Ytb bearing a Yt (b+) child. Some patients with anti-Yta have

received transfusions of Yt (a+) blood with no ill effects while others showed evidence of

decreased red cell survival.

The antigens are sensitive to ficin, papain and DTT. The antibodies are IgG, reacting

optimally at IAT. Some Yta antibodies may cause complement binding, but anti Ytb does

not. Consequently, the clinical significance of Yt antibodies has been the subject of

research.


120

Xg BLOOD GROUP SYSTEM (ISBT 012)

Introduction:78

The Xg blood group system consists of two antigens-Xga and CD99. This is the only blood

group whose genetic locus is assigned to the X chromosome. The Xga antibody was first

described in 1961 in a male who had received many transfusions. An antithetical antigen

to Xga has not been found; thus the phenotypes are Xg (a+) and Xg (a-) .

In females, the Xga and CD99 antigens escape X-chromosome inactivation. The carrier

molecules for both Xga and CD99 are single-pass glycoproteins with an exofacial N-

terminus. The function of the Xga molecule is unknown but CD99 functions as an

adhesion molecule. The gene encoding the antigen is PBDX located on the X

chromosome.

Xga :78

Because the Xg locus is on the X chromosome, females can be XgaXga, XgXg or XgaXg.

Males can be XgaY or XgY. The genotypes XgaXga, XgaX and XgaY all result in the Xg (a+)

phenotype. XgXg females and XgY males have the Xg (a-) phenotype. Because the XG

genetic locus is on the X chromosome, the phenotype frequencies are different. The

prevalence of Xga is 66% in males are 89% in females.

Anti-Xga is an uncommon antibody, is IgG in origin, and detectable by IAT. It is usually

red cell stimulated but some are naturally occurring. Anti-Xga rarely occurs with other

alloantibodies. It is not associated with HTRs or HDFN. The antigen is sensitive to ficin

and papain, but resistant to DTT.

CD99: 78

CD99 is the other antigen of the system. It became part of the Xg system, because the two

genes, MIC2 and XG are adjacent and homologous to each other. The frequency of the

antigen is >99%. Two CD99 individuals have been reported with the antibody. The

antibody is IgG reacting at IAT. It is sensitive to ficin and papain, but resistant to DTT.

There are not enough data to determine whether it is associated with HTR or HDFN.


121

SCIANNA BLOOD GROUP SYSTEM (ISBT 013)

Introduction: 79

The first Scianna antibody was reported in 1962 and called anti-Sm. An antibody defining

a new antigen Bu-a was reported in 1963. At the time of publication, neither group was

aware of the findings of the other so there was no exchange of materials. In 1964, the

possible relationship of the two antigens was described. By 1974, the relationships were

established and the system was named after the original antibody maker.

Scianna Antigens :

Table 35 Scianna Blood Group Antigens. 80

There are five antigens recognized to be part of the Scianna system. Sc1 and Sc3 are high-

incidence antigens, while Sc2 is a low-incidence antigen. Sc3 is expressed on all RBCs

except those of the rare

Sc1- and Sc2- negative phenotype. The antigens are expressed on the ERMAP protein

(Erythroblast Membrane – associated protein). The function of ERMAP is not clear at this

time. Another low-frequency antigen Sc4 or Rd (Radin), is expressed by a variant ERMAP

protein. The high-prevalence antigen, Sc5 or STAR, occurs because of an amino acid

change in the extra cellular portion of ERMAP.

Scianna Antibodies :

The phenotype frequency in Caucasians of the Sc:1, -2 phenotype is 99.7% so anti-Sc1 is

rarely seen. The antibodies are rare and are not associated with HTR or HDFN, but anti

Sc2 has caused a positive DAT.


122

DOMBROCK BLOOD GROUP SYSTEM (ISBT 014) Introduction: 81 The first example of a Dombrock antigen was found in 1965 when anti-Doa was found in

an individual, Mrs. Dombrock, who had been multiple transfused. In 1973, anti-Dob was

described and Dob was noted to be the antithetical to Doa. Three high-prevalence antigens

have been assigned to the Dombrock System : Gregory (Gya), Holley (Hy) and Joseph

(Joa).

The carrier molecule is a GPI-linked, single pass molecule with an unknown function at

this time. The sequence of the protein suggests that it transfers adenosine diphosphate

(ADP) ribose to various protein receptors, but it is not known if this is active on red cells.

In fact, individuals with the absence of the glycoprotein, characterized by the Gy (a-)

phenotype are not clinically affected. Like other GPI-linked antigens, Dombrock antigens

are not expressed on PNH III cells. ART4, the gene that encodes the Dombrock

glycoprotein is found on chromosome 12.

Dombrock Antigens :

Table 36 Phenotypes of the Dombrock System. 82

Table 37 Dombrock system phenotype frequencies : 83

Phenotype Doa Dob Gya Hy Joa Whites (%) Blacks (%) Do (a+b-) + - + + + 18 11 Do (a+b+) + + + + + 49 44 Do (a-b+) - + + + + 33 45 Gy (a-) - - - - - Rare 0 Hy (a-) - +w +w - - 0 Rare Jo (a-) +w +/-w + +w - 0 Rare


123

Doa and Dob are Co dominant alleles that differ in three nucleotide positions that encode

Doa and Dob, respectively. Doa has aspartic acid at position 265 while Dob has

asparagines. The high- prevalence Hy antigen has glycine at position 108 and is associated

with the Do (a-b+) phenotype. Another high-frequency antigen is Joa which has threonine

at position 117. There is a null phenotype in the system called Gy (a-) or Donull. There are

several different mutations that cause the null phenotypes. The antigens are resistant to

enzymes and sensitive to DTT.

Dombrock Antibodies :

Antibodies to Dombrock antigens are not common and often present with other antibodies.

They appear to be clinically significant, but only mild HDN with a positive DAT has been

reported.


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COLTON BLOOD GROUP SYSTEM (ISBT 015)

Introduction: 84

An antibody to a high frequency antigen was reported in 1967 and called anti-Coa. Anti-

Cob was reported in 1970, and the Co (a-b-) phenotype was described in 1974. The Colton

antigens are carried on a multipass membrane glycoprotein. Aquaporin 1 (AQP1)

responsible for water homeostasis and urine concentration. The gene, AQP1, is located on

chromosome 7.

Fig. 22 Structure of aquaporin-1 (AQP-1, channel-forming integral protein [CHIP]). AQP-1 is a multipass protein containing six transmembrane domains,indicated by solid amber cylinders.

The Colton polymorphism is determined by an amino acid substitution at position 45 of

the protein. Alanine is present when the Coa antigen is expressed and valine is present

when Cob is expressed.

Coa is the high-frequency antigen present in 98% of the population, while Cob is the

antithetical antigen present in 8% of the population. A third antigen Co3 is always present

when Coa and / or Cob are expressed. Co3 is not present on Co (a-b-) red cells. These red

cells also lack or have low amount of aquaporin 1; however, there does not seem to be a

medical condition associated with it.


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Colton Antibodies : 84

Antibodies to Colton antigens have caused both mild to moderate HTRs and mild to

severe (rare) hemolytic disease of the newborn. Anti Co3 is only seen in the null

phenotype. The antibodies are IgG in nature and some have been shown to bind

complement. The antigens are resistant to the effect of enzymes and chemicals.


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CHIDO / ROGERS BLOOD GROUP SYSTEM (ISBT 017)

Introduction: 85

The antigens of the Chido / Rogers Blood Group system are located on the fourth

component of complement (C4) instead of a red cell structure. Before the antigens were

associated with C4, they were considered to be like other antigens because they were

detected on red cells with blood grouping reagents and thus were adopted as a system. The

first antigen, Ch was reported in 1967 when the antibody was responsible for cross

matching difficulties. The Rga antigens was later described in 1976 when an antibody was

reacting with 97% of individuals in the British population.

Chido / Rogers Antigens :

Table 38 Chido/Rodgers Blood Group Antigens. 86

The system has nine antigens, which are found on red cells and in plasma. There are six

high-frequency Ch antigens (Ch1 through Ch6) and two high- frequency Rg antigens (Rg1

and Rg2). The ninth antigen, WH, is a hybrid antigen and is associated with Ch : 6,

Rg : 1, -2 phenotype. 87

Chido / Rogers Biochemistry and Genetics : 87

The antigens are located on the C4d fragment of the fourth component of complement

(C4) and adsorbed onto the red cell membrane. The two components to C4, C4A and C4B

are identical in their amino acid sequences, but C4A binds to protein while C4B binds to

carbohydrate. The Chido antigen is found on C4B, while the Rogers antigen is on C4A.

The C4 molecule is a glycoprotein composed of three disulfide – linked polypeptide

chains, ∝, β and γ. The mechanism for the antigens to become bound to the red cells


127

occurs in the course of the classical pathway of complement activation. C4 becomes

activated by one of two mechanisms and binds to the red cell as C4b. The C4b undergoes

proteolytic degradation to split into two fragments, C4c and C4d. C4d, which contains the

antigens, remains attached to the red cell membrane.

The antigens are inherited by two closely linked genes, C4A and C4B, which both encode

the isotypes. The genes are located on chromosome 6.

Ch / Rg Antibodies : 87

The antibodies are usually IgG in nature and react best at IAT induced by blood

transfusion in Chido or Rogers individuals. They usually react weakly and if titration

studies are performed have a high titer with continued weak reactivity. In addition, the

reactions are not reproducible, causing difficult identification. These antibodies are rarely

significant and are not implicated in HTR or HDFN.


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Kx BLOOD GROUP SYSTEM (ISBT 019)

Biochemistry and Genetics: 88

The Kx antigen is carried on the XK protein encoded by the XK gene located on the short

term of the X chromosome. The glycoprotein spans the membrane 10 times with both the

N- and C- terminal domains in the cytoplasm. It has structural characteristics of a

membrane transport protein. The XK protein is linked to the Kell glycoprotein by a single

disulfide bond. Absence of the protein is associated with abnormal red cell morphology

and late-onset forms of nerve and muscle abnormalities also known as the McLeod

syndrome. There is a decrease of Kell antigen expression in individuals missing the Kx

antigen.

GERBICH BLOOD GROUP SYSTEM (ISBT 020) Introduction :89

The Gerbich blood group system was first reported in 1960 and named after the original

person who made the antibody, Mrs. Gerbich. A year later, another anti-Ge was reported

in an individual named Mrs. Yus. The serum of Mrs. Yus was compatible with the red

cells of Mrs. Gerbich, but the serum of Mrs. Gerbich was incompatible with the red cells

of Mrs. Yus. Later a third type called Leach was introduced to the confusion.

Biochemistry and Genetics :

Table 39 Gerbich Blood Group Antigens. 90

The Gerbich blood group system antigens are carried on the single – pass membrane

proteins Glycophorin C (GPC), Glycophorin D (GPD) or both. A single gene on


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chromosome 2, GYPC, produces the highly glycosylated glycophorins. The two

glycophorins are identical except that GPD lacks the 21 amino acids at the terminal

sequence of GPC. Both glycophorins play a role in maintaining red cell integrity by an

interaction with protein 4.1 in the cytoplasm.

Fig.23 Gerbich antigens on glycophorin C (GYPC) and glycophorin D (GYPD). O-linked and N-linked glycans are shown. Also shown is the relationship of the GYPC gene to both proteins.

Three high-incidence antigens are associated with the Gerbich system- Ge2, Ge2 and Ge4.

GPC carries Ge3 and Ge4 while GPD carries Ge2 and Ge3. The five low-incidence

antigens are Ge5 (Wb), Ge6 (Lsa), Ge7 (Ana), Ge8 (Dha) and GEIS. The lack of one or

more of the high-incidence antigens results in three types of Gerbich-negative phenotypes.

The Leach phenotype results from absence of Ge2, Ge3 and Ge4 and is the null phenotype

(Ge:-2,-3,-4). The Gerbich phenotype Lacks Ge2 and Ge3 (Ge:-2,-3, 4) while the Yus

phenotype lacks Ge2 (Ge:-2,3,4). 91

Table 40 Gerbich Negative Phenotypes. 92

Phenotype Type Antibody

Ge : -2, 3,4 Yus Anti-Ge2

Ge : -2, -3, 4 Gerbich Anti-Ge2 or anti Ge3

Ge : -2, -3, -4 Leach types : PL LN Anti-Ge2 or anti Ge3


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Antibodies : 93

Correspondingly, the antibodies produced by Ge-negative individuals can be of three types

: anti-Ge2, anti-Ge3, or anti-Ge4. All three antigens are destroyed by trypsin; in addition,

Ge2 and Ge4 are destroyed by papain. Ge3 is resistant to papain and can be used to

distinguish anti Ge2 from anti-Ge3 (assuming the very rare Ge : -2, -3, -4 phenotype is

excluded). Gerbich antibodies may be immune in origin or can occur without red cell

stimulation. They are not considered clinically significant, but anti-Ge3 has been

associated with HDFN manifesting 2 to 4 weeks after birth.


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CROMER BLOOD GROOUP SYSTEM (ISBT 021)

Biochemistry and Genetics :94

Fifteen antigens comprise the Cromer Blood Group System carried on the glycoprotein

called Decay Accelerating factor or DAF or CD55. The glycorprotein is linked to the red

cell membrane via GPI linkage.

Table 41 Cromer Blood Group Antigens. 95

The antigens are a product of the gene DAF, found on chromosome 1. Complement

regulation is one function of the protein in that it helps to protect red cells from lysis by

autologous complement by inhibition of C3- convertase. Twelve of the antigens are of

high incidence and the remaining three of low incidence. The polymorphisms are the

result of single nucleotide substitutions except for the multiple epitope IFC antigen. Red

cells lacking the IFC antigen (CROM 7) are of the INAB phenotype. Two different single-

point mutations result in a truncation of the protein, which cannot attach to the red cell

membrane and the majority of the DAF protein is lacking.96

Cromer system antibodies are rare. The clinical significance is variable with some

showing decreased survival in transfusion. An example of anti-Tca causing an HTR has

been reported in the literature. Cromer system antibodies have not been shown to cause

hemolytic disease of the newborn possibly because placental tissue is rich in DAF, which

may absorb the antibody. The antibody can be inhibited by plasma and urine from antigen-

positive individuals and platelet concentrates.96


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KNOPS BLOOD GROUP SYSTEM (ISBT 022) Biochemistry and Genetics :97

Knops system antigens are located on CR1, the primary complement receptor on red cells.

The carrier Molecule is a single-pass membrane glycoprotein. The high – incidence

antigens are Kna, McCa, S1a, Yka and S13. The low-incidence antigens are Knb, McCb, and

Vil. There is variable frequency with the antigens between black and white individuals.

Table 42 Knops Blood Group Antigens.98

Antibodies : 99

Knops system antibodies historically were described as “high titer, low avidity” due to

their weak reactions even at high titer. The antibodies are IgG and do not bind

complement. They do not seen to be a cause of HTRs or HDFN.

INDIAN BLOOD GROUP SYSTEM (ISBT 023) Introduction: 100

The Indian antigens are located on CD44 and encoded by the gene, CD44 on chromosome

11. The carrier molecule is a single-pass membrane glycoprotein with a disulfide – bonded

N-terminal domain that acts as a cellular adhesion molecule. Two allelic antigens are Ina

and Inb. The Ina antigen is low frequency in Caucasian, Asians and blacks at 0.1%, but

Indians have a 4% incidence and Arabs up to 11.8%. The antithetical antigen is Inb with a

99% incidence in Caucasians and 96% in Indians. Two missense mutations in the CD44

gene have been reported to encode two antigens – INFI (IN3) and INJA (IN4). They are

both high incidence.

Antibodies to Indian antigens are IgG in nature. They are associated with decreased red

cell survival and positive DAT, but not HDFN.


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OK BLOOD GROUP SYSTEM (ISBT 024) Biochemistry and Genetics: 101

The Ok blood group system was established in 1999, although the antigen was first

described in 1979 when Morel and Hamilton found the antibody in the serum of a

Japanese woman who had been transfused but not pregnant. The Ok antigen is carried on a

single pass type 1 membrane glycoprotein (CD147) with two IgSF (Immunoglobulin super

family) domains. CD147 is encoded by a gene on chromosome 19. The high incidence

antigen Oka nears 100% distribution. Absence of Ok (a) arises from an amino acid change

from glutamic acid to lysine at position 92 on the first IgSF domain. The rare instances of

the Ok (a)-negative phenotype are most frequently seen in individuals of Japanese descent,

but there are two reports in individuals, one each of Iranian and Hispanic descent.

The alloantibody is an IgG antibody reactive at IAT. It is not known to cause complement

binding and is not associated with HDFN. The antibody does appear to be clinically

significant with indications of reduced RBC survival.

RAPH BLOOD GROUP SYSTEM (ISBT 025) Biochemistry and Genetics: 102

The Raph blood group system comprises a single antigen, MER2. It is carried on CDe151,

a glycoprotein, a member of the Tetraspanin family that is encoded by a gene on

chromosome 11. Tetraspanins are proteins with four transmembrane segments. Both the

N- and C- termini lie on the intracellular side of the membrane. Of the two extracellular

loops, one of the domains is longer than the other. It is thought to function as an adhesion

molecule. The antigen is common; about 92% of Caucasian individuals express the

antigen and it shows some variability in strength, particularly decreased expression over

time. Of MER2-negative individuals, there are at least two mutations described. One type

is associated with a truncation of the protein that prevents extracellular expression of the

domains. These individuals are CD151 deficient and have renal failure and other disorders

associated with the deficiency. The other is a single amino acid change from arginine to

cysteine at position 171 and is not associated with renal failure. The extracellular domains

are expressed and the substitution does not appear to affect the role of the protein.


134

Although antiMER2 has not been considered to be clinically significant, a recent report of

clinical significant has appeared in the literature in individuals with the single amino acid

substitution.

JMH BLOOD GROUP SYSTEM (ISBT 026) Biochemistry and Genetics: 103

The JMH (John Milton Hagen) antigen is carried on Semaphorin 7A or CD108, a

glycoprotein bound to the red cell membrane by GPI linkage. SEMA7A, the gene that

encodes Semaphorin 7A is located on chromosome 15. Although Semaphorin 7A has

functional aspects on other cells and tissues, its function in red cells is unknown. There are

four variants to the JMH antigen resulting from single amino acid substitutions in the

Semaphorin domain of the molecule. The JMH antigen is absent on PNH III red cells,

indicating a possible role of other complement proteins.

The antibody has not been demonstrated to be of clinical significance, although there may

be decreased survival in the variant types. It is an IgG antibody, principally, IgG4 reacting

at IAT. It is not associated with HDFN.


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I BLOOD GROUP SYSTEM (ISBT 027) AND Ii BLOOD GROUP

COLLECTION

Introduction:104

The I antigen was first described in 1956 when Wiener el at found a panagglutinating auto

antibody from a patient with CHD (cold hemaglutinin disease). The antibody was called

anti-I and the antigen it detects, I. Anti – i was reported in 1960.

Biochemistry and Genetics : 104

The I blood group system was established in 2002 and is composed of a single antigen-I

encoded by the gene IGnT on chromosome 6. The i antigen remains in the Ii collection

because it is not a product of the IGnT gene. Like ABO, Lewis and P, the Ii antigens are

carbohydrate structures. Synthesis of the antigen occurs by the addition of sugar residues

to a common precursor substance in a branched formation. The I antigen is present on the

interior structures of the oligosaccharides that form the A, B and H antigens. That is, the

antigens are carried on the same glycoprotein and glycolipid chains that carry A, B and H

antigens, but are closer to the red cell membrane. However, the nature of the antigens on

the red cells is not purely linear and same folding occurs, bringing the different antigenic

determinants close to each other. I and i antigens are based on type 2, βGal1-4GlcNAc,

chains. The i antigen is produced from a sequential action of β-3-N-

acetylglucosaminyltransferase and β-4-galactosyltransferase. The I antigen is a branched

form of the i antigen. The enzyme that causes the branching is β-6-N-acetylglucosaminyl-

transferase that attaches a GlcNAcβ16 linkage from Gal to create a branch.

Fig.24 Linear and Branched structures carrying i and I activity.


136

Development of I and i Antigens :

Fig.25 Structure of the I and i antigens. The I antigen is composed of two successive lactosaminyl subunits. I antigen requires the action of GCNT2, a 1-6-Nacetylglucosaminyltransferase. (Gal, Galactose; GlcNAc, N-acetylgalactosamine; R, other oligosaccharide.)

The expression of the antigen occurs as a reciprocal relationship in that fetal and infant red

cells express little I antigen while adult red cells express little i antigen. As development

occurs, the branching of the antigen converts i to I. The process is usually complete by the

age of 18 months. The amount of I antigen on red cells is variable. The adult i phenotype

is uncommon.104

Antibodies in the Ii system : 104

Anti I is present in the sera of most individuals if tests are carried out at 40 C. It may not

always be detectable because of absorption of the antibody. Rarely, clinically significant

auto-anti I will react at temperatures above room temperature. This is usually when

complement binding occurs at room temperature and is carried over to the IAT reading

using polyspecific reagent. Anti-i is rarely encountered. It is usually a cold-reactive IgM

antibody. Auto anti I can also be a cause of cold agglutinin syndrome.

Compound antibodies sometimes occur because of the folding of the chains bringing the

A, B, H, I, P and Leb antigens and antibody to parts of several determinants. The most

common is anti-HI. This cold-reacting agglutinin reacts with cells expressing H and I

antigens, namely Group O and A2 adults.


137

I and Disease :

The adult i phenotype in Japanese is associated with congenital cataracts. The association

does not seem to be as pronounced in whites. Molecular changes in the IGnT gene have

been reported that are associated with the adult i phenotype. It has been reported that a

defect in the I locus may lead directly to the development of congenital cataracts.

GIL BLOD GROUP SYSTEM (ISBT 029) Biochemistry and Genetics: 105

The Gill antigen is of high incidence carried on aquaporin 3 (AQP3), coded by AQP3, on

chromosome 9. AQP3 is a water channel molecule that facilitates the transport of glycerol.

Because of the association of AQP1 with the Colton Blood group system, researchers

looked for a linkage with AQP3 and found the Gill antigen. The monomer spans the

membrane six times with both the amino and Carboxy termini located intracellularly. The

antigen is resistant to enzymes and DTT. The GIL-negative phenotype arises from a

homozygous mutation that generates a premature stop codon. The result is AQP3

deficiency but no disease condition has been reported because of this deficiency.

Little is known about the characteristics of GIL antibody. It is an IgG antibody with

optimal reactivity at IAT. There is some indication of the potential for destruction of

transfused red cells, but the antibody is so rare that further studies are indicated.

Table 43 Effects of Enzyme and Chemical Treatment on various minor blood group

systems.106


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3.3 BLOOD GROUP COLLECTIONS

The ISBT defines a blood group collection as two or more antigens that are related

serologically biochemically or genetically, but which do not fit the criteria required for

system status.107

The following table lists the current antigens that fall into Blood group collections.

Table 44 Collection of Antigens.107

Collection Antigen

ISBT

Number

Name Symbol ISBT

Number

Symbol Frequency

(%)

205 Cost COST 205001 Csa 95

205002 Csb 34

207 Ii I 207002 I Low

(presumed)

208 Er ER 208001 Era >99

208002 Erb <1

208003 Er3 >99

209 GLOB 209002 Pk >99

209003 LkE 98

210 210001 Lec 1

210002 Led 6

211 Vel VEL 211001 Vel >99

211002 ABTI >99


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Cost :107

When the serum of three unrelated individuals was found to have an antibody of similar

specificity, the CSa antigen was found. It was named after two of the patients in which the

antibody was found, Mrs. Co. and Mrs. St. The antithetical antigen was reported in 1987

when the antibody was described. In 1988, the collection was established and named

“Cost”. The incidence of CSa is >98% in most populations, but 96% in blacks. The CSb

antigen has a prevalence of about 34% in all populations. Both antigens are resistant to

enzyme treatment and there is a variable effect with DTT. The antibodies are IgG, reacting

best at IAT, and do not bind complement. Anti-CSa is not associated with transfusion

reactions or HDFN. Only one example of anti-CSb has been reported.

Er: 107

A new high-frequency antibody reported in 1982 and called anti-Era was found in three

unrelated individuals. In 1988, the antithetical antigen was described and called Erb. At the

time, a silent allele was postulated that would present as a null phenotype. In 2003, there

was another report of an antibody to an E-related antigen in an individual with the

phenotype, Er (a-b-). There appears to be apparent heterogeneity with anti-Era in that not

all examples of anti-Era react with Er (a+) red cells. The antibodies are IgG and react at

IAT. They do not cause complement binding and are not associated with HTR. Anti-Era

can cause a positive DAT, but clinical HDFN has not been reported.

Vel :108

Anti-vel was first described in 1952 and named after the antibody maker. The antigen has

a frequency of about 0.02% in all populations except Norwegians and Swedes, where the

incidence is about 0.07%. The strength of the Vel antigen can be variable, making

identification difficult. The antigen is resistant to enzymes and DTT treatment. The

antibody is found as an IgM agglutinating antibody and IgG reacting at IAT. The IgM

form can present as a complement binding hemolysin. Anti-Vel is a clinically significant

antibody and Vel-negative blood should be used for transfusion in patients with antibody.

Since Vel is weakly expressed on cord cells and it is often of IgM form, it generally does

not cause HDFN.


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Another antigen, ABTI, has recently been associated with Vel. It was first reported in

1996 named after the three people in one family who made the antibody. Little is known

outside of these cases, but the antibody is IgG reacting optimally at IAT. There are no data

on clinical significance for HTR or HDFN. ABTI-negative red cells have a weakened

expression of Vel.

The Vel antigen was originally located in the 901 series of high-incidence antigens, but

has now been placed in the blood group collections when the association with ABTI was

established.


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3.4 SERIES OF LOW AND HIGH INCIDENCE ANTIGENS

There are a number of both high and low-incidence antigens that are not associated with a

blood group system. The ISBT working party on terminology has categorized there as

belonging to either the 700 (low incidence) series of antigens or the 901 (high-incidence)

series.

Low-incidence Antigens :109

Table 45 700 Series of Low- Incidence Antigens.109

ISBT Number Name Symbol

70002 Batty By

700003 Christianson Chra

700005 Biles Bi

700006 Box Bxa

700017 Torkildsen Toa

700018 Peters Pta

700019 Reid Rea

700021 Jenson Jea

700028 Livesay Lia

700039 Milne

700040 Rasmussen RASM

700043 Oldeide Ola

700044 JFV

700045 Katagiri Kg

700047 Jones JONES

700049 HJk

700050 HOFM

700052 SARA

700054 REIT


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There are currently 18 antigens in the 700 series of low-incidence antigens. These antigens

occur in less than 1% of the population, do not have a known allele, and have no

association with a blood group collection or system. Most of the antibodies described from

these antigens are only found when a case of HDFN is being investigated. The antigens

are usually not present on screening cells and are thus not detected in pretransfusion

testing. The antibodies thus far that have reports of HDFN are Batty, Biles, Reid, Livesay,

Rasmussen, JVF, Katagiri, JONES. HJk, HOFM and REIT. Of those, only Katagiri and

REIT have reported severe HDFN. Some of the antibodies are found in autoimmune

hemolytic anemia (AIHA).

High-Incidence Antigens :110

Table 46 901 Series of High-incidence Antigens.110

ISBT Number Name Symbol Year Reported

901002 Langereis Lan 1961

901003 August Ata 1967

901005 Jra 1970

901008 Emn 1987

901009 Anton AnWj 1982

901012 Sid Sda 1967

901013 Duclos 1978

901014 PEL 1980/1996

901016 MAM 1993

There are nine antigens in 901 series of high-incidence antigens. They are characterized as

having an incidence of >90%, do not have a known allele, and have no association with a

blood group collection or system. Thus, VEL and ABTI were moved to a collection when

they became associated with each other.

Lan and Anti-Lan :110

The Lan antigen occurs in all but about 1 in 20,000 people. The Lan- negative phenotype

is inherited in a recessive manner. Despite the fact, it is so rare, there are several examples

of anti-Lan in the literature. The antibody has an immune origin, is IgG in nature, and

optimally reactive at IAT. Anti-Lan has been implicated in an immediate HTR, but has not


143

caused severe HDFN, although the DAT may be positive. Individuals with anti-Lan

should receive blood negative for the Lan antigen.

Sda :110

The Sda antigen is one of the more unusual ones. The antigen is also widely distributed in

secretions, excretions, and tissues. Urine contains the largest amounts. The antigen is

famous for its mixed field appearance when tested with anti-Sda. The strength of the

antigen on red cells varies widely with individuals that have Cad (+) red cells, having

marked reaction with anti-Sda. There is a marked depression of antigen expression during

pregnancy and women who are positive for the Sda antigen will appear to be Sd( a-) at full

term. In spite of this, the level of Sda in urine remains at the normal levels. The antigen is

not well developed at birth, and infant cells will often test Sd (a-). Again if the infant has

inherited the Sda gene, the urine will contain the soluble substance.

Many examples of anti-Sda are non – red cell stimulated. The antibody is usually IgM

reacting better at room temperature. If it is found at IAT, it may be a carryover from room

temperature testing. Since most antigen screening cells do not contain the Sda antigen,

most cases of anti-Sda are not found. That is fortunate because the antibody is benign. It is

not known to cause HDFN either.

Other High-incidence Antigens :110

The antigen called Ata was reported in 1967 when the antibody was discovered in the

serum of Mrs. August who had never been transfused, but whose third baby’s cells gave a

weak, positive, direct antiglobulin reaction. The individual and her brother both had

At (a-) red cells. The At (a-) phenotype has only been found in blacks, but it is not

common. The antigen is present on cord cells and resistant to enzymes and DTT. Anti-Ata

has been reported and it appears that pregnancy seems to stimulate the antibody because

many of the antibody makers have been pregnant but never transfused. The antibody class

is IgG reacting at IAT. It does not appear to bind complement or cause HDFN. Two

reports have identified the potential for an HTR.

The Jra antigen was reported in 1970 when seven examples were described.

Approximately half of the known Jr (a-) individuals are of Japanese origin. It appears to be


144

a recessively inherited condition. The antigen is resistant to enzymes and DTT. Survival

studies have indicated that there is shortened survival when Jr (a+) red cells are transfused

to a patient with anti Jra. The antibody can be IgM or the more common IgG. A positive

DAT, but not clinical HDFN has been described.

The antigen called Emm was described in 1987 when the antibody was found in the serum

of four individuals, three of whom had never been transfused. It is thought that the antigen

is carried on a phosphatidylinositol-linked protein because anti-Emm is not reactive with

PNH III red cells (which lack PI-linked proteins). The antigen is resistant to enzymes and

DTT treatment. The antibody has been found to be both IgG and IgM and exhibits some

complement binding.

Another antigen, originally called Anton, but now named Anwj is carried on a CD44

glycoprotein. There is altered expression (weak) of the antigen when an individual has the

InLu phenotype. The antigen shows resistance to enzyme treatment and a variable reaction

DTT. The antibody is IgG and rarely binds complement. It does not appear to cause

HDFN, but there has been one severe case of HTR.

The Duclos antigen was reported in 1978 after the first and only producer of the antibody.

The antigen is not found on Rhnull U-, Rhmod U-, red cells and those of the original

antibody maker. There are little data available on the significance of the antibody.

The antigen PEL was described in 1996 when the antibody was found in the serum of two

PEL-negative French-Canadians. The antigen is presumed to be IgG, but little is known of

the significance.

The MAM antigen, first reported in 1993 and assigned to the 901series in 1999 is resistant

to enzymes and DTT. The antibody is IgG reacting well at IAT. There appears to be a

potential for HTR and HDFN.


145

3.5 TECHNIQUES FOR BLOOD GROUPING AND ANTIGEN TYPING OF RED CELL SAMPLES

Routine blood grouping still relies primarily on haemagglutination reactions between

antigens and antibodies that are read and interpreted either manually or by automated

means. Hemagglutination occurs after sensitization and lattice formation. The principles

behind hemagglutination reactions, the discovery of the biochemistry of blood group

antigens, and the increasing knowledge of the properties of immunuglobulins associated

with blood group group antibodies led to the development of laboratory tests other than

direct agglutination. Antiglobulin methods and enzyme technology were introduced as

routine methods for the detection of antigen – antibody reaction and played a part in the

discovery and expansion of knowledge of blood group systems in general.

Since these advances, new and non traditional laboratory methods have become available

for specialist investigations and as research tools, such as Flow Cytometry, Solid-phase

adherence test, Gel test, RBC affinity column test and analysis of antigens at the molecular

level.111

CONVENTIONAL TUBE TECHNIQUE :

A) SALINE TECHNIQUE :112

Serum / Plasma or known reagent is added to the test tubes followed by an equal volume

of red cells suspended in saline or other appropriate medium and then the contents are

mixed. The tests can be centrifuged immediately and read (immediate or rapid spin

technique) or can be incubated at the appropriate temperature for 10-60 minutes and gently

centrifuged before reading. The saline technique is used for ABO grouping, other red cell

antigen typing and for the detection of type IgM antibodies.

B) INDIRECT ANIGLOBULIN TEST (IAT) :112

Fig. 26 Antihuman globulin antibodies form a bridge between adjacent erythrocytes sensitized with human immunoglobulin (Ig)G or complement components.


146

Red cells are suspended in istotonic saline (NISS), preservation fluids, PBS or LISS.

Saline based IAT:

Serum / plasma is added to the test tubes followed by red cells suspended in the

appropriate NISS medium; the contents mixed and allowed to incubate for 30 minutes

(minimum) to 2 hours (maximum) at 370C. After incubation, the tests are washed in

normal saline to remove any unbound globulins. After the last wash, AHG is added, and

the tube contents are mixed centrifuged and read and re-read after the addition of

sensitized cells to the negative tests.

MICRO COLUMN TECHNIQUES :112

Cards of microcolumns containing a matrix of gel or glass microbeads are available

commercially for micro column techniques. The systems can be manual, semi-automated

or automated.

In the 1980s Dr. Yves Lapierre discovered a gel method to capture hemgglutination, which

was developed by Diamed AG. A LISS IAT method was devised that eliminated the wash

phase and so reduced the potential for error associated with a poor wash technique, as well

as contributing to laboratory standardization and economy of effort. The ‘No-wash’

technique is possible because, on centrifugation, the liquid phase of the reactants remains

in the upper chamber of the microtube and only the cells enter the gel; matrix, which

contains the AHG. Cells sensitized during the incubation phase react with the AHG and

the agglutinates are held at the top of or throughout the gel matrix. Non sensitized cells

form a pellet at the base of the microtube. Because no liquid enters the gel, there is no

danger of neutralization of the AHG, which can happen in a spin-tube method owing to

residual human proteins not removed during the wash phase. Weak positive reactions are

also more robust in the gel (ID-system), as there is no possibility of dissociation of the

antigen-antibody complexes during a too-vigorous wash phase.


147

Fig. 27 Appearance of reaction patterns and grading for gel or column agglutination technology.

Table 47 Comparison of blood grouping methods .113

Feature Tube Gel Solid Phase

End point Agglutination Agglutination Immune

adherence

Indicator Direct

Agglutination/Antihuman

globulin

Antihuman

globulin

Antihuman

globulin-sensitized

indicator cells

Wash step Yes No Yes

Stability 30 minutes 2-3 days 2 days

Process steps 11-14 4 5-8

Special equipment

required

No Yes Yes

Automation

available

No Yes Yes


148

FLOW CYTOMETRY :114

Although not used for routine typing of blood samples, flow cytometry can be applied in a

special circumstances, for example, when investigating the cause of double cell

populations or for identifying weakly expressed antigens. It can also be used to assess the

amount of feto-maternal haemorrhage (FMH) in RhD negative mothers who require anti-D

prophylaxis and for calculating the concentration of anti-D in an immunized RhD negative

antenatal woman.

Fig. 28 Simplified diagram of the concept of flow cytometry.

In flow cytometry, cells are incubated with antibodies conjugated with fluorophores, dyes

that fluoresce under intense light. In the flow cytometer, the cells pass, in single file, past a

laser beam and fluorescence is monitored by photo detectors. As most flow cytometers can

detect light of different wave lengths emitted by two or more different fluorophores, more

than one antigen can be detected at the same time.

Flow cytometry enables large numbers of cells or ‘events’ to be examined in a short space

of time. Its sensitivity is such that a small number of rare events can be detected

accurately, if a sufficient number of total events are counted. This is because the cells

arrive at the point of analysis in a random fashion and a subset of cells will also be

randomly distributed within the suspension. To ensure precision, therefore, enough events

must be recorded to allow the rare events to be detected. For example, if the numbers of

fetal cells is in reality only 0.01% of the maternal sample, then at least 107 events or cells

must be counted in total to give an accurate result, with an acceptable co-efficient of

variation (CV).


149

MOLECULAR BLOOD GROUP GENOTYPING :115

All the clinically significant blood group polymorphisms are now understood at the

molecular level, enabling techniques to be designed to predict blood group genotype from

the DNA sequence. For RhD testing this involves detecting the presence or absence of a

whole gene, but in tests for most other blood groups a single nucleotide polymorphism

(SNP) is targeted. The methods that are commonly employed for SNP detection in blood

group testing involve restriction enzyme digestion of polymerase chain reaction (PCR)

products, PCR with an allele-specific primer, or allelic discrimination by Taqman

Technology.

Table 48 Uses of molecular genetic methods in immunohaematology.115


150

3.6 CLINICAL SIGNIFICANCE OF BLOOD GROUP ANTIBODIES

In immunohaematology, antibodies may be classified as “naturally occurring” or

“immune”. This means that antibody molecules may be present in an individual

regardless of the fact that there has been no known stimulus such as the transfusion of

antigen different blood or feto-maternal haemorrhage. Both ‘naturally occurring’ and

‘immune’ antibodies can be of importance in immunahaematology. Antibodies can be

further divided into categories ‘alloantibody’, raised in response to an antigen lacking on

the individual cells, or ‘autoantibody’ which has specificity against an antigen present on

the individual’s own red cells.116

Fig. 29 Schematic diagram showing the production of polyclonal antibodies of human origin.

Most human antibodies are polyclonal in origin, being of broader specificity than

monoclonal antibodies, which can have single epitope specificity. These antibodies can be

of importance in certain clinical situations as

1) Hemolytic transfusion reaction (HTR), when an antibody destroys antigen-positive

transfused red cells. The most severe HTR are generally caused by antibodies to

antigens of the ABO system, which are IgM in nature and initiate the complement

cascade through to the membrane attack complex (MAC) causing lysis of cells

intravascularly. Other antibodies, IgG in nature, generally either do not initiate

complement activation or only upto the C3 stage, and red cells are cleared

extravascularly.


151

Fig. 30 Activation of the complement cascade through to the MAC, producing ‘pores’ in the red cell, allowing solutes to enter and escape, resulting in cell lysis.

2) Hemolytic disease of the fetus and newborn (HDFN), when an IgG maternally derived

antibody, which is directed against a paternally derived antigen present on the fetal /

neonatal red cells, crosses the placenta and destroys the cells.

3) Hemolytic anemia (HA), when an autoantibody directed against ‘self’ antigens is

produced and causes increased red cell destruction. These antibodies may be non-

specific or may possess a defined specificity, often against a high incidence antigen.

The antibodies may be warm reactive or react optimally in the cold.116

Antibody production and structure :117

Antibodies are immunoglobulins secreted by the progeny of B-lymphocytes that

differentiate in response to antigen stimulation. Multiple clones of B-cells produce

polyclonal antibodies with molecular heterogeneity whereas monoclonal antibodies are

biochemically identical, being produced by a single B-cell clone.

All antibody molecules are similar in overall structure, having a common core

arrangement of two identical light chains, either k (kappa) or λ (lambda) and two identical

heavy chains, which denote the isotype of the antibody. In humans the different isotypes

are IgA, IgD, IgE, IgG and IgM.


152

Fig. 31 Representation of a molecule of IgG to show, indicating antigen binding sites, and line drawings of IgG and IgM, showing the pentamer structure of IgM with 10 binding sites, variable regions of heavy chains and or light chains. Table 49 Characteristics of Serum Immunoglobulins.118

The genes for antibody production are located on chromosome 14 band q32, for the heavy

chains and chromosome 2p11 and 22q11 for k and λ light chains, respectively. Each chain

is separately synthesized before the antibody molecule is assembled. Basic from of IgG

molecule possesses two gamma heavy chains and either k or λ light chains. The antibody-

binding site is made up from the variable regions of the heavy and light chains.

IgM antibodies are in the form of a pentamer.

IgG molecules are flexible and subclasses have different properties and effector functions,

for example the longer hinge region of IgG3 contributes to the flexibility of the molecule;

it is more efficient at complement activation and less molecules per cell are required to

initiate red cell destruction processes.119


153

Table 50 Biologic properties of IgG subclasses.120

Factors affecting the clinical significance of antibodies. All antibodies exert their biological effects by binding to the appropriate antigens. An

antibody is said to be clinically significant when it has the potential to initiate accelerated

destruction of red cells carrying the appropriate antigen. Thus, alloantibodies, whether

naturally occurring or immune and auto antibodies can be of clinical importance.121

Table 51 Factors that influence the pathogenicity of RBC antibodies.122

Antibody specificity :

The following table lists the Serologic behavior of the Principal Antibodies of Different

Blood Group Systems.


154

Table 52 Serologic Behavior of the Principal Antibodies of different Blood Group

Systems.123

The antibody specificity is probably the single most helpful indication as to whether a

particular alloantibody may be capable of promoting accelerated red cell destruction. In

conjunction with its optimal thermal range it may be possible to predict its clinical

significance. Antibodies not detected in tests at 370C are generally not considered to be


155

able to initiate significant red cell destruction, although antibodies within the ABO system

should always be considered of potential importance.

Table 53 Selected blood group antibodies and their clinical importance.124

HAEMOLYTIC TRANSFUSION REACTIONS (HTR) :

HTR may result from intravascular or extravascular destruction of red cells and may be

acute (immediate) or delayed (up to 14 days post transfusion).

Intravascular red cell destruction :125

Fig.32 The destruction of red blood cells (RBCs) intravascularly results in the liberation of haemoglobin (Hb) from the RBC. The Hb combines with haptoglobin (HP), and the HP-Hb complexes are rapidly catabolized in the reticuloendothelial system (RES), resulting in low levels of serum Hp.


156

Fig. 33 Intravascular red cell destruction

Intravascular red cell destruction presents as hemoglobinuria and hemoglobinemia due to

the ultimate hemolysis of the transfused cells. IgM anti-A and –B are most often

implicated. During complement activation, C3a and C5a anaphylatoxins are released and

responsible for many of the signs and symptoms of an acute HTR.


157

Fig. 34 Complement activation may be initiated by antibody binding to the appropriate antigens on the red cells.

C5a is a far more potent mediator of inflammation than C3a, which contributes to the

more serious problems associated with transfusion reactions due to intravascular

destruction initiated by an IgM antibody.

Fig. 35 Biological systems involved in an acute HTR and the potential consquences.


158

Extravascular red cell destruction :126

Fig. 36 The destruction of RBCs extravascularly within the cells of the reticuloendothelial system results in the degradation of hemoglobin and the production of bilirubin.

Extravascular red cell destruction is usually associated with IgG antibodies, which fail to

activate complement or fix complement only up to the C3 stage of the cascade. Red cells

coated with IgG1 and/or IgG3 adhere at the hinge region of the antibody molecule to Fc

receptors on macrophages and are phagocytosed or destroyed by a cytotoxic mechanism.

Phagocytosis is favoured when there is moderate coating of the red cells. If the antibody

does not fix complement, such as virtually all IgG anti-D and some IgG anti-K, -S and –

Fya, the cells are mainly destroyed in the spleen, where conditions of haemo concentration

occur. Free IgG in the circulation prevents significant destruction at other sites because the

circulating IgG molecules compete for the binding sites on macrophages. When

complement components are also present on the red cells, a synergistic relationship

between the two occurs and the cells are destroyed even more effectively. This generally

takes place in the liver where there are abundant phagocytic cells possessing receptors for

both IgG and the C3c component of complement. Antibodies against antigenic

determinants in the Kidd blood group system are often described as ‘complement

dependent’. Recent evidence suggests that only IgM and not IgG, Kidd antibodies are able

to bind complement. Initially, destruction of cells sensitized with complement due to

activation by IgM or IgG antibody is rapid but slows abruptly because the C3b becomes

inactivated and quickly cleaved to C3dg. Macrophages do not have receptors for IgM or

C3dg.


159

Cells heavily coated with IgG1 and/or IgG3 antibodies usually trigger destruction by the

antibody-dependent cytotoxicity mechanism. Lysosomal enzymes released by

mononuclear cells effect the destruction, however, both phagocytosis and cytotoxic

mechanisms can occur at the sometime.

HAEMOLYTIC DISEASE OF THE FETUS AND NEWBORN :127

The corner stone of antenatal care is regular testing for antibodies of potential importance

and monitoring the alloimmunised pregnancy accordingly.

IgM antibodies, whilst efficient at provoking complement activation and so responsible for

most cases of transfusion-related intravascular red cell destruction, do not cross the

placenta and are therefore not implicated in the fetal red cell destruction associated with

HDFN. The most severe manifestation of HDFN is caused by IgG antibodies directed

against D, c or K antigens on the fetal red cells, but any IgG antibody has the potential to

cause the disease with varying severity.

In vitro tests for assessing the ability of the antibody to initiate destruction of sensitized

red cells are useful laboratory tests, such as antibody dependent cell mediated cytotoxicity

(ADCC). This measures the release of 51cr as an indication of target red cells lysed by

lymphocytes or monocytes used in the assay as effector cells. The chemiluminescence test

(CLT) measures adherence and phagocytosis of sensitized red cells by effector cells in the

presence of luminol. The CLT results are expressed as a percentage of the monocyte

response to positive control cells and results over 30% are consistent with moderate to

severe disease. Titres of antibodies implicated in HDFN are not predictive of the outcome

of the pregnancy, but serve as a guide to the level of antibody at any given stage of the

pregnancy. Quantification of levels of anti-D and anti-c can indicate the need for treatment

of the fetus – generally, if the anti-D is >4IU/ml or increase by 50% in comparison with a

previous result, or if the anti-c is >10IU/ml, the fetus may require treatment. Anti-D levels

of >15IU/ml are associated with severe HDFN.


160

There is no method for quantification of anti-K and even a K antibody with a low titre can

cause severe HDFN. Anti-K causes HDFN by affecting early red cell precursors, before

hemoglobin production. Therefore, in HDFN due to anti-K there is little destruction of

circulating mature red cells making measurement of breakdown products of hemoglobin in

the amniotic fluid less informative.

HDFN due to anti-D has been reduced since the introduction of prophylactic anti-D

administration and routine antenatal anti-D administration reduces the incidence even

further. Before the establishment of modern therapy, up to 1% of all pregnant women

developed RhD alloimmunisation. The incidence of RhD sensitization is reduced to about

11 cases per 10,000 births with less than 10% requiring Intra uterine transfusion.

Alloimmunisation due to K antigen accounts for 10% of severely affected fetuses.

Interventions in alloimmunised pregnancies can prevent serious morbidity and mortality.

These interventions are necessary to prevent and / or treat severe fetal anemia, which can

lead to congestive heart failure, intrauterine growth retardation, and hydrops due to hepatic

dysfunction. In utero, bilirubin released from the breakdown of hemoglobin is partly

cleared by the placenta and so it may not be until the infant is born that serious problems

develop, when exchange transfusion may be indicated.

Exchange transfusion removes about 90% of the antigen-positive cells, which may only

survive 2-3 days in the most severe HDN. It also removes up to 50% of the available

intravascular bilirubin and reduces circulating maternal antibody. If these measures are not

taken, there is a risk of Kernicterus, when the bilirubin crosses the blood-brain barrier and

permeates the basal ganglia, resulting in high morbidity and mortality in affected infants.

AUTOANTIBODIES :128

In autoimmune Hemolytic Anemia, autoantibody reacts with all normal red cells,

including the patient’s own. The autoantibody is most often IgG in nature. In cold

antibody HA, the autoantibody tends to be IgM and can activate complement. The

peripheral blood sample generally shows the presence of C3d on the red cells. Paroxysmal

cold hemogolbinuria (PCH) is most often associated with post-viral infection in children.


161

The antibody is a biphasic haemolysin, IgG in nature and with P specificity. Atypical HAs

are known to occur, for example, combining warm and cold antibody involvement and HA

in which the DAT is negative. Some drug-induced HAs are clinically indistinct from warm

antibody immune HA (WAIHA).

In addition to auto antibodies, there may also be alloantibodies present that can cause

destruction of transfused red cells. It is therefore very important to determine if an

alloantibody of clinical importance is present. This can be achieved by adsorption of the

autoantibody to reveal any underlying specific antibody. Antigen-negative blood must be

selected for transfusion in these cases. Determining the specificity to the autoantibody, for

example by elution from the DAT-positive cells, is generally of little help in HA.

TESTS TO ASSESS THE POTENTIAL SIGNIFICANCE OF AN ANTIBODY.129

When an antibody is of questionable importance or is directed against an antigen of high

frequency, it may be necessary to establish the likelihood of red cell destruction in the

event of transfusion or pregnancy. During an alloimmunised pregnancy, tests to

demonstrate the relative strength/concentration of an antibody include quantification

and/or titration end-point estimation. Quantification of anti-D and -c are possible by an

auto-analyser technique, in parallel with standard antibodies of known concentration in

IU/ml or by flow cytometry. It must be noted that titre end points/scores do not correlate

with the outcome of alloimmunised Pregnancies. Other tests, which are based on the in

vivo destruction mechanisms, include the Monocyte Monolayer Assay (MMA) and the

Chemiluminescence test (CLT), which measure monocyte phagocytosis, and Antibody

Dependent Cell-mediated Cytotoxicity (ADCC).

These methods can also be used to assess the biofunctional activity of an antibody in the

plasma of a potential transfusion recipient, when compatible blood is impossible to find,

for example, to distinguish antibodies capable of causing the increased destruction of

transfused incompatible red cells from antibodies that are clinically benign. These assays

are useful if a patient has an antibody to a high incidence antigen or an antibody whose

specificity has not been identified and transfusion is necessary. If a patient has mixture of

antibodies that cause problems in obtaining donor blood negative for all target antigens,


162

these tests can indicate which antibodies need to be considered of primary importance in

terms of preventing a transfusion reaction.

An in vivo method for demonstrating the clinical significance of an antibody is to tag a

small aliquot (0.5 -1.0 ml) of donor red cell with 51cr. The radioactivity is then measured

after 3(baseline), 10, and 60 minutes after injection, in order to provide a measure of risk

should the complete unit be transfused.

Decision – making for transfusion:130

When antibody has been detected and identified as potentially significant, and the patient

requires a blood transfusion, a prudent approach is needed for transfusion therapy.

The following table guides for selecting blood for transfusion of patient with antibodies.

Table 54 Choice of blood for transfusion of patient with antibodies.131


163

Where multiple antibodies exist, the specificities known to be of potential importance need

to be considered and blood that is negative for all such antibodies should be administered.

Consideration may be given to autologous donation or testing of family members to find

compatible blood, or it may be necessary to contact a ‘Rare Donor Registry’.

Patients who are likely to require long term transfusion therapy can be fully phenotyped

for the major blood group systems so that closely matched blood can be selected. When a

patient has been recently transfused, so that serological phenotyping is not possible, the

blood group genotype of the patient can be determined by molecular methods.

Closely matched blood can prevent immunization to important antigens and lessens the

subsequent burden of supplying compatible blood for patients who develop multiple

antibodies.

3.7 COMPATIBILITY TESTING:

ANTIBODY SCREENING AND IDENTIFICATION AS ITS MAJOR COMPONENT.

The goal of pretransfusion compatibility testing is to provide the patient with a beneficial

and safe transfusion.132

Pretransfusion compatibility testing is a series of testing procedures and processes with the

ultimate objective of ensuring the best possible results of a blood transfusion.133

Pretransfusion and perinatal blood testing is performed to prevent transfusion reactions

and hemolytic disease of the fetus and newborn, and must include the key serologic

evaluation of ABO and Rh antigen typing, antibody detection / identification and cross

matching.


164

A consensus on pretransfusion compatibility testing procedures and processes is as

follows :

Table 55 Consensus PRE-TXN Practices, 2001.134

Strict adherence to and application of each parameter of pretransfusion compatibility

testing is imperative to the management of safe blood transfusion therapy.


165

Antibody Detection :

Antibody detection plays a critical role in transfusion medicine. It is a key process in

pretransfusion compatibility testing. It aids in the detection and monitoring of patients who

are at risk of delivering infants with hemolytic disease of the newborn (HDN). It is one of

the principle tools for investigating potential hemolytic anemias. The focus of antibody

detection methods is on “irregular” or “unexpected” antibodies as opposed to the

“expected” antibodies of the ABO system. These unexpected antibodies may be immune

alloantibodies, produced in response to RBC stimulation through transfusion,

transplantation or pregnancy. Other unexpected antibodies may be “naturally occurring”,

produced without RBC stimulation. Naturally occurring antibodies may form as a result of

exposure to environmental sources, such as pollen, fungus and bacteria, which may have

structures similar to some RBC antigens. Another category of antibody is the passively

acquired antibody. Passively acquired antibodies are produced in another individual and

then transmitted to the patient through plasma-containing blood products or derivatives

such as intravenous immunoglobulin (IVIG).

Of greatest concern are the unexpected antibodies that cause decreased survival of RBCs

that posses the target antigen. These antibodies are deemed “Clinically Significant”.

Clinically significant antibodies are usually IgG antibodies that react at 370C or in the

Antihuman Globulin (AHG) phase of the indirect antiglobulin test and are known to have

caused a transfusion reaction or unexpectedly short survival of transfused RBCs.135


166

Table 56 Clinical significance of 370 C. Reactive Antibodies. 136

Usually* Very Unusual( if ever)+ Sometimes

ABO Bg(HLA) Cartwright (e.g.,Yta)#

Rh Ch/Rg(Complement C4) Lutheran (e.g., Lub )#

Kell Leb Gerbich#

Duffy JMH Dombrock#

S,s,U Xga M,N#

P Lea

Vel

LW

Ii

H

Ata

Inb

Mia

Csa

* These antibodies usually cause obvious clinical symptoms and decreased RBC survival.

Sometimes no obvious clinical symptoms occur.

+ These antibodies rarely ( if ever ) cause clinically obvious symptoms, but there are some

data to suggest that some unusual examples of Bg, anti-Kn/Mc/Yk, and JMH cause

shortened RBC survival.

# These antibodies rarely cause acute severe HTR, but when they are “ clinically

significant ” they may cause obvious clinical symptoms (e.g., jaundice); they more often

cause only shortened RBC survival.

Autoantibodies complicate the detection of clinically significant antibodies.

Autoantibodies are directed at antigens expressed on one’s own RBCs. Because they react

with all cells tested, autoantibodies may mask the presence of clinically significant

alloantibodies.


167

The incidence of unexpected alloantibodies depends on the fact the antibody formation is

the result of exposure to a foreign RBC antigen and the patient’s ability to respond to that

exposure. This occurs by allogenic transfusion of RBCs, pregnancy or transplantation. The

incidence of unexpected antibodies in the general patient population, therefore, is low 1.64

in one large study and 0.78 percent in another. It follows, then, that the more frequently a

patient is exposed to foreign RBC antigens, the more likely that patient will produce

unexpected alloantibodies. This is evidenced by a study of multiply transfused sickle cell

patients in which 29 percent of pediatric and 47 percent of adult multiply transfused sickle

cell patients developed clinically significant alloantibodies.137

Table 57 Alloimmunization risk in various disease :138

Disease Number of Patients per study Immunization risk

Range Total Range Median

SCD 34-1044 3409 9.9-46.8 30.0

Children 42-245 596 7.8-29.5 18.5

Thalassemia 39-1434 3424 5.0-28.4 9.7

Hematologic

Myelodysplastic syndrome and

Chronic myeloproliferetive

disease

16-112 231 125-58.6 23.2

Myeloid leukemia 35-209 244 5.7-8.6 7.5

Lymphoid

leukemia

13-193 206 0.0-0.5 0.3

Renal failure 81-405 1296 1.1-14.0 5.9

Transplantation

Organ

35-1132

3007

2.7-9.0

6.2

Hematopoietic stem cell

117-217 885 1.3-9.1 2.3

AIDS 72-81 153 1.4-3.7 2.6

Surgery 374-530 1356 2.1-8.0 5.3


168

Detection of unexpected antibodies is important for the section of donor RBCs that will

have the best survival rate in the patient’s circulation and reduce the risk of hemolytic

transfusion reaction. Antibody screening tests should demonstrate the presence of all

potentially clinically significant alloantibodies in the recipient’s serum / plasma and

indicate the need for further studies. All antibodies encountered in the screening test must

be indentified to determine potential clinical significance and to allow a logical decision to

be made whether there is a need to select antigen- negative units for transfusion.139

3.8 TECHNIQUES FOR ANTIBODY DETECTION & IDENTIFICATION :

I. TUBE TECHNIQUE :

The traditional method of detecting antibodies is an indirect antiglobulin test performed in

a test tube. In this method, the patient’s serum or plasma is tested against RBCs with

known antigens. The test may include an immediate spin phase to detect antibodies

reacting at room temperature. This phase is not required and may lead to the detection of

clinically insignificant cold antibodies. The test must include a 370 C incubation phase.

During this phase, immunoglobulin G (IgG) molecules sensitize antigen-carrying RBCs.

Enhancement media may be added to increase the degree of sensitization. Depending on

the enhancement added, the tubes might be centrifuged and observed for hemolysis or

agglutination following the incubation. To observe for hemolysis, the tube is carefully

removed from the centrifuge so as not to dislodge the RBC button. The supernatant is

observed for pink or red discoloration. To observe for agglutination, the tube is gently

tilted or rolled to dislodge the cell button (tip and roll method). The degree of reactivity is

graded as O (negative, no agglutination present) to W+ (barely visible to the naked eye) to

4+(one solid agglutinate). 140


169

Table 58 Observed agglutination strengths and designated interpretation and score.141

Observation Designation Score* - applicable to

titrations only

One solid agglutinate 4 12

Several large agglutinates 3 10

Many small agglutinates, background not completely clear

2 8

Barely visible, very small or small agglutinates, opaque background

1 5

No agglutination 0 0

mixed field agglutination Mf

Complete haemolysis H

Partial haemolysis PH

* numerical value assigned to degree/strength of agglutination.

The degree of agglutination should be judged only after all the cells have been dislodged

from the bottom of the test tube. The tubes are then washed with saline a minimum of

three times to remove all unbound antibody. AHG (Coomb’s serum) is added to each tube.

The antibody in this reagent will create a bridge between sensitized RBCs, resulting in

observable agglutination. If no antibodies are present, no sensitized RBCs will be

presently and there will be no agglutination with the addition of AHG reagent, the tubes

are centrifuged and examined for hemolysis and agglutination. In this phase, hemolysis

may appear as a loss of cell button mass. Again depending on the enhancement media, the

agglutination reactions may be observed macroscopically only, or macro- and

microscopically. All negative tests will have Coomb’s control cells (check cells) added to

confirm the negative test.142


170

Fig. 37 Steps for perfoming the tube antibody screen test.

RBC Reagents :142

The RBC reagents used in the antibody screen come from group O individuals who have

been typed for the most common, and the most significant, RBC antigens. Group O cells

are used so that anti-A and anti-B will not interfere in the detection of antibodies to other

blood group systems. The cells are suspended at a concentration between 2 and 5 percent

in preservative diluents, which maintains the antigens and prevents hemolysis. The

screening cells are packaged in sets of two or three cells with varied antigen expression.

Within the set, there should be one cell that is positive for each of the following antigens :

D, C, c, E, e K, k, Fya, Fyb, Jka, Jkb, Lea, Leb, P1, M, N, S, s. Other antigens may be

expressed as well. Each set of screening cells will be accompanied by an antigen profile

sheet, detailing which antigens are present on each cell. These profiles are lot-specific and

should not be interchanged.

Table 59 Example of Antigen profile of 3- cell structure set.142


171

Ideally, there will be homozygous expression of many of the antigens within the screening

cell set, allowing for detection of antibodies that show dosage. A cell with homozygous

expression is from an individual who inherited only one allele at a given locus. Therefore,

the cell surface has a “double dose” of that antigen. A cell with heterozygous antigen

expression is from a person who inherited two different alleles at a locus. The alleles

“share” the available antigen sites on the cell surface.

Fig. 38 Homozygous inheritance versus heterozygous inheritance.

Antibodies that react more strongly with a “homozogous” cell are said to show dosage.

Some blood group systems that exhibit dosage include Rh (except D), Kidd, Duffy, MNSs,

Lutheran.

When testing blood donors, it is acceptable to use a pooled screening reagent that contains

cells from at least two different individuals. These reactions should be carefully observed

for mixed field agglutination, as the target antigen may only be expressed on one cell in

the pool.

Using commercially prepared screen cells to detect antibodies is superior to relying

on the cross match alone to ensure compatibility with a donor RBC unit. The screen

cell sets will test for most clinically significant antigens, whereas the cross matched unit of

blood will possess only some of these antigens. The screen cell sets will have cells with

homozygous expression of many antigens, making it more reliable in the detection of

weakly reacting antibodies. Donor units may or may not have homozygous antigen

expression, so it is possible that weak antibodies may not be detected by cross match

alone.


172

Enhancement Reagents :143

Various enhancement reagents or potentiators, may be added to the cell / serum mixture

before the 370 C incubation phase to increase the sensitivity of the test system. These

reagents may also allow for a shortened incubation time.

⇒ 22% Albumin : In an electrolyte solution, negatively charged RBCs are surrounded by

cations, which in turn are surrounded by anions. The effect is to produce an ionic cloud

around each RBC, forcing the cells apart. The difference in electrical potential

between the surface of the RBC and the outer layer of the ionic cloud is called the zeta

potential. Albumin works by reducing the zeta potential, dispersing the charges, thus

allowing the RBCs to approach each other, increasing the chances of agglutination.

Fig. 39 Use of enhancement reagents can lower the zeta potential, allowing for better interaction between RBCs and increasing the possibility of agglutination.

⇒ Low Ionic Strength Solution (LISS): LISS contains glycine in an albumin solution. In

addition to lowering the zeta potential, LISS increases the uptake of antibody onto the

RBC during the sensitization phase. This increases the possibility of agglutination.

⇒ Polyethylene Glycol (PeG): PeG in a LISS solution removes water from the test

system, thereby concentrating any antibodies present. This increases the degree of

RBC sensitization. PeG can cause nonspecific aggregation of cells, so centrifugation

after the 370C incubation is not performed. Generally, PeG test systems are more


173

sensitive than LISS, albumin or saline system. However, in patients with elevated

levels of plasma protein, such as in multiple myeloma, PeG is not appropriate for use

due to increased precipitation of proteins.

AHG Reagent :

The addition of AHG reagent allows for the agglutination of incomplete antibodies.

AABB standards requires that the reagent contain anti-IgG when used for antibody

detection and pretransfusion compatibility testing. Polyspecific AHG reagent (also called

polyvalent or broad spectrum Coombs serum) contains antibodies to both IgG and

complement components, either C3 and C4 or C3b and C3d. It has been suggested that

antibodies to the C3 components, especially C3d are more desirable in the reagent, as

these are more abundant on the RBC surface during complement activation and lead to

fewer cases of false-positive reactions. The presence of complement in the Coombs’ serum

may lead to the detection of clinically insignificant antibodies. Relatively few examples of

clinically significant antibodies, most notably Jka, react with complement alone. To avoid

time consuming investigations of insignificant antibodies, many technologists choose to

use monospecific AHG reagent containing anti IgG only.

Any test that is negative following the addition of the AHG reagent should be controlled

by the addition of Coomb’s control cells. These are Rh-positive cells that have been coated

with anti-D. These antibody-coated cells should agglutinate when added to the negative

test due to the anti-IgG present in the AHG reagent. The addition of Coomb’s control cells

proves that there was adequate washing performed before the addition of the AHG

reagent, that the AHG reagent was added, and that the reagent was working properly. If

the Coombs’ control cells fail to agglutinate, the antibody screen must be repeated.

Use of the tube test remains popular, due to the flexibility of the test that system, use of

commonly available laboratory equipment, and relative in expensiveness. The

disadvantages include the instability of the reactions and subjective nature of grading by

the technologist, the amount of hands-on time for the technologist and problems related to

the failure of the washing phase to remove all unbound antibody.


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Fig. 40 Principle of IAT : spin-tube method.

II. GEL TECHNIQUE :

The antibody screen may also be performed using a micro tubule filled with a dextran

acryl amide gel. The screening cells used for this technique meet the same criteria as for

the tube test but are suspended in LISS to a concentration of 0.8 percent. With this

techniques, the patient’s serum or plasma specimen and screen cells are added to a

reaction chamber that sits above the gel. There are up to six chamber / gel microtubules

contained in a plastic card. The card is incubated at 370 C for 15 minutes to 1 hr., thus

allowing sensitization to occur. The card is then centrifuged for 10 minutes. During this

time, the RBCs are forced out of the reaction chamber down into the gel. The gel contains

anti-IgG. If sensitization occurred, the anti-IgG will meet with the antibody –coated cells,

resulting in agglutination. The agglutinated cells will be trapped within the gel (because of

the action of the anti-IgG and because the agglutinates are too large to pass through the

spaces between gel particles ). If no agglutination occurred, the cells will form a pellet at

the bottom of the microtubule.


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Fig. 41 Principle of the gel test. The ‘No-wash’ ID-System (gel column) for the IAT incorporates the AHG within the gel matrix. Sensitised cells react with the AHG on centrifugation, leaving the liquid reactants, including any unbound globulins in the reaction chamber. Cells free from IgG and/or complement components are centrifuged to the bottom of the microtube. No control of negative results is necessary as no washing is required.

There are numerous advantages to the gel technique. It is reported to be as sensitive as the

PeG tube test method. The omission of the washing and Coomb’s control steps results in

fewer hands-on steps for the technologist to perform. Reactions are stable for up to 24 hrs

and may be captured electronically, leading to standardized grading of reactions and easier

review by a supervisor. Mixed field reactions may be more readily detected with the gel

technique. One of the greatest advantages is the ability to automate many of the pipetting

and reading steps, thereby allowing increased productivity. Disadvantages include the

need for incubators and centrifuges that can accommodate the gel cards.

III. SOLID PHASE TECHNIQUE :145

Fig. 42 Solid phase technique for IAT. The Solid Phase Microplate method for the IAT has the AHG bound to the well of the microplate. Antibody bound to red cells bind to the solid phase AHG and haemagglutination is observed after unbound globulins are washed free.


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Fig. 43 Principle of solid-phase adherence technology with appearance of positive and negative reactions.

With this method, RBC antigens coat microtiter wells instead of being present on intact

RBCs. The patient’s serum or plasma is added to each well in the screen set along with

LISS. Incubation at 370C allows for sensitization. The wells are then washed to remove

unbound antibody. Rather than AHG reagent, indicator cells are added. These cells are

coated with anti-IgG. The wells are then centrifuged for several minutes. If sensitization

occurred, the indicator cells react with the antibody bound to the antigens coating the

microtiter well, forming a diffuse pattern in the well. If no sensitization occurred

(a negative reaction), the indicator cells form a pellet in the bottom of the well.

The solid phase test has been successfully automated. Such instruments may perform

pipetting steps and make determinations of the degree of reactivity by taking multiple

readings of each well. Other advantages include a smaller sample size (when compared

with the tube test), making it ideal in a pediatric setting and a LISS reagent that changes

color when added to serum or plasma. This ensures that an adequate sample is present in

the test system. Among the disadvantages is that with such a small sample and reagent

volume, careful pipetting is necessary when performing the test manually. An inadequate

volume of indicator cells may result in a pattern similar to that of a weak positive reaction.


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Staff should be carefully trained to visually interpret result if automation is not used. Staff

members who have primarily used the tube test previously may interpret the diffuse

positive pattern as a negative reaction and the dense pellet of the negative reaction as a

positive (4+) reaction. Incubators, washers and centrifuges that can hold the wells are

among the special equipment needed for this method. A final disadvantage is the need is

run a positive and negative control with each batch of tests, which adds to the expense of

this method.

3.9 STEPS INVOLVED IN ANTIBODY DETECTION AND IDENTIFICATION :

Interpretation of Antibody Detection (Screening) Results :146

Agglutination or hemolysis at any stage of testing is a positive test result, indicating the

need for antibody identification studies. However, evaluation of the antibody screen

results (and autologous control, if run at this time) can provide clues and give direction for

the identification and resolution of the antibody or antibodies. The investigator should

consider the following questions :

1. In what phase(s) did the reaction(s) occur?

Antibodies of the immunoglobulin M (IgM) class react best at low temperatures and

are capable of causing agglutination of saline suspended RBCs (immediate spin

reading). Antibodies of the IgG class react best at the AHG phase. Of the commonly

encountered antibodies, anti-N, anti-I and anti-P1 are frequently IgM, whereas those

directed against Rh, Kell, Kidd and Duffy antigens are usually IgG. Lewis and M

antibodies may be IgG, IgM or a mixture of both.

2. Is the autologous control negative or positive?

The autologous control is the patient’s cells tested against the patient’s serum in the

same manner as the antibody screen. A positive antibody screen and a negative

autologous control indicate that an alloantibody has been detected. A positive

autologous control may indicate the presence of auto antibodies or antibodies to


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medications. If the patient has been recently transfused, the positive autologous control

may be caused by alloantibody coating circulating donor RBCs.

3. Did more than one screening cell sample react; if so, did they react at the same

strength and phase?

More than one screening cell sample may be positive when the patient has multiple

antibodies, when a single antibody’s target antigen is found on more than one

screening cell, or when the patient’s serum contains an autoantibody.

A single antibody specificity should be suspected, when all cells react at the same

phase and strength. Multiple antibodies are most likely when cells react at different

phases and strengths and auto antibodies are suspected when the autologous control is

positive.

4. Is hemolysis or mixed – field agglutination present?

Certain antibodies, such as anti-Lea, anti-Leb, anti- PPlPk and anti-vel, are known to

cause in vitro hemolysis. Mixed –field agglutination is associated with anti-Sda and

Lutheran antibodies.

5. Are the cells truly agglutinated or is rouleaux present?

Serum from patients with altered albumin-to-globulin ratios (eg. patients with multiple

myeloma) or who have received high-molecular –weight plasma expanders (e.g.

dextran) may cause nonspecific aggregation of RBCs, known as rouleaux. Rouleaux is

not a significant finding in antibody screening tests, but is easily confused with

antibody-mediated agglutination. Knowledge of the following characteristics of

rouleaux helps in differentiation between rouleaux and agglutination :

Cells have a “stacked coin” appearance when viewed microscopically.

Rouleaux is observed in all test containing the patient’s serum, including the

autologous control and the reverse ABO typing.

Rouleaux does not interfere with the AHG phase of testing because the patient’s

serum is washed away prior to the addition of the AHG reagent.

Unlike agglutination, rouleaux is dispersed by the addition of one to three drops of

saline to the test tube.


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Fig. 44 Examples of reactions that may be obtained in antibody screening tests.

Limitations of Antibody Screen :147

Antibody detection tests have proven to be very effective in the detection of potentially

clinically significant antibodies. If the antibody screen is negative, the confidence level is

over 99% that the cross match will also be compatible. AABB standards state that if no

clinically significant unexpected antibodies are detected and there is no record of previous

detection of such antibodies, only serologic testing to detect ABO incompatibility is

required; that is, antiglobulin testing is not required when the cross match is performed.

Failure to detect an antibody may be due to dosage; that is, weak antibody may be

demonstrable only when a homozygous cell for the specificity is tested. Another reason


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for failure to detect a clinically significant antibody is that the antigen is not present on the

screening cells. Low-incidence antigens such as Cw, V, Kpa, Jsa and Wra are rarely present

on the screening cells. However, selection of donor units that possess the low-incidence

antigen is also unlikely. Because the screening cells are group O, passive transfer of an

unexpected ABO antibody in the patient’s serum may not be detected until the cross match

(in the case of non-group O) is completed.

Several factors may influence the sensitivity of the antibody screen.

These include :148

Cell to serum ratio : When antibody is present in the test system in excess (when related

to antigen), false-negative reactions occur as a result of prozone. When the antigen is in

excess, false-negative reactions occur due to post zone. A ratio of two drops of serum to

one drop of cells usually gives the proper balance between antigen and antibody to

allow sensitization and agglutination to occur. Occasionally, when an antibody is weak,

the amount of serum in the test system may be increased to four drops, providing more

antibodies to react with the available antigens. This should be done only when

potentiators have not been included in the test system.

Temperature : The optimal temperature at which an antibody reacts can be a tool used

in detection and identification. When performing pre transfusion compatibility testing,

the focus is on clinically significant antibodies, which generally react at 370 C or with

AHG. Technologists may omit the immediate spin and room temperature phases to

limit the detection of insignificant cold antibodies.

Table 60 Optimal temperature of Reactivity for some common Antibodies.148


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Length of incubation : Antigen / antibody reactions are in dynamic equilibrium. If too

little contact time is allowed, there will not be enough cells sensitized to detect with

routine methods. If the incubation time is allowed to continue for too long, bound

antibody may begin to dissociate from the cell. Incubation time is dependent on the

medium in which the reaction takes place. A saline environment may require an

incubation of 45 minutes to 1 hr; whereas potentiators may shorten the incubation time

to as little 10 minutes.

pH : Most antibodies react best at a neutral pH between 6.8 and 7.2; however, some

examples of anti-M demonstrate enhanced reactivity at a pH of 6.5. Acidifying the test

system may aid in distinguishing anti-M from other antibodies.

ANTIBODY IDENTIFICATION :149

The techniques employed for antibody detection and antibody identification are similar.

Antibody identification methods can be more focused and based on the reactivity patterns

seen in the antibody detection test.

Specimen Requirements :

Either serum or plasma may be used for antibody detection and identification. Plasma is

not suitable for detecting complement – activating antibodies. A 5 to 10 ml aliquot of

whole blood usually contains enough serum or plasma for identifying simple antibody

specificities; more may be required for complex studies. When autologous red cells are

studied, the use of a sample anti coagulated with EDTA avoids problems associated with

the in-vitro uptake of complement components by red cells, which may occur in clotted

samples.

Medical History :

It is useful to know a patient’s clinical diagnosis, history of transfusions or pregnancies

and recent drug therapy when performing an antibody identification. For example, in

patients who have had recent red cell transfusions, the circulating blood may contain

sufficient donor cells to make red cell phenotyping studies difficult to interpret. Special

procedures to separate the autologous red cells for typing may be required.


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Antibody Identification Panels :

Identification of an antibody to red cell antigen(s) requires testing the serum against a

panel of selected red cell samples with known antigen composition for the major blood

groups. Usually, they are obtained from commercial suppliers, but institutions may

assemble their own by using red cells from local sources. Panel cell are group O, (except

in special circumstances), allowing serum of any ABO group to be tested.

Each cell of the panel is from a different individual. The cells are selected so that, taking

all the cells into account, a distinctive pattern of positive and negative reactions exists for

each of many antigens. To be functional, a reagent red cell panel must make it possible to

identify with confidence those clinically significant alloantibodies that are most frequently

encountered; such as anti-D, -E, -K and –Fya. The phenotypes of the reagent red cells

should be distributed such that single specificities of the common alloantibodies can be

clearly identified and most others excluded. Ideally, the pattern of reactivity for most

examples of single alloantibodies will not overlap with any other; e.g., all of the K+

samples should not be the only ones that are also E+. It may also be valuable to include

red cell samples with a double dose of the antigen in question for antibodies that

frequently show dosage. To lessen the possibility that chance alone has caused an

apparently definitive pattern, there must be a sufficient number of red cell samples that

lack, and sufficient red cell samples that express the required antigens.

Table 61 Antibody identification profile sheet: + indicates the antigen is present on the cell; 0 indicates the antigens is not present.150


183

Commercially prepared panels are generally issued early 2 to 4 weeks. Each panel

contains different red cell samples with different antigen patterns; so it is essential to use

the phenotyping listing sheet that comes with the panel in use. Commercial cells usually

come as 2% to 5% suspension in a preservative medium that can be used directly from the

vial. Washing is generally unnecessary unless the media in which the reagent cells are

suspended are suspected of interfering with alloantibody identification.

Panel cells should not be used beyond the expiration date; however, this is not always

practical. Most serologists use-in-date reagent cells for initial antibody identification

panels and, if necessary, use expired reagent cells for exclusion or confirmation of

specificity. Each laboratory must establish and validate a policy for the use of expired

reagent cells.

Basic Antibody Identification Techniques :151

For initial panels, it is common to use the same methods and test phases used in the

antibody detection test or cross match. Some serologists may choose to include an

immediate centrifugation reading and/or a room temperature incubation and reading

without adding an enhancement medium. This may enhance the detection of certain

antibodies (anti-M,-N,-P1,-I,-Lea or–Leb) and may help to explain reactions detected at

other phases. Many institutions omit these steps to avoid finding antibodies that react only

at lower temperature and have little or no clinical significance. Test observation after 370C

incubation may detect some antibodies (e.g. potent anti-D, -K or –E) that can cause direct

agglutination of red cells. Other antibodies (e.g. anti-Lea, JKa) may be detected by their

lysis of antigen positive red cells during the 370C incubation. Some serologists believe that

because clinically significant antibodies will be detected with the IAT, the reading after

370 C can be safely omitted. This omission will lessen the detection of unwanted positive

reactions resulting from clinically insignificant cold reactive auto and alloantibodies.

The phenotypes of the reactive antibody detection cells will provide dues to the specificity

or help exclude specificities. This information is useful for selecting cells that would be

most informative in additional testing.


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If the patient has previously identified antibodies, this may affect panel selection. For

example, if the patient is known to have anti-e, it will not be helpful to test the serum

against a panel of 10 red cell samples, nine of which are e+. Testing a panel of selected e-

red cell samples will better reveal any newly formed antibodies.

Sometimes, the patient’s phenotype influences the selection of reagent cells. For example,

if the patient is D- and the serum is reactive with D+ cells in the screening test, an

abbreviated panel or select cell panel of D- red cell samples may be tested. This can both

confirm the presence of anti-D and demonstrate the presence of absence of additional

antibodies, while minimizing the amount of testing required.

Interpreting Results :152

Antibody screening results are interpreted as positive or negative based on the presence or

absence of reactivity (eg. Agglutination). Interpretation of panel results can be more

complex process combining technical knowledge and intuitive skills. Panel results

generally will include both positive and negative results at different phases of testing, each

of which should be explained by the final conclusion. Determination of the patient’s red

cell phenotype and the probability of antibody specificity can also play roles in the final

interpretation.

Positives and Negatives :

Both positive and negative reactions are important in antibody identification. Positive

reactions indicate the phase and strength of reactivity, which can suggest certain

specificities. Positive reactions also can be compared to the antigen patterns expressed by

the panel cells to help assign specificity. Single alloantibodies usually yield definite

positive and negative reactions that create a clear-cut pattern with antigen-positive and –

negative reagent red cell samples.

Negative reactions are important in antibody identification because they allow tentative

exclusion of antibodies to antigens expressed on the nonreactive cells. Exclusion of

antibodies is an important step in the interpretation process and must be performed to

ensure proper identification of all the antibodies present.


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Exclusion or “Crossing out” :

A widely used first approach to the interpretation of panel results is to exclude specificities

based on non reactivity with the serum tested. Such a system is sometimes referred to as a

“Cross out” or “rule out” method. Once results have been recorded on the worksheet, the

antigen profile of the first non reactive cell is examined. If an antigen is present on the cell

and the serum did not react with the cell, the presence of the corresponding antibody may

be, at least tentatively excluded. Many technologists will cross out that antigen from the

listing on the panel sheet to facilitate the process. After all antigens present on that cell

have been crossed off, interpretation proceeds with the other non reactive cells and

additional specificities are excluded. In most cases, this process will leave a group of

antibodies that still have not been excluded.

Next, the cells reactive with the serum are evaluated. The pattern of reactivity for each non

excluded specificity is compared to the pattern of reactivity obtained with the test serum.

If there is a pattern that matches exactly, that is most likely the specificity of the antibody

in the serum.

However, if there are remaining specificities that have not been excluded, additional

testing may be needed to eliminated remaining possibilities and to confirm the specificity

identified. This requires testing the serum against cells selected for specific antigenic

characteristics. For example, this approach could be employed if the pattern of positive

reactions exactly fits anti-Jka, but anti-K and anti-S have not been excluded. Then serum

should be tested against selected cells, ideally with the following phenotypes : Jk(a-), K-,

S+; Jk(a-), K+ S-; and Jk(a+), K-S-. The reaction pattern with these cells should both

confirm the presence of anti-Jka and include or exclude anti-K and anti-S.

Although the exclusion (Cross out) approach often identifies simple antibody specificities,

it should be considered only a provisional step, particularly if the cross-out was completed

based on the non reactivity of cells with weaker (eg. heterozygous) expression of an

antigen.


186

Probability:

To ensure that an observed pattern is not the result of chance alone, conclusive antibody

identification requires serum to be tested against sufficient regent red cell samples that

lack, and that express, the antigen that corresponds to the apparent specificity of the

antibody.

A standard approach (based on Fisher’s exact method) has been to require, for each

specificity identified, three antigen positive cells that react and three antigen-negative cells

that fail to react. This standard is not always possible, but it works well in practice,

especially if cells with strong antigen expression are available. A somewhat more liberal

approach is desired from calculations by Harris and Hochman, whereby minimum

requirements for a probability (p) value of 0.05 are met by having two positive and three

negative cells, or one positive and seven negative cells (or the reciprocal of either

combination).

Table 62 Probability Values.153

The use of two positive and two negative cells is also an acceptable approach for antibody

confirmation. The possibility of false-negative results with antigen-positive cells must be

considered as well as unexpected positives, i.e. false-positive results due either to the

presence of an additional antibody specificity or an error in the presumptive antibody

identification.


187

Phenotype of Autologous Red Cells :154

Once an antibody has been tentatively identified in a serum. It is often helpful to

demonstrate the presence or absence of the corresponding antigen on the autologous red

cells. For example, if serum from an untransfused individual appears to contain anti-Fya

but the autologous red cells have a negative DAT and type as Fy (a+), the data are clearly

in conflict and further testing is indicated.

Determination of the patient’s phenotype can be difficult if the patient has been transfused

recently generally within 3 months. If a pretransfusion specimen is available, these red

cells should be used to determine the phenotype. Alternatively, the patient’s own red cells

can be separated from the transfused red cells and then typed. The use of potent blood

typing reagents, appropriate controls and observation for mixed-field reactions often allow

an unseparated specimen to be phenotyped. Phenotyping results on post transfusion

samples can be misleading, however, and should be interpreted with caution. If there is

little uncertainty about antibody identification, extensive efforts to separate and type the

patient’s own red cells are not necessary. Compatible antiglobulin cross match, of antigen-

negative donor units provides additional confirmation of antibody specificity. Definitive

testing can be performed on the patient’s red cells after a sufficient period without red cell

transfusion.


188

Fig. 45 Antibody identification flow chart.

In a chronically transfused patient, definitive testing can be performed after an interval

during which only antigen-negative blood has been given. Any antigen-positive red cells

detected after prolonged transfusion of antigen-negative blood would presumably be the

patient’s own.


189

Complex Antibody Problems :155

Not all antibody identifications are simple. The exclusion procedure does not always lead

directly to an answer and additional approaches may be required.

Fig. 46 Approaches for identifying antibodies


190

Variations in Antigen Expression :

For a variety of reasons, antibodies do not always react with all cells positive for the

corresponding antigen. Basic interpretation by exclusion, may result in a given specificity

being excluded because the sample is non reactive with an antigen-positive red cell

sample, despite the presence of the antibody. Technical error, weak antibody reactivity,

and variant or weak antigenic expression are all possible causes. Therefore, whenever

possible, antibody specificities should be excluded only on the basis of cells known to bear

a strong expression of the antigen. Enhancement techniques often help resolve problems

associated with variations in antigen expression.

Zygosity :

Reaction strength of some antibodies may vary from one one red cell sample to another

due to a phenomenon known as dosage, in which antibodies react preferentially with red

cells from persons homozygous for the gene that determines the antigen (i.e. possessing a

“double dose” of the antigen). Red cells from individuals heterozygous for the gene may

express less antigen and may react weakly or be nonreactive. Alloantibodies vary in their

tendency to recognize dosage. Many antibodies to antigens in the Rh, Duffy, MNS and

Kidd systems have this trait.

Variations in Adults and Infants :

Some antigens (e.g. I, P, Lea and Sda) are expressed to varying degrees on red cells from

different donors. This variation is unrelated to zygosity; however, the antigenic differences

can be demonstrated serologically. Certain antibodies (eg. Anti-I, Lea) demonstrate weaker

reactivity with cord red cells than with red cells from adults.


191

Table 63 Antigen Expression on Cord Red Blood Cells. 155

Changes with Storage :

Blood group antibodies may give weaker reactions with stored red cells than with fresh

red cells. Some antigens (eg. Fya, Fyb, M, P1, Kna / McCa, Bg) deterorate during storage

more rapidly than others and the rate varies among red cells from different donors.

Because red cells from donors are often fresher than commercial reagent cells, some

antibodies give stronger reactions with suspensions of donor cells than with reagent cells.

Frozen storage of red cells may result in antigen deterioration that can cause misleading

antibody identification results.

The pH or other characteristics of storage media can affect the rate of antigen

deterioration. For example, Fya and Fyb antigens may be weakened when the cells are

stored in a suspending medium of low pH and low ionic strength. Alternatively, certain

antibodies may demonstrate stronger or weaker reactions with red cells from different

manufacturers using different suspending media. The age and nature of the specimen must

also be considered when typing red cells. Antigens on cells from clotted samples tend to

deteriorate faster than antigens on cells collected in citrate anticoagulants such as ACD or

CPD. Red cells in donor units collected into these anticoagulants generally retain their

antigens throughout the standard shelf life of the blood component. EDTA samples up to

14 days old are suitable for antigen typing; however, the manufacturer’s instructions

should be consulted when using commercial typing reagents.

No Discernible Specificity :

Factors other than variation in antigen expression may contribute to difficulty in

interpreting results of antibody identification tests. If the reactivity obtained with the

serum is very weak and / or if the cross out process has excluded all likely specificities,

alternative approaches to interpretation should be used.


192

Antigens present in common :

Instead of excluding antibodies to antigens on non reactive cells, one can observe what

antigens are common to the reactive cells. For example, if the cells reacting at room

temperature are all P1+, yet not all the P1+ cells react. The antibody could be an anti-P1

that does not react with cells having a weak expression of the antigen. (Sometimes, such

cells are marked on the panel sheet as “+w”). With this in mind, one could use a method to

enhance anti-P1, such as testing at colder temperatures.

If all the reactive cells are Jk (b+), but not all the Jk (b+) cells react, the reactive ones

might all be Jk (a-b+), with a double dose expression of the antigen. Enhancement

techniques, such as enzymes, LISS or PEG, may then help demonstrate reactivity with all

the remaining Jk (b+) cells. Typing the patient’s cells to confirm they lack the

corresponding antigen can also be very helpful.

Inherent variability :

Nebulous reaction patterns that do not appear to fit any particular specificity are

characteristic of antibodies, such as anti-Bga, that react with HLA antigens on red cells.

These antigens vary markedly in their expression on red cells from different individuals.

Rarely, a pattern of clear cut reactive and non reactive tests that cannot be interpreted is

due to the incorrect typing of reagent red cells.

Unlisted Antigens :

Sometimes a serum sample reacts with an antigen not routinely listed on the antigen

profile supplied by the reagent manufacturer; Ytb is one example. Even though serum

studies yield clear-cut reactive and non reactive tests, anti-Ytb may not be suspected. In

such circumstances, it is useful to ask the manufacturer for additional phenotype

information. If the appropriate blood typing reagent is available, reactive and non reactive

red cell samples, as well as the autologous red cells, can be tested. These problems often

have to be referred to an immune homology reference laboratory.


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ABO Type of Red Cells Tested:

A serum sample may react with many or all of the group O reagent red cell samples, but

not with red cells of the same ABO phenotype as the autologous red cells. This occurs

most frequently with anti-H, -IH or –LebH. Group O and A2 red cells have large amounts

of H antigen; A1 and A1B red cells express very little H. Sera containing anti-H or –IH

react strongly with group O reagent red cell samples, but autologous A1 or A1B red cells or

donor cells used for cross matching may be weakly reactive or non reactive. Anti- LebH

reacts strongly with group O, Le (b+) red cells, but reacts weakly or not at all with Le(b+)

red cells from A1 or A1B individuals. Such antibodies should be suspected when the

antibody screen, which uses group O red cells, is strongly reactive, but serologically

compatible A1 or A1B donor samples can be found without difficulty.

Multiple Antibodies :

When a serum contains two or more alloantibodies, it may be difficult to interpret the

results of testing performed on a single panel of reagent red cells. The presence of multiple

antibodies may be suggested by a variety of test results.

1. The observed pattern of reactive and non reactive tests does not fit that of a single

antibody. When the exclusion approach fails to indicate a specific pattern, it is helpful

to see if the pattern matches any two combined specificities. If the typing patterns for

no two specificities fit the reaction pattern, the possibility of more than two antibodies

must be considered. The more antibodies a serum contains, the more complex the

identification and exclusion of specificities will be, but the basic process remains the

same.

2. Reactivity is present at different test phases. When reactivity occurs at several phases,

each phase should be evaluated separately. The pattern seen at room temperature may

indicate a different specificity from the pattern of antiglobulin results. It is also helpful

to look at variability in the strength of reactions seen at each phase of testing.

3. Unexpected reactions are obtained when attempts are made to confirm the specificity

of a suspected single antibody.


194

If a serum suspected of containing anti-e reacts with additional samples that are e-,

another antibody may be present or the suspected antibody may not be anti-e. Testing a

panel of selected e-red cell samples may help indicate an additional specificity.

4. No discernible pattern emerges

When uniform or variable reaction strengths are observed and dosage or other

variation in strength does not provide an explanation, additional approaches and

methods of testing are indicated. Some helpful steps include.

a) If strong positive results are obtained, use the exclusion method with non reactive

cells to eliminate some specificities from initial consideration.

b) If weak or questionable positive results are obtained, test the serum against cells

carrying a strong expression of antigens corresponding to any suspected

specificities and combine this with methods to enhance reactivity.

c) If the patient has not been recently transfused, type the patient’s red cells and

eliminate from consideration specificities that correspond to antigens on the

autologous cells.

d) Use methods to inactivate certain antigens on the red cells e.g. enzyme treatment to

render cells negative for antigens such as Fya, Fyb and S.

e) Use adsorption / elution methods to separate antibodies.

f) Enhance antibody reactivity by using a more sensitive method (e.g. PEG).

Antibodies to High-Incidence Antigens :

If all reagent red cell samples are reactive, but the auto control is nonreactive, an

alloantibody to a high-incidence antigen should be considered, especially if the strength

and test phase of reactions are uniform for all cells tested. Antibodies to high-incidence

antigens can be identified by testing red cells of selected rare phenotypes and by testing

the patient’s autologous red cells with sera known to contain antibodies to high-incidence

antigens. Knowing the race or ethnic origin of the antibody producer can help in selecting

additional tests to be performed. Cells that are null for all antigens in a system (eg. Rhnull

or K0) or modified red cells, (eg. dithiothreitol treated cells) can help limit possible

specificities to a particular blood group.


195

If cells negative for particular high-incidence antigens are not available, cells positive for

lower-incidence alleles can sometimes be helpful. Weaker reactivity with Co (a+b+) cells

when compared with common Co (a+b-) cells, for instance, might suggest anti-Coa.

Antibodies to high-incidence antigens may be accompanied by other antibodies to

common antigens, which can make identification much more difficult. Because the

availability of cells negative for high-incidence antigens is limited, it may be necessary to

refer specimens suspected of containing antibodies to high-incidence antigens to an

immune hematology reference laboratory.

Serologic Clues :

Knowledge of the serologic characteristics of particular antibodies of high-incidence

antigens can help in identification.

1. Reactivity in tests at room temperature suggests anti-H, -I, -P1, -P, -PP1Pk (-TJa), -LW

(some), -Ge (Some), -Sda or –vel.

2. Lysis of reagent red cells when testing with fresh serum is characteristics of anti-vel, -P,

-PP1Pk and –Jk3. It is also seen with some examples of anti-H and –I.

3. Reduced or absent reactivity in enzyme tests occurs with anti-Ch, -Rg, -Inb, -JMH or –

Ge2 and is seen with some examples of anti-Yta .

4. Weak nebulous reactions in the antiglobulin phase are often associated with anti-Kna, -

McCa, -Yka and –Csa. Complement – binding auto antibodies, such as anti-I or anti-IH,

give similar results when polyspecific antiglobulin reagents are used.

5. Antibodies such as anti-U, -McCa, -S1a, -Jsb, -Hy, -Joa, -Tca, -Cra and –Ata should be

considered if the serum is from a Black individual because the antigen-negative

phenotypes occur almost exclusively in Blacks. Individuals with anti-Kpb are almost

always white. Anti-Dib is usually found among Asian, South American Indians, and

Native American populations.


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Interpreting a Positive DAT :

When a patient produces antibody directed to a high-incidence antigen after transfusion,

the post transfusion red cells may have a positive DAT, and both serum and elute may

react with all cells tested. Because this pattern of reactivity is identical to that produced by

many warm-reactive auto antibodies that may also appear after transfusion, these two

scenarios can be very difficult to differentiate. A post-transfusion alloantibody to a high-

incidence antigen would be expected to produce a DAT of mixed-field appearance (i.e.,

some cells agglutinated among many unagglutinated cells) because only the transfused red

cells would be coated with antibody. In practice however weak sensitization and mixed-

field sensitization can be difficult to differentiate. If a pre-transfusion red cell sample is

not available, it may be helpful to use cell separation procedures to isolate autologous cells

for testing. Performing a DAT on autologous cells and / or testing the post-transfusion

serum with DAT-negative autologous cells may help to distinguish autoantibody from

alloantibody.

Antibodies to Low-incidence Antigens :155

Reactions between a serum sample and a single donor or reagent red cell sample may be

caused by an antibody to a low-incidence antigen, such as anti-Wra. If red cells known to

carry low-incidence antigens are available, the serum can be tested against them, or the

one reactive red cell sample can be tested with known examples of antibodies to low-

incidence antigens. A single serum often contains multiple antibodies to low-incidence

antigens; therefore, the expertise and resources of an immunohematology reference

laboratory may be required to confirm the suspected specificities.

Serologic Strategies :

If an antibody to a low-incidence antigen is suspected, transfusion should not be delayed

while identification should not be delayed while identification studies are undertaken. If an

antibody in the serum of a pregnant woman is thought to be directed against a low-

incidence antigen, testing the father’s red cell can predict the possibility of incompatibility

with the fetus, and identifying the antibody is unnecessary. If a newborn has a positive

DAT, testing of the mother’s serum or an eluate from the infant’s cells against the father’s

red cells (assuming they are ABO compatible) can implicate an antibody to a low-


197

incidence antigen as the probable cause; identifying the antibody is usually of little

importance.

Some reference laboratories do not attempt to identify antibodies to low-incidence

antigens because they are often only of academic interest.

Unexpected Positive Results :

When a serum reacts with a panel cell designated as positive for a low-incidence antigen,

further testing to exclude the antibody is usually unnecessary. For every antigen of low-

incidence represented on a panel, there are many more that are not represented and are

also not excluded by routine testing. Reactivity against low-incidence antigens is not

uncommon; although the antigens are rare, antibodies against some of the low-incidence

antigens are much less rare. Presumably, the testing is being performed because the serum

contains some other antibody and reactivity with the cell expressing the low-incidence

antigen is a coincidental finding. This may complicate interpretation of the panel results

but rarely requires confirmation of antibody specificity or typing of donor blood to ensure

the absence of the antigen. If typing is desired, a negative crossmatch with the patient’s

serum is sufficient demonstration that the antigen is absent. Many antibodies to low-

incidence antigens are reactive only at temperatures below 370C and are of doubtful

clinical significance.

When the serum reacts only with red cells from a single donor unit or reagent cell, the

other possibilities to consider are that the reactive donor red cells are ABO-incompatible,

have a positive DAT, or are polyagglutinable.


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Antibodies to Reagent Components and Other Anomalous Serologic Reactions :155

Antibodies to a variety of drugs and additives can cause positive results in antibody

detection and identification tests.

Most of these anomalous reactions are in-vitro phenomenon and have no clinical

significance in transfusion therapy other than causing laboratory problems that delay

needed transfusion. They rarely cause erroneous interpretations of ABO typing that could

endanger the patient.

Ingredients in the Preservative Solution :

Antibodies that react with an ingredient in the solution used to preserve reagent red cells

(e.g. chloramphenicol, neomycin, tetracycline, hydrocortisone, EDTA, Sodium caprylate,

or various sugars) may agglutinate cells suspended in that solution. Reactivity may occur

with cells from several commercial sources or may be limited to cells from a single

manufacturer. The autologous control is often non-reactive, unless the suspension of

autologous red cells is prepared with the manufacturer’s red cell diluents or a similar

preservative. Such reactions can often be circumvented by washing the reagent cells with

saline before testing. The role of the preservative can often be confirmed by adding the

medium to the autologous control and converting a non-reactive test to a positive test. In

some cases, however, washing the reagent cells does not circumvent reactivity and the

resolution may be more complex.

Ingredients in enhancement Media :

Antibodies reactive with ingredients in other reagents, such as commercially prepared

LISS additives or albumin, can cause agglutination in tests using reagent, donor, and / or

autologous red cells. Ingredients, that have been implicated include parabens (in some

LISS additives), sodium caprylate (in some albumins) and thimerosal (in some LISS /

saline preparation). Antibody to ingredients in enhancement media may be suspected if the

autologous control is positive but the DAT is negative. Omitting the enhancement medium

will usually circumvent this reactivity.


199

In some cases, antibodies dependent upon reagent ingredients will show blood group

specificity, eg. Paraben-dependent anti-Jka, caprylate-dependent anti-c. The auto control

may be reactive if the patient’s own red cells carry the antigen, but the DAT should be

negative.

Problems with Red Cells :

The age of the red cells can cause anomalous serologic reactions. Antibodies exist that

react only with stored red cells; they can cause agglutination of reagent red cells by all

techniques and enhanced reactivity in tests with enzyme-treated red cells. Such reactivity

is not effected by washing the red cells and the auto control is usually non reactive. No

reactivity will be seen in tests on freshly collected red cells, i.e. from freshly drawn donor

or autologous blood samples.

The Patient with a Positive Auto control :155

No recent Transfusions :

Reactivity of serum with the patient’s own cells may indicate the presence of auto

antibody. If this reactivity occurs at room temperature or below, the cause is often anti-I or

another cold auto agglutinin. Reactivity of the auto-control in the antiglobulin phase

usually signifies a positive DAT and the possibility of auto antibody. If, in addition, the

serum reacts with all cells tested, auto adsorption or other special procedures may be

necessary to determine whether autoantibody in the serum is masking any significant

alloantibodies. It the serum is not reactive or shows only weak reactivity, an eluate may

demonstrate more potent auto antibody.

A negative DAT but a positive auto control by an IAT is unusual and may indicate

antibody to a reagent constituent causing in-vitro reactivity with all cells, including the

patient’s own. It may also indicate the presence of warm autoantibodies or cold

autoagglutinins such as anti-I, -IH or –Pr reacting by IAT when enhancement media are

used.


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Cold Autoantibodies :

Potent cold autoagglutinins that react with all cells, including the patient’s own, can create

special problems, especially when reactivity persists at temperatures above room

temperature. Cold auto agglutinins may be benign or pathologic.

There are different approaches to testing a serum with a potent cold agglutinin. One

approach is to determine if the thermal amplitude is high enough (usually 300 C or above)

that the antibody has clinical significance. For identification purposes and determination

of thermal amplitude, in-vitro auto adsorption of the serum must be avoided by keeping

the freshly collected blood warm (370C) until the serum is separated. For purposes of

detecting potentially clinically significant antibodies, methods that circumvent the cold

autoantibody are commonly used.

Procedures for the detection of alloantibodies in the presence of cold-reactive

autoantibodies include

1. Prewarmed techniques, in which the red cells and serum to be tested, and saline used

for washing are incubated at 370C before they are combined.

2. The use of anti-IgG rather than polyspecific antiglobulin serum.

3. Cold autoadsorption, to remove auto antibodies but not alloantibodies.

4. Adsorption with rabbit red cells.

Dealing with warm autoantibodies :

Patients with warm-reactive auto antibody present in their sera create a special problem

because the antibody reacts with virtually all cells tested. If such patients are to be

transfused, it is important to detect any clinically significant alloantibodies that the

autoantibody may mask.


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Reactivity of most warm-reactive autoantibodies is greatly enhanced by such methods as

PEG and enzymes and to lesser extent by LISS and albumin. It may be advantageous to

perform antibody detection tests without the enhancement media usually employed. If

tests are non reactive, the same procedure can be used for compatibility tests, without the

need for adsorptions.

Recent Transfusions :

If the auto control is positive in the antiglobulin phase, there may be antibody-coated cells

in the patient’s circulation, causing a positive DAT, which may show mix-field reactivity.

Elution may be helpful, especially when tests on serum are in conclusive. For example,

recently transfused patient may have a positive auto control and serum that reacts weakly

with most but not all Fy (a+) red cells. It may be possible to confirm anti-Fya specificity

by elution, which concentrates into a small fluid volume. It is rare for transfused cells to

make the auto control positive at other test phases, but it can occur, especially with a

newly developing or cold-reactive alloantibody.

If the positive DAT does not have a mixed-field appearance and especially, if the serum is

reactive with all cells tested, the possibility of auto antibody should be considered.

Detection of masked alloantibodies may require allogenic adsorptions.

Accurate phenotyping of red cells may be difficult if the DAT is reactive in any patient,

whether or not there has been recent transfusion. A positive DAT will cause the cells to be

reactive in any test requiring the addition of antiglobulin serum and with some reagent

antibodies (notably those in the Rh system) in a high protein medium. With rare exception,

most monoclonal reagents not tested by an IAT can give valid phenotyping results despite

a positive DAT.

Frequency of Antibody Testing :156

Once an antibody has been identified in a patient’s serum, how frequently should antibody

detection and identification tests be performed? A primary antibody response will produce

detectable antibody as early as 7 to 10 days but typically over a period of 2 weeks to

several months. A secondary immune response produces detectable antibody in a shorter

time, as early as 2 to 7 days and usually within 20 days. Shulman, found that in a small


202

number of patients, “new” antibodies could be detected within 1 to 2 days after

transfusion. AABB standards for Blood Banks and transfusion services requires that, for a

patient who has been pregnant or received red cells within the preceding 3 months,

antibody detection and compatibility tests must be performed on a specimen obtained

within 3 days of the next scheduled transfusion. The transfusion service may consider

testing a fresher specimen if clinical evidence suggests failure of recently transfused red

cells to survive as expected.

If a patient has previously identified clinically significant antibodies, antigen-negative red

cells must be selected for all future transfusions, even if the antibodies are no longer

detectable. In addition, an antiglobulin cross match must be performed using antigen-

negative red cells.

It is rarely necessary to repeat identification of known antibodies. AABB standards states

that in patients with previously identified antibodies, methods of testing shall be those that

identify additional clinically significant antibodies. Each laboratory should define and

validate methods for the detection of additional antibodies in these patients. Depending on

the specificity of the known antibody, repeated testing of the patient’s serum against

routine antibody detection cells is often not informative. It is more useful to test against

cells negative for the antigen(s) to which the patient has antibody and positive for other

major antigens. This allows detection of most additional antibodies that might develop.

Usually, appropriate cells can be selected from available red cell panels. Selection of test

cells may be simplified if the patient’s cells are known to express a given antigen. The

selected cells need not be positive for that antigen because the corresponding antibody

would not be anticipated.


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3.10 SELECTED SEROLOGIC PROCEDURES USEFUL IN ANTIBODY DETECTION AND IDENTIFICATION :

Many techniques and methods may be useful in antibody identification; however no single

method is optimal for detecting all antibodies in all samples.

Table 64 Special Techniques in Antibody Identification: Serum Procedures.157


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Table 65 Special Antibody Identification Techniques: Red Cell Procedures.158

Enhancement Techniques :159

When a pattern of weak reactions fails to indicate a specificity or when the presence of an

antibody is suspected but cannot be demonstrated, enhancement techniques may be useful.

LISS and PEG :

LISS and PEG may be used to enhance reactivity and reduce incubation time. LISS

methods include the use of Low-ionic-strength saline for resuspension of test cells and,

more commonly, the use of commercially available low-ionic-strength additive media.

The use of a LISS additive requires no preparatory stages, but care should be taken to

adhere closely to the manufacturer’s product insert to ensure that the appropriate

proportion of serum to LISS is achieved. Commercially prepared LISS additives may

include other enhancement components besides low-ionic-strength saline. Commercially

prepared PEG additives are also available and may contain additional enhancing agents.


205

Because LISS and PEG enhance autoantibody activity, their use may create problems with

certain samples.

Enzyme Techniques:

Treatment of red cells with proteolytic enzymes enhances their reactivity with antibodies

in the Rh, P, I, Kidd, Lewis and some other blood group systems and simultaneously

destroys or weakens reactivity with other antibodies, most notably those in the Duffy and

MNS systems. The clinical significance of antibodies that react only with enzyme

techniques is questionable. The literature indicates that “enzyme-only” antibodies may

have no clinical significance.

Temperature Reduction :

Some alloantibodies (eg anti-M, -N, -P1, -Lea, -Leb,-A1) that react at room temperature

react better at lower temperatures; specificity may be apparent only below 220C. An auto

control is especially important for tests at cold temperatures because many sera also

contain anti-I or other cold-reactive auto antibodies.

Increased Serum-to-Cell Ratio :

Increasing the volume of serum incubated with a standard volume of red cells may

enhance the reactivity of antibodies present in low concentration. One acceptable

procedures is to mix 5 to 10 volumes of serum with one volume of a 2% to 5% saline

suspension of red cells and incubate for 60 minutes at 370C; periodic mixing during

incubation promotes contact between red cells and antibody molecules. It is helpful to

remove the serum before washing the red cells for the antiglobulin test because the

standard three or four washes may be insufficient to remove all the unbound

immunoglobulin present in the additional volume. Additional washes are not

recommended because bound antibody molecules may dissociate. Increasing the serum-to-

red cell ratio is not appropriate for tests using a low-ionic strength medium or requiring

specific proportions of serum and additive.


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Increased Incubation Time :

For most antibodies, a 15 minute incubation period is insufficient to achieve equilibrium

and the observed reactions may be weak, particularly in saline or albumin media.

Extending incubation to 30 to 60 minutes may improve reactivity and help clarify the

observed pattern of reactions.

Extended incubation may have a negative effect when LISS or PEG are used. If incubation

exceeds the recommended times for these methods, antibody reactivity may be lost. Care

must be taken to use all reagents according to the manufacturer’s directions.

Alteration of pH :

Decreasing the pH of the reaction system to 6.5 enhances the reactivity of certain

antibodies, notably some examples of anti-M. If anti-M is suspected because the only cells

agglutinated are M+N-, modifying the serum to a pH of 6.5 may reveal a definitive pattern

of anti-M reactivity. The addition of one volume of 0.1N HC1 to nine volumes of serum

brings the pH to approximately 6.5. The acidified serum should be tested against known

M-cells as a control for nonspecific agglutination. Similarly, some examples of anti-P may

benefit from a lower pH.

Low pH, however, significantly decreases reactivity of some antibodies. If unbuffered

saline used for cell suspensions and for washing has a pH much below 6.0, antibodies in

the Rh, Duffy, Kidd and MNS systems may lose reactivity. Use of phosphate-buffered

saline can control pH and enhance detection of antibodies poorly reactive at a lower pH.

Techniques to Isolate, Remove or Depress Antibody Reactivity.160

It is sometimes useful to decrease or eliminate the reactivity of an antibody. This can be

done by inhibiting the antibody with specific substances, by physically removing

immunoglobulin molecules or by removing (or weakening) corresponding antigens from

the red cells. Such methods can help confirm suspected specificities and promote

identification of additional antibodies.


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Inhibition Tests :

Soluble forms of some blood group antigens exist in such body fluids as saliva, urine or

plasma or can be prepared from other sources. These substances can be used to inhibit

reactivity of the corresponding antibody. If, for example, a suspected anti-P1 does not give

a definitive agglutination pattern, loss of reactivity after addition of soluble P1 substance

strongly suggests that this is the specificity. A parallel dilution control with saline is

essential.

Inhibition can also be used to neutralize antibodies that mask the concomitant presence of

non neutralizable antibodies. The following soluble blood group substances can be used in

antibody identification tests:

1. Lewis Substances :

Lea and / or Leb substances are present in the saliva of persons who possess the Le

gene. Lea substance is present in the saline of Le(a+b-) individuals, and Le(a-b+)

persons have both Lea and or Leb substances in their saliva. Commercially prepared

Lewis substance is also available.

2. P1 Substance :

Soluble P1 substances is present in hydatid cyst fluid and can be prepared from pigeon

egg whites. P1 substance is available commercially.

3. Sda substance :

Soluble Sda blood group substance is present in various body fluids; the most abundant

source is urine. To confirm anti-Sda specificity in a serum sample, urine from a known

Sd(a+) individual (or a pool of urine specimens) can be used to inhibit reactivity. Urine

known to lack Sda substance, or saline, should be used as a negative control.

4. Chido and Rodgers Substances :

Ch and Rg antigens are epitopes of the fourth component of human complement (C4).

Anti-Ch and Rg react by an IAT with the trace amounts of C4 present on normal red

cells. If red cells are coated in vitro with excess C4, these antibodies may cause direct


208

agglutination. A useful test to identify anti-Ch and –Rg is by the inhibition of the

antibodies with plasma from Ch+, Rg+ individuals.

5. Blood group sugars :

Sugars that correspond to the immunodominant configurations of A, B, H and some

other red cell structures can be used to inhibit antibodies. Inhibiting anti-A or –B may

allow a serum to be tested against non group-O cells.

Inactivation of Blood Group Antigens :

Certain blood group antigens can be destroyed or weakened by suitable treatment.

Table 66 Alteration of Antigens by various Agents.160

Modified cells can be used both in confirming the presence of suspected antibodies and in

detecting additional antibodies. This can be especially helpful if the antigen is one of high

incidence and antigen-negative cells are rare.

Proteolytic enzymes are commonly used. Ficin, papain, trypsin and bromelin, remove

agents such as M, N, Fya, Fyb, Xga, JMH, Ch and Rg. Depending on the specific enzyme

and method used, other antigens may be altered or destroyed. Antigens inactivated by one

proteolytic enzyme will not necessary be inactivated by other enzymes.

Sulfhydryl reagents such as 2-amino- ethylisothiouronium bromide (AET) or dithiothreitol

(DTT) can be used to weaken or destroy antigens Kell system and some other antigens.

ZZAP reagent, which contains proteolytic enzyme and DTT, denatures antigens sensitive

to DTT (eg. all Kell system antigens) in addition to enzyme- sensitive antigens. Glycine-


209

HCI/EDTA treatment of red cells also destroys Bg and Kell system antigens. However,

with the exception of Era antigen, other antigens outside the Kell system that are often

destroyed by sulfhydryl reagents remain intact. Chloroquine diphosphate can be used to

weaken the expression of Class I HLA antigens (Bg antigens) on red cells. Chloroquine

treatment also weakens some other antigens, including Rh antigens.

Adsorption :

Antibody can be removed from a serum sample by adsorption to red cells carrying the

corresponding antigen. After the antibody attaches to the membrane-bound antigens and

the serum and cells are separated, the specific antibody remains attached to the red cells. It

may be possible to harvest the bound antibody by elution.

Adsorption techniques are useful in such situations as :

1. Separating multiple antibodies present in a single serum.

2. Removing auto antibody activity to permit detection of coexisting alloantibodies.

Fig. 47 Steps for performing an autoadsorption.

3. Removing unwanted antibody (often anti A and / or anti-B) from serum that contains

an antibody suitable for reagent use.


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4. Confirming the presence of specific antigens on red cells through their ability to

remove antibody of corresponding specificity from previously characterized serum.

5. Confirming the specificity of an antibody by showing that it can be adsorbed only to

red cells of a particular blood group phenotype.

Fig. 48 Autoadsorption procedure used to evaluate whether underlying alloantibodies are present in a

patient with autoimmune hemolytic anemia.

Adsorption serves different purposes in different situations; there is no single

procedure that is satisfactory for all purposes. The usual serum-to-cell ratio is one

volume of serum to an equal volume of washed red cells. To enhance antibody uptake,

the proportion of antigen can be increased by using a larger volume of cells. The

incubation temperature should be that at which the antibody is optimally reactive.

Pretreating red cells with a proteolytic enzyme may enhance antibody uptake and

reduce the number of adsorptions required for complete removal of antibody. Because

some antigens are destroyed by proteases, antibodies directed against these antigens

will not be removed by enzyme-treated red cells.

In separating mixtures of antibodies, the selection of red cells of the appropriate

phenotype is extremely important and depends on the object of the separation. If none

of the antibodies in the serum has been identified, weakly reactive cells may be used,

on the assumption that they are reactive with only a single antibody. The phenotype of

the person producing the antibody gives a clue to what specificities might be present,


211

and cells intended to separate those particular antibodies can be chosen. If one or more

antibodies have been identified, cells lacking those antigens are usually chosen so that

only one antibody is removed. Adsorption requires a substantial volume of red cells.

Vials of reagent red cells usually will not suffice, and blood samples from staff

members or donor units are the most convenient sources.

Elution :

Elution frees antibody molecules from sensitized red cells. Bound antibody may be

released by changing the thermodynamics of antigen-antibody reactions, by

neutralizing or reversing forces of attraction that holds antigen-antibody complexes

together, or by disturbing the structure of the antigen-antibody binding site. The usual

objective is to recover bound antibody in a usable form.

Table 67 Antibody Elution Techniques.161

Various elution methods have been described. No single method is best in all

situations. Use of heat or freeze-thaw elution is usually restricted to the investigation

of HDFN due to ABO incompatibility because these elution procedures rarely work

well for antibodies on outside the ABO system. Acid or organic solvent methods are

used for elution of warm-reactive auto-and alloantibodies.

Technical factors that influence the success of elution procedures include :

1. Incorrect technique :

Such factors as incomplete removal of organic solvents or failure to correct the

tonicity or pH of an eluate may cause the red cells used in testing the eluate to

hemolyze or to appear “sticky”. The presence of stromal debris may interfere with


212

the reading of tests. Careful technique and strict adherence to protocols should

eliminate such problems.

2. Incomplete Washing :

The sensitized red cells must be thoroughly washed before elution to prevent

contamination of the eluate with residual serum antibody. If it is known that the

serum does not contain antibody, saline washing may not be necessary. Six washes

with saline are usually adequate, but more may be needed if the serum contains a

high-titer antibody. To determine the efficacy of the washing process, supernatant

fluid from the final wash phase should be tested for antibody activity and should be

non reactive.

3. Binding of Proteins to Glass Surfaces :

If the eluate is prepared in the same test tube that was used during the sensitization

phase (eg. in an adsorption / elution process), antibody non specifically bound to

the test tube surface may dissociate during the elution. Similar binding can also

occur from a whole blood sample if the patient has a positive DAT and free

antibody in the serum. To avoid such contamination, the washed red cells should

be transfused into a clean test tube before the elution procedure is begun.

4. Dissociation of antibody before elution :

IgM antibodies, such as anti-A or –M may spontaneously dissociate from the cells

during the wash phase. To minimize this loss of bound antibody, cold (40C) saline

can be used for washing. Although this is not a concern with most IgG antibodies,

some low-affinity IgG antibodies can also be lost during the wash phase. If such

antibodies are suspected, washing with cold LISS instead of normal saline may

help maintain antibody association.

5. Instability of eluates :

Dilute protein solutions, such as those obtained by elution into saline, are unstable.

Eluates should be tested as soon after preparation as possible. Alternatively, bovine

albumin may be added to a final concentration of 6% w/v and the preparation

stored frozen. Eluates can also be prepared directly into antibody-free plasma, 6%

albumin, or a similar protein medium instead of into saline.


213

Elution techniques are useful for:

1. Investigation of a positive Direct Antiglobulin Test.

2. Concentration and purification of antibodies, detection of weakly expressed

antigens and identification of multiple antibody specificities. Such studies are used

in conjunction with an appropriate adsorption technique.

3. Preparation of antibody-free red cells for use in phenotyping or autologous

adsorption studies.

Combined Adsorption Elution :

Combined adsorption-elution tests can be used to separate mixed antibodies from a single

serum, to detect weakly expressed antigens on red cells, or to help identify weakly reactive

antibodies. The process consists of first incubating serum with selected cells, then eluting

antibody from the adsorbing red cells. Both the eluate and treated serum can be used for

further testing. Unmodified red cells are generally used for adsorption and subsequent

elution; elution from enzyme-or ZZAP-treated cells may create technical problems.

Use of Sulfhydryl Reagents :

Sulfhydryl reagents such as DTT and 2-mercaptoechanol (2-ME), cleave the disulfide

bonds that join the monomeric subunits of the IgM pentamer. Intact 19S IgM molecules

are cleared into 7S subunits, which have altered serologic reactivity. The interchain bonds

of 7S Ig monomers are relatively resistant to such cleavage. Sulfhydryl reagents are used

to diminish or detroy IgM antibody reactivity. DTT also destroys certain red cell antigens.

The applications of DTT and 2-ME in immunohematology include :

1. Determining the immunoglobulin class of an antibody.

2. Identifying specificities in a mixture of IgM and IgG antibodies, particularly when an

agglutinating IgM antibody masks the presence of IgG antibodies.


214

3. Determining the relative amounts of IgG and IgM components of a given specificity

(eg. Anti-A or –B).

4. Dissociating red cell agglutinates caused by IgM antibodies (eg. the spontaneous

agglutination of red cells caused by potent auto antibodies).

5. Dissociating IgG antibodies from red cells using a mixture of DTT and a proteolytic

enzyme (ZZAP reagent).

6. Converting nonagglutinating IgG antibodies into direct agglutinins. Commercially

prepared chemically modified, blood typing reagents for use in rapid saline tube, slide,

or microplate tests have been manufactured in this manner.

7. Destroying selected red cell antigens (eg. those of the Kell, Dombrock, Cartwright and

LW systems) for use in antibody investigations.

Titration :

The titer of an antibody is usually determined by testing serial two fold dilutions of the

serum against selected red cell samples. Results are expressed as the reciprocal of the

highest serum dilution that shows macroscopic agglutination. Titration values can provide

information about the relative amount of antibody present in a serum, or the relative

strength of antigen expression on red cells.

Titration studies are useful in the following situations :

1. Prenatal studies :

When the antibody is of a specificity known to cause HDFN or its clinical significance

is unknown, the results of titration studies may contribute to the decision about

performing invasive procedures, eg. Amniocentesis.


215

2. Antibody identification :

Some antibodies that agglutinate virtually all reagent red cell samples may produce an

indication of specificity by demonstrating reactivity of different strength with different

samples in titration studies. For examples, potent autoanti-I may react in the undiluted

state with both adult and cord red cells, but titration may reveal reactivity at a higher

dilution with adult I+ red cells with cord red cells.

Most weakly reactive antibodies lose reactivity when diluted even modestly, but some

antibodies that give weak reactions when undiluted continue to react at dilutions as

high as 1 in 2048. Such antibodies include anti-Ch, -Rg, -CSa, YKa, -Kna, -McCa, -

JMH and other specificities. When weak reactions are observed in indirect antiglobulin

tests, titration may be used to indicate specificity within this group. Not all antibodies

of the specificities mentioned demonstrate such “high titer, low avidity”

characteristics. Thus, although demonstration of these serologic characteristics may

help point to certain specificities, failure to do so does not eliminate those possibilities.

Antibodies of other specificities may sometimes react at high titres.

3. Separating multiple antibodies :

Titration results may suggest that one antibody reacts at higher dilutions than other.

This information can allow the serum to be diluted before testing against a cell panel,

effectively removing one antibody and allowing identification of the other.

Other Methods :

Methods other than traditional tube techniques may be used for antibody identification.

Some are especially useful for identifying individual antibody specificities, for dealing

with small values of test reagents, for batch testing or for use with automated systems.

Such methods include testing in capillary tubes, microplates or by solid phase;

enzyme-linked immunosorbent assays; and column agglutination (eg. Gel techniques).

Other methods useful in laboratories with specialized equipment include

radioimmunoassay, immunofluorescence (including flow cytometric procedures),

immunoblotting and immunoelectrode biosensoring.

3. REVIEW OF LITERATURE -...

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