POSTLAB DISCUSSIONS IN IH LABORATORY BLOOD GENETICS EXPERIMENT 5 TO 7

COMPILED BY: FORTUNE LAPIRA TORRECAMPO, RMT

BASIC GENETIC PRINCIPLES OF BLOOD GROUP SYSTEMS

Genetic Information
Gene basic unit of genetic information. Genes determine the inherited characters. Genome the collection of genetic information.

Chromosomes storage units of genes.

DNA - is a nucleic acid that contains the genetic instructions specifying the biological development of all cellular forms of life

Chromosome Logical Structure

Locus location of a gene/marker on the chromosome. Allele one variant form of a gene/marker at a particular locus.
Locus1 Possible Alleles: A1,A2

Locus2 Possible Alleles: B1,B2,B3

Human Genome
Most human cells contain 46 chromosomes: 2 sex chromosomes (X,Y): XY in males. XX in females. 22 pairs of chromosomes named autosomes.

Genotypes

Phenotypes

At each locus (except for sex chromosomes) there are 2 genes. These constitute the individuals genotype at the locus. The expression of a genotype is termed a phenotype. For example, hair color, weight, or the presence or absence of a disease.

Genotypes Phenotypes (example)

genotypes

phenotypes

Eb- dominant allele. Ew- recessive allele.

Dominant vs. Recessive

A dominant allele is expressed even if it is paired with a recessive allele. A recessive allele is only visible when paired with another recessive allele.

One Locus Inheritance

Female
A|A 1 2 a|a

Male

A| a

a|a

A| a

a|a

heterozygote

homozygote

Mendelian Genetics

Mendels 1st Law

Two members of a gene pair segregate from each other into the gametes, so half the gametes carry one member of the pair and the other half carry the other member of the pair.

Y/y

y/y

Gamete production

Gamete production

all y y/y y Y/y

Y

Calculating Probabilities
We want to predict patterns of inheritance of traits and diseases in pedigrees. E.g., we want to know the likelihood that a dog chosen at random from the study population will have blue eyes.

X-linked Inheritance
Different results obtained from reciprocal crosses between red-eyed and whiteeyed Drosophila.

Explanation: The gene responsible for eye-color is X-linked. Females have 2 X-chromosomes, while males have 1 X-chromosome and 1 Y-chromosome.

Mendels 2nd Law

Different gene pairs assort independently in gamete formation.

This law is true only in some cases.

Gene pairs on SEPARATE CHROMOSOMES assort independently at meiosis.

Medical Genetics
When studying rare disorders, 6 general patterns of inheritance are observed:
Autosomal recessive Autosomal dominant X-linked recessive X-linked dominant Codominant

Mitochondrial

Medical Genetics (cont.)

Autosomal recessive The disease appears in male and female children of unaffected parents. e.g., cystic fibrosis

Medical Genetics (cont.)

Autosomal dominant Affected males and females appear in each generation of the pedigree. Affected mothers and fathers transmit the phenotype to both sons and daughters. e.g., Huntington disease.

Medical Genetics (cont.)

X-linked recessive Many more males than females show the disorder. All the daughters of an affected male are carriers. None of the sons of an affected male show the disorder or are carriers. e.g., hemophilia

Medical Genetics (cont.)

X-linked dominant Affected males pass the disorder to all daughters but to none of their sons. Affected heterozygous females married to unaffected males pass the condition to half their sons and daughters
e.g. fragile

X syndrome

Medical Genetics (cont.)

Codominant inheritance Two different versions (alleles) of a gene can be expressed, and each version makes a slightly different protein Both alleles influence the genetic trait or determine the characteristics of the genetic condition. E.g. ABO locus

Medical Genetics (cont.)

Mitochondrial inheritance This type of inheritance applies to genes in mitochondrial DNA Mitochondrial disorders can appear in every generation of a family and can affect both males and females, but fathers do not pass mitochondrial traits to their children. E.g. Leber's hereditary optic neuropathy (LHON)

Notes
Cystic fibrosis disease affecting the mucus lining of the lungs, leading to breathing problems and other difficulties Huntington disease - or Huntington's chorea is an inherited disorder characterized by abnormal body movements called chorea, and loss of memory. There also is evidence that doctors as far back as the Middle Ages knew of this devastating disease. The incidence is 5 to 8 per 100,000. It takes its name from the New York physician George Huntington who first described it precisely in 1872.

Notes
Hemophilia-illness that impair the body's ability to control bleeding. Fragile X syndrome - is a genetic condition that causes a range of developmental problems including learning disabilities and mental retardation. Usually males are more severely affected by this disorder than females. In addition to learning difficulties, affected males tend to be restless, fidgety, and inattentive. Affected males also have characteristic physical features that become more apparent with age.

Notes -cont
DNA - a pair of molecules joined by hydrogen bonds: it is

organized as two complementary strands, head-to-toe, with the hydrogen bonds between them. Each strand of DNA is a chain of chemical "building blocks", called nucleotides, of which there are four types: adenine (abbreviated A), cytozyne (C), guanine (G) and thymine (T).

Mitochondria, which are structures in each cell that convert molecules into energy, each contain a small amount of DNA.

A chromatid forms one part of a chromosome after it has coalesced for the process of mitosis or meiosis. During either process, the word "chromosome" indicates a pair of two exactly identical ("sister") chromatids joined at the central point of each chromatid, called the centromere.

Notes -cont
Mitosis is the process by which a cell separates its duplicated genome into two identical halves Meiosis is the process that transforms one diploid into four haploid cells. Reciprocal cross a cross, with the phenotype of each sex reversed as compared with the original cross, to test the role of parental sex on inheritance pattern. A pair of crosses of the type genotype A(female) X genotype B(male) and genotype B(female) X genotype A(male).

Hardy-Weinberg Principle
Hardy-Weinberg - original proportions of genotypes in a population will remain constant from generation to generation
Sexual reproduction (meiosis and fertilization) alone will not change allelic (genotypic) proportions. p+q = 1 (always two alleles)

Hardy-Weinberg Principle
Necessary assumptions
Allelic frequencies would remain constant if population size is very large random mating no mutation no gene input from external sources no selection occurring

REVIEW
HOW TO USE THE HARDY WEINBERG PRINCIPLE HUMAN DNA MODERN GENETIC TECHNIQUES

Before proceeding to the ABO BLOOD GROUPING let us recall the Ag Ab reactions

IMMUNOGLOBULINS

drmsaiem

BIOLOGICAL & CHEMICAL PROPERTIES OF Igs

IgG is the major immunoglobulin in human serum, accounting for approximately 75%. Concentration of approximately 1200 mg/dl.
IgG is a monomer consisting of identical pairs of H and L chains linked by disulfide bridges. Four subclasses of IgG have been identified, based on H chain differences: IgG1, IgG2, IgG3, and IgG4.

IgG is the only immunoglobulin that can cross the placenta in humans and protect the infant during the first months of life. IgG molecules are capable of binding complement by the classical pathway (except for the IgG4, which activate by the alternative pathway).

drmsaiem

BIOLOGICAL AND CHEMICAL PROPERTIES OF Igs

IgG is the major antibody produced in the secondary immune response
IgG has a half-life of approximately 21 days Effective antitoxic immunity is exclusively IgG. IgG is the major opsonizing immunoglobulin in phagocytosis; neutrophils have receptors for the Fc fragments of IgGI and IgG3.

BIOLOGICAL AND CHEMICAL PROPERTIES OF Igs

IgM. Represents about 8% to 10% of the total serum Igs . Concentration of I2O mg/dl.

IgM has a pentameric structure consisting of five monomer units linked by a J chain and by disulfide bonds at the Fc fragment.
IgM is easily dissociated by reducing agents, forming five monomeric units.

drmsaiem

BIOLOGICAL AND CHEMICAL PROPERTIES OF Igs

IgM is the first antibody that an immunologically committed B lymphocyte can produce. It has a half-life of approximately 10 days.
IgM is the predominant antibody in the early (primary) immune response to most antigens.

IgM is the only antibody made to certain carbohydrate antigens, such as the ABO blood group antigens on human erythrocytes.
IgM is the most efficient immunoglobulin at activating complement in lytic reactions.

Allo-antibodies & Auto-antibodies

Antigens that initiate the immune cascades results in the formation of either allo-abs or auto-abs. Allo-antibodies are produced after exposure to genetically different or non-self, antigens of the same species. Auto-antibodies are produced in response to self antigens.

Factors Influence Agglutination Reaction

Concentration of Ag-Ab The agglutination reaction is influenced by the, concentration of both Ag and Ab as well as other factors such as pH, temp. ionic strength, surface change, Ab class, red cell Ag dosage, the use of various enhancement media
Under ideal reaction conditions, an equivalent amount of Ag and Ab bind in optimal proportions.

drmsaiem

Factors Influence Agglutination Reaction

Prozone effect: an excess of either Ag or Ab, may lead to unbound immunoglobulin

Postzone effect: surplus (excess of Ag-binding sites)

In prozone or postzone effect the lattice formation and subsequent agglutination may not occur in the test system, leading to the assumption of false negative results.
To correct the problem of excessive Ab, the serum may be diluted with each serial dilution of serum tested against red cells.

To correct the problem of excessive Ag, the serum to cell-ratio in the test system may be in which will tend to increase the number of Abs available to bind with red cell.

drmsaiem

Factors Influence Agglutination Reaction

Effect of pH

The ideal pH is between 6.5 and 7.5, exceptions to this range include some examples of Anti-M, and some Abs of the Pr (SP1) group which show stronger reactivity below pH 6.5.

drmsaiem

Factors Influence Agglutination Reaction

Temperature
Different types of Abs may exhibit optimal reactivity at different temp. IgM Abs usually react optimally at or below 22C. IgG Abs usually require 37C temp. The Ag-Ab reactions are exothermic (heat is given up), therefore, Abs binds to a greater degree at low temperature

drmsaiem

Laboratory Examination of Reaction between Ags and Abs

In vitro testing for the detection of Ags or Abs may be accomplished by a variety of immunologic techniques. Such techniques as agglutination, precipitation, agglutination inhibition, and hemolysis are the most commonly used methods to detect the presence of blood group Ags or Abs. In blood bank testing, agglutination reactions are the major manifestation of the blood group Ag-Ab response. Typing for ABO, Rh, and other blood group Ags is accomplished by agglutination reaction.

drmsaiem

Laboratory Examination of Reaction between Ags and Abs

There are two stages for agglutination to develop; Stage 1: known as sensitization, Ab binding occurs. Antigenic determinants on the red cell membrane combine with the antigen combining site (Fab region) on the variable regions of the immunoglobulin heavy and light chains.
Stage 2: a lattice structure composed of multiple Ag-Ab bridges between Abs and red cell Ags is formed visible agglutination is present during this stage.

drmsaiem

ABO BLOOD GROUP SYSTEM

History: Karl Landsteiner

Discovered the ABO Blood Group System in 1901 He and his five coworkers began mixing each others red cells and serum together and inadvertently performed the first forward and reverse ABO groupings
http://www.nobelpreis.org/castellano/medizin/images/landsteiner.jpg

Why is it important?
ABO compatibility between donor cell and patient serum is the essential foundation of pretransfusion testing It is the only system with expected antibodies Whether they are IgG or IgM, ABO antibodies can activate complement readily
This means that incompatibilities can cause life threatening situations (transfusion reactions)

ABO antigens:
Biochemical & Genetic Considerations

ABO and H Antigen Genetics

Genes at three separate loci control the occurrence and location of ABO antigens The presence or absence of the A, B, and H antigens is controlled by the H and ABO genes

Location
The presence or absence of the ABH antigens on the red blood cell membrane is controlled by the H gene The presence or absence of the ABH antigens in secretions is indirectly controlled by the Se gene

ABO Antigen Genetics

H gene H and h alleles (h is an amorph)

Se gene Se and se alleles (se is an amorph)

ABO genes A, B and O alleles

H Antigen
The H gene codes for an enzyme that adds the sugar fucose to the terminal sugar of a precursor substance (PS) The precursor substance (proteins and lipids) is formed on an oligosaccharide chain (the basic structure)

RBC Precursor Structure

RBC

Glucose Galactose N-acetylglucosamine Galactose

Precursor Substance (stays the same)

Formation of the H antigen

RBC

Glucose

H antigen

Galactose N-acetylglucosamine Galactose

Fucose

H antigen
The H antigen is the foundation upon which A and B antigens are built A and B genes code for enzymes that add an immunodominant sugar to the H antigen
Immunodominant sugars are present at the terminal ends of the chains and confer the ABO antigen specificity

A and B Antigen
The A gene codes for an enzyme (transferase) that adds N-acetylgalactosamine to the terminal sugar of the H antigen
N-acetylgalactosaminyltransferase

The B gene codes for an enzyme that adds D-galactose to the terminal sugar of the H antigen
D-galactosyltransferase

Formation of the A antigen

RBC

Glucose Galactose N-acetylglucosamine Galactose N-acetylgalactosamine

Fucose

Formation of the B antigen

RBC

Glucose Galactose N-acetylglucosamine Galactose Galactose

Fucose

Genetics
The H antigen is found on the RBC when you have the Hh or HH genotype, but NOT from the hh genotype The A antigen is found on the RBC when you have the Hh, HH, and A/A, A/O, or A/B genotypes The B antigen is found on the RBC when you have the Hh, HH, and B/B, B/O, or A/B genotypes

H antigen
Certain blood types possess more H antigen than others:

Greatest amount of H

O>A2>B>A2B>A1>A1B

Least amount of H

 Why do Group O individuals have more H antigen than the other groups?

Group O individuals have no A or B genes to convert the H antigen to A or B antigens.that means more H antigen sites

A A Group O

A A
Group A

Many H antigen sites

Fewer H antigen sites

Most of the H antigen sites in a Group A individual have been converted to the A antigen

ABO Antigens in Secretions

Secretions include body fluids like plasma, saliva, synovial fluid, etc Blood Group Substances are soluble antigens (A, B, and H) that can be found in the secretions. This is controlled by the H and Se genes

Secretor Status
The secretor gene consists of 2 alleles (Se and se) The Se gene is responsible for the expression of the H antigen on glycoprotein structures located in body secretions If the Se allele is inherited as SeSe or Sese, the person is called a secretor
80% of the population are secretors

Secretors
Secretors express soluble forms of the H antigen in secretions that can then be converted to A or B antigens (by the transferases) Individuals who inherit the sese gene are called nonsecretors The se allele is an amorph (nothing expressed) sese individuals do not convert antigen precursors to H antigen and has neither soluble H antigen nor soluble A or B antigens in body fluids

Secretor Status Summary

The Se gene codes for the presence of the H antigen in secretions, therefore the presence of A and/or B antigens in the secretions is contingent on the inheritance of the Se gene and the H gene
A antigen
Se gene (SeSe or Sese) H antigen in secretions
and/or

B antigen

se gene (sese)

No antigens secreted in saliva or other body fluids

ABO Group
Secretors (SeSe or Sese):
A B O

ABH Substances
A
+++ 0 0

B
0 +++ 0

H
+ + +++

AB
Non-secretors (sese):

+++

+++

A, B, O, and AB

Sese + h/h (no H antigen) no antigens in secretions

Lewis (Le)
The Lewis Blood Group System is mentioned here because it is related to secretor status Lewis antigens are plasma antigens formed by tissues and are released into plasma where they adsorb onto the RBCs (they are not an integral part of the RBC membrane) Consists of 2 antigens
Lea Leb

Lewis
Lea and Leb are a single gene (Le) and its amorph (le) Lea is a precursor to Leb The Le gene codes for a transferase, which attaches L-fucose to the precursor chain to form the Lea antigen (designated Le(a+b-) If the H and Se genes are inherited, the Lea is converted to Leb and is designated Le(a-b+) In childhood, both may be on the RBC, Le(a+b+) If a person is lele, they will have no Lewis antigens in plasma or on red blood cells

ABO Subgroups
ABO subgroups differ in the amount of antigen present on the red blood cell membrane Subgroups have less antigen Subgroups are the result of less effective enzymes. They are not as efficient in converting H antigens to A or B antigens (fewer antigens are present on the RBC) Subgroups of A are more common than subgroups of B

Subgroups of A
The 2 principle subgroups of A are: A1 and A2 Both react strongly with reagent anti-A To distinguish A1 from A2 red cells, the lectin Dolichos biflorus is used (anti-A1) 80% of group A or AB individuals are subgroup A1 20% are A2 and A2B

A2 Phenotype
Why is the A2 phenotype important? A2 and A2B individuals may produce an anti-A1 This may cause discrepancies when a crossmatch is done (incompatibility) Whats the difference between the A1 and A2 antigen? Its quantitative The A2 gene doesnt convert the H to A very well The result is fewer A2 antigen sites compared to the many A1 antigen sites

A1 and A2 Subgroups*
Anti-A Anti-A1 Anti-H antisera antisera lectin ABO antibodies in serum # of antigen sites per RBC

A1
A2

4+
4+

4+
0

0
3+

Anti-B
Anti-B & anti-A1

900 x103
250 x103

*Adapted from Flynn, J. (1998). Essentials of Immunohematology

Other A subgroups
There are other additional subgroups of A Aint (intermediate), A3, Ax, Am, Aend, Ael, Abantu A3 red cells cause mixed field agglutination when polyclonal anti-A or anti-A,B is used Mixed field agglutination appears as small agglutinates with a background of unagglutinated RBCs They may contain anti-A1

B Subgroups
B subgroups occur less than A subgroups B subgroups are differentiated by the type of reaction with anti-B, anti-A,B, and anti-H B3, Bx, Bm, and Bel

Other ABO conditions

Bombay Phenotype (Oh) Inheritance of hh The h gene is an amorph and results in little or no production of L-fucosyltransferase Originally found in Bombay (now Mumbai) Very rare (130 worldwide)

Bombay
The hh causes NO H antigen to be produced Results in RBCs with no H, A, or B antigen (patient types as O) Bombay RBCs are NOT agglutinated with anti-A, anti-B, or anti-H (no antigens present) Bombay serum has strong anti-A, anti-B and anti-H, agglutinating ALL ABO blood groups What blood ABO blood group would you use to transfuse this patient??

ANSWER:
Another Bombay
Group O RBCs cannot be given because they still have the H antigen You have to transfuse the patient with blood that contains NO H antigen

ABO Blood Group:

ABO Antibodies

Landsteiners Rule:
Normal, Healthy individuals possess ABO antibodies to the ABO antigen absent from their RBCs

ABO Blood Group System

The ABO Blood Group System was the first to be identified and is the most significant for transfusion practice It is the ONLY system that the reciprocal antibodies are consistently and predictably present in the sera of people who have had no exposure to human red cells

Blood Group Systems

Most blood group systems (ABO and others) are made up of: An antigen on a red cell and the absence of its corresponding antibody in the serum (if youre A, you dont have anti-A) If you do NOT have a particular antigen on your red cells then it is possible (when exposed to foreign RBCs) to illicit an immune response that results in the production of the antibody specific for the missing antigen

ABO
Remember:
The ABO Blood Group System does NOT require the presence of a foreign red blood cell for the production of ABO antibodies ABO antibodies are non-red blood cell stimulated probably from environmental exposure and are referred to as expected antibodies

ABO antibodies
group A serum contains anti-B group B serum contains anti-A group AB serum contains no antibodies group O serum contains anti-A, anti-B, and anti-A,B

Anti-A1
Group O and B individuals contain anti-A in their serum However, the anti-A can be separated into different components: anti-A and anti-A1 Anti-A1 only agglutinates the A1 antigen, not the A2 antigen There is no anti-A2.

Anti-A,B
Found in the serum of group O individuals Reacts with A, B, and AB cells Predominately IgG, with small portions being IgM Anti-A,B is one antibody, it is not a mixture of anti-A and anti-B antibodies

ABO antibodies
IgM is the predominant antibody in Group A and Group B individuals
Anti-A Anti-B

IgG (with some IgM) is the predominant antibody in Group O individuals

Anti-A,B (with some anti-A and anti-B)

ABO antibody facts

Reactions phase: Room temperature Complement can be activated with ABO antibodies (mostly IgM, some IgG) High titer: react strongly (4+)

ABO Antibodies
Usually present within the first 3-6 months of life Stable by ages 5-6 years Decline in older age Newborns may passively acquire maternal antibodies (IgG crosses placenta) Reverse grouping (with serum) should not be performed on newborns or cord blood

Nature of antibodies
Non-red blood cell stimulated (previously discussed) ABO antibodies Red blood cell stimulated Antibodies formed as a result of transfusion, etc Usually IgG Active at 37C Can occur in group O (may occur in group A or B) These antibodies also occur in the other Blood Group Systems

Laboratory Testing:
ABO typing

The Use of Lectins for Antigen Confirmation

Dolichos biflorus = anti-A1 Ulex europaeus = anti-H

90

ABO Blood Groups

ABO Group A
B

Antigen Present A
B

Antigen Missing B
A

Antibody Present anti-B

anti-A

O
AB

None
A and B

A and B
None

anti-A, anti-B, anti-A,B None

Forward & Reverse Typing

Reaction of cellsReaction of serum tested with: tested with:

anti-A

anti-B

A cells

B cells

1 2
3 4

0 +
0 +

0 0
+ +

+ 0
+ 0

+ +
0 0

ABO group O

A
B AB

ABO Ag LOCATION IN THE BODY

Body fluids
Saliva Tears Urine Digestive juices Bile Milk Amniotic fluid

Pathologic fluids
Pleural Peritoneal Pericardial Ovarian cyst

ABO Antigens and Antibodies

ABO antigens based on combinations of three genes: A, B, and O Antibodies are clinically significant and naturally occurring causing most fatal acute HTRs some causing HDFN ABO antibodies neutralized with secretor saliva.

94

Group O
Generally the most common blood group Genotype: OO Antigen: H Antibodies: anti-A, anti-B, and anti-A,B Antibodies are naturally occurring and very strong Anti-A,B (mostly IgG) may cross placenta to cause HDFN

95

Group A
Genotype: AA, AO Antigen: A, H Antibodies: anti-B (primarily IgM) A subgroups A1 (80%) and A2 (20%) most important A1 has more A than A2 (quantitative difference); qualitative differences, too. ~25% of A2Bs form anti-A1 1-8% of A2s form anti-A1 Lectin of Dolichos biflorus agglutinates A1

96

Group B
Genotype: BB, BO Antigen: B, H Antibodies: anti-A (primarily IgM) B subgroups: Not important

97

Group AB
Genotype: AB Antigen: A, B, very little H Antibodies: None B subgroups: Not important A2B: 25% of A2B individuals produce anti-A1

12/11/2011

98

ABO Testing
Cell typing (forward grouping) to determine antigen types on RBCs Serum/plasma typing (reverse grouping or backtyping) to determine type of antibody in serum: Note the opposite reactions If the forward reactions are opposite of reverse, an ABO discrepancy is not present.

99

ABO Grouping Reagents

Forward Grouping Reagent Reverse or Back Tying Cells

100

Forward Grouping Reagent

101

Forward Grouping
Reagent: Monoclonal antibody Highly specific IgM Expected 3+- to 4+ reaction 1 drop Anti-A=Blue; anti-B=Yellow (Acroflavin dye) A and B antigens on patient red cells are agglutinated by known sera (anti-A, anti-B)

102

Reverse or Back Tying Reagent Cells

103

Reverse or Back Typing

Reagent Cells: Human Source Expected 2+ to 4+ reaction 4-5% cell suspension 1 drop Anti-A or anti-B antibodies in patient serum (or plasma) agglutinate with A1 and B antigens on Reagent cells

104

Forward Typing Procedures

To determine what antigens are present on RBCs.

105

Step 1. Label test tubes.

106

Step 2: Make a 2-5% patient red cell suspension.

107

Step 3: Add reagent antisera (1 drop).

108

Step 3A: Add reagent Anti-A antisera (1 drop).

109

Step 3B: Add Anti-B reagent antisera (1 drop).

110

Step 4: Add one drop of 2-5% suspension of patient RBC to each tube.

111

Step 5: Mix and centrifuge (approximately 20 seconds).

112

Group A: 4+ Agglutination with Anti-A 0 Agglutination with Anti-B

113

Group B: 4+ Agglutination with Anti-B 0 Agglutination with Anti-A

114

Group AB: 4+ Agglutination with Anti-A and Anti-B

115

Group O: No Agglutination with Anti-A or Anti-B

116

Back Typing
To determine what antibodies are present in patients plasma.

117

Step 1: Label Test Tubes

118

Step 2: Add two drops of patient serum to each tube

119

Step 3: Add one drop of reagent cells to each test tube

120

Step 3A: Add one drop of Reagent A1 cells

121

Step 3B: Add one drop of Reagent B cells

122

Step 4: Mix and centrifuge (approximately 20 seconds)

123

Group A: 4+ Agglutination with B Cells 0 Agglutination with A1 Cells

124

Group B: 4+ Agglutination with A1 Cells 0 Agglutination with B Cells

125

Group O: 4+ Agglutination with A1 Cells 3+ Agglutination with B Cells

126

Group AB: No Agglutination with A1 and B Cells

127

Exercises
Interpretation of test results

128

Exercises: Interpretation of ABO Testing Results

Forward anti-A Anti-B 4+ 0 4+ 0 4+ 4+ Reverse A1 cells B cells 0 4+ 0 4+ 0 0 Interpretation ABO Group ??

?? ??

4+

4+

??

129

Exercises: Interpretation of ABO Testing Results

Forward anti-A Anti-B Reverse A1 cells B cells Interpretation ABO Group

4+ 0 4+ 0

0 4+ 4+ 0

0 4+ 0 4+

4+ 0 0 4+

A B AB O

130

What can Cause ABO Discrepancies?

Disagreement between the interpretations of forward and reverse grouping Antigen problems Antibody problems

131

Antigen Problems
Lack of expected antigens A subgroup B subgroup Bombay Presence of unexpected antigens Acquired B phenotype Polyagglutinable RBCs, recent marrow transplant, nonspecific agglutination

132

Antibody problems
Lack of expected antibodies Immunodeficiency, neonates, abnormally high concentrations of Ab (prozone) Presence of unexpected antibodies Anti-A1, cold auto-or alloantibodies, rouleaux (false positive)

133

A Subgroups
A1 A2 A3 Ax Aend Am etc

134

A1 vs A2 Phenotypes
Blood Group A1 (80%) A2 (20%) Anti-A + + Anti-A1 lectin + 0

A1 & A2 account for 99% of A group

135

A1vs A2 Phenotypes
Quantitative differences: More antigenic sites on A1 than A2. Qualitative differences between A1 and A2 antigens:
1-8% of A2 individuals produce anti-A1 25% of A2B individuals produce anti-A1.

136

B Subgroups
Very rare and are less frequent than A subgroups. B subgroups demonstrate variations in the strength of the reaction using antiB and anti-A,B

Examples are: B3, Bx, Bm, Bel

137

Acquired B phenotype
Occurs in type A individuals with: Colon cancer, intestinal obstruction, gram negative sepsis Bacteria deacetylate group A sugar (GalNAc); remaining galactosamine crossreacts with reagent anti-B.

138

Acquired B phenotype

139

Acquired B phenotype
AB forward (with weak reactions with reagent anti-B) A reverse Reaction with anti-B is negative, if: Acidify serum Acetic anhydride treatment Auto incubation
140

Acquired B typing result

Forward
Anti-A Interp Anti-B Interp

Reverse
A1 cells B cells

4+ 1-2+ AB

AB

4+

141

Bombay (Oh) Phenotype

Total Lack of H, A, and B antigens Develop strong anti-H, anti-A, and anti-B O forward, O reverse; with positive antibody screen Require other Bombay donors for blood transfusion (Para-Bombay = H antigen in secretions)

142

Reactivity of Anti-H with ABO Blood Groups

O>A2>B>A2B>A1>A1B

143

Blood Type: Antigens vs Antibodies

Blood Type
A B AB O

Antigens on rbcs
A B A,B None

Antibodies in Plasma
Anti-B Anti-A None Anti-A, Anti-B

144

Exercise
Blood Type A B AB Compatible RBCs ___ ___ ___ Compatible FFPs ___ ___ ___ Compatible Whole Blood ___ ___ ___

___

___

___

145

ABO Compatible Blood Components

Blood Type
A B AB O

Compatible RBCs
A, O B, O AB, A, B, O O

Compatible FFPs
A, AB B, AB AB A, B, AB, O

146

ABO Compatible Whole Blood

Blood type A B AB O Compatible WB A B AB O

147

Consequences of ABO incompatibility

Severe acute hemolytic transfusion reactions
One of the most frequent causes of blood bank fatalities Clerical errors

Most frequent HDFN; usually mild.

148

Sources of Technical Errors Resulting in ABO Discrepancies

Inadequate identification of blood samples Cell suspension too heavy or too light Clerical errors A mix-up in samples Missed observation of hemolysis Failure to add reagents Failure to follow manufacturers instructions Uncalibrated centrifuge Contaminated reagents Warming during centrifugation

12/11/2011

149

Resolving ABO Discrepancies

Problems with RBCs Resolution Techniques Rouleaux wash RBCs 4X MF agglutination check tx hx Unusual phenotype (hh) Test with anti-H Disease processes (Acq. B) check patient diagnosis

150

Resolving ABO Discrepancies (Contd)

Problems with serum Resolution Techniques Rouleaux Saline replacement Presence of unexpected Ab Do panel to ID Absence of expected Ab Increase incubation time

151

Other Blood Groups

The Kell Blood Group System

Background information
The Kell blood group system was discovered in 1946. Number of Kell antigens: > 20 These antigens are the third most potent, after those of the ABO and Rh blood groups, at triggering an immune reaction.

Molecular information
The KEL gene is found on chromosome 7 The KEL gene is highly polymorphic, with different alleles at this locus encoding the 25 antigens that define the Kell blood group. The Kell protein is a polypeptide chain of 732 amino acids in length that becomes glycosylated at five different sites. It makes a single pass through the RBC membrane.

Kell Blood Group System

XK gene produces Kx substance, which is a precursor of of Kell Ags Kel genes convert Kx substance into the Kell Ags on RBCs K (Kell) & k (cellano) are produced by allelic genes, this results into 3 phenotypes: K+k- (genotype KK) K+k+ (genotype Kk) K-k+ (genotype kk) Other allelic genes include: Kpa/Kpb, Jsa/Jsb

XK Gene (Chromosome X)

KEL Gene
RBC

Kx

Kell system glycoprotein: Kell Ags reside here.

Kx Substance
Kx substance is present on RBCs & WBCs Kell genes convert Kx substance into the Kell Ags on RBCs Kell genes do not convert Kx on WBCs

 Absence of Kx proteins in RBCs membrane lead to McLeod Phenotype This absence cause:

McLeod Phenotype

abnormal RBCs shape (acanthocytes) & reduced in-vivo survival

Chronic Granulomatous Disease

Absence of Kx proteins in WBCs cause CGD Leukocytes are able to phagocytose but not to kill bacteria Patients with CGD have recurrent bacterial infections Patients who lack Kx on RBCs & WBCs have both Mcleod and CGD

Kell Null (K0) Phenotype

1. K0 is a silent Kell
allele 2. When homozygous K0K0 inherited no Kell system antigens are expressed. 3. Kx antigen expression is enhanced 4. Very rare

Kx

Kell Antibodies
K- individuals produce anti-K when exposed to K+ cells
Frequency of K is low (9%), easy to find compatible blood for the patient with anti-k.

On the other hand frequency of k antigen is 99.9%

Difficult to find blood

Antibodies produced against Kell antigens

Kell Abs
Clinically Significant Yes Abs class

IgG , (rarely) IgM

Thermal range 4 - 37 Transfusion Reactions HDNB Yes

Extravascular Yes

Intravascular Rare

Duffy Blood Group System

The Duffy blood group was discovered in 1950. The Duffy glycoprotein is encoded by the FY gene, found on chromosome 1 , of which there are two main alleles, FYA and FYB. They are codominant. The Duffy gene codes for a glycoprotein also found in other tissues: brain, kidney, spleen, heart and lung. The Duffy glycoprotein is a transmembrane protein Five alleles at Duffy locus, the most important: Fya, Fyb & Fy (Silent Allele) Fya is more immunogenic than Fyb

 Fy(a-b-) blacks do not produce anti-Fya or anti-Fyb following transfusion with Fy(a+) or Fy(b+) blood Fy(a-b-) Caucasians become sensitized following transfusion with Fy(a+) or Fy(b+) blood This suggest that Fy(a-b-) phenotype arises from different genes in the two populations

Different genes

Duffy Antigens
Fya, Fyb antigens are Destroyed by enzymes Abs DO NOT agglutinate enzyme treated cells Moderately immunogenic.

Duffy Antibodies
Duffy Abs
IgG antibodies and can activate complement Anti- Fya is more frequently encountered Anti- Fyb is more frequently found in patients produced multiple alloantibodies
Clinically Significant Yes Thermal range 4 - 37 Abs class

IgG HDNB Yes

Transfusion Reactions Extravascular Intravascular

Yes

Yes

Duffy and Malaria

Black people with the Duffy phenotype of Fy(ab) appear to have resistance to Plasmodium vivax & Plasmodium knowlesi causative agents of Malaria. Duffy antigens appear to be a receptor for the P. vivax organism and when the antigen is not present on the red blood cell membrane P. vivax is unable to access the red blood cell Some areas of West Africa are 100% Fy(ab). Plasmodium falciparum binds to RBCs at integral glycophorin A & B

Kidd Blood Group System

The Kidd blood group was discovered in 1950. The Kidd gene is located on chromosome 18 Three alleles: Jka, Jkb, Jk Codominant Inheritance Jk is a silent allele (amorph) The Kidd protein is an integral protein of the RBC membrane.

Kidd Antigens & Antibodies

Ags are well developed at birth Have tendency to drop to low or undetectable levels following formation.

Abs are of IgG type & can activate complement (Anti-Jka, Anti-Jkb ) Produced following transfusion or pregnancy Can cause HDNB They are also a very common cause of delayed HTRs

MNSs Blood Group System

The antigens M and N are produced by codominant alleles closely linked to the S and s genes, which are also co-dominant. Chromosome 4 contains these linked genes Genes produce two distinct glycophorins or sialyglycoproteins (SGP) on the RBC membrane.

MN Genetics
MN Locus genes produce Glycophorin A (GPA) M-GPAs 1st five aas = Serine-Ser-Thr-ThrGlycine N-GPAs 1st five aas = Leucine-Ser-Thr-ThrGlutamic acid Amino acids (aa) 2, 3 & 4 are the same for both Glycophorin A (GPA) is a glycoprotein also known as MN-sialoglycoprotein

MN Genotypes & Phenotypes

Phenotype M+NM+N+ M-N+ Genotype MM MN NN Frequency % 30 50 20

P Blood Group System

Genetics: These genes code for enzymes that sequentially add sugars to precursor substance. This system is related to the ABO, Le and Ii systems. Genes: P1, Pk, P and lower case p (silent allele) All antigens are expressed on glycolipids on red cells

Phenotypes, Detectable Antigens & Frequencies

Phenotype
P1 P2 Detectable Antigens Frequencies

Whites % 79% 21% Rare

Rare Rare

P1, P P P, Pk Pk
N/A

Pk1 Pk2
p

Pk is the precursor of P.
Rare individuals do not convert Pk into P. Those will have Pk on RBCs.

Anti-P1 Antibodies
Naturally occcurring Abs found in the serum of P2 Individuals

Anti-P1 Abs
Clinically Significant occasionally Thermal range HDNB Abs class

IgM

4 22
Rare 22-37

Yes

Transfusion Reactions Extravascular No Intravascular Rare

Allo Anti-P Antibodies

Allo Anti-P Abs

Naturally occcurring Abs found in the serum of Pk and p Individuals

Clinically Significant Yes Thermal range

4 37S

Abs class
IgM Rare IgG

HDNB
Rare

Transfusion Reactions Extravascular No Intravascular Yes

Auto anti-P Antibodies

It is an IgG biphasic Ab associated with Paroxysmal Cold Hemoglobinuria (PCH) Binds complement at cold temperatures and activates that complement in warm temperatures lysing the red blood cells.

Auto Anti-P Abs

Clinically Significant Yes Biphasic HDNB Abs class

IgG

Binds at 0
Hemolysis 37

Rare

Transfusion Reactions Extravascular Rare Intravascular Yes

Anti Tja Antibodies

Combination of anti-P, anti-P1 & anti-Pk Found in serum of individuals who have no P, P1 & Pk Ags on red cells