immunohematology and blood banking techniques class notes

IMMUNOHEMATOLOGY &

BLOOD BANKING TECHNIQUES

INTRODUCTION:

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IMMUNO HEMATO LOGY

IMMUNOHEMATOLOGY

DEFINITIONS:

Protection against infection or disease caused by foreign

particles.

IMMUNITY:

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4 Study of the cellular

components of the blood. HEMATOLOGY:

Study of the immune system and the immune response.

Study of antigen-antibody reactions in vivo.

IMMUNOLOGY:

Study of antigen-antibody reactions in vitro.

SEROLOGY:

The Immune System

Three functions:

Defense

Homeostasis

Surveillance

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The Immune System

Markers of Self

Markers of Non-Self

Markers of Self:

Major Histocompatibility Complex

Organs of the Immune System

Components:

Cells:

Lymphocytes: T-cells, B-cells & NK cells.

Phagocytic cells: N, E, B, Monocytes,

Macrophages, Dendritic cell, etc.

Chemical mediators:

Complement system.

Chemokines.

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Cells of the Immune System

B Cells

Antibody

Immunoglobulins

Antibody Genes

T Cells

Cytokines

Killer Cells: Cytotoxic Ts and NKs

Phagocytes and Their Relatives

Phagocytes in the Body

Complement

Immunity: Active and Passive

DEFINITIONS:

IMMUNOHEMATOLOGY:

DETECTION,

IDENTIFICATION, and/or

QUANTITATION of antibodies involved primarily with

red cells [although white cells and platelets may also

be involved].

Basically this branch of science related with red cell

antigens and their corresponding antibodies.

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IMMUNOHEMATOLOGY FACILITIES:

TRANSFUSION SERVICE:

Work primarily with patient's blood.

Primary areas of responsibility:

Blood typing. Antibody detection and

identification.

Compatibility testing (crossmatching).

Blood component therapy (hemotherapy).

Transfusion reaction workups.

Autoimmune hemolytic anemia workups.

Hemolytic disease of the newborn (HDN) workups.

Determining Rh immune globulin eligibility.

BLOOD BANK:

Works primarily with donor blood.

Major areas of responsibility:

Donor recruitment. Donor screening. Blood collection. Testing (typing,

infectious disease

screening).

Blood component preparation.

Component preservation. May provide reference

lab services.

May store rare donor blood.

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HISTORICAL OVERVIEW

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1616: Sir William Harvey Described circulation of blood.

1665: Richard Lower, English Physiologist 1st animal-to-

animal [dog to dog] blood transfusion.

1667: Jean Bapiste Denys Unsuccessful transfusion of

animal-to-human [sheep to human] blood transfusion.

1667 1818: Transfusions prohibited.

1818: James Blundell of England 1st successful human-to-

human blood transfusion. This species specific transfusion

had 50% success rate, the rest resulted in death.

1900: Karl Landsteiner Discovered the ABO blood

groups.

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Karl Landsteiner:

Described the ABO

Blood Groups.

Karl Landsteiners discovery:

Discovered that the incompatibility of many

transfusion was due to certain factors on red cells

now known as Antigens.

Postulated two things:

Each species has unique species specific factors on

red dells.

Even in each species has some common and some

uncommon factors to each other.

Introduced the Immunological era of blood

transfusion began the era of scientific based

transfusion therapy foundation of

IMMUNOHEMATOLOGY as a science.

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1927: Landsteiner and Levine discovered M,N and P

system.

1939,40: Levine, Stetson, Landsteiner and Weiner

discovers Rh system and its role in erythroblastosis

foetalis (HDN).

1946-Kell system discovered by Coombs, Mourant and

Race.

1950-51: Duffy, Kidd, Lutheran system discovered.

Landsteiner and Alexanders lead to the discovery of

>800 Blood group systems.

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ANTIGENS:

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ANTIGEN is a substance that either

combines with an antibody or

is processed and binds to a T lymphocyte to

stimulate an IMMUNE RESPONSE.

IMMUNOGEN - An antigen that stimulates an immune

response.

HAPTENS - small chemical substances that must be

bound to a larger molecule to provide sufficient

molecular weight for stimulation of antibody

production.

Proteins Complex carbohydrates

Lipopolysaccharides.

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Properties of Molecules that Contribute to Immunogenicity

PROPERTY DESCRIPTION

Foreignness Non-self more likely to stimulate antibody production

Size >10,000 M.W.

Chemical composition

Proteinbest immune response. Complex carbohydratesecond best immune response. Lipids, Nucleic acid weak immune response.

Complexity More complex molecules produce better immune response.

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Antigen location:

Some antigens protrude from the cell surface, while

others are an integral part of the membrane.

Physical location impacts antibody stimulation as well

as the physical ability of the antigen to react with an

antibody once it is produced.

Red Blood Cell Antigens:

Red blood cell antigens and corresponding antibodies

provide the foundation for blood bank testing.

More than 20 blood group systems that contain

greater than 200 red blood cell antigens.

ABO and Rh antigens are matched between donor and

recipient.

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ANTIBODIES

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Protein.

Produced in response to stimulation with an antigen.

Specific for the stimulating antigen and will react with

that antigen.

IMMUNOGLOBULIN 5 classes, IgG, IgM, IgA, IgD &

IgE.

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Immunological principles:

Primary immunological components are antigens and

antibodies.

Cardinal rule for antigens and antibodies [blood

bank]: Antigens on RBC surfaces & Antibodies in

serum / plasma.

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Primary & Secondary Immune responses:

Antigen-Antibody Reactions: Factors influencing: Each antibody reacts with the antigen that stimulated its

production. Specificity:

Noncovalent bonds. Bonding:

The fit of the antigen and antibody depend on compatible shapes that allow the antigen and antibody to physically

touch - a lock and key mechanism.

Physical fit:

Antigens and antibodies must be present in optimal concentrations; excess antibodies will result in a situation

known as prozone phenomenon.

Concentration of

antigen and

antibody:

Optimal temperature of reactivity for a specific antibody will expedite the combination of antigen and antibody.

Temperature:

Incubation time must be that which is optimal for the specific antibody. General guidelines are a range of 1560 minutes for optimal antigen-antibody attachment.

Time:

A pH range of 7.27.4 is maintained for most antigen-antibody reactions.

pH:

A net negative charge known as zeta potential surrounds the red cells. The reduction of this charge influences the ability

of antigen and antibody to combine.

Surface charge:

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LOCK & KEY CONCENTRATION

AGGLUTINATION

Visualization of Ag-Ab reaction in BB:

2 methods:

Agglutination.

Hemolysis.

Precipitation.

Agglutination involves a particulate antigen or an antigen that

is attached to a particle (such as a red blood cell).

Agglutination occurs in when:

1. An antibody molecule attaches to a single antigen on a

single cell with one antigen-binding site.

2. The free arm of the immunoglobulin molecule attaches

to an antigen on a second red cell. This creates a

crosslink.

3. Multiple cross links create a lattice.

4. The lattice is visualized as agglutination.

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Grading of Agglutination:

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BLOOD GROUP GENETICS

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Chromosomes & Genes:

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Basic Principles:

Genetics: Study of Inheritance.

Inheritance of transmissible characteristics or

traits: such as blood group antigens found on red

blood cells.

Each parent contributes 1/2 of the genetic

information.

The genetic information is contained

on chromosomes composed of DNA

Humans have 23 pairs of chromosomes

a. 22 matched (autosomal) chromosomes and

b. 1pair of sex chromosomes.

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System Common Genes Located on

Chromosome

ABO A, B, O 9

Rh D, C, E, c, e 1

Basic Principles:

Genes are the units of inheritance within the

chromosomes.

Alleles: At each loci, different forms of the genes. E.g.

ABO Blood Group System - A1, A

2, B, and O as common

alleles.

Genotype: Genetic composition for a particular trait.

Homozygous: When the two inherited alleles are

the same. E.g. OO / AA / BB.

Heterozygous: when the two inherited alleles are

different. E.g. AO / AB / BO.

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Basic Principles:

Phenotype: The expression of a genotype.

Dominant: The dominant gene will express itself if

present both in homozygous as well as

heterozygous state. E.g. Rh (D).

Co-dominant: Both the alleles express. E.g. A & B.

Recessive: They express in homozygous state only.

Amorph: No gene product. e.g. O.

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Basic Principles:

Dosage: In some blood group systems, persons

homozygous for an allele have MORE antigen on their

red cells than persons heterozygous for an allele.

Variation in antigen expression due to the number of

alleles present is called DOSAGE.

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BLOOD GROUP SYSTEMS

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HISTORICAL PERSPECTIVE:

In 1901, Karl Landsteiner used his blood and the

blood of his colleagues:

Mixed the serum of some individuals with cells of

others.

Discovered three groups A, B & O.

His colleagues discovered the 4th

group AB.

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LANDSTEINERS LAW / RULE:

ABO antigens on red cells and the reciprocal

agglutinating antibodies in the serum of the same

individual (e.g. A antigens on red blood cells and

anti-B in the serum).

ABO & H SYSTEM ANTIGENS:

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ABO ANTIGENS:

Present on RBC surface.

Also on lymphocytes, thrombocytes, organs,

endothelial cells, and epithelial cells.

Detectable at 5 to 6 weeks of gestation.

Newborns - weaker antigens.

Fully developed by two to four years of age.

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ABO ANTIGENS:

One factor contributing to the difference in ABO

antigen strength between newborns and adults is the

number of branched oligosaccharides.

Adults - greater numbers of branched chains

compared to newborns - more linear chains.

The branched chains permit attachment of more

molecules to determine antigen specificity.

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Phenotype Number of Ag sites

A1 adult 8,10,000 to 1,170,000

A1 cord 2,50,000 to 3,70,000

INHERITANCE:

Simple Mendelian fashion from an individuals

parents.

Each individual possesses a pair of genes.

FOUR genes H, A, B & O.

Hh Chromosome 19 HH / Hh / hh.

hh Bombay phenotype Oh.

A, B & O - Chromosome 9.

A and B genes produce a detectable products while

the O gene is an amorph.

The expression of the A and B genes is codominant.

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INHERITANCE:

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Gene Combination Phenotype

AO A

AA A

BO B

BB B

AB AB

OO O

GENE PRODUCTS & BIOCHEMICAL COMPOSITION OF BLOOD GROUP SUBSTANCES:

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58 Basic common core structure - an oligosaccharide chain attached

to either a protein or a lipid molecule present on cell surface.

GENE TRANSFERASE

H -L-fucosyltransferase

A -3-N-acetyl-D-galactosaminyl

Transferase

B -3-D-acetyl-D-galactosyl

Transferase

O No Transferase.

GENE PRODUCTS & BIOCHEMICAL COMPOSITION OF BLOOD GROUP SUBSTANCES:

The L-fucose is the immunodominant sugar for the H

antigen.

The H antigen serves as a precursor for A and B

antigens.

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SECRETOR STATUS

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Ability to secrete ABH antigens in body secretions.

Chromosome 19: Se / se [amorph]. Gene product L- fucosyltransferase. A & B transferases are found in the

secretions of A / B persons regardless

of their secretor status.

Secretors:

Nonsecretors:

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Saliva, Sweat, Tears, Semen, Serum & Amniotic fluid.

SUBGROUPS OF A & B

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Subgroups of A:

2 major subgroups:

~ 80% A1

~ 20% A2.

2 major subgroups of AB:

~ 80% A1B

~ 20% A2B.

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Group A1 Group A2

Qualitative differences:

Reaction with Anti-A in Forward

Grouping

4+ 4+

Number of Antigen Sites-Adults 10,00,000 2,50,000

Number of Antigen Sites-

Newborn 3,00,000 1,40,000

Quantitative differences:

Reaction with Anti-A1 Positive Negative

Anti-A1 in serum Absent ? Present

-3-N-acetyl-D-galactosaminyl

Transferase Activity Normal Diminished

A1 more antigens on the cell surface more

branched chain oligosaccharides than A2.

2 mutations Pro156Leu / Single nucleotide deletion

1060 reduced enzyme activity of A2.

Routine antisera NO DIFFERENCE in reaction.

The LECTIN Dolichos biflorus is used to obtain an

extract with anti-A1 specificity.

A2 individuals can develop antibodies to the A1

antigen.

Additional A subgroups: Aintr

, A3, A

x, A

m, A

end, A

el,and

Abantu

Fewer antigenic sites on their surface.

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Subgroups of B:

Subgroups of B are very rare and encountered less

frequently than subgroups of A.

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ABO ANTIBODIES:

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Antibodies directed against ABO antigens are the

most important antibodies in transfusion medicine.

It is the only example of a blood group where each

individual produces antibodies to antigens not

present on the red cells.

Newborns have NO ABO ANTIBODIES.

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REVERSE GROUPING of Cord blood / Newborn serum

indicates BLOOD GROUP OF THE MOTHER.

The child will begin antibody production and have a

detectable titre at 3 6 months of age peaks at 5

10 years of age.

Originally thought to be NATURAL ANTIBODIES

formed with no antigenic stimulus.

Proposed mechanism some naturally occurring

substances resemble A & B antigens and stimulate

production of complementary antibodies.

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Conditions with decreased levels of ABO antibodies:

Newborns and young infants Elderly individuals

Age related:

Congenital conditions Congenital hypogammaglobulinemia Congenital agammaglobulinemia

Immunodeficient individuals:

Immunosuppressive therapy Chronic lymphocytic leukemia Bone marrow transplant Multiple myeloma Acquired hypogammaglobulinemia Acquired agammaglobulinemia

Immunosuppressed patients:

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Immunoglobulin class:

ABO antibodies ISOAGGLUTININS Saline agglutinins

with optimal reactivity at 40C.

Mostly IgM.

IgG & IgA.

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Anti AB:

Group O do not have A / B antigens.

Produce anti-A, anti-B and anti-AB.

anti-AB cross-reactive antibody reacts with a

common molecular structure in both antigens.

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Anti-A1:

As per Landsteiners Law, group B and O individuals

produce anti-A.

This anti-A can be separated by absorption

procedures - anti-A and anti-A1.

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Clinical significance of ABO antibodies:

Cause both

HDFN Hemolytic disease of fetus and new born &

HTR Hemolytic transfusion reaction.

HDFN: usually presents itself with a maternal IgG

antibody to an antigen on the surface of the babys red cells.

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ABO HDFN:

Affects 1st pregnancy.

MC: O mother with A baby.

Rh HDFN:

Sensitization occurs in 1st pregnancy and affects subsequent positive pregnancies.

Rh negative mother Rh positive baby.

Hemolytic transfusion reaction:

Occurs when a recipient is transfused with red cells

that are an ABO group incompatible with the

antibodies in his or her serum.

Because of the complement-binding ability of the ABO

antibodies, this is always a life-threatening situation.

As the recipient antibodies react with the

incompatible red cells, complement is activated and

in vivo hemolysis, agglutination, and red blood cell

destruction occurs.

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RH BLOOD GROUP SYSTEM

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INTRODUCTION:

One of the most polymorphic and antigenic blood group

systems.

2nd only to ABO in importance in:

Blood transfusion &

A primary cause of HDFN.

The principal antigen is D and the terms Rh positive or

Rh negative refers to presence / absence of this antigen.

Other 4principal antigens are C, c, E and e.

50 other rare antigens detected.

Rh negative phenotype common in Caucasians 15 17%.

95% Indian population: Rh Positive.

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GENES:

Chromosome 1.

2 genes: RhD & RhCE.

The proteins encoded by these 2 differ by 32 to 35

AAs. That is why RhD is so antigenic in Rh negative

persons.

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NOMENCLATURE:

Discovered in the 1940s.

3 systems:

Fisher & Race: 3 closely linked genes D at one

locus, C / c at the 2nd

locus & E / e at the 3rd

locus.

Modified Weiner terminology: Supposes a single

gene.

ISBT terminology: Assigns each antigen a number

D: Rh1, C: Rh2, E:Rh3, c:Rh4 & e:Rh5.

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D ANTIGEN:

Very antigenic D+ for presence / D- for absence.

Variants: due to a point mutation causing single AA

differences.

1. Weak D [formerly Du, obsolete now]: 1 to 57

types: Type 1 to 3 90% cases.

2. Del: Not detected by routine testing but requires

adsorption-elution studies / molecular RHD

genotyping.

3. Partial D: Due to hybrid genes portion of RHD is replaced by corresponding portion of RHCE gene.

The RBCs type D+ve but make anti-D following transfusion / pregnancy.

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Rh null:

No Rh system antigens.

2 pathways:

Rh-negative person (lacking RHD) who also has an

inactive RHCE gene, referred to as an Rhnull amorph.

More often, inheritance of inactive RHAG gene,

referred to as an Rhnull regulator. RhAG protein is

required for expression and trafcking of RhCE and

RhD to the RBC membrane.

The serum of the people who form these antibodies

agglutinates cells from all people except another

Rhnull.

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Rh ANTIBODIES: Principally RBC stimulated.

Most are of the IgG class, usually the IgG1 or IgG3 subclass.

Can cross placenta.

May occur in mixtures with a minor component of IgM.

The antibodies usually appear between 6 weeks and 6

months after exposure to the Rh antigen.

IgG Rh system antibodies react best at 370C and are

enhanced when enzyme-treated RBCs are tested.

D is the most immunogenic of the common Rh antigens,

followed in decreasing order of immunogenicity by c, E, C,

and e.

30% to 85% of D-ve persons who will make anti-D following

exposure to D+ve RBCs Responders.

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LABORATORY DETERMINATION OF THE ABO SYSTEM

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RBC PRECURSOR STRUCTURE

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Glucose

Galactose

N-acetylglucosamine

Galactose

Precursor

Substance

(stays the

same)

RBC

FORMATION OF H Ag

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Glucose

Galactose

N-acetylglucosamine

Galactose

H antigen

RBC

Fucose

FORMATION OF A Ag

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Glucose

Galactose

N-acetylglucosamine

Galactose

RBC

Fucose N-acetylgalactosamine

FORMATION OF B Ag

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Glucose

Galactose

N-acetylglucosamine

Galactose

RBC

Fucose Galactose

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ANTISERUM

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An antiserum is a purified, diluted and standardized

solution containing known antibody, which is used

to know the presence or absence of antigen on cells

and to phenotype ones blood group.

Antiserum is named on the basis of the antibody it

contains:

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Antisera Antibody present

Anti-A anti-A

Anti-B anti-B

Anti-AB anti-A & anti-B

Anti-D anti-D

Sources:

MONOCLONAL ANTIBODIES

Animal inoculation.

Serum from an individual who has been sensitized to

the antigen through transfusion, pregnancy or

injection.

Serum from known blood group persons.

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Criterias:

Antiserum must meet certain criterias to be acceptable for

use.

Qualities of a good antisera:

Specific: does not cross react, and only reacts with its

own corresponding antigen,

Avid: the ability to agglutinate red cells quickly and

strongly and,

Stable: maintains it specificity and avidity till the expiry

date.

It should also be clear [as turbidity may indicate bacterial

contamination] and free of precipitate and particles.

It should be labelled and stored properly.

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Manifestation of Ag-Ab reaction:

The observable reactions resulting from the

combination of a red cell antigen with its

corresponding antibody are agglutination and/ or

haemolysis.

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Agglutination:

Is the clumping of particles with antigens on their surface,

such as erythrocytes by antibody molecules that form

bridges between the antigenic determinants.

Hemagglutination.

Agglutinogen antigen.

Agglutinin antibody.

Two stages:

1. Sensitization: Abs attach to the Ags on RBC

Sensitized RBC.

2. Agglutination: Ab binding to Ag on >1 RBC Lattice

formation.

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Hemolysis:

Ab Hemolysin.

Complement mediated lysis of the RBC membrane

with release of Hb to stain the plasma.

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Right condition for the RBCs to agglutinate / hemolysis:

1. Ab size:

Zeta potential keeps RBCs 25 nm apart.

IgG Ab max span 14 nm so can only bind to Ag

and sensitize them [can not cause agglutination] in

saline media.

IgM Ab larger and pentameric can bridge a wider

gap cause agglutination.

2. pH: Optimum pH 7.0.

3. Temperature: Optimum temperature varies

depending upon Ab type: IgG 370C, IgM 4 220C.

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4. Ionic strength:

Low ionic strength increases agglutination.

LISS 0.2% NaCl in 7% glucose is used.

5. Number of Ag sites:

Seen that IgG Abs of Rh system fail to agglutinate

RBCs suspended in saline where as IgG Abs of ABO

system can agglutinate because number of ABO

sites are 100 times more in D sites.

6. Centrifugation: at high speed attempts to overcome

the problem of distance in sensitized cells by

physically forcing the cells together.

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7. Enzyme treatment:

Treatment with weak proteolytic enzymes [Trypsin /

Papain] removes surface sialic acid residue on RBC

lowers zeta potential promotes agglutination.

Has a disadvantage destroys some blood group Ags.

8. Colloidal suspension: [Bovine albumin]

Can reduce the zeta potential helps IgG Abs of Rh

system to agglutinate.

9. Ratio of Ag & Ab:

Excess Ab Prozone phenomenon Use serial dilutions

of the antisera.

Avoid heavy RBC suspension as it may mask the presence

of a weak Ab.

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ABO GROUPING TECHNIQUES

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METHODS:

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REAGENTS:

Anti-A antibodies.

Anti-B antibodies.

Anti-AB antibodies (optional).

Group A & B RBCs.

Slides, or Test tubes.

Wooden applicator.

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IMPORTANT THINGS TO FOLLOW:

Verify that patient information on the sample

matches information on the worksheet.

Centrifuge the sample and remove the serum to a

labelled tube.

Prepare a washed 2 - 5% RBC suspension.

Use Patient cell suspension in Forward typing.

Use Patient serum for confirmation Reverse

grouping or backtyping.

At the End, Discard all materials in the isolation

trash containers.

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ABO GROUPING: RULES FOR PRACTICAL WORK:

1. Perform all tests according to the manufacturers

direction.

2. Always label tubes and slides fully and cleanly.

3. Do not perform tests at temperature higher than

room temperature.

4. REAGENT ANTISERA SHOULD BE TESTED DAILY

WITH ERYTHROCYTES OF KNOWN ANTIGENICITY.

This eliminates the need to run individual controls

each time the reagents are used.

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ABO GROUPING: RULES FOR PRACTICAL WORK:

5. Do not rely on colored dyes to identify reagent

antisera.

6. Always add serum before adding cells.

7. Observe for agglutination against a welllighted

background, and record results immediately after

observation.

8. Use an optical aid to examine reactions that appear

to the naked eye to be negative.

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ABO GROUPING: PREPARATION OF RBC SUSPENSION:

Important to the accuracy of testing in the blood

bank.

Can be prepared directly from anticoagulated blood

or from packed red cell (after separating the serum

or plasma).

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ABO GROUPING: PREPARATION OF RBC SUSPENSION:

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Procedure: (as an example preparation of 2% RBC

suspension of 10 ml volume):

Place 1 to 2ml of anticoagulated blood in a test tube.

Fill the tube with saline and centrifuge the tube.

Aspirate or decant the supernatant saline.

Repeat (steps 2 and 3) until the supernatant saline is

clear.

Pipette 10 ml of saline in to another clean test tube.

Add 0.2 ml of the packed cell button to the 10 ml of

saline.

Cover the tube until time of use. Immediately before

use, mix the suspension by inverting the tube several

times until the cells are in suspension.

DIRECT ABO GROUPING:

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DIRECT ABO GROUPING: PRINCIPLE:

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The direct blood grouping also called

Cell grouping / Forward grouping

employs known anti sera to identify the antigen

present or their absence on an individuals RBC.

It can be performed by the

Slide or Test tube method.

DIRECT ABO GROUPING: SLIDE METHOD:

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Make rings on the slide and label one ring as anti- A

and the other ring as anti-B.

First add corresponding antisera to the rings.

Add 10% cell suspension to both rings.

Mix using separate applicator sticks.

Observe the reaction within 2 minutes by rotating

the slide back and forth.

Interpret the results.

Strong agglutination of

RBCs in the presence of

any ABO grouping reagent

constitutes a positive

result.

A smooth suspension of

RBCs at the end of 2

minutes is a negative

result.

Samples that give weak or

doubtful reactions should

be retested by Tube test

ABO grouping.

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DIRECT ABO GROUPING: TEST TUBE METHOD:

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Take two tubes, label one tube anti- A and the second

anti- B.

First add corresponding antisera to the tubes.

Put one drop of the 2-5% cell suspension to both tubes.

Mix the antiserum and cells by gently tapping the base of

each tube with the finger or by gently shaking.

Leave the tubes at RT for 5 minutes.

Centrifuge at low speed (2200-2800 rpm) for 30 seconds.

Read the results by tapping gently the base of each tube

looking for agglutination or haemolysis against a well

lighted white background.


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- Prepare 2-5% cell suspension

- Label Test tubes

- Add 2 drops of Anti sera A, B , and D

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- Add one drop of 2-5% Patient Red Blood Cell suspension.

- Mix the contents of the tubes gently and centrifuge for 15-30 seconds at approx. 900-1000 x g

- Gently resuspend the RBCs buttons and examine for agglutination

TUBE METHOD READING OF RESULTS:

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Interpretation Reaction of cells

tested with

ABO Group Cell Ag Anti-B Anti-A

O No Ag - -

A A - +

B B + -

AB A, B + +

INDIRECT ABO GROUPING:

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INDIRECT ABO GROUPING: PRINCIPLE:

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The indirect blood grouping also called

Serum grouping / Reverse grouping

employs RBCs possessing known antigen to identify

the type of antibodies present or absent in the serum

of an individual.

It can be performed by the Test tube method.

Slide reverse grouping is not reliable.

INDIRECT ABO GROUPING: TEST TUBE METHOD:

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Take two tubes, label A- Cells and B cells.

Put one drop of the serum to be tested each tube.

Add one drop of 2-5% A cells to the tube labeled A cells

and one drop of 2-5% B cells to the tube labeled B cells.

Mix the contents of the tubes.

Leave the tubes at RT for 5 minutes.

Centrifuge at low speed (2200-2800 rpm) for 30 seconds.

Read the results by tapping gently the base of each tube

looking for agglutination or haemolysis against a well

lighted white background.


Agglutination in any tube of RBCs test or hemolysis or

agglutination in serum tests constitutes positive test

results.

A smooth suspension of RBCs after resuspension of

an RBCs button is a negative result.

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INDIRECT ABO GROUPING: INTERPRETATION:

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SERUM TESTED WITH

BLOOD GROUP

A CELL B CELL

Negative Positive A

Positive Negative B

Negative Negative AB

Positive Positive O

INTERPRETATION OF BOTH:

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Interpretation Reaction of serum tested

against

Reaction of cells

tested with

ABO Group O cells B Cells A cells Anti-B Anti-A

O - + + - -

A - + - - +

B - - + + -

AB - - - + +

OTHER METHODS OF BLOOD GROUPING:

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GEL CARDS:

Gel Cards containing Anti-A, Anti-B, and Anti-A1B are used

to test patient or donor red blood cells for the presence or

absence of the A and/or B antigens.

The results of red blood cell grouping should be confirmed

by reverse (serum) grouping, i.e. testing the individuals

serum with known A1 and B red blood cells.

In the Gel Test, the specific antibody (Anti-A, Anti-B, or

Anti-D) is incorporated into the gel. This gel has been pre-

filled into the microtubes of the plastic card. As the red

blood cells pass through the gel, they come in contact with

the antibody. Red blood cells with the specific antigen will

agglutinate when combined with the corresponding

antibody in the gel during the centrifugation step.

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GEL CARDS: INTERPRETATION OF RESULTS

A positive reaction is recorded when red cells are

retained in or above the gel column after centrifugation

A negative reaction is recorded when a distinct button of

cells sediment to the bottom of the column after

centrifugation.

A positive reaction in the Control microtube indicates a

false positive reaction, thus invalidates the tests.

Drying, discoloration, bubbles, crystals, other artefacts,

opened or damaged seals may indicate product alteration.

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PROCEDURE:

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MICROPLATE TECHNIQUE:

Microplate techniques can be used to test for antigens on

red cells and for antibodies in serum.

A microplate can be considered as a matrix of 96 short

test tubes; the principles that apply to hemagglutination in

tube tests also apply to tests in microplate.

Add reagent and patient sample( red cells/ serum)

Incubation,

Centrifugation

Red cell resuspension,

Reading of results.

Interpretation of results.

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ABO GROUPING DISCREPANCIES

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ABO GROUPING DISCREPANCIES: TECHNICAL ERRORS

Most errors are technical in nature & can be

resolve by careful repeating the test procedure.

Common errors are:

1. Contaminated reagents.

2. Dirty glass ware.

3. Over / Under centrifugation.

4. Incorrect serum:cell ratio.

5. Incorrect incubation temperature.

6. Failure to add test specimen / reagents.

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127

ABO GROUPING DISCREPANCIES: NON-TECHNICAL ERRORS

If after careful repeat the same agglutination

pattern is obtained than the causes can be:

1. Missing / Weak reacting Abs.

2. Missing / Weak Abs.

3. Additional Ab.

4. Plasma abnormalities.

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ABO GROUPING DISCREPANCIES: MISSING / WEAK Abs. 1. Age:

Infants before producing own Abs or who possess passively

acquired maternal Abs.

Elderly persons whose Ab levels have declined.

2. Hypogammaglobulinemia:

Lymphoma.

Leukemia.

Immunodeficiency disorders / Use of immnosuppressive drugs.

Following BM transplantation.

RESOLUTION: enhancing reaction in reverse grouping by

incubating test serum with RBCs at RT for 15 mins / at 40C or 16

0C

for 15 mins.

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ABO GROUPING DISCREPANCIES: MISSING / WEAK Ags.

1. Subgroups of A / B Ags. [RESOLUTION: Subgroup the sample.]

2. Diseases like Leukemia: ABO Ags greatly depressed.

[RESOLUTION: Investigate the diagnosis.]

3. Blood group specific substances: Ovarian cysts / carcinomas.

[RESOLUTION: Wash the cells in saline.]

4. Acquired B Ag: Effect of bacterial enzymes & adsorption of

bacterial polysaccharide on to the group A / O RBCs B

specificity weak B Ag reaction in the forward grouping.

[RESOLUTION: Acidify the anti-B reagent to pH 6 rules out

acquired B.]

5. Additives to sera. [RESOLUTION: Wash the cells in saline.]

6. Mixtures of blood: recent BT / BM transplant recipient.

[RESOLUTION: Investigate.]

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ABO GROUPING DISCREPANCIES: ADDITIONAL Ab.

1. AutoAb.: Cold autoAb - spontaneous agglutination of the A & B

cells in reverse grouping. Warm AIHA patients may have RBCs

coated with sufficient Ab to promote spontaneous

agglutination.

[RESOLUTION: Wash RBCs in warm 370C to establish cold Abs. Treat

cells with Chloroquine diphosphate to eliminate bound warm Abs]

1. Anti A1: A2 & A2B individuals may produce naturally occurring

anti-A1 which cause discrepant ABO typing.

[RESOLUTION: Investigate the diagnosis.]

1. Irregular Ab: Irregular antibodies in some other blood group

system may be present that react with antigens on the A or B

cells used in reverse grouping.

[RESOLUTION: Use A & B cells that are negative for corresponding

Ag.]

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ABO GROUPING DISCREPANCIES: PLASMA ABNORMALITIES

1. Increased globulin.

2. Abnormal proteins.

3. Whartons jelly.

All these cause increased rolueaux formation that can be

mistaken as agglutination.

[RESOLUTION: Wash with NS to remove proteins.]

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LABORATORY DETERMINATION OF THE RH SYSTEM

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Direct Slide / Tube testing method.

No Indirect / Reverse grouping.

No naturally occurring Rh antibodies are not found

in the serum of persons lacking the corresponding

Rh antigens.

In performing Rh grouping:

The number of drops,

Time &

Speed of centrifugation shall be determined by

manufacturers directions.

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Rh TYPING: SLIDE TEST METHOD

Place a drop of anti- D on a labelled slide.

Place a drop of Rh control (albumin or other control

medium) or another labelled slide.

Add two drops of 40-50% suspension of cells to each slides.

Mix the mixtures on each slide using separate applicator

sticks, spreading the mixture evenly over most of the slide.

Interpretation or results:

Agglutination of red cells- Rh positive.

No red cell agglutination- Rh negative.

A smooth suspension of cell must be observed in the

control.

Note: Check negative reactions microscopically.

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Rh TYPING: TUBE TEST METHOD

Make a 2-5% red cell suspension.

Mark D on a test tube and add two drops of anti-D.

Place a drop of Rh control (albumin or other control

medium) on another labelled tube.

Add one drop of a 2-5% cell suspension to each tube.

Mix well and centrifuge at 2200-2800 rpm for 60 seconds.

Gently resuspend the cell button and look for agglutination

and grade the results (a reaction of any grade is interpreted

as Rh positive) a smooth suspension of cells must be

observed in the control.

Collect a weakly positive and negative sample to perform

the Du test.

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Rh TYPING: Du TYPING USING IAT

Use the initial Rh D typing tube and control in procedure

above and incubate the Rh - negative or weakly reactive

samples and the control at 370C for 30 minutes.

Wash cells in both test and control tube 3-4 times with

normal saline.

Add one drop of the poly specific antihuman globulin

(Coombs) to each tube and mix well.

Centrifuge at 2200-2800 rpm for 10 second.

Gently suspend the cell button and observe for

agglutination.

Interpretation: the positive result is agglutination in the

tube containing antiD and the control is negative. A

negative result is absence of agglutination in both the test

& control.

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THE ANTI-GLOBULIN TEST

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Introduced in to clinical medicine by Coombs in 1945.

It is a sensitive technique in the detection of

Incomplete antibodies,

Antibodies that can sensitize but which fail to

agglutinate RBCs suspended in saline at room

temperature, mainly IgG.

Complements.

PRINCIPLE:

Anti-IgG / Anti-C3 in antiglobulin serum agglutinates the

incomplete Abs / Sensitizing Abs on neighboring RBCs

/ Complements by cross-linking them.

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AHG Reagent:

It is made by injecting rabbits, sheep or goat with

purified human IgG or C3.

The reagent may be polyspecific or monospecific.

Polyspecific Anti-human Globulin: contains a blend

of Anti-IgG & Anti-C3b, -C3d and sometimes C4

Monospecific reagents: contains Anti-IgG alone or

Anti-C3b,-C3d alone

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Preparation of coombs control check cells (CCC):

Positive Control:

Sensitized O Rh (D) positive cells.

Negative Control:

Sensitized 0 Rh (D) negative cells.

Unsensitized 0 Rh (D) positive cells.

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Preparation of Positive Coombs control check cells (CCC):

Take 0.5 mL of 5-6 times washed and packed 0 Rh (D)

+ve red cells in a test tube.

Add 2-3 drops of IgG anti-D (select a dilution titre

[1:4] of anti-D which coats the red cells but does not

agglutinate them at 37C).

Mix and incubate at 37C for 30 minutes. If there is

agglutination, repeat the procedure using more

diluted anti-D.

Wash 3-4 times and make 5% suspension in saline for

use.

Perform a DAT which should give a 2+ reaction. If no

agglutination occurs, repeat using less diluted anti-D.

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Preparation of Negative Coombs control check cells (CCC):

0 Rh(D) negative sensitized red cells are also prepared

by treating 0 Rh(D) negative cells in the same manner.

The preparation should give a negative direct

antiglobulin test (DAT).

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TYPES:

1. Direct Antiglobulin Test [Direct Coombs Test]

DAT.

2. Indirect Antiglobulin Test [Indirect Coombs Test]

IAT.

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DAT:

It is used to demonstrate whether RBCs have been

sensitized (coated) with antibody or complement in vivo,

as in case of

HDN,

Autoimmune haemolytic anemia,

Drug induced haemolytic anemia, and

Transfusion reactions.

Principle:

Patients erythrocytes are washed to remove free plasma

proteins and directly mixed with AHG, and if incomplete

antibodies are present, agglutination occurs.

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DAT:

The direct antiglobulin test (DAT) detects sensitized

red cells with IgG and/or complement components

C3b and C3d in vivo.

In vivo coating of red cells with IgG and/or

complement may occur in any immune mechanism is

attacking the patient's own RBC's.

This mechanism could be autoimmunity,

alloimmunity or a drug-induced immune mediated

mechanism.

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DAT: Requirements

Requirements:

Test tubes: (10x75 mm)

Pasteur pipettes

Incubator

Centrifuge

Reagent: Anti-human globulin serum.

Specimen: Blood drawn into EDTA is preferred but

oxalated, or clotted, citrated whole blood may be used

(specimen need not be fasting sample).

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DAT: Procedure

Prepare a 5 % suspension in isotonic saline of the red

blood cells to be tested.

With clean Pasture pipette add one drop of the prepared

cell suspension to a small tube.

Wash three times with normal saline to remove all the

traces of serum.

Decant completely after the last washing.

Add two drops of Antihuman globulin.

Mix well and incubate at 370C for 30 mins.

Centrifuge for one minute at 1500 RPM.

Resuspend the cells by gentle agitation and examine

macroscopically and microscopically for agglutination.

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IAT:

1. This test is performed to detect presence of Rh

antibodies or other antibodies in patients serum in

case of the following:

a. To check whether an Rh-negative women (married

to Rh-positive husband) has developed Anti Rh

antibodies.

b. Rh incompatible blood transfusions.

2. To detect Du Ag.

3. In cross-matching to detect Abs that might reduce

the survival of transfused cells.

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IAT: Principle

The serum containing antibodies is incubated with

erythrocytes containing antigens that adsorb the

incomplete antibodies. After washing to dilute the

excess antibody in the serum, the addition anti

globulin serum produces agglutination in the

presence of incomplete antibodies.

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IAT: Procedure

Requirements:

Test tubes: (10x75 mm)

Pasteur pipettes

Incubator

Centrifuge

Specimen: Serum (need not be fasting)

Reagents:

Antihuman globulin

IgG Anti-D serum

Coombs control cells: Make a pooled O Rho (D) positive

cells from at least three different O positive blood

samples. Wash these cells three times in normal saline

(these cells should be completely free from serum with no

free antibodies).

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IAT: Procedure

1. Label three test tubes as T (test serum) PC

(Positive control) and NC (negative control).

2. In the tube labelled as T, add two drops of Test

serum.

3. In the tube PC add two drops of 1:4 diluted IgG

Anti-D.

4. In the tube NC add two drops of NS.

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IAT: Procedure

5. Add two drops of 5 % saline suspension of the

pooled O Rh (D) positive cells in each tube.

6. Incubate all the three tubes for one hour at 37C.

7. Wash the cells three times in normal saline to

remove excess serum with no free antibodies, (in the

case of inadequate washings of the red cells,

negative results may be obtained).

8. Add two drops of Coombs serum (anti human

globulin) to each tube. Keep for 5 minutes and then

centrifuge at 1,500 RPM for one minute.

9. Resuspend the cells and examine macroscopically as

well as microscopically.

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IAT: Interpretation

Observation Conclusion

PC

Agglutination. Correct test.

No agglutination. Incorrect test, repeat.

NC

No agglutination. Correct test.

Agglutination. Incorrect test, repeat.

Test

Agglutination. Positive Patient serum contains Ab.

No agglutination. Negative.

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Factors affecting sensitivity of IAT

Temperature:

Optimal temperature: 370C.

Incubation at higher / lower temperature may give false

positive results.

Serum Cell ratio: Increasing the ratio of serum to cells

increases the antibody coating. Commonly used ratio in

saline suspension is 2:1 but in LISS suspending cells, use

equal volume of serum and 2% cell suspension.

Incubation time:

Saline, Albumin or enzyme technique : 45-60 minutes.

LISS suspended cells - Routine 15 minutes.

Suspension medium: The sensitivity of IAT can be

increased with addition of 22% bovine albumin, enzyme or

by using LISS suspended cells.

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False Negative Antiglobulin test results:

1. Inadequate cell washing.

2. Delay in adding antiglobulin reagent after the

washing step.

3. Presence of small fibrin clots among the cells.

4. Inactive, or forgotten antiglobulin reagent .

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False Positive Antiglobulin test results:

1. Using improper sample (clotted cells instead of

EDTA for Direct Antiglobulin Test, DAT).

2. Spontaneous agglutination (cells heavily coated with

IgM).

3. Non-specific agglutination ("sticky cells").

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CROSS-MATCHING

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Purpose:

1. To select donors blood that will not cause any

adverse reaction like hemolysis or agglutination in

the recipient.

2. Also to help the patient to receive maximum benefit

from transfusion of red cells, which will survive

maximum in his circulation.

However, a cross match will not prevent

immunization of the patient, and will not guarantee

normal survival of transfused erythrocytes or detect

all unexpected antibodies in a patients serum.

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Types of Cross Match:

1. Major.

2. Minor.

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Major Cross Match:

RECIPIENTS / PATIENTS SERUM with DONORs RBCs.

It is much more critical for assuring safe transfusion

than the minor compatibility test.

It is called major because the antibody with the

recipients serum is most likely to destroy the donors

red cells and that is why it is called major cross

match.

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Minor Cross Match:

DONORs SERUM with PATIENTs RBCs.

It is usually thought that any antibody in the donors

serum will be diluted by the large volume of the

recipients blood, so it causes relatively less problem

and so called minor cross match.

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Selection of blood for Cross Match:

When whole blood is to be transfused, the blood

selected for cross- match should be of the same

ABO and Rh (D) group as that of the recipient.

However, Rh positive recipients may receive

either Rh positive or Rh negative blood.

Group A is the second choice of blood because anti-B in Gp

A blood is likely to be weaker than anti-A in Gp B blood.

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Group of

Patient

Choice of Blood

1st 2

nd 3

rd 4

th

A A O --- ---

B B O --- ---

O O --- --- ---

AB AB A* B O

Procedure of Cross Match:

4 PHASES:

1. Saline.

2. Protein.

3. AHG.

4. Enzyme.

1. Saline tube technique at RT: provides the optimum

temperature and medium for the detection of IgM

antibodies of ABO system and other potent cold

agglutinins.

2. Saline 370C: is the optimum for the detection of warm

agglutinin, of which are saline reactive IgG antibodies of

the Rh/ Hr system.

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3. AHG: is highly efficient for the detection of most kinds of

incomplete antibodies.

4. Enzyme technique- is a very sensitive one for the

detection of some low affinity Rh antibodies, which are

not detected by other methods including the antiglobulin

technique.

Procedure:

1. Put 3 drops of patients serum in to a test tube.

2. Put one drop of donors 3% red cells suspension.

3. Mix and centrifuge at 3400 rpm for 15 seconds.

4. Examine for agglutination or haemolysis, if compatible

proceed with the next phase.

5. Mix the contents of the tube and incubate at 370C for 20

30 min.

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Note: potentiators such as a drop of 22% albumin may be

added at this phase to increase the sensitivity of the test.

6. Centrifuge at 3400 rpm for 15 seconds and examine for

agglutination or hemolysis. If there is no hemolysis or

agglutination proceed with the next phase.

7. Wash the contents of the tube 3-4 times with normal

saline.

8. After the last wash, decant all saline and add two drops of

AHG reagent and mix.

9. Centrifuge at 3400 rpm for 15 seconds.

10. Gently re suspend the cells button and examine

macroscopically and microscopically for agglutination or

hemolysis.

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Enzyme cross match can be performed by using different

enzymes:

Bromelin / Ficin / Papain / Trypsin.

Two methods are available to carry out enzyme cross

match:

One stage &

Two stage methods.

The one-stage technique involves enzyme, patients serum

and donors red cell incubated together.

The two-stage technique involves red cells pre-treated with

enzyme and then tested with the patients serum.

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immunohematology and blood banking techniques class notes

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