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Chapter 18: Blood Group Typing and Protein Profiling 

18.1: Blood Group Typing

Blood Groups

  • Blood groups: Defined as antigen polymorphisms present on erythrocyte surfaces.

  • Transfusion reactions occur when an incompatible type of blood is transfused into an individual, which can lead to severe symptoms or even death.

  • Karl Landsteiner discovered the first blood group, known as the ABO system, in the early 1900s, while studying transfusion and transplantation.

  • The International Society of Blood Transfusion currently recognizes 29 blood group systems, which include hundreds of antigen polymorphisms

ABO Blood Group System

  • In the ABO blood group system, two types of antigens, designated A and B, give rise to four blood types:

    • Type A individuals have the A antigen.

    • Type B individuals have the B antigen.

    • Type AB individuals have both A and B antigens.

    • Type O individuals have neither A nor B antigens.

Biosynthesis of Antigens

  • All individuals generate the O antigen, also known as the H antigen.

  • The O antigen is synthesized by fucosyltransferase, a fucose transferase encoded by the FUT genes, which adds a fucose on the end of a glycolipid or glycoprotein.

  • The specificity of this enzyme determines the ABO blood type:

    • The A allele produces the A-transferase, which transfers N-acetylgalactosamine to the O antigen and thus synthesizes the A antigen

    • The B allele produces the B-transferase, which transfers galactose to the O antigen and thus synthesizes the B antigen.

    • The O allele has a mutation (small deletion), which eliminates transferase activity, and no modification of the O antigen occurs.

Biosynthesis of ABO antigens.

Structure of ABO gene and variants. Exons 6 and 7 are shown. T

Secretors

  • Individuals whose A, B, and O antigens can be found in other types of bodily fluids are referred to as secretors.

  • Chromosome 19 contains two homologous genes:

    • FUT1 is expressed in tissues of mesodermal origin and is responsible for the synthesis of the O antigen in erythrocytes.

    • FUT2 is expressed in tissues of endodermal origin; it is responsible for the synthesis of the O antigen in secretions.

Tissue-specific O-antigen biosynthesis by FUT1 and FUT2 gene products.

Inheritance of A and B Antigens

  • A and B alleles are dominant.

  • For AO and BO heterozygotes, the corresponding transferase synthesizes the A or B antigen.

  • A and B alleles are codominant in AB heterozygotes because both transferase activities are expressed.

  • The OO homozygote produces neither transferase activity and therefore lacks both antigens.

  • The inheritance of A and B alleles obeys Mendelian principles.

Blood Group System

Number

Name

Symbol

# of Antigens

Gene Names

Chromosomal Location

001

ABO

ABO

4

ABO

9q34.2

002

MNS

MNS

43

GYPA, GYPB, GYPE

4q31.21

003

P

P1

1

-

22q11.2-qter

004

Rh

RH

49

RHD, RHCE

1p36.11

005

Lutheran

LU

20

LU

19q13.32

006

Kell

KEL

25

KEL

7q34

007

Lewis

LE

6

FUT3

19p13.3

008

Duffy

FY

6

FY

1q23.2

009

Kidd

JK

3

SLC41A1

18q12.3

010

Diego

DI

21

SLC4A1

17q21.31

011

Yt

YT

2

ACHE

7q22.1

012

Xg

XG

2

XG, MIC2

Xp22.33, Yp11.3

013

Scianna

SC

5

ERMAP

1p34.2

014

Dombrock

DO

5

DO

12p12.3

015

Colton

CO

3

AQP1

7p14.3

016

Landsteiner-Wiener

LW

3

ICAM4

19p13.2

017

Chido/Rodgers

CH/RG

9

C4A, C4B

6p21.3

018

H

H

1

FUT1

19q13.33

019

Kx

XK

1

XK

Xp21.1

020

Gerbich

GE

8

GYPC

2q14.3

021

Cromer

CROM

12

DAF

1q32.2

022

Knops

KN

8

CR1

1q32.2

023

Indian

IN

2

CD44

11p13

024

Ok

OK

1

BSG

19p13.3

025

Raph

RAPH

1

CD151

11p15.5

026

John Milton Hagen

JMH

1

SEMA7A

15q24.1

027

I

I

1

GCNT2

6p24.2

028

Globoside

GLOB

1

B3GALT3

3q26.1

029

Gill

GIL

1

AQP3

9p13.3

Forensic Applications of Blood Group Typing

  • The application and usefulness of blood typing in forensic identification are based on the ability to group individuals into four different types using the ABO blood system, allowing individuals to be identified.

  • The probability that any two randomly chosen individuals have an identical blood type is very high.

  • The A and B antigens are very stable and can be identified in dried blood even after many years. They can also be found in semen and other bodily fluids of secretors.

Blood Group Typing Techniques

  • Lattes Crust Assay

    • It relies on the principles of Landsteiner’s experiments. It is an agglutination-based assay that utilizes the A, B, and O indicator cells to test the agglutination reaction with its corresponding naturally occurring serum antibodies in a questioned sample.

    • It is simple and rapid. However, one limitation is that the assay is not very sensitive and requires a large quantity of blood.

  • Absorption–Elution Assay

    • It is highly sensitive and can be used for testing dried bloodstains. This method indirectly detects the presence of antigens.

  • Lattes crust assay. (© Richard C. Li.)

    Diagram of Lattes crust assay results. (a) Indicator cells added before incubation. (b) Strong agglutination: large clumps are observed after incubation. (c) Negative agglutination: a cloudy background may be observed after incubation.

    Absorption–elution assay.

Lattes Crust Assay Procedure

  1. Place small quantities of blood crust from a specimen on a microscopic slide and place a cover slide over the crusts. Prepare slides for A, B, and O cells separately.

  2. Prepare cell suspensions with saline (0.85% NaCl in phosphate buffer, pH 7.4) for the A, B, and O cells separately.

  3. Apply a few drops of the A-cell suspension and allow the cells to diffuse under the cover slip. Repeat this step for B cells and O cells.

  4. Incubate the slides in a moisture chamber at room temperature for 2 h.

  5. Examine results under a microscope.

18.2: Forensic Protein Profiling

Methods

  • Matrices Supporting Protein Electrophoresis: The matrix reduces the effects of diffusion and convection on the macromolecules.

  • Separation by Molecular Weight

    • An electrophoretic method is frequently utilized to resolve various proteins based on their molecular weights.

    • Native electrophoresis can be used to isolate proteins for studying the functions of proteins.

  • Reducing Agents

    • It is common to include reducing agents such as mercaptoethanol (ME), dithiothreitol (DTT), or sodium mercaptoethane sulfonate (MESNA) to denature proteins.

    • Reducing agents cleave the disulfide bonds of proteins.

  • Detergents

    • Detergents disrupt noncovalent interactions within the structures of native proteins.

    • Sodium dodecyl sulfate (SDS): A strong anion detergent that binds to most proteins in amounts proportional to the molecular weight of the protein.

  • Separation by Isoelectric Point

    • It can be used to separate proteins according to their isoelectric points (pI).

    • Isoelectric Points: It is the pH value at which the net electric charge of an amino acid is zero.

    • In IEF electrophoresis, a pH gradient is created in a gel between the electrodes, and a protein sample is placed in a well on the gel.

    • A pH gradient in the gel is established by utilizing materials such as carrier ampholytes or immobilines that are dispersed in the gel.

    • Carrier ampholytes are synthetic amphoteric compounds that contain multiple weak ionizable moieties acting as either acids or bases.

Isoelectric focusing.

Hemoglobins resolved by isoelectric focusing electrophoresis.

Erythrocyte Protein Polymorphisms

  • Erythrocyte Isoenzymes

    • Isoenzymes: Are multiple forms of an enzyme that catalyze the same reaction but differ in their amino acid sequences.

    • Phosphoglucomutase (PGM): An important metabolic enzyme, catalyzes the reversible conversion of glucose-1-phosphate and glucose-6-phosphate.

  • Hemoglobin

    • Fetal Hemoglobin: The dominant form of hemoglobin present in the fetus during gestation.

    • Hemoglobin S: An inherited variant of normal adult hemoglobin.

Serum Protein Polymorphisms

  • Haptoglobin (Hp): The most widely used of the polymorphic serum proteins in forensic biology. It is a protein that binds and transports Hb from the bloodstream to the liver for the recycling of the iron contained in the Hb.

MA

Chapter 18: Blood Group Typing and Protein Profiling 

18.1: Blood Group Typing

Blood Groups

  • Blood groups: Defined as antigen polymorphisms present on erythrocyte surfaces.

  • Transfusion reactions occur when an incompatible type of blood is transfused into an individual, which can lead to severe symptoms or even death.

  • Karl Landsteiner discovered the first blood group, known as the ABO system, in the early 1900s, while studying transfusion and transplantation.

  • The International Society of Blood Transfusion currently recognizes 29 blood group systems, which include hundreds of antigen polymorphisms

ABO Blood Group System

  • In the ABO blood group system, two types of antigens, designated A and B, give rise to four blood types:

    • Type A individuals have the A antigen.

    • Type B individuals have the B antigen.

    • Type AB individuals have both A and B antigens.

    • Type O individuals have neither A nor B antigens.

Biosynthesis of Antigens

  • All individuals generate the O antigen, also known as the H antigen.

  • The O antigen is synthesized by fucosyltransferase, a fucose transferase encoded by the FUT genes, which adds a fucose on the end of a glycolipid or glycoprotein.

  • The specificity of this enzyme determines the ABO blood type:

    • The A allele produces the A-transferase, which transfers N-acetylgalactosamine to the O antigen and thus synthesizes the A antigen

    • The B allele produces the B-transferase, which transfers galactose to the O antigen and thus synthesizes the B antigen.

    • The O allele has a mutation (small deletion), which eliminates transferase activity, and no modification of the O antigen occurs.

Biosynthesis of ABO antigens.

Structure of ABO gene and variants. Exons 6 and 7 are shown. T

Secretors

  • Individuals whose A, B, and O antigens can be found in other types of bodily fluids are referred to as secretors.

  • Chromosome 19 contains two homologous genes:

    • FUT1 is expressed in tissues of mesodermal origin and is responsible for the synthesis of the O antigen in erythrocytes.

    • FUT2 is expressed in tissues of endodermal origin; it is responsible for the synthesis of the O antigen in secretions.

Tissue-specific O-antigen biosynthesis by FUT1 and FUT2 gene products.

Inheritance of A and B Antigens

  • A and B alleles are dominant.

  • For AO and BO heterozygotes, the corresponding transferase synthesizes the A or B antigen.

  • A and B alleles are codominant in AB heterozygotes because both transferase activities are expressed.

  • The OO homozygote produces neither transferase activity and therefore lacks both antigens.

  • The inheritance of A and B alleles obeys Mendelian principles.

Blood Group System

Number

Name

Symbol

# of Antigens

Gene Names

Chromosomal Location

001

ABO

ABO

4

ABO

9q34.2

002

MNS

MNS

43

GYPA, GYPB, GYPE

4q31.21

003

P

P1

1

-

22q11.2-qter

004

Rh

RH

49

RHD, RHCE

1p36.11

005

Lutheran

LU

20

LU

19q13.32

006

Kell

KEL

25

KEL

7q34

007

Lewis

LE

6

FUT3

19p13.3

008

Duffy

FY

6

FY

1q23.2

009

Kidd

JK

3

SLC41A1

18q12.3

010

Diego

DI

21

SLC4A1

17q21.31

011

Yt

YT

2

ACHE

7q22.1

012

Xg

XG

2

XG, MIC2

Xp22.33, Yp11.3

013

Scianna

SC

5

ERMAP

1p34.2

014

Dombrock

DO

5

DO

12p12.3

015

Colton

CO

3

AQP1

7p14.3

016

Landsteiner-Wiener

LW

3

ICAM4

19p13.2

017

Chido/Rodgers

CH/RG

9

C4A, C4B

6p21.3

018

H

H

1

FUT1

19q13.33

019

Kx

XK

1

XK

Xp21.1

020

Gerbich

GE

8

GYPC

2q14.3

021

Cromer

CROM

12

DAF

1q32.2

022

Knops

KN

8

CR1

1q32.2

023

Indian

IN

2

CD44

11p13

024

Ok

OK

1

BSG

19p13.3

025

Raph

RAPH

1

CD151

11p15.5

026

John Milton Hagen

JMH

1

SEMA7A

15q24.1

027

I

I

1

GCNT2

6p24.2

028

Globoside

GLOB

1

B3GALT3

3q26.1

029

Gill

GIL

1

AQP3

9p13.3

Forensic Applications of Blood Group Typing

  • The application and usefulness of blood typing in forensic identification are based on the ability to group individuals into four different types using the ABO blood system, allowing individuals to be identified.

  • The probability that any two randomly chosen individuals have an identical blood type is very high.

  • The A and B antigens are very stable and can be identified in dried blood even after many years. They can also be found in semen and other bodily fluids of secretors.

Blood Group Typing Techniques

  • Lattes Crust Assay

    • It relies on the principles of Landsteiner’s experiments. It is an agglutination-based assay that utilizes the A, B, and O indicator cells to test the agglutination reaction with its corresponding naturally occurring serum antibodies in a questioned sample.

    • It is simple and rapid. However, one limitation is that the assay is not very sensitive and requires a large quantity of blood.

  • Absorption–Elution Assay

    • It is highly sensitive and can be used for testing dried bloodstains. This method indirectly detects the presence of antigens.

  • Lattes crust assay. (© Richard C. Li.)

    Diagram of Lattes crust assay results. (a) Indicator cells added before incubation. (b) Strong agglutination: large clumps are observed after incubation. (c) Negative agglutination: a cloudy background may be observed after incubation.

    Absorption–elution assay.

Lattes Crust Assay Procedure

  1. Place small quantities of blood crust from a specimen on a microscopic slide and place a cover slide over the crusts. Prepare slides for A, B, and O cells separately.

  2. Prepare cell suspensions with saline (0.85% NaCl in phosphate buffer, pH 7.4) for the A, B, and O cells separately.

  3. Apply a few drops of the A-cell suspension and allow the cells to diffuse under the cover slip. Repeat this step for B cells and O cells.

  4. Incubate the slides in a moisture chamber at room temperature for 2 h.

  5. Examine results under a microscope.

18.2: Forensic Protein Profiling

Methods

  • Matrices Supporting Protein Electrophoresis: The matrix reduces the effects of diffusion and convection on the macromolecules.

  • Separation by Molecular Weight

    • An electrophoretic method is frequently utilized to resolve various proteins based on their molecular weights.

    • Native electrophoresis can be used to isolate proteins for studying the functions of proteins.

  • Reducing Agents

    • It is common to include reducing agents such as mercaptoethanol (ME), dithiothreitol (DTT), or sodium mercaptoethane sulfonate (MESNA) to denature proteins.

    • Reducing agents cleave the disulfide bonds of proteins.

  • Detergents

    • Detergents disrupt noncovalent interactions within the structures of native proteins.

    • Sodium dodecyl sulfate (SDS): A strong anion detergent that binds to most proteins in amounts proportional to the molecular weight of the protein.

  • Separation by Isoelectric Point

    • It can be used to separate proteins according to their isoelectric points (pI).

    • Isoelectric Points: It is the pH value at which the net electric charge of an amino acid is zero.

    • In IEF electrophoresis, a pH gradient is created in a gel between the electrodes, and a protein sample is placed in a well on the gel.

    • A pH gradient in the gel is established by utilizing materials such as carrier ampholytes or immobilines that are dispersed in the gel.

    • Carrier ampholytes are synthetic amphoteric compounds that contain multiple weak ionizable moieties acting as either acids or bases.

Isoelectric focusing.

Hemoglobins resolved by isoelectric focusing electrophoresis.

Erythrocyte Protein Polymorphisms

  • Erythrocyte Isoenzymes

    • Isoenzymes: Are multiple forms of an enzyme that catalyze the same reaction but differ in their amino acid sequences.

    • Phosphoglucomutase (PGM): An important metabolic enzyme, catalyzes the reversible conversion of glucose-1-phosphate and glucose-6-phosphate.

  • Hemoglobin

    • Fetal Hemoglobin: The dominant form of hemoglobin present in the fetus during gestation.

    • Hemoglobin S: An inherited variant of normal adult hemoglobin.

Serum Protein Polymorphisms

  • Haptoglobin (Hp): The most widely used of the polymorphic serum proteins in forensic biology. It is a protein that binds and transports Hb from the bloodstream to the liver for the recycling of the iron contained in the Hb.