ABO Blood Typing from Forensic Materials - Merits and demerits of
detection methods utilized in our laboratories, and biological
significance of the antigens
By: Wiraya Nakkeamkeao
Adviser: Dr.Sirilak Teeraputon
Researcher
Katsuji Nishi, Steven Rand,
Tokiko Nakagawa, Aiko Yamamoto,
Shigeru Yamasaki, Yoshio
Yamamoto, Aki Kobayashi,
Masateru Kane, Akira Morimoto,
Hedwege Spalthoff and Beate
Annuss
Journal of Forensic Medicine and Toxicology ,2005
Introduction
- In forensic science and medicine the ABO system has been
a major focus
- A, B and O(H) antigens on erythrocytes are also associated
with other cells and tissues throughout the body
- A, B and O(H) antigens are known to be considerably
stable to the such violent conditions as heating or drying
- The merits and demerits of the ABO blood-grouping
method
Objective
- The merits and demerits of the ABO blood grouping methods
- Discuss the biological significance of the ABH and its related
antigens on basis of the data showing the distribution of these
antigens in the animal organs
http://www.enh.org/uploadedimages/antigens.jpg
Review
ABO Blood Groups
Blood Type Genotype RBC Antigen
Present
Serum
Antibody
A
AA, Ai
A, H
Anti-B
B
BB, Bi
B, H
Anti-A
AB
AB
A, B, H
None
O
ii
H
Anti-A, Anti-B
Review
Subgroups A
Group Anti-A Anti-B Anti-AB Anti-A1
A1
+
-
+
+
A2
+
-
+
-
Anti-A1 = Lectin (Dolichos biflorus)
Forensic material
"Forensic material" means:
(a) Sample ; blood, semen or
(b) Hand prints, finger prints, foot prints
or toe prints; or
(c) Taken from or of a person’s body by a forensic
procedure
ABO blood grouping method
Method
 Hemagglutination
 Absorption - Elution
 Histochemical techniques
 ABO genotyping method
Hemagglutination method
“Hemagglutination” mean
A reaction outside the body between like antigen and
antibody
- Cell grouping
- Serum grouping
Hemagglutination method
Principle
Hemagglutination method
Hemagglutination method
 ABO grouping can be easily performed from corpses when
fresh blood can be obtained
 Storage periods of blood or in the postmortem time of
corpses increases
1. Changes in the agglutinability
2. Deformability
3. Antibody binding capacity
4. Osmotic fragility
5. Morphology
6. ATP content
Hemagglutination method
Developed Hemagglutination method
- Glutalaldehyde or heat,and storage in acidic-dextrose
(ACD) solution, blood bank condition
- Blood bank condition and Incubation of the aged
erythrocytes in a saline solution containing 5 mM adenine, 20
mM inosine and 5 mM glucose for 60 min at 370 C
Hemagglutination method
Scanning electron microscopic observation of erythrocytes. Erythrocytes stored for 8 week
in blood bank condition were incubated in the solution containing adenine and inosine. A; before
incubation, B; after incubation.
http://www.geradts.com/anil/ij/vol_006_no_002/papers/paper001.html
Hemagglutination method
Table. Relationships between ATP content and agglutinability of
erythrocytes stored 8 weeks
Incubation
period (min)
ATP content
(mg/dl)
0
Agglutinability*
anti B
Anti N
Anti D
6.6
32
2
1
30
14.9
64
4
1
60
21.9
128
8
2
120
24.9
128
8
2
Hemagglutination method
It is often seen that the hemolysis of aged erythrocytes
Michailow's method
1. 1 ml of blood sample was gently shaken for 5 min in 1ml of 40%
saccharose solution
2. adjusted to pH 6 with 25% citric acid, and then 5 ml of 7%
saccharose solution (pH 6) was added.
3. The sample was left standing for 5 min,then washed three times
with physiological serum (inactivated group AB human serum)
4. Used to prepare a 2% red blood cell suspension
Absorption Elution method
Principle
Absorption Elution method
ABO typing of erythrocytes
- Blood Stained
* Often encountered discrepant results when determining
the blood group
- Making autopsies of bodies who have died due to fire
* Glycolipids and sialoglycoproteins
Absorption Elution method
ABO typing from body fluid
Table. Types of organs and tissues expressing the ABH antigen
Absorption Elution method
ABO typing from body fluid
Table. Types of organs and tissues expressing the ABH antigen
Absorption Elution method
ABO typing from body fluid
- ABH and related antigens are expressed in the secretory
organs of many species of animals
- The ABH and their related antigens were expressed in the
salivary glands, kidneys and reproductive organs of animals as
well as in those from humans
- The emphasize that the species identification prior to ABO
blood grouping is indispensable
Absorption Elution method
ABO typing from other tissue
 Skin
 Nail
 Hair
 Bone
www.cc.gatech.edu & www.emedicinehealth.com
Histochemical method
- Material of body
- Staining ; Fluorescent Dyes
- Microscopic
http://www.histochem.org/
Histochemical method
ABO Blood grouping from materials obtained from humans
 The antigens are easily and certainly detected by means of
immunohistochemical technique using
*Antibodies against ABH antigens
*Lenctins specific to blood group A, B, or O
 It is a great merit that we can microscopically observe the
distribution of antigens and reaction sites in the tissue
Histochemical method
ABO blood group incompatible cases
ABO group determination in the blood group incompatible case. The transfused A
typed red blood cells indicated by arrows were easily detected inside blood vessels of the
kidney with anti A antibody (photo A), and a large amount of O typed red cells shown by
arrow inside blood vessels and collecting tubules were stained by anti H antibody (photo B).
http://www.geradts.com/anil/ij/vol_006_no_002/papers/paper001.html
Histochemical method
Hair
ABH antigens in hair by animmunohistochemical methodusing the AvidinBiotin-Peroxidase technique.
Figure . ABO grouping from hair. A-antigen was detected only in the
medulla of the blonde hair showing mosaic pattern distribution. The
positive and negative sits in the medulla are observed by turns.
http://www.geradts.com/anil/ij/vol_006_no_002/papers/paper001.html
Histochemical method
Typing of Blood Group A Subgroup
- Monoclonal anti H antibody usually can not bind to the
secretory cells, epithelium of Hassall's corpuscles or blood
vessel of A1 individuals
- These cells of A2 individuals reacted strongly with H antibody
- A2 individuals may therefore be most clearly discriminated
from A1 individuals by the difference in the reactivity of
antibodies against A and H antigens in the tissue sections
Histochemical method
Typing of secretor status
- Compared the histochemical properties of mucous
cells of sublingual and submandibular glands from blood
group A and AB Japanese non-secretors with those from
German non-secretors
- Anti A-antibody, HPA and HAA lectins
- Proved the ethnical difference in the expression of
blood group antigens
Histochemical method
ABO Blood grouping from materials obtained from
criminal scene
- ABH antigens are expressed in the secretory organs of
many species animals
- Distribution of ABH antigens on organs and cells in each
animal or human is revealed and the reaction sites on the
tissue sections can be microscopically
Histochemical method
Table . Expression patterns of ABH antigens and lectin binding in red cells,
endothelial cells,and secretory cells of various animal species
Histochemical method
Table . Expression patterns of ABH antigens and lectin binding in red cells,
endothelial cells, and secretory cells of various animal species
Histochemical method
Figure. ABO grouping from mummified skin. Endothelial cells of blood vessel in the mummified skin
obtained from the leg (photo B) of a skeleton body (photo A) showed reactivity with corresponding antibody
as shown in photo C. The PCR product of DNA obtained from the mummified skin could not be amplified .
http://www.geradts.com/anil/ij/vol_006_no_002/papers/paper001.html
ABO genotyping
Method
1. DNA extraction
2. Master mix preparing and PCR reaction
3. Amplification
4. Gel electrophoresis
5. Ethidium staining
6. Gel document
ABO genotyping
http://wheat.pw.usda.gov/~lazo/docs/cotton/image/pcr.jpg
ABO genotyping
- DNA extracted and fresh blood
and formalin-fixed paraffin
embedded tissues.
- Amplified by PCR
Figure. Electrophoretic pattern of the PCR product using pairs of primers for ABO genotyping.
M;174/Hae III, 1;Human, 2;Japanese Monkey, 3;pig, 4;Cat, 5;Rabbit, 6;Chinese Hamster,
7;Rat(Wistar),8;Rat(Fischer), 9; Mastomys, 10;Mouse, and 11: Goldfish
Discussion
- ABH and related antigens may be associated with the chemoreceptor system
- ABO polymorphism might appear in contact with the external
environment and play a role in the relationships of the species with
pathogens, although the antigen might be involved in cell-cell
recognition in the embryo in lower vertebrate species
Conclusion
Conclusion
References
1. ABO Blood สื บค้นข้อมูลเมื่อวันที่ 8 กรกฎาคม 2549
http://www.mie.utoronto.ca/labs/lcdlab/biopic/fig/10.14.jpg
2. ABO Blood สื บค้นข้อมูลเมื่อวันที่ 8 กรกฎาคม 2549
http://www.enh.org/uploadedimages/antigens.jpg
3. ABO Blood Typing from Forensic Materials - Merits and demerits of detection methods utilized in our
laboratories, and biological significance of the antigens สื บค้นข้อมูลเมื่อวันที่ 15 มิถุนายน2549
http://www.geradts.com/anil/ij/vol_006_no_002/papers/paper001.html
4. ศาสตราจารย์แพทย์หญิงทัศน์ยานี จันทนยิง่ ยง , เวชศาสตร์ทการธนาคารเลือด , เมษายน 2541 ,
พิมพ์ครั้งที่ 1 ,ธรรกมลการพิมพ์ ,กรุ งเทพ
5. Forensic science สื บค้นข้อมูลเมื่อวันที่ 1 กรกฎาคม 2549
http://www.barascientific.com/bscnews/forum/Forensice/forensic.php
THE END