ABO Blood Typing from Forensic M - aterials Merits and demerits of de tection methods utilized in our lab

oratories, and biological significan ce of the antigens

By: Wiraya NakkeamkeaoAdviser: Dr.Sirilak Teeraputon

Researcher

Katsuji Nishi, Steve n Rand,

Tokiko Nakagawa, Aiko Yamamoto,

Shigeru Yamasaki,Yoshio

Yamamoto, Aki Kobayashi,

Masateru Kane, Aki ra Morimoto,

Hedwege Spal thoff a ndBeate

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

ReviewABO Blood Groups

Blood Type

Genotyp

e

RBC Antige

n Presen

t

Serum Antibo

dy

A AA, Ai

A, H Anti-B

B BB, Bi

B, H Anti-A

AB AB A, B, H NoneO ii H Anti-A,

Anti-B

ReviewSubgroups 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

Hemagglutinatio

n Absorption -

Elution Histochemical

techniques ABO

genotyping method

MethodMethod

Hemagglutination method

A reaction outside the body between like antigen and antibody

- Cell grouping

- Serum grouping

“Hemagglutination” mean

Hemagglutination methodPrinciple

Hemagglutination method

Hemagglutination method ABO grouping can be easily pe

rformed from corpses when f resh blood can be obtained

S torage periods of blood or in the postmortem time of corpse

s increases

1. C hanges in the agglutinabili ty 2. Deformability 3 . A ntibody binding capacity

4 . O smotic fragility 5 . M orphology

6 . ATPcontent

Hemagglutination method

Developed Hemagglutination method - G lutalaldehyde or heat,an

- d storage in acidic dextrose (AC D) solution, blood bank conditio

n - B lood bank condition

and Incubation of the aged erythroc ytes in a saline solution containi

ng 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 weeks 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

Incubation period (min)

ATP content (mg/dl)

Agglutinability*

anti B Anti N Anti D

0 6.6 32 2 1

30 14.9 64 4 1

60 21.9 128 8 2

120 24.9 128 8 2

Table. Relationships between ATP content and agglutinability of erythrocytes stored 8 weeks

Hemagglutination method 1 1. ml of blood sample was gentl

ii i i i ii i i i iii5 1 4 0 %iiiiiii iiiiiiii

2 . 6 2 5 %adjusted to pH with ci i i i ii i i iiiiiiii, 5 7 %

sesolution(pH6) was added. 3 . The sample was left standing for

5min,then washed three times with phy siologicalserum(i nact i vat ed gr oup AB human ser um) 4 . U 2sed to prepare a % red bloo

d cel l suspensi on

MichailowMichailow 's method 's method

It is often seen that the hemolys is of aged erythrocytes

Absorption El ution methodPrinciple

Absorption Elu tion method ABO typing of er ABO typing of er

ythrocytesythrocytes- Blood Stained * O ften encountered discrepant result

s when d etermining the blood group- M aking autopsies of bodies who

have died due to fire * G lycolipids and sialoglycoprot

eins

Absorption Elu tion method

ABO typing fro ABO typing fro m body fluid m body fluidTable. Types of organs and tissues

expressing the ABH antigen

Absorption Elu tion method ABO typing fro ABO typing fro

m body fluid m body fluidTable. Types of organs and tissues expressing the ABH antigen

Absorption Elu tion method ABO typing fro ABO typing fro

m body fluid m body fluid- ABH and related antigens are

expressed in the secretory organs of many species of animals

- The ABH and their related anti gens were expressed in the sali

vary glands, kidneys and reprod uctive organs of animals as well as in those from humans

- The emphasize that the specie s identification prior to ABO blo

od grouping is indispensable

Absorption Elu tion method ABO typing fro ABO typing fro

m other tissue m other tissue Skin Nail Hair Bone

www.cc.gatech.edu & www.emedicinehealth.com

Histochemic al method

http://www.histochem.org/

- Material of body

- Staining ; Fluores

cent Dyes

- Microscopic

Histochemic al method ABO Blood grouping from mate ABO Blood grouping from mate

rials obtained from humans rials obtained from humans

The antigens are easily and cert ainly detected by means of

immunohistochemical techniqu e using

* A ntibodies against ABH antigen s

* L enctins specific to blood group A, B, or O

It is a great merit that we can m icroscopically observe the

distribution of antigens and re action sites in the tissue

ABO blood group incom ABO blood group incom patible cases patible cases

Histochemi cal method

ABO group determination in the blood group in compatible case. The transfused A typed red bl

ood cells indicated by arrows were easily detec ted inside blood vessels of the kidney with anti

A antibody (photo A), and a large amount of O t yped red cells shown by arrow inside blood ves

sels and collecting tubules were stained by ant i H antibody (photo B).

http://www.geradts.com/anil/ij/vol_006_no_002/papers/paper001.html

Histochemi cal method ABH antigens in h

-air by animmuno his tochemical methodu

-sing the Avidin Bioti- n Peroxidase techni

.

HaiHairr

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

Histochemi cal method Typing of Blood Gro Typing of Blood Gro

up A Subgroup up A Subgroup

- M onoclonal anti H antibody usuall y can notbind to the secretory cells,

epithelium of Hassall's corpuscles or blood vessel of A1 individuals

- T hese cells of A2individuals react - ed strongly with H antibody A2 in

dividuals may therefore be most cle arly discriminated from A1 individu al s by the di ff erence i nthe reac

ti vi ty of anti bodi es agai nst Aa ndHanti gens i nthe ti ssue secti

ons

Histochemica l methodTypingTyping of secre of secre tor status tor status

- C ompared the histochemi cal properties of mucous cells

of sublingual and submandibul ar glands from blood group A a - nd AB Japanese non secretors

-with those from German non secretors

- A - nti A antibody, HPA an d HAA lectins

- P roved the ethnical diffe

rence in the expression of bloo d group antigens

Histochemi cal method ABO Blood grouping from ABO Blood grouping from

materials obtained from cri materials obtained from cri minal scene minal scene

- ABH antigens are expressed in the secretory organs of man

y species animals - D istribution of ABH antigen

s on organs and cells in each a nimal or human is revealed an

d the reaction sites on the tiss ue sections can be microscopic

ally

Histochemi cal methodTable . Expression patterns of ABH antigens and lectin binding in red cells,

endothelial cells,and secretory cells of various animal species

Histochemi cal methodTable . Expression patterns of ABH antigens and lectin binding in red cells,

endothelial cells, and secretory cells of various animal species

Histochemic al 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 sho

wn 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

Method1. DNA extraction

2. Master mix preparing and PCR reaction

3. Amplification4. Gel electrophoresis5. 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 produ

ct 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(Wist ar),8;Rat(Fischer), 9; Mastomys, 10;Mouse, and

11: Goldfish

Discussion - ABH and related antigens may b

-e associated with the chemo recepto r system

- ABO polymorphism might appea

r in contact with the external enviro nment and play a role in the relation

ships of the species with pathogens, although the antigen might be invol

- ved in cell cell recognition in the em bryo in lower vertebrate species

Conclusion

Conclusion

References1. ABO Blood สื�บค้�นข้�อมู�ลเมู��อวั�นที่�� 8 กรกฎาค้มู 2549 http://www.mie.utoronto.ca/labs/lcdlab/biopic/fig/1014. .jpg

2. ABO Blood สื�บค้�นข้�อมู�ลเมู��อวั�นที่�� 8 กรกฎาค้มู 2549 http://www.enh.org/uploadedimages/antigens.jpg3. ABO Blood Typing from Forensic Materials - Merits and demerits of detection methods utilized in our laboratories, and biological significance of the antigens สื�บค้�นข้�อมู�ลเมู��อวั�นที่�� 15 มู�ถุ�นายน2549 http://www.geradts.com/anil/ij/vol_006_no_002/papers/paper001.html4 . ศาสืตราจารย�แพที่ย�หญิ�งที่�ศน�ยาน� จ�นที่นย��งยง , เวัชศาสืตร�ที่การธนาค้ารเล�อด , เมูษายน 2541 , พ�มูพ�ค้ร�)งที่�� 1 ,ธรรกมูลการพ�มูพ� ,กร�งเที่พ5. Forensic science สื�บค้�นข้�อมู�ลเมู��อวั�นที่�� 1 กรกฎาค้มู 2549

http://www.barascientific.com/bscnews/forum/Forensice/forensic.php

THE END