By: Wiraya Nakkeamkeao

1. 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

2. 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

3. 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

4. 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

5. Review ABO Blood Groups

6. Review Subgroups A Anti-A1 = Lectin (Dolichos biflorus)

7. 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

8. ABO blood grouping method Method • Hemagglutination • Absorption - Elution • Histochemical techniques • ABO genotyping method

9. Hemagglutination method “Hemagglutination” mean A reaction outside the body between like antigen and antibody - Cell grouping - Serum grouping

10. Hemagglutination method Principle

11. Hemagglutination method

12. 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

13. Hemagglutination method DevelopedHemagglutination 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

14. 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

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

16. 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

17. Absorption Elution method Principle

18. 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

19. Absorption Elution method ABO typing from body fluid Table. Types of organs and tissues expressing the ABH antigen

20. Absorption Elution method ABO typing from body fluid Table. Types of organs and tissues expressing the ABH antigen

21. Table 2-1. 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

22. Absorption Elution method ABO typing from other tissue • Skin • Nail • Hair • Bone www.cc.gatech.edu &www.emedicinehealth.com

23. Histochemical method - Material of body - Staining ; Fluorescent Dyes - Microscopic http://www.histochem.org/

24. Histochemical method • 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 ABO Blood grouping from materials obtained from humans

25. 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

26. Histochemical method Hair ABH antigens in hair by animmuno-histochemical methodusing the Avidin-Biotin-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

27. 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

28. Histochemical method Typingof 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

29. 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

30. Histochemical method Table . Expression patterns of ABH antigens and lectin binding in red cells, endothelial cells,and secretory cells of various animal species

31. Histochemical method Table . Expression patterns of ABH antigens and lectin binding in red cells, endothelial cells, and secretory cells of various animal species

32. 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

33. ABO genotyping Method 1. DNA extraction 2. Master mix preparing and PCR reaction 3. Amplification 4. Gel electrophoresis 5. Ethidium staining 6. Gel document

34. ABO genotyping http://wheat.pw.usda.gov/~lazo/docs/cotton/image/pcr.jpg

35. 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

36. Discussion - ABH and related antigens may be associated with the chemo-receptor 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

37. Conclusion

38. Conclusion

39. References 1. ABO Blood สืบค้นข้อมูลเมื่อวันที่ 8 กรกฎาคม 2549 http://www.mie.utoronto.ca/labs/lcdlab/biopic/fig/10.14.jpg 2. ABO Blood สืบค้นข้อมูลเมื่อวันที่ 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 antigensสืบค้นข้อมูลเมื่อวันที่ 15 มิถุนายน2549 http://www.geradts.com/anil/ij/vol_006_no_002/papers/paper001.html • ศาสตราจารย์แพทย์หญิงทัศน์ยานี จันทนยิ่งยง , เวชศาสตร์ทการธนาคารเลือด , เมษายน 2541 , พิมพ์ครั้งที่ 1 ,ธรรกมลการพิมพ์ ,กรุงเทพ • Forensic science สืบค้นข้อมูลเมื่อวันที่ 1 กรกฎาคม 2549 http://www.barascientific.com/bscnews/forum/Forensice/forensic.php

40. THE END