Directorate of Forensic Scence, MHA, Gort. of India 10 4, BLOOD GROUPING 4.4. Collection of Fresh Blood Sample: 6 + Gterize the bulb of a finger with an alcohol swab and let it dry. hile compressing the finger bulb. 2. Prick it with @ disposable needle wl in a tube with normal saline. 3. Collect the blood orpm for one minute. 4. Centrifuge the contents at about 300 matant and resuspend the button of cells in fresh normal 5. Discard the supe! saline. 6. Repeat steps 4 and 5 twice and finally prepare 2% or 0.2% (aS per requirement) cell suspension in normal saline. n be stored for upto a week in lI suspension (50%) cal prior to use. ould be washed in normal saline ection Intensities Note: Packed red cell refrigerator. The cells sl 4.2. Grading of the Agglutination Ri Macroscopic agglutination: C-Complete, all cells forming one big clump. } V-Visual, all cells forming few visible big clumps. Microscopic agglutination: 44+4-Very large agglutination clumps with few free cells. 44-Smaller agglutinates with more free cells. +-Agglutinates of 5- 10 cells with many free cells. W-Agglutinates of 3- 5 cells with many free cells. N- No agglutination. 4,3. Preparation of Anti- H Lecti ropaeus in 10ML of normal saline for overnight. 1. Soak about 2g seed of Ulex eu! n hour. Centrifuge at 3,000rpm for 5 2. Macerate seeds and agitate paste for at minutes and discard sediment.Laboratory Procedure Manual- Forensic “orensie Serolo = W 3. If supernantant is cloud F ; use the supernatant. ly, centrifuge at about 10,000rpm for 15minutes and 4. Check the specificity of the lectin wi i ir 7 F: . ith group O cells & titrate. Anti- H lectin with minimum titre of 32 should be used for grouping reactions. 4,4, Grouping of Fresh Blood [Link] Grouping ‘ABO blood group can be determined from the fresh blood samples by detection of antigens on RBC's (forward method) or detection of antibodies in serum (reverse method). [Link]. Forward Method 4. Take aclean, dry cavity tile and mark two cavities as A and B. 2. Put one drop of anti-A serum and anti-B serum in the above marked cavities respectively. 3. Add one drop of about 2% cell suspension in each cavity and mix the contents thoroughly. 4, Rotate the tile for 5 minutes and examine the contents macroscopically as well as microscopically for agglutination. Agglutination in cavity Antigen on RBC's Blood group A B + ~ A A 7 + B B + + AB AB : c Nil ° 4 ils 2. Reverse Method the whole blood sample clot and then remove the serum carefully. 2. Take one clean and dry cavity tile and mark two cavities as A and B. 3. Put one drop of serum in each cavity. 4. Add one drop of 2% cell suspension of A and B cells in the respective cavities. Mix the contents and rotate the tile for five minutes. 5, Examine the contents for the presence of agglutination macroscopically as well as microscopically.Directorate of Forensle Science, MHA, Gort of India 12 Agglutination in cavity Antibody present Blood " B B : +Anti BA AB . + - Anti A - . Nil AB t + Anti A, Anti B ‘ e : jutinated by anti- A serum, anti- B serum or in case of Bombay blood group, RBC's are not agglutinated by ani &EEe A anti: H lectin. Presence of anti- H in the serum is indicative of 4.4.2 Rh Typing 1, 1) Take one clean and dry cavity tle and mark five cavities as C, ©, D, E & e. 2. Place one drop of anti C serum, anti c serum, anti D serum, anti E serum and anti e serum in the respective cavities. 3, Add one drop of 2% cell suspension in each cavity and mix the contents thoroughly. Rotate the tile for 5 minutes at room temperature. 4, Examine for agglutination both macroscopically as well as microscopically. 5, If there is no agglutination, keep the tile at 37° C for 15 minutes, rotate the tile and again examine for agglutination. Presence of agglutination indicates the presence of respective antigen. 4.4.3. MN Typing 1. Take a clean, dry cavity tile and mark two cavities as M and N. 2. Wash red blood cells three times with normal saline. Properly we f ee tin fashed cells are absolutely critical in MN typing in order to avoid false positive "ecu 3. Place one drop of the appropriate antiserum in the wells. 4. Add one drop of 2.0% suspension of red blood cells. 5. Rotate the tile at room temperature for five minutes. 6. Examine for agglutination both macroscopically as well as microscopically Agglutination in cavity Antigen on RBC's group urination inca Antigen on RBC's Blood grouy + + M ; : W N ‘ + M,N MN -Laboratory Procedure Manual- Forensic Serology 13 4.4.4. Lewis Typing 1. Label two test tubes: Lea and Leb. 2. Place one drop of anti Lea serum and anti Leb serum in the respective tubes. 3. Add one drop of 2.0% cell suspension in the both tubes and mix. 4. Allow tubes to incubate at room temperature for 30 minutes. 5. Centrifuge at about 5000rpm for 15 seconds. 6. Gently shake the tube until the cell button just detaches itself from the bottom of the tube in one or more agglutinated clumps or until the cell button disperses. 7. Read and grade the results macroscopically as well as microscopically. Agglutination in cavity Antigen on RBC's Blood group Lea’ Leb + - Le® Le atb- : + Le? Le abt : as Nil Le a-b- 4.5. ABO Grouping of Body Fluid Stains and Tissues If microbial growth is present over the stains, the stains can be kept at 100°C for one hour to destroy the microbes and then typing can be performed. The polyclonal antisera are preferred over monoclonal antisera for typing the body fluid stains & tissues. If polyclonal antisera are not available, monoclonal antisera from different sources should be pooled and validated before analyses. 4.5.1. Reverse Grouping of Bloodstains: Lattes test This method is based on the identification of antibody in the blood stains. It is useful in the identification of mixture of blood stains. 4. Take about 0.5 [Link] bloodstain and add 2 drops of normal saline. Keep for an hour. 2. Squeeze the stain and then remove it. Centrifuge the extract and take the supernatant. 3. Take one clean dry cavity tile and mark two cavities as Aand B. 4. Put one drop of bloodstain extract in each cavity.= ag Porn Senet ME, Core of ng respective cavitie, 5. Add one drop of 2% cell suspension of A and B cells " the resp 8. Mix the contents and rotate the tile for five minutes. sath scopically as | 6. Examine the contents for the presence of agglutination macroscop! well as microscopically. lood group Aqalutination in cavity Antibody present 8 A B A = aes Anti B B i 7 Anti A AB 2 7 Nil ae S + + Anti A, Anti B Precaution: The antibodies are less stable in comparison to antigens and can sensitive also. be typed only in the fresh stains. The method is less ee . Absorption Elution Method 1. Take three clean and dry test tubes and mark them A, Band H. . Cut the blood stain and put about 2 sq. mm or 2- 5mm long threads in each test tube. 3. Dip the fabric in anti- A serum, anti- B serum and anti- H lectin respectively and keep at 4°C for overnight. 4, Then remove the antiserum and give 3-4 washings with ice chilled normal saline. 5. After the last wash remove whole of the normal saline and add one drop of fresh normal saline. 6. Plug the test tubes with cotton swab and keep in water bath at §6-60°C for 15- 20 minutes. 7. Add one drop of 0.2-0.5% A, B and O indicator cells in the respective tubes and keep at 4°C for half an hour. 8. Centrifuge, shake and examine the contents for agglutination both macroscopically and microscopically. Aaglutination in cavity Blood group A B H + - - Ort A 7 + -Or+ B + + ort AB z - + °Laboratory Procedure Manual- Forensic Serology 15 Precaution: 1. Washing should be done with ice chilled normal saline. 2. At the time of elution, only one drop of normal saline is required. 4.5.3. Absorption Elution Method: Howard Martin 1. Take a cellulose acetate sheet (minimum thickness 0.4mm) and mark three areas as A, B and H. Glue one cm long bloodstained thread with acetone/fingernail polish to each of 3 areas. 2. Allow the threads to fix on the sheet for 15 minutes. 3. Put one drop of the anti- A serum, anti- B serum and anti- H lectin respectively on the fixed threads 4, Place the sheet in a moist chamber and allow it to absorb overnight at 4°C in the refrigerator. 5. Remove from the refrigerator, rinse off the excess antisera and blot the threads dry with a paper towel by inverting the plate face down on a paper towel and rubbing the back of the glass plate with another towel. 6. Place in the refrigerator at 4°C for a ininimum of 2 hours. Longer wash times will not have a negative effect on the results provided the temperature does not exceed 4°C. Shorter wash times may result in incomplete rinsing of unbound antibody. 7. Remove from the wash bath and blot dry with a paper towel as previously described. 8. Add one drop of appropriate 0.2- 0.5% indicator cells to each thread. 9. Place in a moist chamber and elute in a 56°C incubator for 20 minutes. 40. Place the sheet in a moist chamber and rotate on a VDRL rotator for 30 minutes. 1 Read results roscopicall 4.5.4. Absorption Elution: Ammonia Method ‘Ammoniacal elution is especially useful when typing bloodstains on substrates which do not lend themselves to Howard-Martin absorption elution typing or very old, insoluble bloodstains.Aiwayy 4.5.6 Grouping of Keratinized Tissues (Hair Shaft or Nail) 4, Clean the sample by dipping in mixture of ethanol: ether (1:1). 2. Dry and flatten them upto 4-5 times by hammering. The keratinized tissue should be flattened thoroughly to increase the surface area. 3. Take about 1 cm of the flattened strand or 10 mg of nail in each tube marked A, B and H and follow the Absorption elution method employing papainised indicator cells. 4.5.7. Grouping of Calcified Tissues (Bone or Dentine) 1. Pulverize the tissue to obtain a very fine powder. 2. Put 5-10 mg of the tissue in each tube and follow absorption elution method employing papainised indicator cells. 4.5.8. Mixed Agglutination Method 1. Take three clean and dry test tubes and mark them A, B and H. 2. Cut the blood stain and put about 2 sq. mm or 2- 5mm long threads in each test tube. 3. Dip the fabric in anti- A serum, anti- B serum and anti- H lectin respectively and keep at 4°C for overnight. 4. Then remove the antiserum and give 3-4 washings with ice chilled normal saline. 5, Alter the last wash remove whole of the normal saline and add one drop of 0.2-0.5% A, B and O indicator cells in the respective tubes. 6. Plug the test tubes with cotton swab and keep in water bath at 50°C for 10 minutes. :Directorate of Forensic Seience, MHA, Govt of India 18 7. Keep tubes at 4°C for half an hour, centrifuge, shake and examine the contents for agglutination attached to fabrics, both macroscopically and microscopically. A Blood group A + - -or+ A - & -or+ B + + -or+ AB si " + O Precaution: Washing should be done with ice chilled normal saline. ~_ 4.6. ABO Groping of Saliva and Semen : The persons who secrete ABH substances in their saliva, semen or vaginal secretions and urine etc. are called secretors and ABO blood group can be detected from their body fluids and stains. All secretors secrete H substance in the body fluids irrespective of their blood group. ABO grouping from these body secretions should be done by Absorption- Inhibition as well as Absorption- Elution method as detailed earlier. 4.6.1. Typing of Fresh Saliva Fresh saliva has enzymes which degrades the ABH substances. Hence, keep the tube containing fresh saliva in boiling waterbath for about 10 minutes to idestrey the enzyme. Before typing, dilute the fresh saliva 2-3 times with normal saline. 4.6.2. Preparation of Extract from semen or saliva stains 4. Cut about 0.5- [Link] of the stain and dip in 4-5 drops of the normal saline. 2. Keep at 4°C for a minimum of two hours. [Link] out the stain fabric and centrifuge the content ‘ is at abot minutes. Use the supernatant for grouping test. ut SogdrmIn fore