This document discusses various methods for examining blood stains, determining if they are human or animal, and identifying their blood group. Physical examination of the stain provides initial clues. Chemical tests like benzidine and phenolphthalein can detect the presence of blood. Additional confirmation is done through physicochemical tests like thin layer chromatography and electrophoresis. Microscopic analysis of crystals formed by reagents helps identify hemoglobin and confirm human blood. Spectroscopy examines the absorption bands of oxyhemoglobin, methaemoglobin, and carboxyhemoglobin. Immunological tests like precipitin and latex agglutination inhibit reactions between human blood extract and antibodies. The enzymatic method analyzes isoenzyme patterns through electrophoresis to differentiate human from

1. Examination of Blood Stains
Toxicology & Forensic
Serology
1
Dr. Aamir Ali Khan
Assistant Professor MLT
PhD Biochemistry

2. Blood Stains
Toxicology & Forensic
Serology
1
• The points to bedeterminedregardingblood stains are:
– The natureof stain
– Ifblood stainseither human/animal
– Ifhumanblood ,to whichgroup it belong(A,B,AB,O)
The method used to determinethesefactsareas follows

3. PhysicalExamination:
Thevalueablecluescanbe achievedbyvisitingcrime
scenes
Toxicology & Forensic
Serology
3
– Clothing andstainson it
– Size,shape&directionofblood spot
– Character of stain
– Age of stain
– Condition of body
– Other causesof stain

4. Chemical Examination of Blood
• Benzidine Test:
• The benzidine test is most commonly used for the detection of
blood in the feces, and was therefore singled out for investigation.
• whenbenzidinesolution isadded to thesuspectedsolutionagreenish
bluecolor is obtainedifblood is present
– Benzidinesolution iscombinationof10% benzidine in
glacialaceticacidandhydrogen peroxide
• Testcanremainpositiveif1:500,000dilutionof blood is made
• Benzidineiscarcinogenicinnaturethat’swhysome labs
preferother tests

5. Chemical Examination of Blood
5
• Phenolphthalien(Kastle-Meyer Test):
• The Kastle-Meyer colour test works on a similar principle to Luminol, with an
activecompoundwhichreactswithhydrogenperoxideinthepresenceofblood.
In this case the chemical involved is phenolphthalein. This will react with
hydrogenperoxideinthepresenceofhaemoglobintoproduceapinkcolour.
• Phenolphthalien is reducedby zinc dust in a strongly
alkaline medium, which is oxidized by
nascentoxygenlibratedbyperoxidaseon H2O2, give pinkor purplecolor
ifblood present
– Phenolphthalien reagent: 2 g phenolphthalein, 25g KOH dissolve in 100 g H2O, add
15 gpowderedzincandheat untilpinkcolor disappear

6. Physicochemical Examination
6
• The most testfor confirmation ofblood stains are
• Thin Layer Chromatography (TLC):
– An appropriatequantityofsampleextract,standard haematin
chloride andcontrol sampleplacedon prepared gel
• Electrophoresis:
– Agar plateis use andblood sample,standard&other body
secretionsrun on the plate

7. Microscopic Examination
Toxicology & Forensic
Serology
7
• The hemoglobin of the RBCs under various staining methods give important
information
• Takayama’sHaemochromogen CrystalTest:
• Takayama test is a confirmation test used to detect blood spots. Based on
the research results this test can still be used to identify dried blood spots
on clothing aged 20 years, and able to detect positively the presence of
blood with the formation of pink crystals.
• The dried powdered from stain extract placed on the slide and cover with cover slip,
then two drops of takayama’s reagent are added on slide and examine under
microscope after5 minutes.Ifblood presentpink featherycrystalof haemochromogen
are formed

8. Review of Forensic Medicine and
ToxicologyToxicology & Forensic
Serology
8

9. • Haemin Crystal Test (Teichmann’s Test):
• A confirmatory test for blood based on the formation of distinctive haematin
crystals that are viewed under a microscope. The reagents typically used are
sodium chloride and glacial acetic acid.
• The principle is to convert the haemoglobin of the stain into haemin, which in the
presenceofhalogen,is convertedintosaltandformbrown rhombic crystal.
• Darkbrowncrystalareproduced.
9
Microscopic Examination

10. Spectroscopic Examination
1
0
Toxicology & Forensic
Serology
• The spectroscopic testis one oftheconfirmatory test
for blood, itssensitivity 1:5,000
• Ifthesamplecontainsfreshoxygenatedblood i.e
oxyhaemoglobin,two dark absorption bandswillbe seen
• If stain is old or has been exposed to any extent,
methaemoglobin will have been formed, a darker
pigmentformed when blood is decomposing

11. • Carboxyhaemoglobin:
• It has a spectrum so much like that of oxyhaemoglobin that
the scaled spectroscope is necessary to define the degree of
shiftto theright
1
1
Toxicology & Forensic
Serology

12. Review of Forensic Medicine and Toxicology
1
2
Toxicology & Forensic
Serology

13. Detection of specie Origin
1
3
Toxicology & Forensic
Serology
• Blood is thoroughly examinedto confirmitshuman
origin to rule out
– The claimthatblood is animal blood
– Torule out animalblood havingsameblood group antigen
to human blood
– In caseofprohibitedanimalspeciesshooting andidentify the
blood ofendangered species

14. Method for detection of blood origin
• Immunological Method
• Precipitin test:
• The precipitin test rests on the fact that when a. suitable animal is injected with
foreign protein, its. serum, when mixed with a solution of the foreign. protein,
will form a. precipitate.
• theprecipitinwhenmixedwithhumanbloodserum eveninminutequantityform precipitate
1
4

15. Immunological Method
– Haemagglutination-inhibition test: Ifanti-humanglobulin is
mixedwithextractofahumanblood stainandsubsequently tested
againstsensitizedred cells,theagglutinationreaction willbe inhibited
1
5

16. Immunological Method
– LatexTest: A salineextractofblood stainwhen react with
dilutedlatexparticlesensitizedwithantiserum,clump willform
showing positive reaction.
1
6

17. Enzymological Method
• Blood stains of human and several other species gives
specificisoenzyme
• Electrophoretic pattern of which enables differentiation
betweenhumananddifferentanimal Species
• The most common enzymes are peroxidase, lactate
dehydrogenase (LDH), malate dehydrogenase (MDH)
and esterase.
Toxicology & Forensic
Serology
1

18. 1
8
Toxicology & Forensic
Serology

20. Summary
Stains
Blood Stain
Physical
Origin of
blood
Chemical Physicochemical Microscopic Spectroscopic
Immunological
Method
Enzymatic
Method
1
9
Toxicology & Forensic
Serology

21. Reference
Toxicology & Forensic
Serology
2
• Text book of Medical Jurisprudence,Forensic medicineandToxicology, BV Subrahmanym Seventh edition