Body Fluids in Forensic Analysis

1. Body Fluids
Dr. Sangeetha Balakrishnan
23 February, 2017

2. Presumptive/Preliminary/Screening/
Field/Indicative Tests for Blood
1. Luminol Test
2. Fluorescein Test
3. Phenolpthalein Test

3. Luminol Test at a Crime Scene
0 to 30 s

4. Fluorescein Test at a Crime Scene

5. Phenolpthalein Test
• Can detect blood diluted to 1 part in 10
million.
• Gives false positives for certain vegetable
extracts.
• How is it done?:
(i) Suspected blood stain (on a cotton swab) +
water + Phenolpthalein colourless
(ii) Add hydrogen peroxide immediate
PINK colour!

6. Confirmatory Tests for Blood
1. Teichmann Test
2. Takayama Test (Hemochromogen Test)
• Both are called Microcrystal Tests.
Blood + Crystallising Reagent
Characteristic Shaped
Crystals.

7. Teichmann Test
• Blood + Teichmann’s Reagent (heat) Hb
hemin react with halides brownish-
yellow rhomboid crystals
• Microscopic Observation
• Teichmann’s Reagent: Potassium bromide +
Potassium chloride + Potassium iodide in
Acetic acid.

8. Teichmann Test

9. Takayama Test
(Hemochromogen Test)
• Suspected blood stain on a glass slide + heat +
add pyridine in Sodium hydroxide + reducing
sugar red feathery crystals of pyridine
ferroprotoporphyrin
• Very sensitive test.
• Even very old blood stains give +’ve results!

11. Saliva
• Saliva is produced in the mouth.
• Function: preliminary digestion of food.
• Contains: water, proteins, enzymes, salts.
• Alpha amylase: enzyme in saliva.
• Alpha amylase breaks down starch in food.
• It is also present in other body fluids, but in
low concentrations.

12. Preliminary Test - Saliva
• Principle: Starch + Iodine Deep blue colour
• Suspected saliva sample + water/saline
incubate at body temperature.
• If the suspected sample is indeed saliva, it will
contain alpha amylase!
• Alpha amylase will break down starch into
simpler components. (digestion)
• Add Iodine solution.
• Absence of starch (it has been broken down)
No Deep Blue colour!

14. Drawbacks of the Saliva
Preliminary Test
• Not particularly sensitive.
• Not specific to saliva.
• Use saliva sample for DNA testing.
• Detection of saliva at scene of crime:
Shine UV light fluorescence!

15. Saliva – Confirmatory test
• Phadebas Amylase Test
Developed by Pharmacia Diagnostics.
Qualitative and quantitative test.
Phadebas: a synthetic biochemical substrate.
• The substrate has starch microspheres.
• The microspheres are chemically bonded to a blue
coloured dye.
Phadebas substrate + suspected saliva (in water)
salivary amylase digests starch starch microspheres
break down blue dye is released!

16. Phadebas Amylase Test

17. Urine
• Main components:
(i) Creatinine
(ii) Urea
Presumptive Test – Urine
1. Jaffe Test
• Based on the detection of creatinine
• Suspected urine sample + picric acid + 5% NaOH
immediate orange colour

18. Presumptive Test – Urine … cont’d
2) Assay for Urea
• Bromothymol Blue is a pH-indicator dye.
• Colour: Yellow-green at pH 6
Aqua blue at pH > 7.6
(i) Whatman Filter Paper + Bromothymol Blue (1
drop) allow to dry
(ii) Add a drop from the suspected urine sample.
(iii) Add a drop of Urease.
(iv) If urea is present (meaning: urine is present),
urease will degrade urea to ammonia gas.
(v) This will cause the pH to increase.
(vi) Aqua blue colour!

19. Semen
• Produced by the male sex organ.
• Semen = Spermatozoa + Seminal fluid
(cellular component) (fluid component)
Spermatozoa
(Sperm)
Seminal Fluid
Male reproductive cells Contains: proteins, salts,
organic materials.
Flavin: UV fluorescence!
Consist of: head, tail and mid
piece
Produced in prostate gland
Seminal Acid Phosphatase
(SAP)
Prostate Specific Antigen (p30
protein)

20. Presumptive Test - Semen
• Based on Seminal Acid Phosphatase (SAP).
• There are other acid phosphatases in the
body!
• Brentamine Spot Test or Walker Test
• Suspected Semen Sample + Brentamine Fast
Blue B Intense purple colour within 2
minutes

21. Brentamine Fast Blue B added to
suspected semen samples

22. Confirmatory Test - Semen
1. Christmas Tree Stain Test
• Basis: Sperms analysed in lab are NOT motile.
• Hence identification should be in the presence of other
cellular material!
• A pair of dyes is used:
(i) Picroindigocarmine (PIC)
(ii) Nuclear Fast Red
• PIC: stains the tails green-blue-grey
• Nuclear Fast Red: stains the heads (with DNA) bright
crimson.

23. Christmas Tree Stain Test

24. Confirmatory Test – Semen … cont’d
2. Detection of Prostate Specific Antigen or p30
protein:
Oligospermia (low sperm count)
Aspemia (no sperms)
Suspected semen sample + Reagent Intense
purple.

25. Fecal Stains
• Basis: Human waste has bilirubin.
• Bacteria in body, break down bilirubin to
urobilinogen.
Presumptive Test: Edelman’s Reagent
Fecal stain bilirubin urobilinogen
urobilin shine UV light + Edelman’s reagent
Green fluoresence.

26. Confirmatory Test – Fecal Stains
• Microscopy:
• To look for animal and plant cells present due
to digestion.

27. Sweat
• Suspected sweat sample + Crystal Violet -
deep purple
• Fatty acids in sweat react with the dye.