Body Fluids

Dr. Sangeetha Balakrishnan23 February, 2017

Presumptive/Preliminary/Screening/Field/Indicative Tests for Blood

1. Luminol Test2. Fluorescein Test3. Phenolpthalein Test

Luminol Test at a Crime Scene

0 to 30 s

Fluorescein Test at a Crime Scene

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!

Confirmatory Tests for Blood

1. Teichmann Test2. Takayama Test (Hemochromogen Test)

• Both are called Microcrystal Tests.

Blood + Crystallising Reagent Characteristic

Shaped Crystals.

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.

Teichmann Test

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!

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.

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!

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!

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!

Phadebas Amylase Test

Urine• Main components: (i) Creatinine(ii) Urea

Presumptive Test – Urine1. Jaffe Test• Based on the detection of creatinine• Suspected urine sample + picric acid + 5% NaOH

immediate orange colour

Presumptive Test – Urine … cont’d2) 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!

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)

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

Brentamine Fast Blue B added to suspected semen samples

Confirmatory Test - Semen1. 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.

Christmas Tree Stain Test

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.

Fecal Stains

• Basis: Human waste has bilirubin.• Bacteria in body, break down bilirubin to

urobilinogen.

Presumptive Test: Edelman’s ReagentFecal stain bilirubin urobilinogen urobilin shine UV light + Edelman’s reagent

Green fluoresence.

Confirmatory Test – Fecal Stains

• Microscopy:• To look for animal and plant cells present due

to digestion.

Sweat

• Suspected sweat sample + Crystal Violet -deep purple

• Fatty acids in sweat react with the dye.