Various Tests in Forensic Biology : Blood, Semen, Saliva & DNA Analysis

 Introduction 

In many criminal cases, the most reliable evidence is not always a weapon or fingerprints—it is often biological evidence. Even a tiny drop of blood, a small saliva stain, or invisible touch DNA can help forensic scientists identify a suspect and solve a case. This is where forensic biology testing becomes crucial. Forensic biology includes a wide range of laboratory tests used to detect, identify, and analyze biological materials such as blood, semen, saliva, and other body fluids, followed by DNA profiling to establish identity. 

In this article, we will explore the various tests performed in forensic biology, including both traditional serology methods and modern DNA-based techniques.

Identification Of Blood Stains

There are two types of test we perform for identification of blood stain on evidence Presumptive & Confirmative 

A] Presumptive Examination :  Presumptive blood assays are designed to detect traces of blood.These  tests indicate the possible presence of blood, but are not confirmatory.

1.  These examination is based on the basic principle of the oxidation–reduction reaction catalyzed by the heme moiety of the hemo globin. hemoglobin that catalyzes oxidation of colorless reagents → colored products. These examination are very sensitive and can detect blood in samples with 10^5−10^6 -fold dilutions. 

      

Human adult hemoglobin. Four subunits, two α and two β chains, of human adult hemoglobin are shown. Each hemoglobin subunit contains a heme moiety. (© Richard C. Li.)

These Presumptive Examination Includes Various Chemical Test these chemicals are 

oxidized-reduced by hemeglobin present in blood and give color changes for positive result. Such chemical test are listed below.

1. Benzidine Test : Oxidation of benzidine by heme group produces a Blue-green color. 
2. Phenolphthalein Test : The phenolphthalin assay for blood identification is also known as the Kastle–Meyer test. Phenolphthalin, a colorless compound, is catalyzed by heme with hydrogen peroxide as the oxidant . The oxidized derivative is phenolphthalein, which appears pink under alkaline conditions.
3. Leucomalachite Green Test : Malachite green is a triphenylmethane dye. The leuco base form of malachite green is colorless and can be oxidized by the catalysis of heme to produce a green color. The reaction is carried out under acid conditions with hydrogen peroxide as the oxidant  

      There are other  test also exist such as Luminol Test & Fluorescein Test. 

B] Confirmatory Examination 

There are two type of confirmatory tests we perform :  Takayama Test & Teichmann Test 

 Takayama Test 

 A bloodstain is treated with pyridine and glucose (a reducing sugar that is capable of reducing ferric ion) under alkaline conditions to form pink feathery crystals of pyridine ferroprotoporphyrin also known as Hemochromogen. 

 

(Microcrystal assays using the Takayama method) 

Teichmann Test  

 In 1853, Teichmann documented a method of forming crystals of blood specimens. When blood specimens are treated with glacial acetic acid and salts, and subsequently heated, hematin chloride (ferriprotoporphyrin chloride), a prismatic brown-colored crystal, is formed. Hematin is a heme derivative; its iron is in the ferric (Fe+3) state. This hematin assay has a similar sensitivity and specificity as hemochromagen assays. The hematin assay has the advantage of being more reliable than hemochromagen assays for aged blood samples. 

(Microcrystal assays using the Teichmann method) 

Summary Table 

Test Name	Principle	Reagent Used	Positive Result Appearance
Benzidine Test (obsolete due to carcinogenicity)	Oxidation of benzidine by heme group	Benzidine + Hydrogen peroxide	Blue-green color
Phenolphthalein Test (Kastle–Meyer Test)	Reduced phenolphthalein → oxidized to pink phenolphthalein	Phenolphthalein + H₂O₂	Intense pink color
Leucomalachite Green Test	Heme catalyzes oxidation of colorless leucomalachite → green	Leucomalachite green + H₂O₂	Green color
Hemastix Test	Peroxidase activity of hemoglobin	Reagent strip	Green to blue color
Luminol Test	Chemiluminescence due to oxidation	Luminol + H₂O₂	Blue glow in darkness (very sensitive)
Fluorescein Test	Fluorescein oxidized by heme	Fluorescein + H₂O₂	Greenish fluorescence under UV light

Takayama Test  (Hemochromogen Test)	Pyridine + Glucose + NaOH	Hemochromogen (pyridine    ferroprotoporphyrin)	Pink feathery          crystals
Teichmann Test (Hemin Test)	Glacial acetic acid + NaCl	Hemin (brown rhomboid crystals)	Brown                   rhomboid crystals under microscope

Note: Luminol and fluorescein are used at crime scenes for detecting trace or cleaned blood.

Identification Of Semen Stains 

Semen is a viscous, whitish fluid composed of:

• Spermatozoa (sperms): Produced by testes.
• Seminal plasma: Secretions from seminal vesicles, prostate, bulbourethral glands.
• Main constituents:
• Proteins (acid phosphatase, semenogelin)
• Fructose (energy source for sperms)
• Choline
• Citric acid
• Enzymes (prostate-specific antigen — PSA)

Examination of Suspected Semen Stain

A. Visual Examination

Feature	Observation
Color	Grayish-white when fresh, turns yellowish or stiff on drying
Texture	Stiff and crusty on cloth/fabric
Odor	Faint, characteristic seminal odor
Location	Commonly on clothing, undergarments, bedding, body parts

Sometimes stains may not be visible to the naked eye.

Use of Alternate Light Source (ALS)

• UV or blue light (350–450 nm) reveals semen as bluish-white fluorescence.
• Helps detect faint or washed-off stains before chemical testing.

B. Preliminary (Presumptive) Tests for Semen 

(i) Acid Phosphatase (AP) Test

Principle:
Semen contains high levels of acid phosphatase enzyme secreted by the prostate gland.
This enzyme hydrolyzes phosphate esters → forms a colored product with specific reagents.

Reagent:
α-naphthyl phosphate + Fast Blue B salt

Procedure:
A small cutting or swab from the suspected stain is treated with the reagent.

Positive Result:

• Immediate appearance (within 30 seconds) of deep purple color → indicates high AP activity (likely semen).
• Weak/slow reaction → may indicate vaginal secretions or old/washed stains.

(ii) Barberio’s Test (for Spermine Crystals)

Principle:
Spermine hydrochloride in semen reacts with picric acid to form yellow needle-shaped crystals.

Reagent:
Saturated aqueous picric acid.

Positive Result:
Yellow needle-shaped crystals under microscope.

Also not fully specific; confirmatory test must follow.

C. Confirmatory Examination 

(i)  Microscopic Examination

  Principle: Direct microscopic examination of a stained extract for presence of sperm cells.

Procedure:

1. Take small portion of the stain → soak in saline/water.
2. Centrifuge or smear a drop on a clean slide.
3. Stain with Christmas Tree Stain (Nuclear Fast Red + Picroindigocarmine).

Observation:

Component	Color in Christmas Tree Stain
Sperm head (nucleus)	Red
Sperm tail	Green/Blue

Result Interpretation:

• Presence of spermatozoa → confirms semen.
• Absence does not rule out semen (e.g., in azoospermic males, vasectomized men, or degraded samples).

(ii) Prostate Specific Antigen (PSA/p30 Test)

Principle:
PSA (p30) is a protein secreted by the prostate gland — specific to human semen.

Method:

• Immunochromatographic test (RSID–Semen): Works like a pregnancy test kit.
• ELISA or Immunodiffusion: Detects PSA antigen–antibody reaction.

Positive Result:
Visible colored band or precipitin line → confirms human semen.

Highly specific confirmatory test (even in azoospermic or degraded samples).

Summary Table 

Stage	Purpose	Test/Method	Result/Observation
Visual	Locate stain	UV/ALS	Bluish-white fluorescence
Presumptive	Indicate semen	Acid Phosphatase Test	Purple color
Microscopic	Confirm semen	Spermatozoa under microscope	Sperm cells visible
Immunological	Confirm human semen	PSA/p30 test	Positive line or precipitin

Identification, and Examination of Saliva Stains

1. Introduction

Saliva is a biological fluid secreted by the salivary glands in the mouth.
In forensic investigations, saliva stains can serve as valuable evidence in:

• Sexual assault cases (bite marks, kissing, oral contact)
• Chewing gum, cigarette butts, drinking glasses
• Envelopes, stamps (licked areas)
• Food remains, clothing, and skin of victims or suspects

Detection and identification of saliva help in linking a suspect to a crime scene through DNA profiling.

Examination of Saliva Stains

A. Visual Examination

Characteristic	Observation
Color	Usually colorless or slightly yellowish
Texture	Stiff or crusty on fabric after drying
Odor	Odorless
Location	Bite marks, lips, face, clothing near mouth or genital area, cigarettes, food

Saliva stains are often invisible to the naked eye.

Detection by Alternate Light Source (ALS)

• Under UV or blue light (350–450 nm), saliva may show bluish-white fluorescence.
• Useful for locating trace stains before testing.

B. Presumptive Tests for Saliva

These tests detect amylase enzyme activity — the primary biochemical marker of saliva.

(i) Starch–Iodine Test

Principle:
Salivary amylase breaks down starch → reducing blue color intensity of starch-iodine complex.

Procedure:

• Apply starch solution on suspected area.
• Add iodine solution (produces blue color).
• If saliva is present → blue color fades rapidly due to starch hydrolysis.

Result:

• Disappearance of blue color = Positive for saliva.

Not specific — other amylase-containing materials (food, sweat) can interfere. 

(ii) Phadebas Test (Most Common Modern Presumptive Test)

Principle:
Detects α-amylase activity using starch linked to a blue dye.
When amylase breaks the starch–dye bond → dye is released → blue color appears.

Procedure:

• Add Phadebas tablet or solution to extract from stain.
• Incubate at 37°C for ~30 minutes.

Positive Result:
Blue coloration → indicates presence of salivary amylase.

Advantages:

• Sensitive and quick (detects minute quantities).
• Used widely in forensic labs.

(iii) RSID–Saliva (Rapid Immunochromatographic Test)

Principle:
Detects human salivary α-amylase using antigen–antibody reaction.

Result:
Colored test line appears (similar to pregnancy test kit).

(iv) Microscopic Examination

Identification of Buccal Epithelial Cells

• Smear prepared from suspected stain.
• Stained with Nuclear Fast Red or Hematoxylin–Eosin.
• Squamous epithelial cells from mouth are observed under the microscope.

Result:
Presence of nucleated epithelial cells → indicates oral origin (saliva).

However, epithelial cells may also be found in other body fluids, so not conclusive alone.

Summary Table 

Stage	Purpose	Test / Method	Positive Result
Visual / ALS	Locate stain	UV / Blue light	Fluorescence
Presumptive Test	Detect amylase activity	Starch–Iodine, Phadebas	Fading blue / Blue color
Confirmatory Test	Confirm human saliva	RSID–Saliva, ELISA	Test line / color change
Microscopy	Identify cells	Buccal epithelial cells	Flat nucleated cells

Refrences : Forensic biology 2nd edition (richard li)

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