Influence of the Luminol Chemiluminescence Reaction on the

Confirmatory Tests for the Detection & Characterization of Bloodstains in

Forensic Analysis

Julio Abreu, William Chauvin, Conor McMenamin, John Vitale, Andrew Youngstrom

BIOL 1115Dr. Missy McElligot16 February 2012

Introduction• Detection of crime scenes

• Focus police work

• Luminol

• Detection of invisible blood stains

• Evaluation of whether or not luminol negatively affects blood and DNA testing

 

Objectives

• Verify effect luminol has on blood and DNA testing

• Evaluate dependence on incubation time

• Evaluate dependence on luminol concentration

              

Method of use in CSI

• Luminol applied to a surface via spray bottle

• Detects the presence of minor, unnoticed or hidden bloodstains

• Diluted down to a level of 1:106 (1 μL of blood in 1 L of solution)

• Used to trace blood trails or splatters

• Blood has distinct slow blue glow

Luminol Reaction• Luminol dissolved in a base to deprotonate nitrogens• Luminol reacts with an oxidizing agent

• Removes N2

• Replaces the Nitrogen with Oxygen

• Protons released in the form of blue light (photons) as the molecule falls to the ground state

Testing

• Human gamma-globulines in an immune-hematological assay

• Gamma globulines protect against bacterial and viral infectious diseases

• Assay measures activity of immune-related parts of blood

• X and Y chromosomes quantified using Plexor HY System

• Plexor® HY assay - DNA detection down to 6.4pg 

• Plexor® HY System determines the concentration of total human DNA and male human DNA simultaneously

• Tests for false-negative results that may occur

Methods: Investigation 1

MethodsGroup 1:

• 21 samples

• 100 µL of blood mixed with 200 µL of luminol solution

• Luminol concentrations of 0.50%, 2.50%, and 5.0% p/v 

• Samples incubated and measured at: 1, 4, 6, 8, 12, 24, 48, 72 hours

• Immune-hematological assays for gamma-globuline detection performed on the samples

• DNA extraction from samples and quantified

Methods: Investigation 2

Methods Group 2:

• 21 samples

• Serial dilution of DNA and luminol solution

• 50 to 0.032 ng/µL 

• Luminol concentrations of 0.50%, 2.50% and 5.0% p/v 

• Incubated for 8 months

• Quantitated using real time PCR on a Bio-Rad IQ5 qPCR instrument

Figure 1. DNA quantitation of the Group 1 samples with luminol                 solutions at varying concentrations.

Figure 1• Group 1 - varied shorter incubation time

• Incubation time and luminol concentration did not affect DNA concentration 

Results

DNA amount after incubation of 4 µL of DNA and 4 µL of luminol solution at varying concentrations by eight months.

Table 1•  Group 2 - long constant incubation time for all samples

• DNA amount after 8 month incubation• DNA can be detected even at low solution concentration levels

Results

Conclusion• Luminol impacts the identification of the blood splatter

• Does not impact DNA identification

• After long periods of incubation in the luminol solution, DNA concentration remains unaffected

• Important because luminol should not affect or otherwise impact the identification of DNA profile

Significance to Biology• Biochemistry

• Iron within hemoglobin of red blood cells acts as a catalyst for the luminol solution to begin a redox reaction

• Reaction reduces the luminol and produces photons

• Emits a blue glow

• DNA

• Autosomal DNA: all chromosomes that are unrelated to the X,Y sex chromosomes

• Makes up genetic "fingerprint"

Article Citation

Santos, V.R.D., W.X. Paula, and E. Kalapothakis. "Influence of the Luminol Chemiluminescence Reaction on the Confirmatory Tests for the Detection and Characterization of Bloodstains in Forensic Analysis." Forensic Science International (2009): 196-97. Web.

Influence of the Luminol Chemiluminescence Reaction on the

Confirmatory Tests for the Detection & Characterization of Bloodstains in

Forensic Analysis

Julio Abreu, William Chauvin, Conor McMenamin, John Vitale, Andrew Youngstrom

BIOL 1115Dr. Missy McElligot16 February 2012