Forensic Science International: Genetics Supplement Series 2 (2009) 196–197

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Forensic Science International: Genetics Supplement Series

journal homepage: www.elsevier.com/locate/FSIGSS

Research article

Influence of the luminol chemiluminescence reaction on the confirmatory tests

for the detection and characterization of bloodstains in forensic analysis
V.R.D. Santos a,b,*, W.X. Paula b, E. Kalapothakis a
a
Laboratório de Biotecnologia e Marcadores Moleculares, Departamento de Biologia Geral/Genética, Universidade Federal de Minas Gerais, Brazil
b
Divisão de Laboratório, Instituto de Criminalı´stica de Minas Gerais, Brazil

A R T I C L E I N F O A B S T R A C T

Article history: Preliminary tests for the detection of stains at crime scenes aim to focus the police work making them
Received 28 August 2009 more efficient in the combat of criminality. The application of the luminol chemiluminescence reaction
Accepted 2 September 2009 (3-aminoftalhidrazida) in presumptive tests for the detection of bloodstains is known for more than 40
years in forensic science. This reaction is based on the emission of light through the chemical reaction of
Keywords: luminol mixed with hydrogen peroxide and a hydroxide in the presence of a catalytic molecule (iron
Luminol from the hemoglobin) (Laux [1]).
DNA
This work evaluates the luminol interference and its effect on subsequent serological and DNA
Forensic science
testing. Samples prepared with blood and different concentrations of luminol solution containing
luminol, peroxide of hydrogen and sodium carbonate, were analyzed. Additionally, samples of serial
dilutions of standard DNA mixed with luminol solution were also analyzed. Although presumptive tests
with luminol do not establish the characterization and identification of stains at crime scenes,
preliminary results indicated that it is suitable for the detection of invisible bloodstains for forensic
analysis, with few detrimental effects on the serological tests and subsequent DNA recovery and typing.
ß 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 2. Materials and methods

The physical evidence of violent crimes is constituted by
vestiges left at crime scene such as weapons or fragments of
explosives. However, frequently it is constituted only by invisible
vestiges of fingerprints, footprints, traces of ink and tools, blood,
sperm, saliva and fibers that the offender leaves behind or carry
with him, becoming silent witnesses. Preliminary tests for the
detection of stains at crimes scene attempt to make the police work
more efficient in the crime combat. The application of luminol
chemiluminescence reaction in presumptive tests for the detection
of blood stains is well known for more than 40 years in forensic
science [2,3]. The objective of this work was to verify the influence
of luminol on the confirmatory serological tests based on the
identification of blood through the detection of human gamma-
globuline [4] and DNA quantification and typing.
* Corresponding author at: Divisão de Laboratório, Instituto de Criminalı́stica de
Minas Gerais - Rua Juiz de Fora, 400, Barro Preto, Belo Horizonte, Minas Gerais,
Brazil.
E-mail addresses: valeriarosalina@terra.com.br (V.R.D. Santos),
xavierdepaula@yahoo.com.br (W.X. Paula).
The samples were analyzed and prepared as follows: Group 1:
21 samples of 100 mL of blood mixed with 200 mL of fresh luminol
solution (SL solution) [1] at concentrations of 0.50% p/v, 2.50% p/v
and 5.0% p/v; the samples were identified according to the luminol
concentration and time of incubation, which varied from 30 min to
72 h; Group 2: 21 samples prepared from serial dilutions of
standard DNA from the Plexor HY System (Promega Corporation),
varying from 50 to 0.032 ng/mL were mixed with luminol solution
at the same concentrations described above and incubated for
eight months; the samples were also identified according to the
luminol concentration and time of incubation.
Analysis of the human gamma-globulines was done by
immune-hematological assay using 5 mL of the SL solution,
Coombs’ serum and Controcel sensitivized red cells, according to
the manufacture instructions (Diamed Latin America). Blood DNA
was purified by proteinase K treatment followed by organic
extraction—phenol/chloroform (Sambrook & Maniatis, 1989). DNA
was quantitated by Real Time PCR using Plexor HY System
(Promega) on a Bio-Rad IQ5 qPCR instrument. STR analysis was
performed by DNA amplification using PowerPLex16 System
(Promega) and fragments analyzed by capillary electrophoresis
on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems)
using the GeneMapper ID v3.2 software.

1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.fsigss.2009.09.008
 V.R.D. Santos et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 196–197 197

gamma-globuline detection were performed on the samples of

Group 1 and all of them gave positive results (data not shown). The
DNA extracted from Group 1 samples were quantitated and Fig. 1
shows the results.
The samples from Group 2 that contain serial dilutions of
standard DNA mixed with three different concentrations of the
luminol solution were stored at room temperature for eight
months. Table 1 shows the quantitation results for both autosomal
and Y chromosomes.

4. Discussion

Although the presumptive tests with luminol do not establish

Fig. 1. DNA quantitation of the Group 1 samples. The graphic shows the DNA the characterization and the identification of stains at crime
amount in ng/mL (axis Y) after the incubation of samples with luminol solution at scenes, preliminary results indicated that this test is suitable for
concentrations C1 (0.50% p/v), C2 (2.50% p/v) and C3 (5.0% p/v) (axis X).
the study of potential blood spots in forensic analyses and luminol
does not affect the confirmatory immune hematological tests. The
Table 1
incubation time with luminol solution, as well as the different
Quantitation of the samples from ‘‘Group 2’’.
luminol concentrations did not affect the DNA concentration, as
Sample DNA DNA volume Results Ratio observed on samples from ‘‘Group 1’’: the samples showed an
number quantitation (mL) used for auto/Y average of 3–4 ng/mL after incubation periods varying from 1 h to
(ng/mL) quantification Autosomal Male 72 h (Fig. 1). The incubation time, storage conditions and other
DNA DNA
factors had collaborated for the reduction on DNA recovery from
50C1 25 1 0.28 0.175 1.6 the samples, however those factors did not affect the detection of
50C2 25 1 0.721 0.275 2.6
genetic profiles, once all samples showed enough amount of DNA
50C3 25 1 0.661 0.87 0.8
10C1 5 1 15.8 17 0.5
for microsatellite genotyping of the nuclear DNA.
10C2 5 1 0.116 0.0701 1.7
10C3 5 1 13.9 18.4 0.8 5. Conclusions
2C1 1 1 0.371 0.472 0.8
2C2 1 1 0.0905 0.0592 1.5
2C3 1 1 0.404 0.469 0.9 In the experimental conditions of this study, the results indicate
0.4C1 0.2 1 0.0919 0.114 0.8 that the treatment with luminol did hinder the detection of blood
0.4C2 0.2 1 0.0822 0.109 0.8 on the analyzed samples as indicated through the human gamma-
0.4C3 0.2 1 0.122 0.105 1.2 globuline assays. The amount of DNA recovered after organic
0.08C1 0.04 1 0.0253 0.0124 2
0.08C2 0.04 1 0.0214 0.0307 0.7
extraction was sufficient for microsatellites typing of the nuclear
0.08C3 0.04 1 0.0303 0.0452 0.7 autosomal DNA.
0.016C1 0.008 1 0.0183 0.0159 1.2
0.016C2 0.008 1 0.0125 0.00992 1.3
Conflict of interest statement
0.016C3 0.008 1 0.0139 0.008 1.7
0.0032C1 0.016 1 0.0234 0.0153 1.5
0.0032C2 0.016 1 0.016 0.0896 1.2 None.
0.0032C3 0.016 1 0.00513 0.00529 1

DNA amount in ng/mL after incubation of 4 mL of DNA and 4 mL of the luminol
solution at concentrations C1 (0.50% p/v), C2 (2.50% p/v) and C3 (5.0% p/v) by eight
months; 1 mL of DNA solution was used for the quantitation assays. Samples 10C1
and 10C3 showed high DNA quantitation results when compared with the other
samples.
3. Results
After the incubation time with the three different concentra-
tions of luminol solution, the immune-hematological assays for
References
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