“Blood-Enhancement Reagents, Luminol, Bluestar®, Fluorescein, and Hemascein™ :
a quantitative comparison of properties essential for crime scene investigations”
Peter Bilous, Marion Fossum, Cassandra Hallmark
Department of Chemistry & Biochemistry
Eastern Washington University Cheney, Washington, USA 99004
Crime Scenes: Latent (non-visible) bloodstains are
detected using light-emitting blood enhancement reagents
Importance: To visualize bloodstain patterns for
crime scene reconstruction To locate bloodstains for DNA analysis
Detecting Latent Bloodstains
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Chemiluminescent Reagents The haem group of hemoglobin acts as a
catalyst which accelerates the hydrogen peroxide (H2O2) mediated oxidation of chemiluminescent compounds
This reaction results in light emission
www.bluestar-forensic.com
Luminol: Grodsky - 1951
Luminol, sodium carbonate, and sodium perborate
Weber - 1966 Luminol, sodium hydroxide, and
hydrogen peroxide Bluestar®: Blum - 2000
Bluestar® is a luminol-based commercial product
Both Bluestar® and H2O2 are provided as tablets for uniformity & convenience
Luminol and Bluestar®
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www.bluestar-forensic.com
Fluorescent Reagents The haem group of hemoglobin acts as a
catalyst which accelerates the hydrogen peroxide mediated oxidation of fluorescent compounds
An external light source of 415 - 480 nm is used to excite the oxidized fluorescent compound, resulting in the emission of yellow-green light
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Fluorescent Reagents Fluorescein: Cheeseman (1995) –
evaluated its potential for crime scene investigations
Hemascein™: Hemascein ™ (2009/10) is a
fluorescein-based commercial product which offers uniformity & convenience
www.abacusdiagnostics.com
Howard & Nessan J For. Ident. 60:682 (2010)
Painted wall + Fluorescein
1. To establish optimal conditions for testing dilute liquid blood samples using a fluorometer
2. To compare the light emission characteristics of Luminol**, Bluestar®, Fluorescein, and Hemascein™
3. To determine and compare the limits of detection/sensitivity of the four reagents
4. To determine and compare the stability of the four reagents
** Washington State Patrol (WSP) Crime lab formulation (2010)
Objectives
Serial dilutions of canine blood were prepared using de-ionized(DI) water
Chemiluminescent/fluorescent “working solutions” were mixed with diluted blood samples and the light emissions quantified using a BioRad fluorometer Optical Filters for Luminol & Bluestar®
460 ± 5 nm emission filter Optical Filters for Fluorescein & Hemascein™
460 ± 5 nm excitation filter 520 ± 5 nm emission filter
Materials & Methods
BioRad VersaFluor
Figure 1 Chemiluminescent Reagents: Light Emission Characteristics
0
1000
2000
3000
4000
0 60 120 180 240 300
RFU
Time (Seconds)
1:10 000 Blood dilution
Bluestar
WSP-Luminol
Visual limit of detection ~100 RFU
Table 1 Chemiluminescent Reagents: Limits of Detection/Sensitivity
Blood Dilution Bluestar* WSP-Luminol*
1:1 000 10 370 16 860
1:10 000 3 700 3 090
1:50 000 770 420
1:100 000 230 290
* Maximum light emission values (RFU) Visual limit of detection ~ 100 RFU
Figure 2 Chemiluminescent Reagents: Stability
0
5000
10000
15000
20000
25000
0 30 60 90 120 150 180
RFU
Time (Seconds)
1:1 000 Blood Dilution
WSP-Luminol
Bluestar
• Tests performed 49 days after preparation of reagent • Reagents stored in the dark at 4ºC
Figure 3 Fluorescent Reagents: Light Emission
0
4000
8000
12000
16000
20000
0 60 120 180 240 300
RFU
Time (Seconds)
1:800 000 Blood Dilution
Hemascein
Fluorescein
Visual limit of detection ~ 150 RFU
Table 2 Fluorescent Reagents: Limits of Detection/Sensitivity
Blood Dilution Fluorescein* Hemascein*
1:80 000 16 000 20 000
1:800 000 2 110 8 050
1:8 000 000 540 610
1:80 000 000 ND 180
• Light emission values (RFU) at 30 sec • ND = Not detected • Visual limit of detection ~ 150 RFU
Figure 4 Hemascein: Effect of Reagent Concentration
• Hemascein working solution as per kit instructions = 100% • Dilutions of Hemascein prepared with DI water • Blood and H2O2 concentrations were constant • Decrease in light intensity with dilution of Hemascein • Importance of proper working solution preparation
0
3000
6000
9000
12000
15000
0 60 120 180 240 300
RFU
Time (Seconds)
1:320 000 Blood Dilution
90 %
75 %
Figure 5 Hemascein: Effect of H2O2 Concentration
0
4000
8000
12000
16000
20000
0 60 120 180 240 300
RFU
Time (Seconds)
1:320 000 Blood Dilution
A = 0.1% v/v H2O2 B = 0.3% v/v H2O2
A
B
Figure 6 Fluorescent Reagents: Stability of Working Solution
0
2000
4000
6000
8000
10000
0 30 60 90 120 150 180
RFU
Time (Seconds)
1:1 000 000 Blood Dilution
Hemascein
Fluorescein
• Tests performed 51 days after preparation of reagents • Reagents stored in the dark at 4ºC
Summary: WSP-Luminol & Bluestar®
WSP-Luminol and Bluestar® have comparable light emission characteristics
WSP-Luminol and Bluestar® have similar limits of blood detection, less than 1 in a 100 000
Only WSP-Luminol retained activity after 49 days of storage at 4ºC
Our data indicates that the WSP-Luminol formulation is a possible alternative to other Luminol formulations
A study is in progress to determine the impact, if any, of the WSP-Luminol formulation on DNA integrity
Summary: Fluorescein & Hemascein™
Hemascein™ exhibited stronger light emission signals than that of Fluorescein, and had a lowest limit of blood detection , ~1:80 million
Maximum light emissions achieved with Hemascein™ working solution at the recommended strength
Maximum light emissions achieved using lower H2O2 concentration
Summary: Fluorescein & Hemascein™ continued Only Hemascein™ retained activity after
51 days of storage at 4ºC Hemascein’s™ low limit of detection,
long shelf-life, and ease of preparation make it an excellent option for the detection of trace quantities of blood
A study is in progress to apply the findings of this study to the detection of latent bloodstains
Acknowledgements
EWU Department of Chemistry & Biochemistry, Forensic Science Students:
Marion Fossum Cassandra Hallmark Kelsey Shears
Dr. W. Holleman Abacus Diagnostics
Questions ?
Peter Bilous, PhD Director, Forensic Science Program
Department of Chemistry & Biochemistry Eastern Washington University
Cheney, Washington, USA 99004 [email protected]