“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 pbilous@ewu.edu