Technical Note

Hemaglow™

Hemaglow™ is a commercially available luminol preparation which is reported to produce luminescence with blood, produce fewer false positives, and remain effective for at least two months if stored properly. The manufacturers sent a sample to our department for testing for validation. The scope of this lecture will be to detail the comparisons made between conventionally prepared luminol in an aqueous solution and Hemaglow™.

According to the Merck Index, Luminol is a compound that reacts with the copper, iron peroxides and cyanide. Blood contains both iron and peroxides, which when combined with luminol, react to produce luminescence that is visible in low light. As luminol reacts with substances not specific to blood, luminol solutions should not be used as a "presumptive test" for blood. All luminol solutions can and do produce numerous false positive reactions. Opinions stating that a stain was blood because when he was sprayed with a luminol solution produced luminescence should be avoided!

The initial experimentation at our laboratory involved spraying known blood samples with two different solutions (an aqueous luminol prepared with hydrogen peroxide and Hemaglow™). When initially using the Hemaglow™ an odor consistent with isopropyl alcohol could be detected. Subsequent instrumental analysis confirmed the presence of alcohol. The Michigan State Police Crime Laboratory, as well as a few other laboratories around the U.S. still use conventional serology for screening and eliminations before or instead of doing DNA analysis. The alcohol in the Hemaglow solution lyses or destroys the red blood cells preventing any further serological analysis. The forensic laboratory in your area should be contacted to determine if their serologists still do any form of conventional serology before using Hemaglow™.

Blood samples sprayed with aqueous luminol preparations had been tested and it has been determine that they do not interfere with PCR DNA analysis. However, no data was available for Hemaglow. Megan Clement from LabCorp, Inc, a DNA laboratory in Research Laboratory Park, North Carolina, graciously accepted several blood samples that we had treated with Hemaglow and offered to attempt DNA testing. She confirmed several weeks later that on the whole blood sample that we treated with Hemaglow, she was able to extract DNA without difficulty. However, on the dilute blood sample we submitted, she was unable to extract any DNA.

Sensitivity testing was done between fresh and two week old aqueous luminol solutions and Hemaglow™. Serial dilutions of blood were made up from 1/10 to 1/100,000 on a strip of filter paper. A fresh luminol solution (prepared just before experimentation) composed of .1 gram of luminol, 5.0 grams of sodium carbonate Na2CO3 and 90 milliliters of distilled water combined with an equal amount of 3% hydrogen peroxide (H2O2) was made. The comparison results are show below:

Aqueous Luminol:
1/10 dilution strong reaction
1/100 dilution weaker reaction
1/500 dilution weak reaction
1/1000 dilution very weak reaction

Hemaglow™:
1/10 dilution strong reaction
1/100 dilution weaker reaction
1/500 dilution weak reaction
1/1000 dilution no reaction

A second comparison was made with between a 2 week old luminol solution combined with equal amount of a fresh preparation of 3% hydrogen peroxide (H2O2) and a different bottle of Hemaglow™. The results of the comparison are shown here:

Aqueous Luminol:
1/10 dilution strong reaction
1/100 dilution weaker reaction
1/500 dilution weak reaction
1/100 dilution no reaction

Hemaglow™:
1/10 dilution strong reaction
1/100 dilution weaker reaction
1/500 dilution extremely weak reaction
1/1000 dilution no reaction

Again, as with the previous comparisons, Hemaglow™ reactions produced sustained luminescence that didn’t require re-spraying. The aqueous luminol required several re-sprays of the reagent onto the sample to sustain the same level of luminescence.

A side note about Hemaglow™: One of our lab scientists received a pink nightgown with no visible stains. The nightgown was possibly material in a homicide investigation and allegedly had been washed by the suspect. The nightgown was sprayed with Hemaglow™ and the several luminescent areas resulted and were photographed. The sample was then put away for several weeks. The nightgown was removed from the envelope and several red brown stains were now visible on the nylon material. The serologist on the case suggested that alcohol in the Hemaglow™ fixed the blood. This caused the lysed red brown cells in the stain to become fixed on the material.

Separate comparisons were made to demonstrate false positives. The manufacturers state that Hemaglow™ produces fewer false positives than an aqueous luminol solution. Numerous substances were tested with both solutions. The results of the comparisons are listed below:

+ strong luminescence on entire substance
+- slight dot luminescence on certain areas of substance
- no luminescence produced

Conclusions:

• Hemaglow™ is not as sensitive to dilute amounts of blood as luminol. (10 fold loss of sensitivity)
• Hemaglow™ only requires mixing the pre-made luminol solution with a small bottle of activator before using. (little preparation time)
• Hemaglow™ does not adversely affect DNA testing on whole blood samples.
• Hemaglow™ should not be used if the forensic lab to which you submit evidence still uses conventional serological techniques to test blood samples.
• Hemaglow™ produces as strong of a reaction to whole blood as that of luminol, even after the non-activated solution has been sitting for 1 year.
• In actual field use of Hemaglow™, no false positives developed after tracking a bleeding suspect to a residence one block away from the crime scene. At the residence, Hemaglow™ produced strong reactions to dripped blood which was left on the back stairs, even after the blood was diluted with rain.
• Hemaglow™ has a place, as do many other reagents, in the crime scene “kit” of forensic laboratory. Testing and experience should guide the analyst in the choice of the proper reagent.

Note: Caution should be used in interpreting any luminescent reaction produced with either an aqueous luminol solution or with Hemaglow™. One should “practice, practice, practice” with the reagent and “test, test, and re-test” before using any luminol solution, including Hemaglow™, on any forensic samples!!!

Comments about luminol:

Dried plant samples do not produce false positive reactions with luminol or HemaGlow™.

Acknowledgements:

I would like to thank Tani Watkins, John Lucey, Kyle Hoskins and Bill Lugten. These individuals are analysts at the Bridgeport Laboratory who, without their assistance, this project could not have been completed. I would also like to thank Megan Clement from LabCorp, Inc. for doing the DNA extractions on the Hemaglow™ samples.

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