Review: STR Analysis Following Latent Blood Detection by Luminol, Fluorescein, and BlueStar

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Emily C. Lennert

 

Category

Chemistry

Keywords

DNA, short tandem repeat, STR, profile, luminol, chemiluminescence, fluorescein, fluorescence, BlueStar, latent, blood, bloodstain

Article Reviewed

  1. Jakovich, C. A. STR analysis following latent blood detection by luminol, fluorescein, and BlueStar. Journal of Forensic Identification. 2015, 65(4), 693-698.

Additional References

STR Analysis. http://nij.gov/journals/267/pages/extending-str.aspx (accessed August 29, 2016).

Disclaimer

The opinions expressed in this review are an interpretation of the research presented in the article. These opinions are those of the summation author and do not necessarily represent the position of the University of Central Florida or of the authors of the original article.

For more information on STR analysis, refer to Review: Next generation sequencing and its applications in forensic genetics.

Summary

Luminol and fluorescein are common methods of latent blood detection at a crime scene. However, each method has limitations. Luminol requires total darkness to visualize latent blood, and the period of chemiluminescence (i.e. when compounds illuminate due to a chemical reaction) is relatively short. Fluorescein may be used in partial darkness and the fluorescence lasts longer compared to luminol; however, the method requires more time and preparation material compared to luminol. BlueStar is a newer method that the manufacturers claim has a long-lasting reaction and is easier to use than luminol and fluorescein which makes it a superior choice. While studies have been conducted to determine whether luminol and fluorescein interfere with short tandem repeat (STR) DNA analysis, no studies have been published regarding the effect of BlueStar on STR analysis. This study evaluated the three latent blood detection methods to determine if BlueStar interferes with STR analysis, as well as to compare the three methods to determine whether a significant difference in effect on STR analysis existed.

Blood was obtained from a known volunteer for use in this study. Blood was applied to several objects prior to transfer to carpet samples. First, blood was applied to the sole of a shoe using a sponge; the shoe was pressed to butcher paper until blood was faintly visible, then pressed evenly onto a piece of carpet. Next, blood was applied to a golf club and machete, which were wiped with a cloth and rinsed with water prior to being pressed onto the carpet samples. A drop of blood was then applied to the volunteer’s hands, which were rubbed together and pressed onto the carpet samples. Carpets were then cut and labeled “luminol”, “fluorescein”, or “BlueStar” and allowed to dry for at least 5 days.

After drying, each sample’s respective reagent was applied. Luminol and BlueStar were prepared and used according to manufacturer instructions. Fluorescein was prepared and used following the San Diego Sheriff’s Crime Laboratory’s protocol. In a darkroom, the respective reagent was sprayed over the sample until the appropriate reaction was observed. Luminol and BlueStar were observed with the “naked eye”, while fluorescein required the use of orange safety goggles and a specialized alternative light source to observe a reaction. For this test the Omnichrome forensic light source was used and set to 450 nm. After reagent application, samples were allowed to dry for 24 h prior to DNA analysis.

Each carpet was swabbed in three areas for each reagent sample, for a total of nine areas. An unused swab was used as a negative control, and a cutting of a known blood stain was used as a positive control. A blank of each reagent was also prepared. Swabs were placed in separate 1.5 mL tubes and digested with 500 L digest buffer and 15 L of 10 mg/mL protein kinase K for 2 hours at 56C. After digestion, DNA was extracted according to San Diego Sheriff’s Crime Laboratory protocol, which called for DNA purification by organic extraction and ultrafiltration. DNA was then quantified, and human DNA was detected in all nine samples. Following extraction and quantification, DNA was amplified using AmpFISTR Profiler Plus and AmpFISTR COfiler PCR Amplification Kits. Samples were then prepared for capillary electrophoresis analysis and subsequently typed by ABI Prism 310 Genetic Analyzer.

Full profiles were obtained for all samples, regardless of reagent, with the exception of one sample which showed small quantities of DNA during quantitation. Due to low DNA yield, a full profile could not be obtained for that one sample. The authors concluded that, since full profiles were obtained regardless of reagent, there did not appear to be any significant difference in the effects of luminol, fluorescein, and BlueStar on STR analysis.

The authors noted some limitations to this study. Namely, the study did not evaluate the sensitivity of each reagent for latent blood detection. Additionally, the study only examined carpet samples and did not examine a full range of possible substrates that may be encountered, such as linoleum or wood.

Scientific Highlights

  • Luminol, fluorescein, and BlueStar were compared for their potential effects on STR analysis.
  • The results confirmed previous studies that have indicated no impact of luminol or fluorescein on STR analysis.
  • BlueStar did not appear to affect STR analysis, with full profiles being obtained from samples treated with BlueStar.

Relevance

Presumptive tests are often used to detect latent blood at a crime scene. It is crucial that these tests be easy to use, effective, and not impact subsequent DNA analysis.

Potential Conclusions

BlueStar may be a viable option for latent blood detection, with no impact on STR analysis, and may therefore provide an alternative to luminol and fluorescein.