Review: Effects of Storage Conditions on Forensic Examinations of Blood Samples and Bloodstains Stored for 20 Years

Emily C. Lennert





blood, bloodstain, leuco-malachite green, anti-human Hb, HBB, actin-beta, ACTB, glyceraldehyde-3-phosphate dehydrogenase, GAPDH, 18S rRNA, 18S, DNA degradation, DNA, STR typing

Article Reviewed

Hara, M.; Nakanishi, H.; Yoneyama, K.; Saito, K.; Takada, A. Effects of storage conditions on forensic examinations of blood samples and bloodstains stored for 20 years. Legal Medicine. 2016, 18, 81-84.


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.


Storage conditions play an important role in the integrity of forensic evidence over time. Previous studies, cited by the author, have indicated that short tandem repeat (STR) alleles may be detected in bloodstains stored at room temperature for over 20 years. The authors of this study sought to investigate the effect of different storage conditions on several blood identification tests, DNA degradation, and STR typing.

Blood samples were obtained from six volunteers; samples contained heparin as an anticoagulant. A portion of the sample was set aside for analysis as whole blood, while another portion was used to prepare bloodstains. Bloodstains were prepared by absorbing the blood onto cotton gauze. Bloodstains were then stored for 20 years at room temperature, 4˚C, -20˚C, and -80˚C. Whole blood samples were stored at -20˚C and -80˚C for 20 years. Whole blood samples were also analyzed when the sample was fresh, i.e. without aging, to serve as controls.

Blood identification tests included leuco-malachite green reagent testing, anti-human Hb immunochromatography, and four mRNA based assays: HBB, actin-beta (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S rRNA (18S). Leuco-malachite green reagent testing gave a positive result for blood if the reagent turned a blue-green color after a 1 min incubation period at room temperature. An OC-Hemocatch S device was used according to manufacturer instructions for anti-human Hb testing by immunochromatography. To perform the mRNA based assays, total RNA was extracted using a RNeasy Mini Kit. To evaluate DNA degradation, DNA was extracted and purified using a EZ1 DNA Investigator Kit according to manufacturer instructions. The ratios of 129 base pair (bp) and 305 bp DNA fragments to 41 bp DNA fragments were calculated to assess DNA degradation. A StepOne-Plus Real-Time PCR System was used to quantify DNA fragments. Finally, for STR typing, an AmpFISTR® Indentifiler™ Plus PCR Amplification Kit was employed to perform STR typing according to manufacturer instructions, using 28 polymerase chain reaction (PCR) cycles. STR fragments were analyzed using an ABI 310 Genetic Analyzer and GeneMapper™ ID software.

Table 1 within the study shows the results for the blood identification tests. All samples, i.e. all aged bloodstains, aged blood, and fresh blood, showed positive results for the leuco-malachite green reagent test as well as for the anti-human Hb immunochromatography. Four mRNA based assays were performed. HBB was undetected in the aged blood samples, but was detected in the aged bloodstains as well as in the fresh blood control. ACTB was undetected in one of six room temperature bloodstains, as well as in all aged blood samples. However, ACTB was detected in the remaining bloodstains stored at room temperature, as well as all bloodstains stored at or below 4˚C and the fresh blood control. Similarly, GAPDH was undetected in one of six room temperature bloodstains, and was undetected in five of six blood samples stored at -20˚C as well as five of six blood samples stored at -80˚C. GAPDH was detected in the rest of the bloodstains stored at room temperature, all bloodstains stored at or below 4˚C, and all fresh blood control samples. 18S was detected in all samples, regardless of type or storage condition. Figure 1 within the study displays the stability of each mRNA. No statistically significant difference in stability was observed between HBB, ACTB, and GAPDH mRNAs for bloodstains stored at -20˚C and -80˚C, indicating that these mRNAs were all stable below 20˚C.

The relationship between storage condition and DNA degradation is displayed in Figure 2 within the study. Based on the 129:41 and 305:41 bp fragment ratios, it was determined that bloodstains stored at room temperature and 4˚C showed significantly high degradation of DNA when compared to fresh blood. Upon examining STR typing, it was determined that one locus was undetected in one bloodstain stored at room temperature, and four loci were undetected in another bloodstain stored at room temperature. In all other blood and bloodstain samples, all loci were detected and minimum degradation was observed.

Scientific Highlights

  • While leuco-malachite green reagent testing and anti-human Hb immunochromatography were unaffected by storage conditions, mRNA assay results were highly dependent on the storage conditions.
  • Samples stored as whole blood produced negative results for most mRNA assays.
  • Samples stored at room temperature and 4˚C showed significant DNA degradation when compared to fresh blood samples.
  • Samples stored at room temperature had undetected loci during STR typing.


In some cases, it is required that evidence must be stored or that aged evidence must be re-examined. In these cases, it is of vital importance that the storage method for the evidence does not damage the evidence.

Potential Conclusions

Blood samples should be stored as bloodstains at below -20˚C to maintain the integrity of the sample.