A one-tube method for simultaneous extraction of hemoglobin and DNA from a minute bloodstain

An established approach to forensic bloodstain examinations is to first confirm that the suspected bloodstain is human blood and then to perform tests to determine individual identity. Although the confirmation of human blood step may be skipped in order to secure sufficient sample size for individual identification when the amount of bloodstain available for analysis is very limited, the validity of such partial examinations is likely to be challenged in court trials. Therefore, in order to perform complete examination of minute bloodstains, we developed a one-tube method in which hemoglobin and DNA were simultaneously extracted for use in confirmation of human blood and individual identification, respectively. Blood was diluted in 5 steps to produce 1- to 500-fold dilutions, and 1-μl aliquots were applied to tips of cotton-wool swabs. Prepared swabs were held at room temperature for periods up to 28 days. All swabs emitted light in a darkened room after being sprayed with luminol. Using 1.5-ml microcentrifuge tubes, the sprayed swabs were soaked in 100 μl of blood extraction buffer, one of the reagents in a DNA extraction kit designated for forensic samples. Following incubation, a 3-μl aliquot of the resulting blood extraction fluid (bloodstain dissolved in blood extraction buffer) was subjected to immunoblotting against human hemoglobin A in order to confirm the presence of human blood. The remaining blood extraction fluid was used for DNA extraction. After the quantification of DNA, TH01, F13A01 and amelogenin loci, and mtDNA were amplified by PCR. All samples tested positive for human blood by immunoblotting. The amount of DNA extracted using this method was comparable with that of other extraction protocols. TH01 and the amelogenin loci gave similar results in that both loci were amplified in up to 5- or 20-fold dilutions of blood that had been held for 7 days and in 1- or 5-fold dilutions held for 28 days. F13A01 was less successful in that the locus was amplified in 1- or 5-fold dilutions held for 7 days, while amplification was only possible for 40% of samples at 1-fold dilution (no dilution) held for 28 days. In contrast, amplification was possible for mtDNA at concentrations as low as 500-fold dilution held for 28 days. Thus, the one-tube method allowed both confirmation of human blood and individual identification from minute bloodstains on single swabs ; however, only mtDNA can be amplified in aged and/or diluted bloodstains.