Highly sensitive HLA-DNA typing from formalin-fixed and paraffin-embedded tissue samples

Nucleotide sequences have been determined for more than 1700 different alleles at the core of the human leukocyte antigen (HLA) system. The highly polymorphic character of these genes affects adaptive immune response and is also useful for forensic applications. HLA typing from formalin-fixed and paraffin-embedded tissue provides abundant useful information for both clinical settings and forensic investigations. This study, which investigated the potential use of DNA from formalin-fixed and paraffin-embedded tissue samples in an HLA PCR sequence-specific primer and probe (SPP) system, showed that tissue fixed in formalin for less than 3 days and embedded in paraffin can serve as a useful source of DNA for PCR-SPP typing kits.     

Allelic structure and distribution of two STR loci, D8S580 and D22S442, in the Japanese population

The allelic frequency and structural characteristics of two STR loci D8S580 and D22S442 were investigated using blood samples from 143 unrelated healthy Japanese individuals. Thirty-eight alleles in D8S580 locus and 13 alleles in D22S442 locus were identified. The discrimination power, heterozygosity, and the polymorphic information content of those loci displayed high values (0.98, 0.88, and 0.87 in D8S580 and 0.97, 0.86 and 0.85 in D22S442), and their frequency distributions met Hardy-Weinberg equilibrium expectations. The allelic pattern of D8S580 was complex and differentiated into three groups (group I: alleles 184-194bp; group II: alleles 203-223, 235, 239, 243, 252 and 255bp; group III: alleles 227-286bp). Most of their alleles contained five categories of repeat units (A: aaaag; B: aaag; C: aagg; D: caag; E: agaa). On the other hand, D22S442 contained only two types of repeat units (A: agga; B: aggg). The present study, hence, proves that both D8S580 and D22S442 are highly polymorphic and represent stable genetic markers applicable to forensic investigations.     

The structure, frequency, and forensic application of the STR locus D16S543 in the Japanese population

D16S543 is a complex STR locus consisting of five types of repeat units. The frequency distribution and genetic characteristics of this locus in Japanese were investigated using blood samples from 124 unrelated Japanese and 15 families. Alleles were detected using denatured polyacrylamide gels followed by automated analysis on an ABI 373 sequencer using Genescan software 672. Twenty-one alleles were identified, ranging in size from 281 to 489 bp. An allelic ladder containing the 21 alleles was constructed and used as a typing standard. The repeat unit arrays allowed the 21 alleles to be classified into three distinct groups, including alleles 1 to 7 in group I, alleles 8 to 14 in group II, and alleles 15 to 22 in group III. The alleles in group II were characterized by the insertion of one repeat unit of CAGG, one of AAAG, and three of AAGG, while the group III alleles differed from those of groups I and II by the insertion of a total of 32 repeat units ranging in 5 types. Within each group, the alleles differed from each other only in one 5' side tetranucleotide AAGG. The power of discrimination (Pd) and the estimated heterozygosity were calculated to be 0.989 and 0.934, respectively. Typing of this locus was successfully applied in four old forensic materials. The study presented herein demonstrates that D16S543 is a highly polymorphic and applicable locus in Japanese.     

Estimation of the age of human bloodstains by electron paramagnetic resonance spectroscopy: long-term controlled experiment on the effects of environmental factors

In this study, we examined the efficacy and limitations of electron paramagnetic resonance (EPR) for estimating the age of human bloodstains. At 77K, human bloodstains give four striking EPR signals in the g=6.2 (g6), 4.3 (g4), 2.27 (H) and 2.005 (R) regions due to ferric high-spin, ferric non-heme, ferric low-spin and free radical species, respectively. We found that plotting double logarithms of the EPR intensity ratio of H/g4 versus days past bleeding gave a linear correlation up to 432 days with an error range within 25% of the actual number of days under controlled conditions. However, environmental factors such as differences of absorbent, light exposure and fluctuations of storage temperature affected the changes of these EPR-active compounds, which result in misestimation of the time since bleeding occurred. Therefore, one should take such factors into account in estimating the period since bleeding by this method.     

Estimation of postmortem interval from hypoxic inducible levels of vascular endothelial growth factor

Estimation of the postmortem interval (PMI) is one of the most important tasks in forensic medicine. Five autopsy organ tissues such as brain, lungs, heart, liver, and kidneys were taken at the time of forensic autopsy from 19 known PMI cases with a range of postmortem intervals ranging from 1 to 120 h (the mean was 25.81 h), and the time-course of vascular endothelial growth factor (VEGF) expression was measured. The human hepatoma-derived Hep 3B cell line was used as a control. The levels of VEGF increased linearly with the PMI up to 20 h in lung (r = 0.95 and in kidney (r = 0.89), and up to 15 h PMI in liver (r = 0.88). The VEGF levels fell after 24 h PMI, and then remained stable. In brain, the levels of VEGF started to increase after 24 h PMI and increased linearly with PMI up to 40 h in brain (r = 0.94) and then begin to fall. In heart, there was no clear correlation between the PMI and VEGF level. Some variations occurred in selected cases, such as the infant and asphyxial deaths. In conclusion, measurement of hypoxia-inducible levels of VEGF in various body organs appears to be a useful method of estimating the PMI up to 24 h in forensic medicine and pathophysiology. This method is also probably applicable in ischaemia in clinical and basic medicine.     

Allele frequencies for 15 STR loci in Tibetan populations from Nepal

Samples from 105 unrelated healthy Sherpa in Namche Bazaar and 111 unrelated non-Sherpa in Kathmandu valley from Nepal were used to obtain allele frequency data for 15 short tandem repeat (STR) loci (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX and vWA) included in the AmpFLSTR Identifiler kit. No deviations from Hardy-Weinberg equilibrium were observed, but only after applying a Bonferroni correction in the case of D5S818 in the Sherpa population and D7S820 in the Kathmandu population. Genetic parameters of forensic interest were calculated and genetic differentiation between the two populations tested.