Scientists from ARP Sciences may encounter cases as old as 140 years past death. When so much time has passed, samples are generally fragmented and environmentally challenged. Direct references for the cases are rarely available, so it’s usually necessary to rely on mitochondrial DNA (mtDNA) analysis. mtDNA sequencing provides a high level of success compared to other testing technologies – in fact, its success rate is over 90 percent, much higher than other sequencing techniques like short tandem repeat on the Y-chromosome (Y-STR) and autosomal short tandem repeat (auSTR) testing, which have a success rate just under 50 percent. Why such a significant difference? Nuclear testing relies on the single copy of the nuclear genome, which degrades with age to the point where it can no longer deliver reliable results. Each mitochondrion in the cell has its own mtDNA, meaning an increased likelihood that sequencing will be able to extract usable information.

Mitochondria don’t always hold the answers, though. Sometimes, the laboratory receives small, unidentifiable bone fragments. The first question, of course, is: are they human remains? To find out, the lab uses 12S rRNA gene testing. Since the implementation of this testing protocol, 45 percent of samples tested were found to be non-human – helping investigators work out which ones need continued investigation.

Samples at the laboratory vary from the highly challenging to the quite ordinary. Routine samples may include human remains and reference samples. The former are usually bones, teeth, tissue, hair, dentures, envelopes and clothing (with body fluid stains like saliva and blood extracted from the latter two). Reference samples include bloodstain cards and buccal swabs, from both subjects and family members.

Bloodstain cards became standard in the early 1990s, shortly after Operation Desert Storm. At that point, the US Department of Defense began collecting blood specimens from military service personnel. A quality control process for extracting DNA from the cards, which are solely used to identify remains, had to be developed and implemented. The DNA must be collected in such a way that its integrity is maintained and the test results are meaningful and legally defensible – so how do they do it? Samples must be labeled and stored separately in appropriate containers, and personnel must change their tools and personal protective equipment between each collection. Despite the rigorous protocols, though, not every forensic sample leads to a new identification; samples might have degraded, remains commingled, there might not be appropriate references, and it’s possible that a submitted sample may not be of human origin.

But even with these caveats, advancements in forensic science have come so far so fast that there may never be another truly unknown soldier. In 1972, an Air Force pilot was shot down in Vietnam. The remains of this Vietnam War Unknown were laid to rest in the Tomb of the Unknown Soldier at Arlington National Cemetery. This would once have been the end of the soldier’s story – but in 1998, his remains were exhumed. With mtDNA testing, AFMES was able to identify the remains of First Lieutenant Michael J. Blassie, United States Air Force. The crypt of the Vietnam Unknown is now vacant.

Robin Stombler is President of Auburn Health Strategies, Arlington, USA.