Shannon Ryan, an Armed Forces DNA Identification Laboratory analyst, sands a vertebrae bone, Feb. 20, 2019. Credit: Air Force Staff Sgt. Nicole Leidholm
Since DNA was first used in forensic science in the late 1980s, it has opened doors for criminal investigators and genealogists to solve cases that have been cold for decades. For the U.S. military, it's been essential in carrying out the age-old motto, "no one left behind."
Today, the identification of fallen and missing service members comes down to DNA. The Armed Forces DNA Identification Laboratory, which is part of the Defense Health Agency's Armed Forces Medical Examiner System, is the DOD's only human remains testing laboratory. It performs DNA tests on service members who've died in current operations, as well as those who have been missing for decades. The lab works in tandem with the Armed Forces Repository of Specimen Samples for the Identification of Remains, which maintains millions of blood samples from folks who've served over the past 32 years.
Next-Gen Methods Help to Clear Cold Cases
The DNA Identification Lab — known as AFDIL — has two main missions, the largest of which is past accounting. This section's nearly 100 analysts support the Defense POW/MIA Accounting Agency by providing the DNA tests that support the identification of remains of service members who died in previous conflicts such as World War II, Korea and Vietnam.
The process begins out in the field. DPAA-trained anthropologists and archaeologists are sent to locations across the world to find the remains of service members where they were last reported to be.
"It could be on the side of a mountain. It could be in a rice paddy. They do all of that recovery, then the remains go back to the DPAA labs," explained Dr. Tim McMahon, AFMES' DNA Operations director. "They will then start to sort those using anthropological methods."
The samples those experts procure are then sent to AFDIL, where analysts use an extensive set of tools to process them. One of those pieces of technology is called next-generation sequencing.
For a long time, McMahon said the lab wasn't able to test unknown service members from World War II and Korea whose bodies had been chemically treated during the embalming process because those chemicals didn't fit with conventional testing methods. But now, thanks to advancements in next-generation sequencing generated by AFDIL, the lab is able to get results from chemically treated, highly degraded samples.
"[A lot of] those bones have been in the environment for 80 years," McMahon said. "When we extract that bone, we get bacterial DNA, fungal DNA, parasitic DNA and the very degraded human DNA."
"A lot of the samples we receive have been exposed to a lot of really nasty environmental conditions. Sometimes they're covered in jet fuel. Sometimes they've been burned, buried, or lost in salt water," explained Katelin Hollowell, a DNA analyst for the past accounting mission. "All of those things factor into how much DNA is present in the sample and can be recovered."
With next-gen sequencing, analysts take the damaged DNA and bind it with human mitochondrial DNA-RNA, which are ribonucleic acid probes that have a metal molecule bond to them. Then, using magnets, the laboratory can pull the damaged human DNA/mtDNA-RNA probe away from the non-human DNA. The mtDNA-RNA probes are used like bait to perform a process called enrichment.
"These probes are relatively human-specific, so they will bind predominantly to human [mitochondrial] DNA," McMahon explained. "It allows us to enrich — or pull — that human DNA out of a vast pool of bacterial, fungal and parasitic DNA that is commingled with our sample."
McMahon said when they started the next-gen sequencing in 2016, they were able to do four samples a month with a 24% success rate. Now, they're processing more than 100 samples a month with a 65% success rate.
"We're still the only laboratory in the world that can do this," the director said, although he made it clear that the lab is happy to share its cutting-edge methods with others across the country and world.
"We publish everything. We provide our results in our standard operating procedures if people ask for them," McMahon said. "There's no need for anyone to reinvent the wheel. The same work that we do for our fallen military members, if we can assist the local jurisdictions in returning these unknown human remains or cold case-type scenarios to their loved ones, we want to get that stuff out there."
Once scientists have been able to process a readable DNA sample from the remains, they'll compare it to DNA from the families of missing persons. McMahon said the lab uses three genetic tests on DNA — mitochondrial DNA tests, which trace maternal ancestry; autosomal short tandem repeat tests, which can verify family connections (mother, father, brother or sister); and Y chromosome short tandem repeat tests, which trace paternal lineage from male offspring.
This means that the tests scientists can run will depend on the family member who donated his or her DNA for comparison. For example, if a sister of a service member donated her DNA, it would only get tested twice. If a service member's brother donated, his DNA would be tested three times.
"Because they had the same mother, [that brother] has the same mitochondrial DNA as the missing service member," McMahon explained. "Because they shared the mother and father, we can use that nuclear or autosomal short tandem repeat to determine that they're siblings. And then, because he shared the same father, that brother has the same Y chromosome as the missing service member. So, a brother, we'd have to test three times."
Crucial For Comparison: Familial DNA References
How, exactly, do experts get DNA from family of missing service members? Those samples can be acquired one of two ways. The first is through genealogical searches, which are conducted by service casualty office representatives to find family members of missing persons. Once family members are identified, those representatives send them a kit to do a cheek swab to donate their DNA. The families then forward those samples to the DNA Lab for analysis.
The second way samples are collected is through DPAA-hosted regional family member updates, which are held a few times a year for families of missing service members.
"The family member updates are where family members of our missing heroes get briefings from their service casualty officers, DPAA representatives and AFDIL," McMahon said. "I interact with the families by giving a DNA update, and then we will actually collect references in person from eligible family members."
The samples are then entered into a family reference database. McMahon said the database currently maintains DNA from relatives of 92% of the original 8,157 service members missing from Korea and from relatives of 86% of the original 2,641 Vietnam-era missing service members. For World War II, he said, it's a little more complex.
"If you take the original 73,678 World War II missing, AFDIL has family reference samples on file for 17% of the missing service members," McMahon said. But he clarified that they have a higher percentage of family references for specific battle sites. "At the Battle of Tarawa, there are 486 service members still missing in action. For those 486, there are active recoveries going on in Tarawa, and I have an 88% coverage of references."
For service branch representatives, getting to know the families of the missing is part of the job.
"I've talked to these families a lot over the years, so I know a lot of them," said Allen Cronin, the past conflicts branch chief for Air Force Mortuary Affairs Operations, which leads the effort to identify service members for the Air Force. When a missing service member is positively identified, representatives such as Cronin notify the families and work with them on their wishes for their loved one's burial. They also travel with DPAA and DNA Lab experts to the families' homes to present the details of how experts came to their conclusion.
"There are a lot of questions that some [family members] have, and I can't answer them. I'm not a scientist – I don't do DNA," said Cronin of his role as a service casualty representative. "So, I ask the director next door [at the AFMES DNA Lab] if I can take the person who made the identification with me. Then, sitting there with the family, I don't have to say, ‘Well, I'll go back and see if I can get the answer.' They're going to get the answer right there."
According to the DPAA, in fiscal year 2023, the agency recovered the remains of 127 service members: 88 from World War II, 35 from Korea and four from Vietnam. Nearly 81,000 American service members remain missing.
Identifying Our Current Fallen
The DNA Identification Lab's second mission is current day operations, which supports AFMES forensic pathology investigations division work in identifying service members and civilians who die in current theaters of operation, during training or on land under federal exclusive jurisdiction.
For AFMES, any case that comes up after 1991 is considered a current day operation. That's when DNA began to be more commonly used to identify people in the U.S. It's also the year the first Gulf War occurred. There have been no missing service members since.
All military personnel and deployable contractors since 1991 have a DNA blood reference card on file with the Armed Forces Repository of Specimen Samples for the Identification of Remains. McMahon said they log about 200,000 new cards a year and have more than 9 million cards on file.
"They're used for the identification of that service member if they're killed in a current theater of operation or training accident," McMahon said. However, because the cards are considered a medical record and are maintained for 50 years from the date of collection, they can be used otherwise, as well.
"For example, if we have a retired service member who passes away and the local jurisdiction can't do fingerprint or dental [identification], and there are no living relatives to provide a DNA reference, AFDIL may be able to assist in the best interest of the service member," McMahon continued. "If their crime lab develops a DNA profile from those unknown human remains and sends it to me, I can pull the card. I will process it here to make the comparison. If they match, I will issue them what we call a ‘believe to be' report with a statistic. But we do not release the DNA profile information."
Analysts say the current-day samples are fresher and better quality than most of the DNA samples tested in past accounting. They're more like those tested in a crime lab.
"They're usually easier to process and get a quick turnaround time of ID, which is what the families want," said Miranda Frady, a DNA analyst for current day operations. "It's streamlined. We have a process that works for us, and we try to get those out as quickly as possible to the medical examiner."
Frady, who has been at the DNA lab for a decade, said the work is incredibly rewarding.
"These loved ones have given the ultimate sacrifice, so it's very important for me to be able to bring that information back to their families," she said. "I play a small part in helping to provide some closure for them."
Current day analysts also help with peacetime losses, such as the 1952 crash of an Air Force C-124 on Mount Gannett, Alaska. That joint mission is known as Operation Colony Glacier and involves a team of service members and civilians who travel to the wreckage every June to conduct search and rescue operations.
"We've been doing recoveries there since 2012," McMahon said. "Our [current day] DNA section has assisted the medical examiners with identifying 47 of the 52 missing."
"We also help military law enforcement agencies, other federal agencies and military hospitals," Frady explained. "In the event of a mass fatality or something like that, we can provide DNA testing."
Quality Control is Key
While Frady, Hollowell and their colleagues work on DNA samples, other analysts, such as Ashley Doran-Roth, do behind-the-scenes work in quality control to make sure the lab's instruments and reagents – the chemicals and kits needed to test samples — are working properly so successful identifications can continue.
"If something breaks down, we have to go and fix it," Doran-Roth said. "We've got to make the reagents, label them, deliver the to the labs and track everything."
She said the day-to-day for QC analysts often changes based on what's going on in the world and what types of samples are being tested the most.
"For example, when 9/11 happened, [the lab] helped a lot with identifications there. So, we have to … figure out, ‘OK, we have more of this type of sample. We need to do these kinds of reagents,'" she explained.
The overall mission at the DNA Lab is some of the most important work that can be done for service members and their families – bringing closure when it's needed.
"We all work together — I think that's the important thing," Doran-Roth said. "Even though we're in different sections, we're all intermixed, and we all help each other to do this common mission, which is to bring home our service members."
Republished courtesy of DOD