It all happened so fast. On Father’s Day of 1967 newly appointed Army Staff Sergeant James (Jim) Van Bendegom, just 19 years old, said goodbye to his family and headed off for his first tour of duty in Vietnam. One month later he was gone.

The confirmation that the entire Van Bendegom family sought came not long after, and it was the outcome they all dreaded most. They were shown the photograph of POWs and knew immediately that Jim was NOT one of the prisoners in the photograph. Several POWs infamously released during Operation Homecoming in 1973 were able to confirm that Jim had died of his battle wounds shortly after capture.

As for what happened to his remains, that remained a seemingly unanswerable question.

The waiting was over, so the family thought. It was finally time to grieve, to mourn Jim’s death, and to plan a service to allow them to move on. His brother Mike is still moved to tears when describing the event that took place over 40 years ago, an occasion that felt hollow. “We had nothing but an empty table with a flag. There was no casket, no remains. When the service ended we took that flag down and folded it up for my Mom to keep.”

A precious few details about Jim’s death slowly emerged and helped his family to accept their profound loss. They learned years later that after Jim was carried off the battlefield to a military hospital, a friend and member of his battalion helped care for him in the short time before he died from his gunshot wounds. At one point the Van Bendegoms were contacted by the U.S. government and asked to submit their DNA as reference samples—information that might one day prove valuable if soldiers’ remains were recovered from Vietnam. They complied and mailed off vials containing DNA swabbed from the inside of their cheeks. Time continued to pass, and life went on for the family. Jim’s three brothers raised families of their own, though never straying far from their parents and hometown of Kenosha.

Meanwhile, on the other side of the world, U.S. military initiatives aimed at recovering the remains of Vietnam War casualties were making progress. A collection of human remains, commingled bones believed to contain fragments of American soldiers, were turned over by the Vietnamese government. The collection ended up at the U.S. Defense POW/MIA Accounting Agency (DPAA) Laboratory in Hawaii where they would eventually undergo analysis and DNA testing. DNA technologies had improved in recent years to the point where some of the more challenging samples were now amenable to definitive analyses.

On October 17, 2014, Jim’s mother once again received news that would transform the Van Bendegom family. One of the bone fragments in the mix of remains recovered from Vietnam, a single bone from the radius of an arm, was identified as belonging to Jim. A comparison of the DNA extracted from the bone sample was an unequivocal match to the DNA samples provided by his brothers. Jim was coming home.

Final Rest for a Military Hero

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“The military was very gracious,” recalls Mike, from the moment his family was contacted with the news. A representative met with them to discuss their options, which included the opportunity to bury Jim at Arlington National Cemetery with full military honors. They arranged for his remains to be flown to General Mitchell Airport, the closest commercial airport to the family’s home in Kenosha, and covered the cost of it all, as well as the burial and service. One of the most touching moments of the experience was the reaction by the airline pilot when the plane landed and the casket containing Jim’s remains was unloaded.

“The pilot quietly saluted in front of the plane that was full of people,” says Mike. It was the kind of gesture that meant everything to a family who had endured so much leading up to that moment.

The Van Bendegoms chose to have Jim buried next to his father, who had passed away in 2002, never knowing that his son would one day come home. A lengthy motorcade of military personnel escorted the family along a 30-mile stretch of interstate highway from the airport in Milwaukee to their church in Kenosha. There they were met by an outpouring of community support that included the local chapter of the Veterans of Foreign Wars and many childhood friends of Jim’s.

Army Staff Sergeant James Lee Van Bendegom was finally laid to rest on Veteran’s Day, 2014, in a graveside service with full military honors. A most fitting holiday and day of remembrance, it provided the closure his family had been seeking for nearly 50 years.

The painstaking case of lost and found for the Van Bendegom family is only one of many that is playing out around the world. There may eventually be thousands more. Over fifteen hundred Americans who fought and died in the Vietnam War have yet to be identified; and that is only one war. Government officials estimate there are another 7800+ unidentified lost in the Korean War, and 125+ missing soldiers who fought and died in the Cold War. More than 73,000 service personnel from World War II still remain unidentified today.

The agency responsible for gathering the information and providing a potential means of identifying the remains of American soldiers killed in war is the U.S. government’s Defense POW/MIA Accounting Agency (DPAA). This organization’s mission is to provide the fullest possible accounting for all missing military personnel to their families. As Jack Kull, Policy Advisor, Personnel Accounting Policy Directorate of DPAA, succinctly puts it, “The sheer magnitude of the task is huge. We have people lost in every corner of the globe.”

Yet, Kull and his team are wholly committed to what they do. Speaking at a recent monthly gathering for family members still waiting for the recovery and identification of those lost at war he stated, “It is a privilege to do this work. It is not just a job, it is a mission, and we pledge to do everything humanly possible to find your loved ones.”

This work, this mission, relies on cutting-edge forensic science, and continual advances in the technology have translated into more families finally gaining closure after decades of waiting.

The scientific process begins with the recovery of human remains, typically bones, from a site linked to the loss of missing military personnel. The remains are first sent to one of the DPAA’s two main laboratories at Hickam Air Force Base in Hawaii or Offutt Air Force Base in Nebraska where a range of preliminary analyses are first conducted. Larger remains may provide identifying clues from dental impressions; chest radiographic comparisons may be made if original chest x-rays are available. Those samples deemed workable for DNA analysis are then sent on to the Armed Forces DNA Identification Laboratory, or AFDIL, located at the Dover Air Force Base in Delaware.

The only Department of Defense lab in the United States tasked with performing DNA testing on human remains, AFDIL is a division within the Armed Forces Medical Examiner System. Their work includes not only DNA identification of remains from past wars, known as the past accounting division, but present day military operations as well.

Popular media—through TV, movies and serial crime shows—often portray the process of DNA testing as quick and simple; nearly effortless. Routinely used as evidence in criminal courtrooms, and now used to exonerate convicted criminals, DNA is often the game changer that can throw a case wide open or just as quickly slam the door shut. Forensic scientists use DNA as a critical tool in the quest to link someone to a criminal scene or act. Equally as complex as the loops and whorls revealed by an inked fingertip, the genetic code that is responsible for making each of us unique individuals provides another level of identity, one that is often more accessible.

Yet, the underlying science that makes DNA identification a mainstay in forensic analysis is not quite so simplistic. Nor is the process quick. Suzanne Shunn, a forensic analyst at AFDIL, works every day to identify the remains of American war casualties. She explains that her job requires time and teamwork. The science can be frustratingly slow, as the stakes are priceless when it involves positively identifying samples recovered from long ago. “It’s important to understand how much effort is expended to make sure our results are undisputedly accurate. Each and every case involves many different analysts and requires the coordination of the entire team.”

Forensic DNA Analysis: Tools of the Trade

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So, what does forensic DNA analysis actually entail? And what kinds of technologies underlie the series of steps that together have the power to forever impact families affected by disasters, crimes, and war?

Step one in the process requires extracting DNA from a sample, actually teasing out the deoxyribonucleic acid that encapsulates an individual’s unique genetic code. Some of the more common sources of DNA include blood, a hair follicle, or a swab from inside the cheek. These samples most often prove easy to work with as they can be processed to yield whole cells (a blood cell, hair cell or cheek cell) and the intact DNA can then be isolated from the cells.

When it comes to isolating DNA from human remains recovered from a battlefield, often embedded underground or deep below the ocean’s surface, the DNA has most likely endured trauma. The bone cells trapped within a remain have often been subjected to harsh environmental conditions for an extended period of time. Since DNA is sensitive to changes in temperature, in particular heat, as well as exposure to ultraviolet rays from the sun, bones left exposed to the elements for decades can pose a challenge to the successful extraction of DNA. In general, the fragile coils of DNA that reside inside the nucleus of every cell are subject to degradation simply over the course of time.

The positive identification of Jim Van Bendegom’s remain exemplifies a successful case of particularly challenging forensic DNA analysis. His bone sample was recovered from Vietnam, a region of the world where the soil is highly acidic and thus especially damaging to DNA. The low pH tends to degrade the DNA, requiring scientists to work around this technical wrench using more sensitive technologies.

When war remains first arrive at AFDIL, they are logged in by an Evidence Custodian and then assigned to a DNA Analyst for extraction. The scientific protocol they use for DNA extraction is outlined in the following steps:

The addition of demineralization buffer (step #5), deserves special mention. A significant technological advance was implemented in the year 2006 due to the development of a new and improved version of this buffer. Modifications to the chemical solution served to maximize the amount of DNA isolated from bone cells once they were free of interfering proteins. Before improvement to the buffer, technicians needed approximately 2.5 grams of starting bone material in order to extract a workable amount of DNA. Today, a starting sample of only 0.2 grams of bone are necessary to achieve the same results.

“This meant a twenty-fold reduction in the amount of bone required for testing, thanks to this discovery,” says Dr. Timothy McMahon, Deputy Director of Forensic Services for AFDIL. “This opened up a much larger number of samples submitted to us that we could work with.” In more recent wars such as Vietnam and Korea which involved high impact crashes, explains McMahon, there was much more fragmentation of bone resulting in significantly smaller pieces of remains. AFDIL is proud to note that the majority of forensic labs around the world now use this improved demineralization buffer.

Once a clean DNA sample has been successfully extracted from a bone fragment, AFDIL analysts begin by examining mitochondrial DNA. There are two different types of DNA present in our cells—autosomal or nuclear DNA which resides in the nucleus of a cell, and mitochondrial DNA which is located in the cell’s mitochondria. Mitochondria are often referred to as the “powerhouses” of the cell as their role is to provide energy for various cellular functions. Because there are hundreds to thousands of copies of mitochondrial DNA in an individual cell, versus one single copy of nuclear DNA per cell, there is typically more mitochondrial DNA for analysts to process. This natural abundance increases the chances of obtaining useful DNA information from an unidentified sample.

Another advantage of analyzing mitochondrial DNA is its natural structure—a circular molecule. This circular nature makes it more resistant to exonucleases, or enzymes that naturally chew up and break down DNA. This means there may be enough intact mitochondrial DNA, even in degraded samples, for analysis. Mitochondrial DNA offers the benefits of being an abbreviated, yet equally informative version of the DNA present in a cell’s nucleus; its smaller size and particular structure endow it with a durability and resistance to degradation over time.

And finally, yet another feature of mitochondrial DNA that lends itself to more effective forensic analysis is that it serves as a lineage marker—it is transmitted to subsequent generations, but only through the maternal line. This means that all siblings share the same mitochondrial DNA as their biological mother. In cases of identification where close family members’ DNA samples, such as a parent, child or sibling, are not available, the shared maternal lineage provided through mitochondrial DNA can be extremely valuable. This is often the case for identification of samples that remain unsolved over long periods of time, and the availability of extended family members’ DNA as a reference sample may be the deciding factor.

Forensic DNA analysts examine a particular region of mitochondrial DNA called the control region to begin to try to identify a DNA sample. Only 1,600 base pairs total in size, the control region is roughly ten percent of the overall mitochondrial DNA. Within this section of DNA sequence are two much smaller regions that are highly variable in their sequence, hence they’re called “hypervariable” or HV1 and HV2. The regions arise through random mutation, and because they are punctuated by DNA differences among individuals, they essentially serve as genetic fingerprints.

Once mitochondrial DNA is isolated from a bone fragment, analysts need to amplify or create many more copies of the hypervariable regions to generate an ample supply of that section of DNA. Amplification is achieved through what has become a standard technology in molecular biology, and one that truly revolutionized the field in the early 1990s: the polymerase chain reaction (PCR). PCR gets its name from the critical enzyme that makes this reaction possible, DNA polymerase, and the “chain reaction” that results from a repeating cycle of three essential steps. In the first step the DNA sample is subjected to a high temperature which promotes DNA to uncoil and the two strands of the double helix to separate, or denature, into two separate single strands.

The temperature in the reaction is then lowered, and two selective and short DNA sequences called primers are introduced to the reaction. Once added, the primers seek out and find their complementary match on the target DNA sequence. They naturally bind to this complementary region due to the properties of nucleotides that support the matching up of nucleotide partners A and T (adenine and thymine) and C and G (cytosine and guanine).

The choice of primers introduced into a DNA sample is critical to amplification and analysis of the target region of interest. In the case of samples from the remains of war casualties, the mitochondrial DNA’s HV regions are what may hold the key to a positive identification; therefore, DNA primers that bracket that region along the mitochondrial genome are used for PCR. According to McMahon, the success rate for amplifying the HV regions within a sample’s mitochondrial sequence is about 90 percent, if the sample has not been chemically modified through preservation efforts in an earlier stage of remains recovery.

In the final step of a PCR cycle, Taq polymerase, the enzyme that drives the reaction, goes to work once the temperature is again raised to a value optimal for Taq’s activity. The polymerase binds to the primed sections of DNA and adds single nucleotides to synthesize a matching second strand of DNA based on the nucleotide sequence it is reading on the first stand. The extension from the primer as it moves along the strand completes the first cycle of PCR.

The final product after one round of PCR is an exact duplicate of the original section of DNA. And because PCR is as an exponential process, continuing this three-step cycle through multiple rounds serves to create an impressive number of copies. After as few as 30 cycles there are as many as a billion copies of the original target DNA—a bountiful supply of DNA that aids subsequent steps of the analysis. The pattern of nucleotides within a sample’s mitochondrial DNA HV region is next “read” like words on a page through a molecular method called sequencing. That sequence is then compared to a published reference sequence that serves as the standard for forensic DNA analysis. In forensic-speak, these differences are referred to as an individual’s “mito-type” or mito-profile; this information is what is reported back to the DPAA in the hopes of matching it to a reference sample already in their database.

More Tools, More Success

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Mitochondrial DNA analysis is not the only tool used by forensic DNA scientists when attempting to make an identification. If those results do not provide conclusive information, they may then turn to nuclear, or autosomal DNA. Unlike mitochondrial DNA, autosomal DNA is unique to each individual, which makes it such a powerful and effective approach for forensic science. Because there is much greater variability among individuals’ autosomal DNA it offers a higher level of discrimination when comparing differences and looking to make a conclusive match to another DNA sample. McMahon points out that a limitation of using mitochondrial DNA alone is that Caucasians all share a 7.7 percent similarity within the control region of mitochondrial DNA. This commonality can interfere with a conclusive match if no other analyses are conducted.

Forensic DNA analysis of autosomal DNA involves targeting a number of specific and well defined locations (or loci) found throughout the 22 pairs of autosomal chromosomes and sex determining chromosomes (X and Y). Each locus consists of a very short nucleotide sequence, usually a mere 2-7 base pairs in length, called a short tandem repeat (STR). Because STRs do not encode genetic information that is essential for life, they naturally vary among individuals in their number of repeats.

Similar to the approach used for amplifying the HV regions of mitochondrial DNA, specific DNA primers designed to seek out and find STRs within an autosomal DNA sample are added to the PCR mix. The autosomal DNA sample is amplified using PCR technology, and the specific number of repeats at each STR can be identified through visualization of the different sized DNA fragments.

STR analyses are typically described like this: if a particular STR in an individual is deemed “18/19 heterozygous” that means that the STR at that locus on one chromosome is repeated 18 times, and the same STR on the matching chromosome is repeated 19 times. The numerical designations for each locus are combined and comprise an individual’s STR profile, similar to a mito-type. Everyone shares half of their STR profile numerical values with their biological mother and half with their biological father.

Over the course of several decades of forensic DNA research efforts, scientists have defined a group of particular STRs that best serve as a set of standards for identification of individuals. In the case of criminal forensics, 13 different STRs sprinkled throughout the chromosomes are referred to, along with a supporting database, as the Combined Indexing System (CODIS for short). Established in 1998, CODIS is used by U.S. criminal labs and has been popularized by its frequent mention in crime shows and popular media. McMahon makes the important distinction that AFDIL utilizes CODIS STRs for their identification procedures in addition to several other defined STR loci. He also emphasizes that the Armed Forces Medical Examiner System database is completely separate from the CODIS database used in criminal forensics.

Another effective and related tool AFDIL analysts may rely on to help solve a case of identification is Y chromosomal DNA analysis using Y-chromosome specific STRs. All three DNA approaches proved to be necessary in the case of positively identifying Jim Van Bendegom’s remains; a valuable combination when used together, they offer the highest degree of discrimination. The availability of three different forensic DNA technologies has aided analysts in cases whereby testing one or two types of a sample's DNA did not yield a conclusive analysis.

“It’s very important to have many different tools in your tool belt—or different ways to attack a human identification challenge because of the various ages and conditions of samples,” says McMahon.

Once DNA testing has revealed an unidentified sample’s mito-type and/or autosomal and Y chromosome STR profiles, the information can then be compared with reference samples present in the AFDIL database. A final report that includes all these instructive DNA differences is sent back out to the DPAA lab, and based on their review they may recommend that DNA analysts search against a specific database of family reference samples. By the end, these searches “kick out the potential results—no matches, one possible match, or many possible matches.” And, depending on the results, additional analyses may be run as forensic scientists hone in on the information needed to make a conclusive identification.

Critical to AFDIL’s success, yet challenging to its speed, are the standards the agency must uphold. The Defense Science Board, established by the Department of Defense, mandates that all DNA identification procedures be immune to errors, and thus every analysis must be conducted in duplicate. So, per protocol, two separate DNA extractions are obtained from the same sample and the data analysis is done by two independent analysts. Every case goes through multiple levels of review; in order for a report to be sent back out to the DPAA those two results must match exactly.

Major advances in forensic DNA analysis are serving to impact the certainty of identification. As McMahon says, “We’re constantly trying to improve how we do things and evaluating any technologies that are three to five years out.”

One major technological breakthrough, Next Generation Sequencing (NGS), was recently incorporated into AFDIL’s toolbox. So innovative, AFDIL is currently the only forensics lab in the U.S. using this technology. As Lieutenant Colonel Alice Briones, Deputy Chief Medical Examiner and doctor of forensic pathology, explains, NGS is able to sequence the smallest and most degraded forensic samples, typically those that cannot be analyzed through conventional mitochondrial DNA analysis or autosomal DNA STR analysis. Although the process takes longer to run and is extremely labor intensive, the results it can provide are pivotal to the most challenging cases of forensic DNA analysis.

Briones realizes that NGS is more than just a complex DNA technology; it represents a means with which to bring closure to families who may have never received it. “The work we do brings the science, the past, and the present communities all together. We use it to bring our service members home,” she says. “I feel that as a scientist, what more honorable mission can we do?”

As Director of the Department of Defense DNA Registry, Briones leads the group of AFDIL DNA analysts, and she understands that the seemingly anonymous vials of DNA material do mean something. Everyone is driven, and feels appreciative, to be able to possibly attach an actual name to a sample. “When we’re able to provide the information that results in a positive identification you know that you’re providing something personal—that someone is coming home. And there are no words to adequately describe what that is like.”

Late on the night of November 30, 1950—the coldest reported that year—Air Force Captain Claude Albert Batty, Jr took off from the U.S. base in Iwakuni, Japan heading to his target area in North Korea. Flying with his co-pilot and navigator, the 731st Bomber Squadron, they aimed their B-26C “Invader” bomber into enemy territory. What was only their fourth night mission in the Korean War proved to be their last.

Batty was an experienced Air Force pilot. Following training experience during WWII alongside his older brother and fellow pilot, Jack, the pair went overseas to Japan and Korea as members of the U.S. occupying forces. Batty had recently retired from the Air Force and joined the reserves when he was called back into service. Stationed at George Air Force Base in Riverside, California at the outbreak of the Korean War, he was forced to leave behind his wife and 10-year-old daughter. Yet the Missouri native never questioned his call to duty.

Night intruder missions were particularly perilous. Flying just 5 to 10 feet above the ground, in complete darkness, only the best pilots were assigned this job. Courage, gutsiness and a certain willingness to take a risk were necessary; Batty trained for just this kind of mission and never questioned his ability.

Perhaps that is why it was so difficult for his squadron, and his family, to accept the news that the plane disappeared. Perhaps that is why members of his family, his sister leading the charge, have continued to push for information and answers about what happened since the day they learned he went down.

Dona Reeves-Marquardt, Batty’s sister, was 18 years old when her brother was reported missing. She recalls the “world shaking and everyone going to pieces” the day their family received notification by telegram.

“We went to work immediately after Claude was declared MIA,” explains Dona. “My other brother contacted Claude’s group based in Japan and was able to obtain a general map of the area in North Korea they were ordered to target.”

This investigative effort is pushing up on nearly 70 years, and the progress that’s been made has required the help of many, including the families of Batty’s two co-flyers on the doomed mission as well as DPAA officials. They still don’t know exactly what happened to the plane, only that once it flew through the “radio silence zone” mission contact was lost—the radio remained silent. Batty was never heard from again, and there were no Americans who witnessed the plane’s fate.

“My mother was in contact with the mothers of the other two boys. But that connection dissolved over time as they all grew older,” says Dona. “When she died at age 97 she still thought that Claude would one day come home. She was still waiting.”

If they could pinpoint the location of the downed bomber they might then be able to recover remains. So Dona, with the help and support of her husband Lewis, continue to press on. Little by little they are reconstructing the circumstances of her brother’s disappearance. They’ve learned more details about his target area in western North Korea, north of the Chong'chong River up to the Yalu, which served as the border with China. A more recent trip to the Maxwell Air Force Base in Birmingham, Alabama yielded a valuable clue when they uncovered a “morning report” filed by the 732nd Squadron which flew the following morning, December 1, 1950. In the report was a description of evidence that a downed plane may have placed a call for help on the ground; included were the longitude and latitude of that sign. Dona provided those geographical quadrants to their military contact, another piece in the excruciatingly difficult puzzle they continue to try and solve.

At a recent Family Meeting Update organized by the DPAA in Austin, Texas where Dona and her husband now live, she met once again with the service casualty officer assigned to her brother’s case. Dona has attended many of these meetings and remains vigilant in her own investigation into where her brother’s plane may have gone down. “We go to these meetings, because we simply can’t give up,” Dona says. “But time is of the essence—all the witnesses who might have seen something or know something are disappearing.”

Integral to the positive identification of Claude’s remains, if ever recovered, are DNA reference samples. So, as part of their effort to do everything possible to bring Claude home, Dona submitted blood samples, as did several other members of their family. Those samples, along with Claude’s DNA extracted from a hair comb the family sent to the DPAA, now sit in a freezer in the agency’s laboratory and are ready for analysis should the need arise.