The largest-ever DNA identification project is underway to name those who perished more than 40 years ago in the Vietnam War. The Vietnamese government is investing about $25 million and an international consortium involving QIAGEN is supporting the Vietnamese scientists to overcome the considerable challenges of the vast project. Skeletal remains are in a bad condition due to the decades that have passed and the tropical climate, but state-of-the-art molecular technologies are ready for this challenge.
In almost every Vietnamese living room there is a small wooden shrine. Sometimes it is decorated with flowers or golden ornaments, sometimes there is not much more to see than framed photographs of deceased loved-ones and a small flickering candle. In each and every case it is a sacred place. Fruits and food are offered on the altar to make the spirits of ancestors feel at home. And the rich scent of incense sticks lingers in the air.
In Vietnam the date of someone’s death is considered to be more important than their birthday. Because only at the end of an individual’s life is it possible to know what a person has achieved. An old proverb says that for the Vietnamese “a country is composed as much by the people who laid its foundations as the living who perpetuate it”.
Only by bearing all this in mind is it possible to grasp the severity of the fact that more than 40 years after the end of the Vietnam War, an estimated 500,000 victims of the conflict are still missing. Their remains lie in anonymous mass graves or undetected in rain forests or fields. Almost every day, construction workers and farmers uncover the skeletal remains of individuals whose families never had any certainty about their fate. And therefore do not know on what date to celebrate and remember.
Now the Vietnamese government is starting a historically unprecedented forensic program to identify the missing ones with the help of DNA analysis and other methods. If everything goes as scheduled, about 70,000 victims will be identified by 2020. This so-called “Project 150” has ambitious goals, but there is no alternative, says Dr. Huyen Tran Thi Thanh, Deputy Director of the DNA Identification Center at the Vietnam Academy of Science and Technology (VAST). “The relatives of the war victims are getting old and the victims’ bones are drastically degrading under the harsh environmental conditions,” explains Dr. Tran. “There’s an urgent need for such a project. Time is running out.”
The researchers, however, face serious logistical and technological challenges. And that is why an international consortium under the leadership of the German biotech company Bioglobe and including the renowned International Commission of Missing Persons (ICMP) and QIAGEN is assisting the Vietnamese scientists in assigning names to the dead and giving certainty and inner peace to the survivors. Before the project even started, the consortium consulted the Vietnamese government, set up an expert network and defined the necessary technology and training protocols.
Finally in spring 2016, a delegation from the Vietnam Institute of Science and Technology visited Europe for three months, and received extensive training in analysis tools and workflow processing. “I feel honored to be part of such an important undertaking,” says Christian Starke, project manager and forensic specialist at QIAGEN, who was deeply involved in planning and networking the project. “It is obvious that everyone involved is highly motivated.”
Over the past decades, Vietnamese society had already tried to identify as many war victims as possible, with numerous organizations searching for the remains of Vietnamese war victims. “Long before ‘Project 150’ was officially approved, our research team had already started to apply DNA analysis to identify individual missing soldiers following family requests,” Dr. Tran says. But only now have the authorities equipped the laboratories with high-throughput technologies capable of supporting a comprehensive project aimed at all victims of the war. In addition, they will start to establish a vast database with reference samples from relatives of the missing persons to match the DNA results from skeletal remains.
The rapid establishment of the reference database is paramount because many of the victims’ direct relatives are already very old and the sample material from the missing is continually degrading. Yet the matching will also require substantial efforts, as on average an estimated three to six reference samples will be required to determine the identity of one missing person. In other words, at least 1.5 million reference profiles need to be generated and stored in the database.
So far, scientists have collected a variety of samples from these relatives – hair, fingernails, saliva or blood. But in order to analyze tens of thousands of samples annually, the processes will have to be highly coordinated and highly standardized, as Christian Starke explains: “Our automation guarantees ease of use and reproducible results, helping the laboratories to save probably the most precious factor in this project, which is time.”
Another major challenge for the Vietnamese scientists is the condition of the sample material from the missing persons itself. “I saw bones that were as soft as peat,” says Prof. Dr. Wolfgang Höppner, founder of Hamburg-based Bioglobe and head coordinator for this project. Since the skeletal remains lie in humid soil, often directly under the surface, they are extremely vulnerable to microbial and plant damage, and are in a state of advanced decomposition. “The time span and extreme conditions mean that the DNA in the bones is significantly degraded and contaminated,” says QIAGEN’s Christian Starke.
Just a couple of years ago, it would have been impossible to extract DNA from such material, but recent innovations have given the investigators new tools to work with. In workshops in Hamburg, at QIAGEN’s headquarters in Hilden and at ICMP facilities in Bosnia, the Vietnamese forensics specialists were trained in how to utilize a state-of the art DNA analysis workflow, which relies mostly on QIAGEN technologies.
First, the bone samples are pulverized by means of the TissueLyser II, which simultaneously grinds multiple biological samples to a fine powder through high-speed shaking in stainless steel grinding jars. Afterwards, the powder is lysed (decalcified and protein digested), and the lysate is put in an EZ1 Advanced XL system for automated DNA extraction. As the samples are in a bad shape, the DNA extraction is of great importance. Finally, QIAGEN’s Investigator 24plex QS Kits are used to generate so-called STR profiles.
These kits cover 24 different non-coding positions in the human DNA – a considerably higher number of markers is used than is otherwise common in forensics. The tests, also used by the US federal agency FBI, thereby increase the value of the comparisons with the profiles of close and even distant relatives. At the same time, the specially designed test using very short DNA sequences raises the probability of being able to generate a profile, even for heavily degraded samples.
Following the extensive training period in Europe, the Vietnamese team will now work with actual bone samples from the victims and ramp up operations. “In the upcoming months, the increased working capacity and human resource quality will help to increase our staff’s effectiveness,” explains Dr. Tran.
The high degree of motivation comes as no surprise to Thomas Parsons, ICMP’s Director of Forensic Science: “Hats off to the Vietnamese government that they are willing to allocate the resources and address this open wound in their society.” Before joining the ICMP, Thomas was Head Scientist of the U.S. Armed Forces DNA Identification Laboratory and advised the U.S. National Institute of Justice on DNA identification in response to the 9/11 attacks in New York City. It is fair to say that nobody understands the complexities and challenges that come with building a large DNA identification lab system better than he does. ICMP experts have helped to identify victims of war, natural catastrophes and major accidents, thus helping people in Europe, Iraq, Africa and South America.
“The Bosnian war killed 40,000 people and we’ve identified just 70 percent after 20 years,” he says. Almost as important as technology and sufficient financial resources are a strategic approach and appropriate communication systems. “There are so many steps involved,” he explains. “First, a pilot study has to be done to get an impression of the DNA material we are dealing with. That has important ramifications for the DNA labs. Not everything works out of the box.”
Anthropologists have to analyze mass graves and put individual bodies back together. Specialists need to develop a system for sample recovery and submission. And the project needs to establish a data workflow to ensure that all the samples are analyzed anonymously. “It’s difficult to get all the different modules to work well together,” Thomas says. But having experienced the investigations on the Balkans, he also knows that all the effort is well worthwhile. “You have to work with the different organizations and groups, educate them about the technological process, get to know their concerns, and empower them,” he says. If everything works well, “a process like this can have an enlightening effect for a country. Especially for a post-conflict society it is important to deal with these questions, which can simmer on for generations, so there is no re-occurrence of the conflict.”
All the parties involved in “Project 150” are aware of the huge task ahead – and there is no guarantee of success in view of the challenging conditions. The first groups of scientists from the Vietnam Academy of Science and Technology have completed their training. Back home, they will train co-workers on the new equipment. As a next step, Thomas Parsons and the ICMP propose a wide-scale systematic pilot study of DNA preservation and a meeting of stake holders to clearly define variables and set out an action plan. Everyone knows this is just the beginning of the end of a generation-old trauma. “But it’s a milestone that we can even think about a project on this scale,” Thomas Parsons says.