Case summary:

In October 2019, two adult female decedents were processed by the same funeral home in Guilford, Connecticut. According to traditional Jewish funeral practices, the bodies of both women were prepared for burial according to tahara (ritual purification, no embalming) by a chevra kadisha (sacred burial society), wrapped completely head-to-toe in a tachrichim (simple white shroud), and interred in a simple wooden casket (aron). Decedent #1 was emaciated at the time of death due to a long illness; Decedent #2 was reportedly obese. Pallbearers at the funeral of Decedent #1 remarked that her casket seemed exceedingly heavy based on her low body weight at the time of death. A few months later, family members of Decedent #1 visited her gravesite, noticed that the date-of-birth on her headstone was incorrect, and suspected that there may have been an accidental “burial switch” at the cemetery. Consequently, the families of both women convened and petitioned the funeral home to exhume Decedent #1 to investigate the matter. In June 2020, human remains were exhumed for DNA testing to confirm the identity of the woman interred in Decedent #1’s burial plot. A small section from the diaphysis of the exhumed decedent’s right femur was collected for DNA testing, along with family reference samples (buccal swabs) from two of Decedent #1’s biological daughters and the sole biological daughter of Decedent #2. Traditional forensic DNA casework approaches for human skeletal remains are time-consuming and labor-intensive, often taking weeks-to-months to obtain genetic testing results. Rapid DNA technology offers the capability to provide a more timely resolution (< 2 hours processing time) and closure to families on such sensitive issues. Bone samples collected during the exhumation and buccal swab (family) exemplars were processed on the ANDE 6C Rapid DNA System (ANDE Corporation, Waltham, Massachusetts USA) using the FlexPlex® 27 assay (which incorporates the 20 core CODIS loci mandated by the FBI for casework) and the patented I-Chip™ and A-Chip™ (for bone samples and reference swabs, respectively). Kinship analysis was performed using the Claimed Relationships module of ANDE FAIRS™ software (ANDE Corporation).

Introduction

The death of a loved one is a tragedy that is difficult to accept and overcome. When a funeral home or cemetery personnel are suspected of mishandling the burial or gravesite of a deceased person (due to negligence or malfeasance), the family’s heartache and grief are even further amplified. Although formal investigations of alleged “burial switches” via exhumation and DNA testing can provide definitive resolution, traditional (standard) forensic DNA casework methods for decomposed or skeletonized human remains are labor-intensive and time-consuming (which can prolong the family’s anguish and delay closure). Rapid DNA testing platforms offer the potential for a reliable, quick resolution to investigations involving an allegation of mistaken burial in the wrong cemetery plot.

In this case, two adult female decedents were processed by the same funeral home on the same day in October 2019. Decedent #1 was scheduled to be interred at a cemetery in Guilford, Connecticut; the other adult female (Decedent #2) was to be transported to New Jersey for burial. The bodies of both women were prepared according to traditional Jewish burial customs --- i.e., with tahara (ritual purification, no embalming) by a chevra kadisha (sacred burial society), wrapped completely head-to-toe in a tachrichim (simple white shroud), and with ‘funeral stones’ (typically shards of earthenware clay pottery) placed over their eyes. After being wrapped (head-to-toe) in the shroud, the deceased is placed in a simple wooden casket (aron) and there is no “viewing” prior to burial. Decedent #1 barely weighed 100 lbs at the time of her death due to a prolonged battle with cancer; Decedent #2 was reportedly obese. The pallbearers at Decedent #1’s funeral (all of whom were strong males in their mid-20s) commented that the casket seemed excessively heavy, especially given the emaciated state of the decedent at the time of her death (but the family was too grief-stricken at the time to consider any potential mistakes or to inquire with the funeral director). A few months later, upon visiting Decedent #1’s gravesite, the family noticed that the date-of-birth (DOB) on her headstone was incorrect. With some additional investigation, it was discovered that the DOB etched into the headstone above Decedent #1’s burial plot was actually the DOB of Decedent #2. The families of both Decedent #1 and Decedent #2 worked together to request a formal inquisition into the matter, to investigate if the burials were accidentally switched.

Materials and Methods

In June 2020, the remains of an adult female (Decedent #1) interred at a cemetery in Guilford, Connecticut were exhumed for the purpose of DNA testing. The total burial period at the time of exhumation was approximately 9 months. Post-exhumation, height measurements were taken, the tachrichim (white shroud) was unwrapped, and the traditional placement of ‘funeral stones’ (clay pot remnants) atop the eyes of the decedent was confirmed. A portion of the diaphysis of the decedent’s right femur was removed using an oscillating Mopec® 1000 Autopsy Saw and sterile sectioning blade (Mopec, Madison Heights, Michigan USA); the femur section was stored in a sterile 50-ml polypropylene conical tube for storage and transport. Adhered (decomposed) soft tissue was removed from the femur section using a sterile scalpel. The bone was then surface-cleaned via submersion in 10% sodium hypochlorite (NaOCl) with vigorous shaking for 30 seconds, thorough rinsing with molecular grade water, and brief immersion in absolute ethanol (EtOH). After drying overnight (at ambient temperature) in a dead-air hood, the femur section was pulverized using a pre-sterilized hammer and steel mortar-and-pestle; 0.5g of pulverized femur was immersed in ANDE™ Bone Solution (proprietary) with Proteinase K (20 mg/mL); and the sample was incubated overnight to demineralize the bone matrix and facilitate DNA release.

For comparison, familial reference samples (buccal swabbings using sterile cotton swabs) were collected from the following individuals: 1) the two biological daughters of Decedent #1; and 2) the sole biological daughter of Decedent #2. These exemplars were processed separately from the femur samples collected during the exhumation of Decedent #1. All samples were processed on the ANDE 6C Rapid DNA System (ANDE Corporation, Waltham, Massachusetts USA) using the FlexPlex® 27 assay (which incorporates the 20 core CODIS loci mandated by the FBI for casework) and the patented I-Chip™ and A-Chip™ (for bone samples and reference swabs, respectively). Kinship analysis was performed using the Claimed Relationships module of ANDE FAIRS™ software (ANDE Corporation).

Results and Conclusions

Complete (full) DNA profiles were obtained from the exhumed remains (femur), from the buccal swabs of Decedent #1’s two biological daughters (A-B), and from the sole biological daughter (C) of Decedent #2 (Table 1).

Table 1. Overview of autosomal STR typing results for the exhumed remains of Decedent #1 and the putative (P) daughters’ (A,B,C) reference samples (ANDE 6C System, FlexPlex® 27 assay).

Using the genetic typing results shown in Table 1 and the Claimed Relationships module of ANDE FAIRS™ software (ANDE Corporation), match statistics were calculated and the following conclusions were drawn:

• The exhumed remains (Decedent #1) cannot be excluded as being the biological mother of the reference sample donated by putative Biological Daughter (A). The Combined Maternity Index is 719,842,881. The relative chance of Maternity is 99.99999986% as compared to an untested, unrelated woman in the NIST general population.

• The exhumed remains (Decedent #1) cannot be excluded as being the biological mother of the reference sample donated by putative Biological Daughter (B). The Combined Maternity Index (CMI) is 8,214,305,007. The relative chance of Maternity is 99.99999999% as compared to an untested, unrelated woman in the NIST general population.

• The exhumed remains (Decedent #1) are excluded as being the biological mother of the reference sample donated by the putative Biological Daughter (C), due to the absence of an obligate maternal allele at the following tested loci: D3S1358, D2S441, D10S1248, Penta E, D18S51, D6S1043, D7S820, D5S818, D8S1179, SE33, and FGA (see Table 1).

This case study demonstrates proof-of-concept that the ANDE 6C Rapid DNA System can be effectively used to generate DNA profiles from exhumed human remains in alleged “mistaken burial” cemetery investigations. In this case, the remains of two adult females processed by the same funeral home on the same day were actually interred in the correct burial plots; however, the DOB information on their headstones was transposed. The postmortem interval (PMI) for the exhumed remains in this case was approximately 9 months. Future studies should explore PMI limits for this technology, as well investigate buried remains from a broad range of burial environments (e.g., different climates, varied soil pH ranges).

Future Directions for Rapid DNA Technology and Unidentified Human Remains (UHRs)

ANDE Corporation’s "Rapid DNA" platform is revolutionizing forensic DNA testing capabilities in that it allows testing to be performed in the field, as opposed to being restricted to stationary brick-and-mortar laboratories. There are numerous scenarios encountered in forensic casework that may result in mass fatalities, including bombings/explosions, natural disasters, fires, terrorist attacks, war conflicts, aviation crashes, and other mass transit accidents (e.g., high speed passenger trains, subways). Human remains recovered in these situations often are severely damaged, dismembered, fragmented, commingled, in varying states of decomposition, or even skeletonized. Although many national, state, and regional crime laboratories exist throughout the country with extensive expertise and training in forensic DNA identification, these fixed “brick-and-mortar” laboratories are not amenable to field-based operations and testing. Hence, although these laboratories are willing (and competent) partners in disaster victim identification (DVI) efforts, the logistics of coordinating subcontracts and transporting biological samples long distances for genetic testing can be tedious. Additionally, traditional DNA casework methods for skeletal remains are both labor- and time-consuming, increasing the amount of time that families of decedents must wait for a positive identification.

Credit: ANDE

Rapid DNA technology offers a potential solution to meet the high-throughput and onsite testing demand of mass fatality events. The ANDE 6C Rapid DNA System is field-deployable and can generate forensic DNA profiles in less than two hours using its patented I-Chip™ and FlexPlex® 27 assay (which includes the 20 core CODIS loci mandated by the FBI for casework) [1]. The instrument was specifically engineered for field deployment and onsite testing operations. In addition to its relatively small size (dimensions 75cm x 45cm x 60cm) and weight (117lbs), it is operational at altitudes as high as 3048 meters (10,000 feet) and was ruggedized to account for vibration and shock during transportation, meeting the strict requirements of U.S. Military Standard 810G (MIL-STD-810G) [2]. Moreover, both the I-Chip™ and accompanying reagents are stable at room temperature for up to six months [1]. Complementing the ANDE 6C Rapid DNA instrument’s compact size, fast run times, and temperature-stable reagents is kinship analysis software. This software, called FAIRS™, has two secure modules which compare DNA profiles obtained from unidentified victims to direct reference samples or family member exemplars: 1) the Claimed Relationships module, and 2) the Familial Search module. When algorithms embedded in the software determine a match, a formal report containing the decedent’s genetic profile and the associated match statistic calculations are automatically generated for the case file. Hence, this comprehensive human identification system is particularly well-suited for location-based testing at accident sites, in disaster zones, and/or in temporary buildings or tents constructed by Disaster Mortuary Operational Response Teams (DMORTs).

The Department of Homeland Security (DHS) Science and Technology (S&T) Directorate is currently exploring the possibility of implementing Rapid DNA technology in mass fatality responses, especially since it is already being used to support immigration investigations and to combat transnational human trafficking [3-5]. DHS S&T has developed numerous drills and mock disaster victim identification exercises in order to: 1) assess if the instruments can be shipped in an expeditious and efficient manner for disaster response operations, and 2) to identify and mitigate potential logistical challenges that might be encountered during complex deployments. Additionally, the American Society of Crime Laboratory Directors (ASCLD) recently formed a Rapid DNA Disaster Victim Identification (DVI) Subcommittee, the goal of which is to determine best practices and develop policies for mass fatality applications (www.ascld.org). Additionally, although the Scientific Working Group on DNA Analysis Methods (SWGDAM) has not yet developed formal guidelines for Rapid DNA analysis of DVI casework samples, the FBI has recently formed a working group to facilitate discussions on this issue.

Recent high-profile (real world) human identification successes using Rapid DNA technology likely will expedite discussions among the DHS S&T, ASCLD, SWGDAM, and FBI working groups, and may facilitate formal implementation of the technology in mass fatality response efforts. One of the aforementioned high-profile cases involved the 2018 ‘Camp Fire’ in Paradise, California, which claimed the lives of 85 people and is the deadliest wildfire in California history. In collaboration with Butte County’s Sheriff’s Office and the California Department of Justice (DOJ), ANDE Corporation deployed a team to assist with identification efforts. Remains from 69 victims were recovered; however, due to the intensity and duration of the fire, traditional methods of identification (e.g., fingerprints, odontology, surgical hardware) were only feasible for 22 of the decedents. Using a mobile (recreational) vehicle and three ANDE 6C Rapid DNA instruments, a variety of sample types from the victims’ remains (e.g., bone, muscle, brain tissue, liver) and ̴ 300 family reference samples were tested. DNA profiles were obtained from 62 of the 69 victims recovered (89.9% of samples tested). Of the 62 victims for which DNA profiles were successfully generated, 58 victims (93.5%) were able to be positively identified via comparison to exemplars from biological relatives [6-14]. In 2019, less than a year after the wildfires, another tragedy struck in California. Thirty-three passengers and one crew member aboard the dive boat Conception perished when it caught fire off the coast of Santa Cruz Island. All 34 victims were identified within 10 days using ANDE Rapid DNA technology, much faster than the identification effort would have taken using traditional laboratory-based testing [15-16].

In addition to the human remains from the California wildfires and the Conception boat fire that were successfully processed for DNA, the ANDE Rapid DNA platform has been subjected to extensive research with a broad spectrum of biological samples. During developmental validation, a total of 101 samples across seven different DVI-relevant tissue types were processed for accuracy and concordance. Included in the study were 18 bones (femora, humeri, ribs), 3 molar teeth, 24 liver samples, 34 skeletal muscles, 2 brain cortices, 11 lung tissues, and 9 kidney samplings [1]. In another study, ten deceased (donated) human subjects were exposed to the environment (surface deposition) at an approved outdoor research facility, and various tissue samplings were collected at regular time intervals over the course of a 1-year study period. In general, successful DNA typing of soft tissues (brain, muscle, liver) was limited to relatively short postmortem time intervals (1-11 days), attributed to accelerated decomposition during environmental exposure compared to hard tissues. The majority of the bone/tooth samples tested yielded useful DNA profiles from the first day of collection through the end of the 1-year experimental period [17]. Both of these research studies --- as well as the casework performed during the California wildfires and the Conception boat fire --- demonstrate proof-of-concept for DVI applications and support the use of this technology as a way to expedite human identification and family reunification efforts.

Recently, ANDE published a comprehensive developmental validation for the processing of casework and disaster victim identification (DVI) samples using I-Chips™ and the ANDE 6C Rapid DNA Identification system [18]. This technology will undoubtedly have substantial impact on future forensic genetic identification efforts in law enforcement, DVI, military, and homeland security applications.