THE SURVIVABILITY OF HUMAN SCENT AFTER EXPOSURE TO NINHYDRIN.

1. THE SURVIVABILITY OF HUMAN SCENT AFTER EXPOSURE TO NINHYDRIN. Kevin Chow* (Lisa M. Harvey, Ph.D.), Victor Valley College, Dept. of Biology, 18422 Bear Valley Rd., Victorville, CA 92395

2. Introduction Bloodhounds have been used for centuries to help humans in their endeavors to find missing persons, run-away slaves, marauding Indians or fleeing felons.1 Traditionally, the handler would collect an article the person had recently touched, such as a piece of clothing from a missing person or a knife used to commit a murder. This article would be presented to the dog, which after taking a good sniff, would match the scent on the article to the scent on the ground and follow the trail to the perpetrator. As more and more police organizations apply the techniques of forensic science to crime scene analysis, items found at crime scenes have taken on greater significance. For instance, a toothbrush can provide DNA, a shirt may contain hair or fibers, and fingerprints may be left behind on a weapon. Evidence is now carefully handled because of the potential evidentiary value it may contain. One important piece of evidence that may be left behind at a crime scene is a finger or palm print. These prints are formed when the sweat of a person is left behind on a surface. If the individual’s sweat print is left on a porous surface, ninhydrin can be applied in order to “lift” the print. Ninhydrin is an organic compound that will react with α amino group within a protein and form a purple color known as Ruhemann’s purple.2 Although Ninhydrin has been used for a number of years to “lift” or develop prints left by a person’s sweat, there have been no previous studies to demonstrate whether this organic compound may contaminate human scent. If the Ninhydrin were able to destroy or contaminate human scent then the scent could not be used to locate an individual, or worse, the scent may be altered and the wrong person may be identified by the dog and incarcerated. The purpose of the present study was to look at the survivability of human scent after being saturated with Ninhydrin and stored for a prolonged period.

3. Methods Bloodhounds The present study was utilized five well-trained bloodhounds. The bloodhounds were similarly qualified using the methodology described by William Tolhurst.3 The Tolhurst method employs a “hide and go seek” type of trailing scenario. The dogs were taught to associate the scent on an article with that of an individual. Eventually, the dogs learned to discriminate between the scent on the article and multiple scents are laid down on the ground in order to find an individual. The age of the dogs at the time of the study ranged from 2.5 to 9 years with an average age of 4.4 years. Positive and Negative Trails Each bloodhound had been previously trained to identify a positive verses a negative trail. During the testing, the handlers were asked to judge whether their dog presented them with a positive or a negative trail and whether the final identification was correct or wrong. The final identification or “tag” was distinguished by the handler, as each individual dog may present their tag in various ways. For example, one dog may jump on the person they are identifying, while another dog may sit in front of the person, yet another may place a paw on the individual. A positive trail was accomplished when the bloodhound was able to match the smell of an individual on an article or scent pad with a trail laid by that same person. A trail was considered negative after the scent of an individual on an article or scent pad was presented to the bloodhound, and the dog was not able to find the scent of the person from the article or pad anywhere on the ground in the dog’s general vicinity. The scent on the articles and scent pads that were presented to the dogs for a negative was collected from an individual who was known to have never been in the area in which the dog would trail. If a negative is correctly performed by the dog there will be no trail. Instead the dog will typically stand in place or circle the immediate locale briefly and then stop, thereby letting the handler know there is no trail and the scent is a negative. During the current study, if any dog trailed off of the negative scent, that dog was later eliminated from the data for that trial.

4. Scent Collection Scent was collected by having every individual press their full-palm firmly onto a white letter size envelops. Each person was then asked to fold the envelop in half and sealed in a Ziploc bag. The first Ziploc bag was placed into a second Ziploc bag by a researcher and sealed shut. The Ziploc bags were put into an individual brown paper bag, which was stapled shut for storage. The bags containing the scent pads were marked with shapes, in lieu of names, in order to carry out a double blind study. The bags were stored at room temperature for approximately 48 hours before ninhydrin application. Scent Contamination During the contamination with ninhydrin he envelops were handled by a crime scene technician who was not present on the days that the dogs trailed and had never been to the sight previously. The envelops were removed from the brown bags and both Ziploc bags and hung in a fume hood. Ninhydrin was sprayed on both sides of the envelops until they were saturated (Ninhydrin Spray, Lynn Peavey Co.). They were left to development and dry until the purple palm prints were exposed. The envelops were placed back into the Ziploc bags and stapled such in the brown paper bags again. The bags were stored at room temperature for approximately 4 weeks prior to presenting them to the dogs for trailing. Day of experiment On the actual days of trailing, three groups of trail layers were formed. Each group contained three people for a total of nine trail layers. All trails were walked using a “spoke-wheel” pattern. The spoke-wheel pattern was formed by three trail layers walking side-by-side to the middle of the field (approximately 100 yards) and then splitting off in three different directions. One individual went to the left, one to the right and the last individual remained in the middle. Each of the trail layers walked approximately 100 yards to the edge of the field and then hid behind a tree, bush or side of a building.

6. Once the trail layers were in place the handler/bloodhound pairs were brought to the field and given a brown paper bag containing an envelop in a Ziploc bag. The Ziploc bag contained an envelop with either the trail layer’s scent or a control scent. The control scent was that of a person who was never present at the sight of the trails and sprayed with ninhydrin. The handler presented the dog with the envelop and then commanded the bloodhound to trail. If the bloodhound did not going anywhere, it was a negative trail and the trail was then terminated according to the handler. If the handler indicated the dog was trailing it was considered a positive trail. The handler was asked to decide whether his/her dog performed a positive or negative trail, and if a positive trail was done whether the dog made a correct identification or not. Weather The weather during all seven trial periods was very consistent. During the first set of trails the temperature ranged between 65F and 75F. The wind speed was between 0-3mph in a northwesterly direction and humidity was 5%. On the second day of trails the temperature ranged from 68F to 76F with wind speeds up to 5mph. The humidity reached a high of 10%. The weather condition during the third trial was very similar with temperatures ranging from 64F in the early morning to 72F during midmorning. The wind blew up to 8mph in a northwesterly direction and humidity levels rose to 12%. At the start of the fourth set of trails the temperature was 60F and increased to 72F by the last trail. The wind speeds ranged between 3 and 5 miles per hour with humidity up to 10%. The fifth trial had an average temperature of 65F during the trails and a maximum wind speed of 6mph. The humidity peaked during the trails at 13%. Statistical Analysis The dog’s trails were scored as “Correct” if the dog presented a positive trail and made an accurate identification. The dog’s trails were considered “Wrong” when the handler indicated the dog presented a negative trail, when in fact it should have been positive since scent was present on either the pad or article and the person who touched the pad or article was also hidden nearby. The trail was also considered “Wrong” if the dog trailed, but did not make a correct identification at the end of the trail. A goodness of fit test was used to demonstrate that the dogs performed better than chance. A probability of p<0.05 was considered statistically significant. The Wilcoxon signed rank test was used to test for differences between experimental groups.

7. Results The performance of each dog was assessed using a goodness of fit test. The results indicate that all five dogs were consistent in their abilities to trail people after smelling either an article or a scent pad. Each dog ran no less than three and no more than nine trails per trial. Overall, the bloodhounds performed better than chance. Out of 114 trails using ninhydrin saturated envelops, the bloodhounds were able to trail and correctly identify 102 people. There was no significant difference in the ability of each dog to correctly identify the trail layer (Wilcoxon sign ranks test, p<0.01). The combine total correct finds for dogs was 89.5% (random score = 50%; χ2 = 12.9, df = 4, p<0.05) Dog 1 was able to correctly trail and identify the hidden persons 91.3% of the time utilizing envelops sprayed with ninhydrin and stored for at least four weeks. Dog 2 correctly identified the trail layers 95.6% of the time with the ninhydrin saturated envelops. After sniffing the Ninhydrin envelops, dog 3 was able to trail and identify the correct person 87.0% of the time. After smelling the all of the Ninhydrin soaked envelops it was presented with the fourth bloodhound used in this study identified 81.6% of its trail layers. The fifth bloodhound accomplished a 91.3% accuracy rate sniffing the envelops. (Figure 1) Dog 1 was a well-trained bloodhound that had been utilized on a number of criminal cases and missing person cases for multiple police agencies. Dog 2 was also a well-trained bloodhound that had been utilized by multiple police agencies. At the time of the experiment dog 2 was one year older than dog 1, but had approximately the same hours of experience and training. Dog 3 had a great degree of training, but had never run a search and rescue or police case prior the day of the trails. Dog 4 had been utilized by several police agencies over a nine year period. Dog 5 was the youngest dog participating in the experiment. Dog 5 had been utilized somewhat by local law enforcement agencies, but not nearly as much as the other dogs.

10. Discussion The results of the present study demonstrate that there is no significant difference between the bloodhound’s ability to trail and identify the correct person if the scent was previously saturated with Ninhydrin spray. Even though the envelopes were sprayed with Ninhydrin and stored for a prolonged period, the scent was not affected and the dogs were still able to trail. It would appear that scent can still be collected, stored and used in order to locate a missing person or an unknown criminal, even if the item has been previously sprayed with Ninhydrin in order to lift fingerprints or palm prints. Over 25 years ago, Lewis Thomas suggested: if human’s odortype is genetically controlled, then one should be able to train dogs to cross-match humans for organ transplants.4 Other experimenters have also suggested that scent may be genetically controlled and dogs, therefore, should not be able to distinguish between the scents of monozygotic twins.5 Several studies have been carried out in order to examine the difference in odortypes in both animals and humans.6-8 These studies have attempted to ascertain whether there is a correlation between an individual’s odortype and their genetically derived MHC, which is used for determination of organ histocompatibility. MHC demonstrates considerable polymorphism, and is the main contender of an individual’s genetic scent odortype.9,10 Studies have established that MHC is used as a chemosensory signal in order to identify an individual by his or her phenotype. MHC odortypes have not only been well documented in mice and rats, but also in humans.9,11,12 Studies have also demonstrated that various animals, as well as humans, can smell and recognize their own kin, and this recognition is most likely determined by the MHC genes.12,13 Changing a single base in the genetic code of MHC is thought to be able to alter an individual’s MHC odortype.7The slight alteration, and the extreme polymorphism of MHC, may account for the ability of the bloodhound to differentiate between individuals in a population. The more changes in base pairs, the greater the alteration in odortype, and the easier it is for the dog to scent discriminate.

11. The question then is: “What is the dog actually smelling as it runs along sniffing the ground?” Studies to date have put forward two potential hypotheses. The first hypothesis suggests that an individual’s MHC reflects the immune response against one’s bacterial flora on the skin, urinary tract and gut. Therefore, a person’s unique MHC would correspond to one’s own distinctive bacterial flora, and that flora would not change over time.7 A study on germ-free mice have determined they can still be distinguished by their odortype, thereby suggesting that one’s unique odor is not based on bacterial flora.14 A second suggested hypothesis states that an individual’s unique MHC antigens carry volatile compounds that can then be transported to the urine, sweat and/or saliva.15 This genetically specific “cocktail” of volatile compounds is released from degraded MHC onto the body’s surface and given off as scent that can be detected by the bloodhound. In contrast to these hypotheses, an earlier study suggests that an individual does not have one unique scent, but that a person’s scent varies depending upon where on the body the scent was collected.16 For instance, scent would be different if it was collected in the crook of the elbow verses collected from the palm of the hand. Current research on odortypes and MHC would be directly opposed to these findings since an individual’s MHC would not be unique in different body parts. Also, in the same research, a single bloodhound was used, and this dog was able to perform better than chance. Our research would suggest that bloodhounds can identify a person by their individual odor, and that odor is based mainly on genetics, which is uniform throughout nucleated cells. During the present experiment the question proposed was whether or not the chemical components of ninhydrin destroy parts of scent that deals with MHC. Ninhydrin is an organic compound commonly known for development of fingerprints in law enforcement. Ninhydrin reacts with protein and binds free amino acids and compounds within amino groups.17 Since MHC is thought to be one of the compounds contributing to human odortype, it may then react with ninhydrin in order to yield the purple product. The actual reaction that occurs when using ninhydrin is the binding of α-amino acids with hydrindantin molecules, a molecule in equilibrium with ninhydrin and diketohydrindole which forms a purple compound commonly known as Ruhenmann’s purple. It has an absorption maximum at 570nm and can be detected by a spectrophotometer.17

12. The rate-determining step in the ninhydrin reaction appears to involve a nucleophilic-type displacement of an OH group of ninhydrin hydrate by a nonprotonated amino group. The reaction of R-amino acids with ninhydrin entails two molecules of ninhydrin for each molecule of amino acid to form Ruhemann’s purple. For the R-amino acids, both polar and steric parameters are involved during the nucleophilic displacement of an OH group of ninhydrin by an NH2 group in the first step of the reaction. It may be assumed that decarboxylation is not the rate-determining step because decarboxylation would be expected by is unimolecular and not subject to steric hindrance.17The products of the reaction of R-amino acids with ninhydrin are CO2, an aldehyde and Ruhemann’s purple. Since the present study was able to demonstrate that an individual’s odortype was not destroyed after spraying with ninhydrin it would appear that human scent is not particularly made up of proteins and/or amino acids. Although MHC appears to play an integral role in the production or “mixing” of the volatile components that make up a human’s scent, it does not appear to be part of what the dog’s are actually smelling. The study shows the forensic importance that ninhydrin does not mask human scent proving that scent from fingerprint analysis can be used without doubt as evidence. Our findings demonstrate that there is no significant difference to the bloodhounds trailing results even when the scent is covered by ninhydrin. Without doubt, scent that is saturated with ninhydrin may still be used as viable evidence in bloodhound trails. Conclusion The present study demonstrates that human scent is still viable for bloodhound use even after the porous object has been saturated with ninhydrin and stored for a prolonged period of time. This study also demonstrated that there is no significant difference in the bloodhound’s ability to trail and accurately identify the correct person when the scent has been saturated with ninhydrin spray.

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