Pitfalls in Evidence Sampling

DNA fingerprinting or DNA profiling (as it is now known) was first developed by Alec Jeffreys in 1985 who found that in the human genome, some regions contained DNA sequences that were repeated over and over again, next to each other. He also discovered that the number of repeated unit could differ from individual to individual allowing human identity testing. Since that time, DNA typing methods has been commonly used in criminal cases (to identify a suspect or a victim or to absolve an innocent individual) as well as in the identification of missing persons or in paternity testing. Today, the most commonly used DNA repeat regions used are microsatellites also known as Short Tandem Repeats (STR). These loci in which the repeat unit is at least two bases but no more than seven in length, are amplified by PCR (Polymerase Chain Reaction) in a multiplex fashion (multiple primers) reducing sample consumption. Nevertheless, it is of great importance to make the distinction between the samples containing large quantities of high quality DNA and those containing minute amounts of DNA and/or poor quality molecules. If for the first type of samples, the occurrences of errors or pitfall are rare, in the second type, the interpretation of the allelic profiles should be done with care and caution. Two types of damage are mainly likely to affect DNA over time: hydrolytic and oxidative damage. Hydrolytic damage results in deamination of bases and in depurination and depyrimidination, whereas oxidative damage results in modified bases.  Various kinds of samples can be typed with the PCR-based methodologies such as: blood samples and blood stains, cigarette buts, human hairs with a special mention of the possibility of analysis of single hair, urine samples and urine stains, fingernail scraping, bite marks, all kinds of touched objects; such as tools, clothing, firearms, parts of vehicle, food, condoms, glass, bottles, lip cosmetics, wallets, jewelry, paper, cables, stones and construction material, FTA cards can be used to collect blood or saliva in order to assure a better preservation of the DNA molecules by the specific fixation on the treated card paper, teeth and bone tissues as well as burnt tissues. Touched objects provide a wide scope for revealing the offender’s DNA profile in investigations of offences including theft, burglary, vehicle crimes, street robbery, drug cases, homicide, rape and sex offences, clandestine laboratories, armed robbery, assaults, crime. The positive DNA identification from those samples allowed the creation of national offender databases. One of the best methods to collect trace samples is the use of swabs after having identified as precisely as possible the areas to target. The first step is to swab the hole defined surface by one or several moistened swab multiple times with some pressure and rotation given to the swabs. The second step is to complete the swabbing by the application of dry swabs to recapture the moisture containing hydrated cells. The moistening agent can be sterile water, 0, 01% sodium dodecyl sulphate or isopropanol. The quantities of cellules retrieved depend also of the physical characteristics of the surface and the use of different moistening agents for different surfaces may facilitate collection. The quality of the swabs is also important, the quality should be DNA-free; cotton swabs are the most frequently used but other types such as foam may also be considered. The best way to retrieve DNA containing material from worn clothing or from touched surfaces without collecting in the same time inhibitory factors present on this material (staining chemicals and/or color denim). By pressing a strip of tape multiple times over a target area, the most recently deposited material , with fewer inhibitory factors, are collected. In our experience, this method is not often used and should be replaced by a easiest way to collect DNA such as cutting away stain fragment samples. To isolate relevant target cells from other over-whelming cell types, laser microdissection techniques were used. The different cell types can be recognized by morphological characteristics, various chemical staining or fluorescence labeling techniques. These methods allow to establish a clear DNA profile from few cells present in a mixture samples that otherwise had not be detected while swabbed by the major component and not detectable in the profile. As cells could be lost during the transfer, it would be preferable to use actually laser microdissection methodology is directly used on the initial collection material. For samples containing enough DNA of high molecular weight, the classical technics of DNA extraction can be performed without pitfall, appropriate technologies were developed to increase the chance to obtain useful profiles from very minute DNA samples such as the low copy number (LCN) procedure with extra cycles or low template DNA (LTDNA) methods. Minute samples or trace DNA refers to samples where only 100pg to 200pg of DNA could be extracted. Every DNA contamination will be also amplified and can false the result and on the other hand the excess of DNA produced by the PCR will be present either on the machines used but also in the surrounding environment such as the air and the work surfaces. The number of cycles used during the PCR of the STR loci is increased to 34 compared to the standard 28 cycle reactions. In molecular anthropology and in ancient DNA work, the number of cycles could be increased up to 60 in order to maximize the success of amplification. The efficiency of the amplification reaction can also be increased by the addition of chemical adjuvants such as bovine serum albumin (BSA) which is known to prevent the inhibition of the activity of Taq polymerase by sequestering phenolic compounds which otherwise scavenge the polymerase. No methods can actually eliminate completely artifact product during the amplification step in particular when the DNA is degraded or present in minute amounts but their occurrence should be statistically evaluated. To be able to develop such an approach it is of importance to understand the factors that may cause each type of artifact and the accurate data regarding the frequency and scale of their occurrence. For each profile interpretation, the sampling of biological material found at the crime scene must be replaced into context and the possibility of pitfalls should be taken into account such as the possibilities of material transfer, the difficulties of the amplification process and the possibility of artifacts affecting the true result. This interpretation carefulness is of particular importance when the analyses are performed on degraded or very low quantities of DNA and has to consider imperatively the four most common features which can occur in those cases: allele drop-out, allele drop-in, stutter bands, contamination and decreased heterozygote balance. Strict interpretation guidelines can give reliable and robust result and minimize these pitfalls. One of the most used methods to eliminate incorrect genotypes is to replicate the amplifications reactions and to generate consensus profiles. A particular mention must be made for DNA mixture interpretation. In fact mixed samples are by definition composed of one or more major contributors with high quantities of DNA and with a minor contributor present only at trace levels, in other cases, the contributors are all present at trace levels. A profile can be falsely identified as a false mixed samples when high stutter peaks are present indicating that the DNA is coming from multiple individuals although it truly derive from a single source. In mixed samples, the high probability of drop-in, drop-out and increased stutter bands avoid the precise determination of the number of contributors and the separation of the genotypes at any given locus. Contaminations are the major pitfall in the analyses of DNA in the forensic field either in producing valuable profiles or in accurate interpretation of the results. This is a major issue when the samples are degraded or when the DNA molecules are present in minute amounts. Contaminations may appear in every step of the analysis process from the sampling on the crime scene to the laboratory work. The contaminations can occur on the body itself or during the sampling of the evidences, at the scene of the crime, during the transportation of the body to the mortuary, at the autopsy room and after, of course, during the laboratory procedures. At the crime scene, one of the more frequent situation where contaminations of the crime scene can occur if the individuals who entered the scene speak or caught and handle evidences over the corps before the arrival of the forensic investigative team. To avoid the possibility of contaminations one should perform analyses about the persistence of DNA on different kinds of surfaces in various environmental conditions, improve and standardize the sample collection methodologies in order to improve the targeting of the samples and to decrease unwanted underlying DNA, collect the profiles of all the persons involved in the collecting and laboratory steps to recognize a contamination coming from these professionals, To avoid breathing, talking and of course coughing during the sampling step in restricting the access of non specialist investigators to the scene,  use full-body scene suit (to avoid contamination by cell shedding coming from exposed areas of skin), hood, hair net, gloves and mouth masks by all the investigators in charge of the sampling step, avoid direct touching of the evidences containing the DNA and changing gloves and masks regularly at the crime scene and obviously in the laboratories, use of DNA-free plastic ware and consumables.

There have always been two mayor goals of the method of DNA fingerprints – one is to obtain highly discriminating genetic profiles from minute amounts of DNA and for highly degraded samples – and – another one – is to avoid the possibility of contaminations due to the crime scene work, the sampling step or the laboratories procedures.

Acknowledgements:

The Police Department;

www.politie.nl and a Chief Inspector – Mr. Erik Akerboom     ©

 Bibliography:

1.    Criminal Investigations – Crime Scene Investigation.2000

2.    Forensic Science.2006

3.    Techniques of Crime Scene Investigation.2012

4.    Forensics Pathology.2001

5.    Pathology.2005 

6.    Forensic DNA Technology (Lewis Publishers,New York, 1991).

7.    The Examination and Typing of Bloodstains in the Crime Laboratory (U.S. Department of Justice, Washington, D.C., 1971).

8.    „A Short History of the Polymerase Chain Reaction". PCR Protocols. Methods in Molecular Biology.

9.    Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press.2001

10.   "Antibodies as Thermolabile Switches: High Temperature Triggering for the Polymerase Chain Reaction". Bio/Technology.1994

11.   Forensic Science Handbook, vol. III (Regents/Prentice Hall, Englewood Cliffs, NJ, 1993).

12.   "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes". In Kieleczawa J. DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett. 2006

13.   Nielsen B, et al., Acute and adaptive responses in humans to exercise in a warm, humid environment, Eur J Physiol 1997

14.   Molnar GW, Survival of hypothermia by men immersed in the ocean. JAMA 1946

15.   Paton BC, Accidental hypothermia. Pharmacol Ther 1983

16.   Simpson K, Exposure to cold-starvation and neglect, in Simpson K (Ed): Modem Trends in Forensic Medicine. St Louis, MO, Mosby Co, 1953.

17.   Fitzgerald FT, Hypoglycemia and accidental hypothermia in an alcoholic population. West J Med 1980

18.   Stoner HB et al., Metabolic aspects of hypothermia in the elderly. Clin Sci 1980

19.   MacGregor DC et al., The effects of ether, ethanol, propanol and butanol on tolerance to deep hypothermia. Dis Chest 1966

20.   Cooper KE, Hunter AR, and Keatinge WR, Accidental hypothermia. Int Anesthesia Clin 1964

21.   Keatinge WR. The effects of subcutaneous fat and of previous exposure to cold on the body temperature, peripheral blood flow and metabolic rate of men in cold water. J Physiol 1960

22.   Sloan REG and Keatinge WR, Cooling rates of young people swimming in cold water. J Appl Physiol 1973

23.   Keatinge WR, Role of cold and immersion accidents. In Adam JM (Ed) Hypothermia – Ashore and Afloat. 1981, Chapter 4, Aberdeen Univ. Press, GB.

24.   Keatinge WR and Evans M, The respiratory and cardiovascular responses to immersion in cold and warm water. QJ Exp Physiol 1961

25.   Keatinge WR and Nadel JA, Immediate respiratory response to sudden cooling of the skin. J Appl Physiol 1965

26.   Golden F. St C. and Hurvey GR, The “After Drop” and death after rescue from immersion in cold water. In Adam JM (Ed). Hypothermia – Ashore and Afloat, Chapter 5, Aberdeen Univ. Press, GB 1981.

27.   Burton AC and Bazett HC, Study of average temperature of tissue, of exchange of heat and vasomotor responses in man by means of bath coloremeter. Am J Physiol 1936

28.   Adam JM, Cold Weather: Its characteristics, dangers and assessment, In Adam JM (Ed). Hypothermia – Ashore and Afloat, Aberdeen Univ. Press, GB1981.

29.   Modell JH and Davis JH, Electrolyte changes in human drowning victims. Anesthesiology 1969

30.   Bolte RG, et al., The use of extracorporeal rewarming in a child submerged for 66 minutes. JAMA 1988

31.   Ornato JP, The resuscitation of near-drowning victims. JAMA 1986

32.   Conn AW and Barker CA: Fresh water drowning and near-drowning — An update.1984;

33.   Reh H, On the early postmortem course of “washerwoman’s skin at the fingertips.” Z Rechtsmed 1984;

34.   Gonzales TA, Vance M, Helpern M, Legal Medicine and Toxicology. New York, Appleton-Century Co, 1937.

35.   Peabody AJ, Diatoms and drowning – A review, Med Sci Law 1980

36.   Foged N, Diatoms and drowning — Once more.Forens Sci Int 1983

37.   "Microscale chaotic advection enables robust convective DNA replication.". Analytical Chemistry. 2013

38.   Sourcebook in Forensic Serology, Immunology, and Biochemistry (U.S. Department of Justice, National Institute of Justice, Washington, D.C.,1983).

39.   C. A. Villee et al., Biology (Saunders College Publishing, Philadelphia, 2nd ed.,1989).

40.   Molecular Biology of the Gene (Benjamin/Cummings Publishing Company, Menlo Park, CA, 4th ed., 1987).

41.   Molecular Evolutionary Genetics (Plenum Press, New York,1985).

42.   Human Physiology. An Integrate. 2016

43.   Dumas JL and Walker N, Bilateral scapular fractures secondary to electrical shock. Arch. Orthopaed & Trauma Surg, 1992; 111(5)

44.   Stueland DT, et al., Bilateral humeral fractures from electrically induced muscular spasm. J. of Emerg. Med. 1989

45.   Shaheen MA and Sabet NA, Bilateral simultaneous fracture of the femoral neck following electrical shock. Injury. 1984

46.   Rajam KH, et al., Fracture of vertebral bodies caused by accidental electric shock. J. Indian Med Assoc. 1976

47.   Wright RK, Broisz HG, and Shuman M, The investigation of electrical injuries and deaths. Presented at the meeting of the American Academy of Forensic Science, Reno, NV, February 2000.