Under the Microscope: Samples Required For DNA Testing - To The Bone!

 

Amplification techniques, such as polymerase chain reaction (PCR), etc., now allow minute samples to be  tested, the larger the sample, the better the chance of a successful DNA test. Post-mortem material is inferior to live blood and tissue for DNA fingerprinting if any significant post-mortem change has proceeded enough to break down nuclear chromatin. Plain blood samples should be taken, however,  though leucocytes may have already disintegrated. Some 5 ml of blood is taken into an EDTA tube, which extracts metallic ions and not only prevents clotting, but inhibits enzymes in blood or micro-organisms, which may break down DNA during storage. The best material is said to be - muscle or the spleen and, if decomposition is advancing, the bone marrow is recommended. At least 0.5 g of tissue should be cut from the parenchyma of an organ and placed in a small plastic tube with no fixative or preservative.  This should be frozen at -20°C if there is likely to be the slightest delay in transmission to the laboratory. In sexual offences associated with homicide, as much material as can be obtained from the vagina should be - collected, either fluid by pipette or multiple swabs from vagina, rectum and mouth. These again should be frozen if there is more than a few hours delay in transit to the laboratory.

One of the classic problems of forensic pathology, the identification of a whole or partial skeleton, involves techniques  and expertise that span a number of disciplines from  anatomy to radiology from archaeology to dentistry. It  often overshadows the equally important need to identify  intact or decomposed corpses, but the disproportionate amount of space occupied by skeletal identification in most  textbooks can be partly justified by the fact that bones can survive for decades, centuries or millennia, so that the cumulative reservoir of material is vast. Recognition is usually easy, by the shape, texture and especially weight of the objects. Greater' difficulty may be experienced when simulated human 'bones' are found, such as the 'radius'. This originated from a medical student's anatomical skeleton and was made from plaster coated with a plastic polymer. Visually it was indistinguishable from a real bone, but the abnormal lightness indicated the true nature. The recognition of species is important, but deciding on a human origin is usually easy, unless marked fragmentation. Many animal bones are found by the general  public and police who are not qualified to decide whether they are human. Extensive rebuilding programmes and the renovation and demolition of old sites reveal many caches of bones, bur in the authors' experience the majority are of animal origin, often originating from butchers' debris. First, the size is assessed and many small, slender bones excluded on obvious grounds. Even a turkey thigh cannot be mistaken for a metatarsal, nor a beef rib for human. The gross anatomy is then studied, and even if the makes no pretensions to being a comparative anatomist, the majority of intact animal bones can be recognized from their lack of correspondence to any human bone. Difficulties arise  with smaller bones from some animals, especially the hands  and feet, where digits, metatarsals and metacarpals require  careful study to distinguish them from the human. Several  errors have been made in respect of bear paws, which closely  resemble the human hand.

When bones are incomplete or fragmentary, the problems escalate rapidly. If the ends of longer bones are present, then their non-human shape may be more readily  determined, but cylindrical segments of the central shaft  have little in the way of distinguishing features, apart from  size. Burnt bone fragments offer similar problems, added to  which is the possibility of heat distortion and shrinkage.

The test is essentially seeking plasma constituents within the bone, the recognition being carried by techniques such as electrophoresis or gel diffusion. DNA  can now identify human tissue, if not the alternative species. The drawback of such serological tests is that they cannot be applied to bones that no longer have extractable proteins, and these include burnt or cremated bone and bone that has been dead for some years. The length of time for which identifiable protein persists is variable, but a negative result is usually to be expected after 10 years following death, though DNA techniques may be more sensitive. The accuracy of determination of the sex of skeletal remains  varies with the age of the subject, the degree of fragmentation of the bones and biological variability. The determination of sex is statistically the  most important criterion, as it immediately excludes approximately half the population whereas age, stature and race each  provide points within a wide range of variables. Obvious sex  differences do not become apparent until after puberty. usually in the 15-1 8-year period, though specialized measurements on the pelvis can indicate the sex even in fetal material.  Sex and age are linked, especially where body size and weight  are concerned. Similarly, race confuses sexing, for example,  the size of the supraorbital ridges in a normal negroid female  may exceed those in the average Caucasian male.  The accuracy of sexing is hard to estimate, as various  loading factors exist. Krogman comments that he scored  100 per cent accuracy using the whole skeleton, 95 per cent  on pelvis, 92 per cent on skull, 98 per cent on pelvis plus  skull, 80 per cent on long bones and 98 per cent on long  bones plus pelvis. He admitted, however, that, as most  anatomy department material has a sex ratio of about 15: 1 in  favour of men, marked bias could be introduced by assigning  all doubtful bones to the male category.

In general, adult female  skeletal measurements are about 94 per cent that of the male  of the same race, but different measurements may vary from  91 to 98 per cent.

 

 

Acknowledgements:

www.politie.nl  Politiekorpschef  @Janny Knol©

www.aived.nl    AIVD – @Erik Akerboom ©

www.politie.nl WEB Politie - @Henk van Essen©

https://www.police-nationale.interieur.gouv.fr/ @ Stephane Folcher ©

 

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.