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Anthropology Physical/ Biological Anthropology Forensic Anthropology

Paper No. : 01 Physical/ Biological Anthropology Module : 30 Forensic Anthropology

Prof. Anup Kumar Kapoor Department of Anthropology, University of Delhi

Development Team

Principal Investigator

Paper Coordinator

Content Writer

Content Reviewer Prof. Barun Mukhopadhyay Indian Statistical Institute, Kolkata

Prof. Subho Roy Department of Anthropology ,University Of Calcutta

Dr. Jyoti Ratan Ghosh Visva-Bharti Santiniketan, West Bengal

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Description of Module

Subject Name Anthropology

Paper Name 01 Physical/Biological Anthropological

Module Name/Title Forensic Anthropology

Module Id 30

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Contents of this unit 1. Introduction 2. Skeleton as a Forensic Evidence

A. Is it bone or not? B. Is it human? C. How long since death? D. What is the race or ancestry? E. What is the Sex?

i. The skull has many features that vary in the two sexes ii. The pelvis has many features that vary in the two sexes

F. What is the Age? i. Estimation of age from sutures of the skull ii. Estimation of age from the ossification in the epiphysis and their fusion with diaphysis iii. Estimation of age from pubic symphysis iv. Estimation of age from teeth

a. Temporary or deciduous teeth b. Permanent teeth

G. What is the stature? H. Facial reconstruction I. DNA from skeleton as personal identification

3. Blood as a Forensic Evidence A. Is the sample blood?

i. Presumptive tests a. Phenolphthalein test b. Luminol test

ii. Confirmatory Tests for Blood a. Takayama test or hemochromogen test

B. Is the sample human blood? i. Immunohematology ii. Electrophoresis iii. Molecular marker

C. What is the Sex? D. Genetic Markers in Blood E. DNA from blood for personal identification F. Bloodstain Pattern Analysis

4. Fingerprint as a Forensic Evidence A. Classification of fingerprints patters

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B. Minutiae C. Types of fingerprint commonly found in crime scenes

i. Visible ii. Plastic iii. Latent

D. Latent fingerprints i. Ninhydrin ii. Iodine

E. Collection of fingerprint from a crime scene F. Comparison of fingerprints for personal identification

5. DNA as a Forensic Evidence A. What are the sources of DNA? B. Nuclear DNA

i. Restriction fragment length polymorphism (RFLP). a. Visualization of VNTRs b. Polymerase chain reaction (PCR)

ii. Short tandem repeats (STRs) C. Identification of Sex from DNA

a. Amelogenin b. Y-STRs

D. Mitochondrial DNA (mtDNA) E. Interpretation of DNA profile

Learning objectives Ø What is forensic anthropology? Ø What are the different types of evidences? Ø Importance of skeleton in personal identification Ø Importance of blood in personal identification Ø Importance of fingerprints in personal identification Ø Importance of DNA in personal identification

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1. Introduction Forensic science is the application of the techniques of science to legal matters, both criminal and civil (Bell et al., 2008). Forensic science includes a number of disciplines and sub-disciplines such as forensic anthropology, forensic engineering, forensic medicine, forensic ecology, forensic pathology, forensic chemistry, etc.

Forensic anthropology is the application of knowledge and techniques of physical anthropology to study questions of medicolegal importance. According to Houck and Siegel, 2010, forensic anthropology is the application of the study of humans to situations of modern legal or public concern. The objective of forensic anthropologists is to assist in the identification of human remains and personal identification including age, sex, ethnicity, stature and unique features, if any.

There are different types of evidences including skeleton, body fluids like blood, fingerprints, etc., that can be found in a crime scene and are used by forensic anthropologist for personal identification.

2. Skeleton as Forensic Evidence It is easy to identify a skull, complete skeleton, bones as part of a complete skeleton or individual intact bones, but becomes problematic if the bone is fragmented. Because, things such as plastic or pieces of tree may look like bone and thus create confusion. So, unlike readily identifiable human bones, indifferent bones, and especially for fragmented bone, the key question is, is it bone? Next a forensic anthropologist tried to answer a number of questions like, is it human? Are the bones male or female? What was the age at time of death? What is the race? etc. The answers to these questions are very important, because this will lead to the personal identification of the remains and, possibly, to determine the cause and manner of death.

A. Is it bone or not? The presence of cortex (outer layer cortex) and cancellous (spongy interior part) tissue differentiate bone from other materials.

B. Is it human? Ø Skull with rounded vault and flat face indicate that it is a human skull. Ø Randomly scattered osteons in cancellous tissue is indicative of human bone. Ø Dentition and some features of the teeth like filings or caps also indicate that they are of human. Ø Precipitin test is also used for identifying as to species.

C. How long since death? It is very difficult to determine the time since death because number of factors including temperature, soil type, whether preserved in a coffin or not, etc. affect the decomposition rate of a body.

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Ø Presence of flesh and odor are good indicator of recent death. Ø Recent bones will have a “greasy” feel. Ø Older bones will be lightweight and have a weakened surfaces or ends. Ø Severe tooth wear and erosion into the dentin is indicative of ancient remains.

D. What is the race or ancestry? It is very difficult ascertain race or ancestry from skeletal remains because no pure race exist today. However, forensic anthropologists are routinely called to assess the skeletal remains for clues as to that person’s ancestral affiliation to help investigator for personal identification.

Table 1. Racial characteristics of the Skull Trait Mongoloid Caucasoid Negroid Skull length Long Short Long Skull breadth Broad Broad Narrow Skull height Middle High Low Sagittal contour Arched Arched Flat Face breadth Very wide Wide Narrow Face height High High Low Orbital opening Rounded Rounded Rectangular Nasal opening Narrow Moderately wide Wide Nasal bones wide, flat narrow, arched Narrow Lower nasal margin Sharp Sharp Troughed Facial profile Straight Straight Downward slant Palate shape moderately wide moderately wide Wide

Broad U-shape V-shape U-shape Shovel-shaped 90%+ <5% <5% incisors General form large, smooth Rounded, large Smooth, elongated (Source: Pickering and Bachman, 2009; pp. 83) E. What is the Sex? Several elements of the skeleton can be used by a physical anthropologist for determination of sex in adults, but the pelvis and skill are the most reliable elements for determination of sex. For example,

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i. The skull has many features that vary in the two sexes:-

Table 2. Sexual characteristics of the Skull Trait Male Female Supraorbital ridge (ridge above the eyes) Robust Gracile Occipital protuberance (base of skull) Robust Gracile Mastoid processes (bony process behind ear canal) Long, broad Short Chin U-shaped, square V-shaped

(Source: Pickering and Bachman, 2009; pp. 89)

(Source: Juniordentist website) Figure 1. Human skull; a,.male b, female

ii. The pelvis has many features that vary in the two sexes:-

Table 3. Sexual characteristics of the Pelvis

Trait Male Female Symphysis High, narrow Low, wide Sub pubic angle V-shaped U-shaped, round Obdurate foramen Large, ovoid Triangular Sciatic notch Narrow, deep Wide, 70–90 degrees Sacroiliac articulation Large, straight Small, oblique Ilium High, vertical Wide Sacrum Long, narrow, straight Short, broad, curved Pelvic inlet Heart-shaped Circular, elliptical Acetabulum Large Small

(Source: Pickering and Bachman, 2009; pp. 89)

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a, female b, male Figure 2: Human pelvis; a, female b, male

(Source: Wikipedia website) F. What is the Age? There are no skeletal features that allow a specific age of the specimen, such as 30 years of age. However, age determination can be done with a range, for example, 45 to 47 years, or 60+ years.

i. Estimation of age from sutures of the skull Closer of suture gives an idea about the approximate age of the specimen. For example, the presence of large fontanels and wide open sutures in skull indicated that it belongs to an infant. Fusion of the sagittal suture is indicative of about 35 years of age. The Spheno-Parietal suture begins to close about thirty years, but is not usually completely closed until about seventy years.

ii. Estimation of age from the ossification in the epiphysis and their fusion with diaphysis An estimation of age can be possible by analyzing the times of appearance of ossification in the epiphysis and their fusion with diaphysis of bone. For example, appearance of ossification at the capitulum of humerus, distal radius, distal tibia and fibula indicate one year of age in both sexes. At about twenty-one years of age ossification appears at the medial end of Clavicle in both sexes.

Fusion of the epiphyses of greater tubercle of the head of humerus indicates about four years of age in female and about five years of age in male. Fusion of the epiphyses of the distal phalanges of second, third, and fourth toes indicates about fifteen years of age in both sexes.

iii. Estimation of age from pubic symphysis The morphological changes of the pubic symphysis are considered one of the most reliable estimators of age at death. For example, in post-adolescent and early adult life, the sub-epiphysial symphysial face presents a transversely billowed (wavy) surface with an increasing sharp dorsal margin and a beveled ventral area. In the letter part of the third decade the billowed texture is lost and a typical ovoid symphysial face results. Toward the end of the fifth decade a narrow beaded rim develops on the

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margins. During the sixth decade erosion of surface and breakdown of ventral margin begin to modify the configuration of the symphysial face.

Figure 3: Age changes in pubic symphysis in adult white males

(Source: Allthingsaafs website)

iv. Estimation of age from teeth An examination of the state of eruption of the teeth in infants will offer a reliable indication of age in most cases, since the respective periods of eruption of the different teeth are fairly constant.

Approximate period at which the different temporary and permanent teeth appear:-

a. Temporary or deciduous teeth

Table 4. Estimation of age from temporary teeth Period Teeth 7-9 months Upper central incisor 6-8 months Lower central incisor 9-10 months Lateral incisor 18 months Canine About 12 month First molar About 2 years Second molar

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A, Temporary B, Permanent Teeth Figure 4. Human teeth; a, temporary b, permanent teeth

(Source: The University of Queensland website) b. Permanent teeth

Table 5. Estimation of age from permanent teeth Period Teeth 7th year Central incisor 8th year Lateral incisor 11th year Canine 9th year First premolar 10th year Second premolar 6th year First molar 12th year Second molar 17th -21st year or later Third molar G. What is the stature? Living stature is directly related to the length of bone, specially, the long bone of the lower limbs. However, to estimate stature, the only difficulty is that sex and ethnicity must be known to correctly, because the prediction equations are dependent on sex and ethnicity. For example, a White male with a femur length of 55.88 cm. would be estimated to have been between 189 cm and 196 cm tall. This can be done by the following regression equation.

For white males: Stature = {(2.38 x Humerus length) + 61.41 +/− 3.27}

H. Facial reconstruction One of the widely used practices in forensic anthropology for personal identification is facial reconstruction based on the features of the skull. In this process, the soft tissues of a face are reconstructed on a skull to create a likeness of that individual.

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I. DNA from skeleton as personal identification DNA profiling can play an important role in personal identification (details will be discuss later under the subheading of ‘DNA as forensic evidence’), especially when the bones are very fragmentary and other ways of personal identification is not possible.

3. Blood as Forensic Evidence In crime of violence body fluids are routine encountered as viral evidence. Blood is the most common form of biological material that is recovered from crime scenes. Along with other evidence, blood stain analysis may be used to place a person at a crime scene, or possibly to completely exclude that person. A. Is the sample blood? There are many stains such as rust, paint, wine etc. which look like blood. Thus, chemical tests are essential to confirm whether the sample is blood or not.

i. Presumptive tests Presumptive tests are generally used at the crime scene and merely confirm that the substance in question is most likely blood. For example,

a. Phenolphthalein test: At first a clean cotton swab dip in distilled water and then rubbed on the suspected stain. A drop of phenolphthalein solution is then added to the swab’s tip. A drop of hydrogen peroxide is then added; if the tip turns pink, the test is presumptively positive for blood.

Figure 5: Phenolphthalein test for presence of blood (Source: Centralia College website)

b. Luminol test: Luminol (3-aminophthalhydrazide) is sprayed in crime scene which reacts in the presence of haemoglobin, when an oxidizer is applied. The reaction, however, results in a blue-white to yellow-green luminescence (light emitted as a by-product of a chemical reaction) if blood is present.

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Figure 6. A footwear impression in blood that has been enhanced with luminol reagent (Source: Bureau of Criminal Apprehension website)

ii. Confirmatory Tests for Blood a. Takayama test or hemochromogen test

The commonly used confirmatory test for blood is the Takayama test or hemochromogen test. In this test a small sample of the presumptive stain is placed under a cover slip and heated lightly. Then pyridine under alkaline conditions in the presence of a reducing sugar is added with a pipette in it. If blood is present, salmon-colored crystals will form and can be viewed through a microscope.

B. Is the sample human blood? There are a number of tests that can be used to identify human blood, for example,

i. Immunohematology: Antigen – antibody agglutination test, precipitation test

ii. Electrophoresis: For identification of red cell enzyme, serum protein

iii. Molecular marker: Most stable DNA isolation and test for human kind RFLPs

C. What is the Sex? Presence of drumstick in WBC indicates female sex and its absence indicate male sex.

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Figure 7: Presence of drumstick in WBC

(Source: Stellenbosch University website)

D. Genetic Markers in Blood Blood possess a number of genetic markers both in cell and serum including ABO blood group, Rh, MNS, PGM, and HP etc. for personal identification.

E. DNA from blood for personal identification Once it is confirmed as human blood, DNA can be extracted from WBC for DNA profiling and personal identification (details will be discussed later under the sub-heading of ‘DNA as forensic evidence’).

F. Bloodstain Pattern Analysis Often found at the scenes of violent crimes, the analysis of bloodstains can provide vital clues as to the occurrence of events. For example, Venus blood will show fine droplets with striking surface right angled star like. Artery blood generally will show oblique pattern due to pressure.

Figure 8: Showing how bloodstain pattern analysis works

(Source: Howstuffworks website)

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4. Fingerprint as Forensic Evidence Fingerprints have long been considered as the backbone of forensic identification, even in the age of DNA technology. This is because of the fact that fingerprints are matchless to the individual and thus, assumed that a match between a suspect print and an evaluation print is sufficient to individualize the evidence and link it to a common source.

A. Classification of fingerprints patters There are four types of pattern namely, Arch, Loop, Whorl and Composite. The arches are either plain arch or tented arch. Loops are further subdivided in two types depending on their openings as ulnar loop and radial loop. Whorls are also further subdivided into plain whorl, central pocket loop, double loop, and accidental.

A. Arch B. Loop C. Whorl

Figure 9: Fingerprint patterns, a. Arch, b. Loop, c. Whorl (NIST scientific and technical databases website)

B. Minutiae Along with fingerprint pattern, the ridge of each fingerprints also contain unique features which allow them for personal identifications. These ridge characteristics are collectively known as minutiae. This includes the patterns of the pores on fingerprints, bifurcations, island, short ridge, termination, enclosure, etc.

Figure 10: Minutiaes

(Source: Center for imaging science website)

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C. Types of fingerprint commonly found in crime scenes Three types of fingerprints are commonly observed in crime scenes, namely,

i. Visible - it is clearly visible, does not require processing and can be found on substance such as paint or blood.

ii. Plastic – it is also clearly visible, does not require processing and can be found on soft surface, such as tar, putty.

iii. Latent - it requires treatment to be visualized and can be found for example, on human skin.

D. Latent fingerprints A latent fingerprints is one left on an objects surface when it is touched. The oldest and still most common method to make visible latent fingerprint is to use fingerprint powder of contrasting colour by a soft fiberglass brush to create contrast between the ridge pattern and the background. Once a latent print becomes visible, it is photographed.

Figure 11: latent prints; a, use of fine powder to reveal latent fingerprints b, paper treated with Ninhydrin reagent c, site treated with fluorescent dye stain

(Source: forensic science simplified website)

A number of chemical methods are also available for the visualization of latent prints, for example,

i. Ninhydrin: Ninhydrin is used to develop latent prints (purple colour) on porous surfaces like paper by reacting with amino acids in latent print residue.

ii. Iodine: Fumes from iodine crystals develop latent prints on surfaces that are impractical for traditional dusting or have residue such as grease. The print developed are visible only for few hours and then become fade, so it should be photographed immediately once observable.

E. Collection of fingerprint from a crime scene Fingerprints that are clearly visible can be photographed and the print can be used for comparison. However, for latent fingerprint photograph can be taken only after it become visible by physical or

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chemical treatment. Sometimes it is possible to lift the latent fingerprint. This can be done by placing a lifting tape on the fingerprint. The lifting tape is then placed on a latent lift card to preserve the print.

Figure 12: Use of lifting tape to lift fingerprint (Source: CHIEF website)

F. Comparison of fingerprints for personal identification The comparison of fingerprints collected from the suspect and from the crime scene for identification is not only made by comparing the patterns or by merely the presence or absence of minutiae, but is done by comparing the presence, kind, number and, especially, the arrangement of the pattern and minutiae characteristics that create a unique pattern.

Figure 13. Comparison of minutiaes

(Source: SPIE website) Now days this comparison can be done by automated fingerprint identification systems (AFIS). AFIS is a computer programming which digitized the images of a fingerprint and then encode the details of the minutiae for searching in the database. This system does not identify a person uniquely but rather

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provide a list of potential matches for the fingerprint examiner to evaluate and make the final determination.

5. DNA as Forensic Evidence The development and application of human genetics has reformed forensic anthropology in the last two decades. Now a day’s DNA profile makes it possible to associate a DNA sample to a specific person with a high degree of confidence and thus providing a new, powerful identification tool that supplements fingerprints and other methods of identification to the forensic investigation.

There are two types of DNA, namely the nuclear DNA (located in the nucleus) and mtDNA (located in the mitochondria). However, both are used for DNA profiling in personal identification.

A. What are the sources of DNA? DNA samples can be obtained from a number of biological materials including saliva, tissue, blood, hair follicles, semen and bone from a scene of crime.

Figure 14: Nuclear DNA (National Library of Medicine website)

B. Nuclear DNA The humans nuclear DNA contains approximately 3.2 billion base pairs and organized into 23 chromosomes. A large portion of this DNA is similar and is required for the protein synthesis.

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However, there are segments of DNA that vary from person to person and are called hyper variable regions. These regions are commonly used as markers for personal identification. There are several techniques that make DNA typing possible for forensic identification like restriction fragment length polymorphism (RFLP).

i. Restriction fragment length polymorphism (RFLP). In RFLP analysis a restriction enzyme is used to cut the DNA sample into a number of pieces of variable size. The number and the size of the DNA fragment depend on the variation in the number of times a sequence of base pairs in DNA is repeated. From a forensic anthropological viewpoint, these fragments are extremely polymorphic since the number of repeat sequence varies from person to person. Because the polymorphism comes from the number of repeats, this kind of DNA typing is categorized as VNTR (variable number of tandem repeat). These are length polymorphisms with a core sequence are between 10 and 100 base pairs in length and in one person, this sequence may be repeated fifty times on a given chromosome, while in another person it may repeat ten times. In forensic science, four to six of these highly polymorphic VNTR loci are analyzed, and the results are used to generate DNA profiles for comparison and personal identification.

a. Visualization of VNTRs The technique by which these VNTRs can be identified is called Southern blotting electrophoresis. In order to visualized the VNTRs, the DNA fragments are loaded into a well at the base of a slab of electrophoresis gel. An electrical current is then applied, which separates the VNTRs based on length and charge of DNA fragment. After that the DNA fragments are stained with radioactive single-strand DNA sequences for visualization and identification.

Figure 15: VNTR locus

(Source: University of Arizona website)

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Figure 16: VNTR alleles (Source: University of Arizona website)

b. Polymerase chain reaction (PCR)

RFLP gives good result when there is large amount of high quality DNA. But, the DNA sample come from a crime scene may not be large in amount and more frequently they are too fragmented to be useful. In this situation PCR technique is used primarily to increase the amount of DNA by amplification. PCR is based on the natural process of DNA replication. PCR uses DNA polymerase to rapidly replicate a specific DNA sequence millions of times.

Figure 17: Polymerase chain reaction

(Source: National Center for Biotechnology Information website)

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ii. Short tandem repeats (STRs)

STRs are currently the most commonly analyzed genetic polymorphism in forensic genetics. There are thousands of STRs that can potentially be used for forensic analysis. STR loci are spread throughout the genome including both Autosome and sex chromosomes. STRs have a core unit of between 2 and 6 base pairs and the repeats typically range from 50 to 300 base pairs. STRs have greater advantages over VNTRs, which make STRs more attractive for forensic comparison purposes. For example, STR markers have greater variability in a population, thus giving rise to high degrees of association of evidence with a suspect. There are many STRs to choose from for forensic purposes. Moreover, STRs are much less sensitive to degradation of the DNA due their smaller repeat size. Similar to that of VNTRs, STRs are also identified by electrophoresis and the results are then used to generate DNA profiles for comparison and personal identification.

C. Identification of Sex from DNA There are two approaches to sex identification from a DNA sample.

a. Amelogenin: Presence of one electrophoresis band for Amelogenin locus indicate female sex and presence of two electrophoresis band for Amelogenin locus indicate male sex.

b. Y-STRs: The presence of Y-STRs, a sex-specific DNA marker indicates male sex and its absence indicates female sex.

D. Mitochondrial DNA (mtDNA) MtDNA located in mitochondria and inherited maternally. The human mtDNA is a circular molecule and consisting of 16,569 nucleotides. There are a number of situations where mtDNA becomes a valuable genetic marker, for example, it is very helpful when the amount of cellular material available for analysis is very small or if the source does not contain nucleated cells. Moreover, mtDNA is also advantageous for the study of highly degraded human remains where standard STR typing is not possible. Due to its maternal inheritance, mtDNA is very useful for personal identification of missing person or person in mass disasters, especially when there are no direct relatives to use as a reference sample, because, all maternal relatives share the same type of mtDNA. Analysis of mtDNA is generally performed by comparing the sequences of its hyper-variable regions.

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Figure 18: mtDNA (Source: Nature website)

E. Interpretation of DNA profile Once it has been established that two DNA profiles are the same, the significance of the match has to be estimated. This significance of the match or probability is calculated by the frequencies of each allele using population data base and indicates the probability of a chance occurrence of a given DNA profile within a chosen population. Thus, in order to reduce the chance occurrence of similar DNA profile, forensic experts are commonly use multi-locus genotype using large number of STR loci for identification. Because, this multi-locus genotype becomes so rare that it is improbable that two people in the world would have the same exact type. This helps the forensic experts to conclude in DNA profile cases that the match of the DNA type from the evidence and the suspect can be used for identification, except in the case of identical twins.

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