Side Determination and Fragment Identification

Fragment identification is a hierarchical decision tree. The first decision is the broadest: is this bone at all, or is it mineralised tendon, antler, shell, or other calcified tissue? The second decision: if it is bone, is it human? The third: if human, which skeletal element does it belong to? The fourth: which region of that element? The fifth: which side?

At the coarsest level, cortical bone has a characteristic microstructure visible under a hand lens or at low magnification. The compact outer cortex has concentric rings (osteons or plexiform structures) visible in cross-section. The inner cancellous (trabecular) surface has a lattice-like structure. This immediately distinguishes bone from antler (which lacks a true cortex and has a spongy core with a thin periosteal layer) and from dense shell or mineralised tendon.

Thickness of the cortex relative to total cross-sectional diameter is a powerful discriminator between anatomical regions and between adult and sub-adult bone. Midshaft femoral cortex is thick (approximately 6 to 9 mm cortical wall on each side, with the medullary cavity occupying roughly one-third of the total diameter). Rib cortex is thin (2 to 3 mm on each side, with a substantial medullary space). Cranial vault bone has an outer table, a diploic cancellous layer, and an inner table, a sandwich structure immediately distinguishable in cross-section from long-bone cortex. Vertebral body cortex is very thin over a primarily trabecular interior. These thickness patterns allow assignment to an element class from even a small fragment with a visible cross-section.

The Owsley-Mann fragment identification protocol, documented across a series of papers by Douglas Owsley (Smithsonian Institution) and Robert Mann (US Army DPAA Central Identification Laboratory, now DPAA Offutt) in the early 1990s, systematised the macroscopic and low-magnification criteria for element identification from fragmented commingled remains, particularly in mass-grave and mass-disaster contexts. The protocol prioritises: (1) preserved articular surfaces, which carry unambiguous element-specific morphology; (2) muscle attachment ridges and fossae whose shape and position are element-specific; (3) cross-sectional shape at defined shaft positions; (4) cortical thickness and cancellous architecture at defined shaft positions. In the DPAA Central Identification Laboratory in Hawaii (which processes the remains of US military personnel missing from World War II, Korea, Vietnam, and subsequent conflicts, often from commingled aircraft-crash or mass-grave contexts), the Owsley-Mann protocol is the standard initial triage for each fragment before histology or DNA is requested.

In Indian casework, fragment identification from mass-disaster recovery has been applied at the Nithari serial killings case (Noida, 2006-2007), where commingled skeletal remains of multiple victims were recovered from a septic tank. The CFSL Kolkata team applied standard element-identification criteria to separate individual skeletal inventories from the commingled assemblage, using size, preservation pattern, and element-specific morphology before DNA profiling assigned fragments to individual victims.

The question of human versus non-human identity is the first gate of any forensic bone examination. No biological profile, no crime investigation, no court action should proceed until human identity is confirmed. In most forensic anthropology services, the majority of submissions are ultimately identified as non-human: deer bones in the UK countryside and Scotland; pig and bovid bones from butchery waste in urban India; bear and deer bones in North American wilderness recoveries; crocodile and large-fish bones in tropical riverine recoveries in Africa and South Asia.

Macroscopic criteria for human versus non-human discrimination operate across four domains: overall size and proportion, cortical bone architecture, articular surface morphology, and specific anatomical features that are human-unique.

Size and proportion: the human femoral shaft has a midshaft cross-section that is roughly oval with a mediolaterally flattened shape in the proximal third (platymeria) and more circular in the midshaft. A midshaft femoral diameter of 25 to 35 mm is consistent with adult human; a diameter above 40 mm suggests a large non-human mammal (horse, bovid, large deer). However, size alone is unreliable: a juvenile human femur overlaps dimensionally with an adult dog femur. Proportion (the ratio of cortical thickness to total diameter) is more discriminating.

Cortical bone architecture at the macroscopic level: human long-bone cortex in adults has a relatively thick wall (cortical-index above 0.35 in most elements) compared with many domesticated animals, whose bones have been selected for rapid growth and thus thinner cortex. However, bird bones (which are frequently submitted as "possible human" due to their thin, lightweight construction) are distinguished by their hollow medullary cavity with internal bony struts (pneumatic bones in many species) and by their extremely thin, nearly papery cortex. A human cortex fragment has a consistent thickness of several millimetres; a bird limb-bone fragment has cortex that is less than 1 mm thick in many species.

Plexiform bone is a diagnostic feature of rapid-growth large mammals (bovids, horse, deer): its characteristic ladder-like alternating layers of woven bone and lamellar bone, visible in cross-section without magnification as alternating pale and dark bands, are not found in human bone. Human bone is predominantly Haversian (osteonal) in the adult cortex, with no plexiform pattern. The presence of plexiform bone in a cortical cross-section is a reliable indicator of a large non-human mammal. This criterion is robust enough to be stated in court without histological confirmation in most jurisdictions, provided the cross-section is unambiguous.

Articular surface morphology: the shape and proportions of articular surfaces are highly element-specific and species-specific. The glenoid fossa of the human scapula is pear-shaped; the glenoid of a large dog is more circular. The human talus has a characteristic trochlear surface with medial and lateral facets that is highly specific; ungulate tali (deer, pig, bovid) have a deeply grooved trochlear surface adapted for their locomotor pattern. These articular-surface differences are reliably discriminating when the articular surface is preserved.

Specific human-unique features: the human cranial vault has three distinct layers (outer table, diploe, inner table) with a total thickness of 5 to 10 mm in most regions. No common domestic or wild animal produces a three-table cranial vault in this thickness range. A three-table bone fragment in this thickness range is virtually diagnostic for human, though large primates (gorilla, chimpanzee) would produce a similar pattern and should be excluded in zoo or wildlife-research contexts. The nuchal torus, the mastoid process morphology, and the shape of the zygomatic arch also have human-specific proportions that differ from common animals.

Histological examination of bone in thin cross-section under light microscopy is the most reliable method for distinguishing human from non-human bone when macroscopic criteria are ambiguous. The technique requires sectioning a small piece of the fragment (usually 5 to 10 mm of shaft), grinding or decalcifying it to approximately 50 to 100 micrometres thickness, mounting it on a slide, and examining it under polarised or transmitted light at 40x to 100x magnification.

Human osteons are characterised by a specific morphology. They are roughly circular in cross-section, ranging from 150 to 350 micrometres in mean diameter (most between 200 and 300 micrometres), with a central Haversian canal of 20 to 70 micrometres diameter. The lamellar structure (the concentric rings of collagen around the Haversian canal) is visible under polarised light as alternating bright and dark rings. The cement line (the boundary between the osteon and the surrounding interstitial bone) is smooth to slightly scalloped. Secondary osteons (those that have replaced earlier bone) have scalloped cement lines and slightly smaller diameters than primary osteons.

Non-human osteons differ systematically. Deer and bovid bone has elongated, elliptical osteons with irregular cement lines, reflecting the plexiform microstructure visible at lower magnification. Pig bone has a microstructure that is closer to human than most other domestic animals: its osteons are roughly circular and overlap dimensionally with human osteons. Pig bone is the most common cause of false positives in non-human histological examination, particularly from fragmentary cremated bone, which is why pig bone is used in forensic research as the best non-human proxy for human burning experiments. In a courtroom context, a histological opinion distinguishing human from pig bone on a cremated fragment requires a stronger qualification than distinguishing human from deer. Dog bone osteons are typically smaller and more irregularly arranged than human. Bird bone, in the cortical regions where it exists, may have osteons but they are much smaller and the overall pattern reflects avian bone microstructure, which is distinct.

Osteon population density (the number of complete osteons per square millimetre of cortex) correlates with age in the adult skeleton. The Kerley 1965 method and its subsequent revisions (including the Ahlqvist-Damsten 1969 revision and the Stout-Paine 1992 short-bone method) quantify osteon density, osteon fragmentation, and non-Haversian canal percentage across the cortex of a long-bone cross-section to estimate age at death. The method is most reliable for skeletons in the 20 to 60 year age range and carries a 95 per cent confidence interval of approximately 10 to 12 years on either side of the point estimate. In the UK, histological age estimation has been used in cold-case investigations where the skeleton is too degraded for morphological age estimation (the pubic symphysis and sternal rib ends are absent or damaged). In India, histological osteon-counting is practised at the AIIMS New Delhi department of forensic medicine and has been cited in court as a corroborating age indicator.

Foetal and perinatal bone fragments present a specific fragment-identification challenge that deserves separate treatment. In cases of alleged infanticide, neonatal death, or the discovery of buried or concealed neonatal remains (a category of case that is documented across India, the UK, the US, and across most legal systems, typically charged under laws governing concealment of birth, child destruction, or homicide depending on gestational age and circumstances), the analyst may receive small, fragile, and poorly preserved bone fragments that must be identified as specifically as possible.

The most common ambiguity in foetal skeletal identification involves rib fragments versus vertebral neural arch pieces. Both are thin, curved, and similarly sized in foetal and perinatal specimens. The distinguishing features are as follows: rib fragments have a consistent curvature in two planes (the rib curves in the transverse and the sagittal plane simultaneously), a smooth external convex surface, an internal surface with a costal groove along the inferior border (visible with a hand lens), and a cortex that, in cross-section, is plano-convex. The vertebral neural arch is flattened in cross-section, has two distinct cortical surfaces (periosteal and articular), and often shows the beginning of the articular facet morphology at the preserved ends. The neural arch is also slightly more opaque and denser in section than a rib fragment of similar size.

Long-bone diaphyseal fragments in foetal remains are typically thin-walled and cylindrical, with a smooth periosteal surface and a proportionally wide medullary canal relative to the thin cortex. These are distinguishable from rib fragments by their circular cross-section. Skull fragments in foetal remains are incompletely ossified and often show the granular woven bone texture of intramembranous ossification at the margins, with a thin and single-layered table (the three-table diploe structure does not develop until childhood).

In mass-grave or charnel-pit contexts that contain both adult and foetal remains (a combination seen in historical burial grounds, in disaster recoveries, and occasionally in concealed burial sites in criminal cases), the challenge is compounded by differential preservation: foetal cortical bone dissolves faster in acidic soil than adult cortical bone, so foetal elements may survive only as calcium phosphate staining in the soil profile while adult elements are still structurally recognisable. The analyst should document the presence of foetal-bone staining or mineral deposits even when no structural fragment is recovered.

1. Step 1: Is it bone?
   Examine the fragment under a hand lens. Look for the characteristic cortical-cancellous structure or Haversian canal pattern. Rule out antler (spongy core, no true cortex), mineralised tendon (parallel fibre bundles, no osteons), and dense shell (lamellar but no Haversian canals).
2. Step 2: Is it human?
   Assess cortical thickness, overall proportions, and plexiform bone presence. Thick, round-section cortex without plexiform layers is consistent with human. Plexiform ladder-banding excludes human. Very thin cortex (under 1 mm) with internal struts indicates bird. If ambiguous, proceed to Step 3.
3. Step 3: Histological section if macroscopic is inconclusive
   Request thin-section histology. Human osteons are 200-300 µm, roughly circular, with a smooth cement line. Deer/bovid osteons are elongated. Pig osteons are circular but differentiated by overall pattern. Document uncertainty: 'consistent with human' is appropriate when histology supports but does not exclude pig.
4. Step 4: Element identification from preserved landmarks
   Articular surface: element-specific. Muscle attachment sites: element-specific. Cross-sectional shape at defined shaft positions: oval vs circular vs plano-convex. Cortical thickness ratios: thick-walled (femur) vs thin-walled (rib) vs sandwich (cranium). Assign to element class; record confidence level.
5. Step 5: Side determination from asymmetric features
   Apply the element-specific siding criteria. For the clavicle: conoid tubercle and costal tuberosity are inferior surface landmarks; sternal end is pyramidal. For the femur: linea aspera is posterior, head is medially directed, medial condyle is longer. For the os coxae: hold with crest superior; acetabulum faces laterally; pubic symphysis medial and anterior.
6. Step 6: Age (adult vs sub-adult) and minimum number of individuals
   Check for epiphyseal fusion lines (sub-adult indicator), cortical remodelling degree (higher in older adults), and bone density (lower in elderly). If multiple same-element same-side fragments of the same or different age class are present, the minimum number of individuals (MNI) increases accordingly.

When macroscopic and histological assessment does not produce a confident identification, two further lines of evidence are available: plain radiography and DNA profiling. A third option, geometric morphometric comparison to a reference skull or long-bone dataset using landmark coordinates, is available in specialist laboratories with the requisite software (FORDISC 3.1, MorphoJ, or geometric morphometric pipelines) and is covered in the emerging methods module.

Plain radiography of a fragment reveals internal architecture that may not be visible on the surface: the trabecular pattern of cancellous bone, the thickness profile of the cortex across the full diameter, any pathological changes (lytic lesions, sclerosis, periosteal reactions), and the presence of any residual growth plate or epiphyseal scar. Radiographic comparison to a reference atlas (such as the Spalteholz photographic atlas or the Hamann-Todd collection radiographic archive at the Cleveland Museum of Natural History) can resolve human-vs-non-human ambiguities in some cases. In the UK, the National Crime Agency forensic anthropology consultant network uses radiographic assessment as a standard second tier for ambiguous submissions, and the Faculty of Forensic and Legal Medicine recommends it in its guidance on bone identification consultations.

The 2005 McCann scene in Praia da Luz, Portugal (the disappearance of Madeleine McCann), is frequently cited in forensic anthropology teaching as a case where animal bones from the excavation of a search area temporarily complicated the investigation. The bones, identified through macroscopic and histological examination as non-human, required professional osteological assessment before the investigation could continue. The incident illustrates that forensic anthropology consultations on the human-vs-non-human question are not limited to laboratory settings: they arise at search scenes when investigators recover fragments and need immediate guidance.

DNA profiling from a fragment provides the definitive answer to the human-vs-non-human question. Extraction of nuclear DNA and PCR amplification using human-specific primers (STR multiplex kits calibrated only on human loci) will not produce a profile from non-human bone. A partial profile that matches no known reference sample (human DNA present but not identified) still confirms the material is human. Mitochondrial DNA, because it exists in thousands of copies per cell, is more likely to be recovered from degraded fragments than nuclear DNA and can be typed for a human species-specific sequence that definitively excludes animal origin. In India, the DNA profiling services at the CFSL New Delhi, CFSL Kolkata, and the FSL Hyderabad all maintain the capability to confirm human origin from fragmentary bone, and this confirmation is routinely sought in cases where the forensic anthropologist's macroscopic and histological assessment is qualified as "consistent with human but not confirmed."

In reported case series from the US (Tersigni-Tarrant and Shirley 2013 review of JPAC/DPAA casework) and the UK (Buck 2019 review of Home Office forensic anthropology submissions), the rate of initially submitted "possible human" fragments that are ultimately confirmed as non-human ranges from 50 to 80 per cent across different submission contexts. This base rate has an important implication for the sequencing of forensic response: macroscopic and histological assessment by a forensic anthropologist should precede DNA profiling, because it eliminates the majority of non-human submissions without consuming limited and expensive DNA laboratory capacity.