ASTM E3316-22

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3316 − 22 An American National Standard
Standard Guide for
Forensic Examination of Hair by Microscopy
This standard is issued under the fixed designation E3316; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope E1732Terminology Relating to Forensic Science
E2917Practice for Forensic Science Practitioner Training,
1.1 This guide covers procedures used by forensic labora-
Continuing Education, and Professional Development
tory personnel in the forensic examination of hair by
Programs
microscopy, including microscopical comparisons and classi-
fication of hair samples. 2.2 Other Standards:
ISO 17025Testing and calibration laboratories
1.2 This guide addresses instrument setup, hair collection,
sample handling techniques, and the use of various micro-
3. Terminology
scopes in the examination and comparison of hair.
3.1 Definitions—For definitions of terms used in this guide,
1.3 This guide addresses the benefit of following micro-
refer to Terminology E1732.
scopical examinations with DNA analysis.
3.2 Definitions of Terms Specific to This Standard:
1.4 This standard is intended for use by competent forensic
3.2.1 anagen,n—theactivegrowthphaseofahairfolliclein
science practitioners with the requisite formal education,
the hair growth cycle.
discipline-specific training (see Practice E2917), and demon-
strated proficiency to perform forensic casework. 3.2.1.1 Discussion—The root from a pulled anagen hair is
elongated and is usually fully pigmented (1).
1.5 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
3.2.2 ancestral group, n—a biogeographic designation of
responsibility of the user of this standard to establish appro-
human populations (for example, Asian, African, European)
priate safety, health, and environmental practices and deter-
whose hair can share similar morphological and microscopic
mine the applicability of regulatory limitations prior to use.
traits.
1.6 This international standard was developed in accor-
3.2.2.1 Discussion—The racial terms Caucasoid,
dance with internationally recognized principles on standard-
Mongoloid, and Negroid should not be used as these terms are
ization established in the Decision on Principles for the
nolongeracceptableinthefieldofanthropology(thefieldfrom
Development of International Standards, Guides and Recom-
which these designations originated) (2).
mendations issued by the World Trade Organization Technical
3.2.3 association, inclusion, n—the result of a comparison
Barriers to Trade (TBT) Committee.
between two hair samples in which the characteristics of the
questioned hair are present in the known sample without any
2. Referenced Documents
exclusionarydifferences,andthereforethedonoroftheknown
2.1 ASTM Standards:
hair sample can be included as a possible source of the
E620Practice for Reporting Opinions of Scientific or Tech-
questioned hair.
nical Experts
3.2.3.1 Discussion—A microscopical association of hair
E1459Guide for Physical Evidence Labeling and Related
cannot identify the definitive source of a questioned hair to the
Documentation
exclusion of all others, and the number of individuals who
E1492Practice for Receiving, Documenting, Storing, and
could be included as a possible donor of a specific hair is
Retrieving Evidence in a Forensic Science Laboratory
unknown and cannot be reliably estimated.
3.2.4 buckling, n—an abrupt change in the shape and
orientation of a hair shaft with or without a slight twist.
ThistestmethodisunderthejurisdictionofASTMCommitteeE30onForensic
Sciences and is the direct responsibility of Subcommittee E30.01 on Criminalistics.
Current edition approved May 1, 2022. Published October 2022. DOI: 10.1520/
E3316-22.
2 3
For referenced ASTM standards, visit the ASTM website, www.astm.org, or Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM 4th Floor, New York, NY 10036, http://www.ansi.org.
Standards volume information, refer to the standard’s Document Summary page on Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
the ASTM website. this standard.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3316 − 22
3.2.5 catagen, n—the transitional phase of the hair follicle hair sample, and therefore the donor of the known sample
betweentheactivegrowthphase(anagen)andtherestingphase cannot be included as a possible source of the questioned hair.
(telogen) in the hair growth cycle (1). 3.2.17.1 Discussion—This result is reached in a compara-
tive hair examination when differences are noted in the
3.2.6 classification, n—the systematic arrangement of hairs
macroscopic or microscopic characteristics between the ques-
into categories (for example, human, animal, somatic origin,
tioned and known hairs; however, the differences are insuffi-
ancestry) based on shared traits.
cientforanabsoluteexclusionofapersonasapossiblesource.
3.2.7 cortex, n—the primary anatomical region of a hair
This could be due to the natural variation that occurs in hairs
between the cuticle region and the medullary region composed
as a biological specimen, the effect that time or environment
of elongated and fusiform cells.
can have upon a hair, or the reference sample does not capture
3.2.8 cortical fusi, n—small air spaces that form between
the complete variation of the individual’s hair.
the cortical cells in the hair shaft and under transmitted light
3.2.18 exclusionary difference, n—a difference in a feature
appear as tiny, dark structures.
or property between compared items that is substantial enough
3.2.9 cortical texture, n—the relief or definition of the
to determine that they did not originate from the same source.
margins of the cortical cells when viewed using transmitted
3.2.19 follicular tag, n—tissue from a hair follicle that is
light microscopy.
still attached to the root end of a hair which has been forcibly
3.2.10 cross-sectional shape, n—the shape of a hair shaft removed.
when cut at a right angle to its longitudinal axis.
3.2.20 fungal tunneling, n—air pockets in a hair shaft
3.2.10.1 Discussion—When viewed longitudinally with
caused by fungal growth.
transparent light, the apparent cross-sectional shape is deter-
3.2.21 imbricate, n—a term that describes a scale pattern in
mined by slowly focusing through the hair (optical cross-
which the scales overlap and the edges have an irregular wavy
sectioning). When viewed longitudinally between crossed
pattern; this pattern is typical of human hair.
polars, the cross-sectional shape can be determined by observ-
3.2.22 inconclusive, n—the result of a comparison between
ing the interference colors.
two hair samples in which similarities and differences were
3.2.11 cuticle, n—the outermost region of a hair composed
observedinthecharacteristicsoftheprovidedstandardandthe
of layers of overlapping scales.
questioned hair to the extent that the known source of the
3.2.11.1 Discussion—The dimension of the cuticle as mea-
standardcouldnotbeincludedorexcludedasapossiblesource
sured from its outer margin to the cortex is often described in
of the questioned hair.
relative terms (for example, thin, medium, thick).
3.2.23 individualization, n—a term indicating an individual
3.2.12 cuticle, cracked, n—a cuticle with linear breaks that
can be discriminated to the exclusion of all other sources.
are perpendicular to the length of the shaft.
3.2.23.1 Discussion—Hairs cannot be individualized by
3.2.13 cuticle, looped, n—afeatureinwhichthedistaledges
means of microscopical hair comparison.
of the cuticular scales are curved away from or cupped toward
3.2.24 inner cuticle margin, n—the border between the
the hair shaft.
cortex and the visible cuticle.
3.2.14 cuticle, serrated, n—a cuticle in which the outer
3.2.25 keratin, n—a class of sulfur-containing fibrous pro-
margin has a notched appearance like a saw blade.
teins that forms the foundation of outgrowth tissue from the
3.2.15 decompositional changes, n—alterationintherootor epidermis, such as hair, nails, feathers, and horns (1).
the proximal end of a hair that can include discoloration,
3.2.26 medulla, n—the core of the hair shaft that is com-
postmortem root banding, or a tapered or brush-like appear-
posed of vacuoles and cells that can be air- or fluid-filled.
ance as well as fungal tunneling along the length of the shaft.
3.2.26.1 Discussion—The medulla (if present) occurs in a
continuous, discontinuous, or fragmented pattern along the
3.2.16 exclusion/elimination, n—the result of a comparison
between two hair samples in which exclusionary differences length of a hair and appears translucent or opaque.
are observed in the characteristics of the questioned hair that
3.2.27 monilethrix,n—ahairdisorderthatresultsinperiodic
are not present in the known hair sample, and therefore the
nodes or beading along the length of the hair with intervening,
donor of the known sample can be excluded as a possible
tapering constrictions that are not medullated.
source of the questioned hair.
3.2.28 ovoid bodies, n—oval-shaped, heavily-pigmented in-
3.2.16.1 Discussion—This result is reached in a compara-
clusions usually found in the hair cortex.
tive hair examination when exclusionary differences (for
3.2.29 pigment aggregation, n—clusters of pigment gran-
example,color,characteristicsindicativeofancestry)arenoted
ules.
in the macroscopic or microscopic characteristics between the
questionedandknownhairs.Inthesecircumstances,thesource 3.2.30 pigment density, n—the relative abundance of pig-
of the known hairs, as represented by the known sample, is ment granules in the hair cortex as described along a con-
eliminated as a possible source of the questioned hair. tinuum (for example, sparse, medium, heavy).
3.2.17 exclusion with limitations, n—the result of a com- 3.2.31 pigment distribution, n—the pattern or arrangement
parison between two hair samples in which the characteristics of the pigment granules in the hair shaft, such as uniform,
of the questioned hair differ from those present in the known peripheral, one-sided, variable, or central.
E3316 − 22
3.2.32 pigment granules, n—small particles in hair com- 3.2.47 trichonodosis, n—a condition characterized by ap-
posed of melanin that impart color. parent or actual knotting of the hair.
3.2.32.1 Discussion—Melaninisanaturalpigmentofwhich
3.2.48 trichoptilosis, n—a condition characterized by longi-
two forms, eumelanin (brown to black) and phaeomelanin
tudinal splitting or fraying of the hair shaft.
(reddish brown to yellow), determine the color of human and
3.2.49 trichorrhexis invaginata, n—a genetic disease char-
animal hair.
acterized by a segment of bulbous, dilated hair enfolded into a
3.2.33 pili annulati, n—a hair disorder causing hairs to concave hair terminal, recalling the appearance of a bamboo
appear ringed or banded due to the alternating light and dark
node; if the hair breaks at the bulbous end, the hair has a
bands in the hair shaft; the dark bands are a manifestation of “golf-tee” shaped end.
abnormal air spaces in the cortex.
3.2.50 trichorrhexis nodosa, n—a condition characterized
by the formation of nodes; the hair is weaker at the node and
3.2.34 pili torti, n—a hair disorder characterized by the hair
subject to breakage.
shaft being flattened and twisted 180 degrees numerous times
alongitsaxis;itisusuallyfoundatirregularintervalsalongthe
3.2.51 trichoschisis,n—aconditioninwhichthehairreadily
shaft.
breaks or splits along transverse cracks.
3.2.35 postmortem root banding, n—the appearance of an
4. Significance and Use
opaque band near the root/proximal end of a hair potentially
4.1 Amicroscopical hair examination is conducted to deter-
observed in anagen or catagen hairs that have been removed
mine if the item is a hair; from a human; from a particular
from a decomposing body; the possibility of other conditions
somatic region; characteristic of a broad geographically-
causing the same or similar characteristics cannot be elimi-
assigned ancestral group; characteristic of a particular growth
nated (3).
phase; damaged; symptomatic of disease, condition, or disor-
3.2.36 root, n—the structure that anchors a hair to a follicle
der; forcibly removed; chemically altered (for example, dyed
and from which cells divide and produce the hair shaft.
or bleached); suitable for microscopical comparison; suitable
3.2.36.1 Discussion—The portion of follicular tissue sur-
for DNA analysis; and similar to or different from a known
rounding a root structure is the sheath.
sample (4-9).
3.2.37 sample, known, n—asampleforwhichtheidentityof
4.2 Mostoften,hairsfromtheheadandpubicregionsofthe
the donor is established and which is used for comparison
body are used for microscopical comparisons.There is usually
purposes.
moreinterpersonalvariabilityinthecharacteristicsofheadand
pubic hairs than in the hairs from other somatic regions. Head
3.2.38 sample, limited, n—a sample of known hairs that is
hairs usually show more interpersonal variation than pubic
insufficient in quality or quantity to reflect a representative
hairs.Hairsfromothersomaticregionsmayalsobecompared,
range of characteristics or traits.
but these comparisons are usually limited and less frequently
3.2.39 sample, representative, n—a collection of hairs from
conducted.Accordingly, this guide primarily considers human
a specific somatic region that reflects the range of characteris-
head and pubic hair comparisons.
tics of a person’s hair in that area.
4.3 Microscopical hair comparisons are not a means of
3.2.40 scales, n—overlapping, plate-like structures com-
individualization (10). This limitation is to be stated in any
posed of keratin that form the cuticle.
communication (for example, reports, testimony) when an
3.2.41 shaft, n—the portion of the hair emerging from the association is reported.
hair follicle.
4.4 Additionalanalysescanbeperformedonhairsthathave
been chemically altered (for example, dyed hair) or have trace
3.2.42 shaft form, n—the shape of the hair both longitudi-
materialsonthesurface(forexample,glitter).Suchtechniques
nally (for example, curly, straight) and cross-sectionally (for
example, round, flattened). are beyond the scope of this document.
3.2.43 shaft thickness, n—the diameter of the hair.
5. Summary of Guide
3.2.43.1 Discussion—This is expressed either numerically
5.1 This guide includes a summary of techniques for col-
or in relative terms, such as fine, medium, or coarse.
lecting hair samples, a description of the instrumentation used
in the microscopical examination of hair, a description of the
3.2.44 shouldering, n—avariationofthehairformalongthe
shaft, resulting in an irregular and often asymmetrical change microscopical examination procedure, a section on interfacing
of cross-sectional shape. with subsequent DNA analysis of hair, and examples of the
types of microscopical hair examination results. Interpretation
3.2.45 somatic region, n—anareaofthebody,suchashead,
and significance will be addressed in more depth in a separate
pubic, or leg; synonymous with “body area”.
document for trace evidence.
3.2.46 telogen,n—therestingphaseofthehairfollicleinthe
6. Sample Collection
hair growth cycle.
3.2.46.1 Discussion—During this phase, the hair has 6.1 Questioned Sample:
stoppedgrowingandtherootbecomeskeratinizedandbulbous 6.1.1 Loose hairs are collected from an object by picking
(club-like) in shape (1). them off individually. Embedded hairs or hairs adhering to a
E3316 − 22
personorobjectareinspectedpriortoremovalandthelocation 6.2.3.2 A known pubic hair sample or a sample from any
from which these hairs are recovered is documented. Use other somatic region is recommended to consist of at least 25
hairs obtained by a combination of pulling and combing from
caution when interpreting the significance of the location from
the sampled region.
whichthehairwasrecoveredsincehairscouldberedistributed
6.2.4 Acomparisoncanstillbeperformedwithlessthanthe
on an item or among items that are packaged together. Take
recommendednumberofhairs,butthiscanincreasethechance
care not to contaminate, crush, or break the hairs.
of a false exclusion.
6.1.2 Hairs are collected from clothing, bedding, or other
large surfaces using adhesive lifts, scraping, or vacuuming. Be
7. Microscopes
aware that the adhesive from the lifting material can interfere
with the examination of surface treatments that might be 7.1 Stereomicroscope:
present on the hairs. Vacuuming or adhesive lifting can be 7.1.1 Because of the increased working distance and ability
useful in collecting hairs from larger areas or specific areas to observe a sample in three dimensions, a stereomicroscope
with a magnification range of 10× to 60× or greater is used for
such as carpets, vehicle seats, etc.
the initial examination of unmounted and mounted hairs.
6.1.3 Anew comb is used when retrieving evidence from a
person’s head or pubic region. Place a clean piece of paper, to
7.2 Transmitted Light Microscope:
catch loose hairs and debris, under the area to be combed and
7.2.1 It is necessary to examine the microscopic character-
include the paper in the evidence package with the comb.
istics of hairs with a high-quality compound transmitted light
microscope with a range of objectives permitting observations
6.1.4 Since hairs can be very small, package them in a
from approximately 40× to 400×. Quality objectives are
mannerthatpreventstheirloss(forexample,inapaperfold,on
important, but highly corrected plan apochromats are not
a sticky note, on a gel lifter).
necessary (12).
6.2 Known Sample:
7.2.2 A polarized light microscope can enhance the ability
6.2.1 Known hairs are collected from specific somatic
toseecertainfeaturesanddeterminethecross-sectionalshapes
regions of relevant people for comparison to questioned hairs.
of the hairs.
These hairs are collected as soon as possible relative to the
7.2.3 Equip the microscope with a high-intensity light
occurrence of the crime. Hairs collected a period of time after
source, suitable for photomicrography, and a daylight correc-
the occurrence of the crime can appear different than they did tion filter (if necessary).
at the time of the crime since hairs can change due to a variety
7.3 Comparison Microscope:
of factors (for example, age, health, environment, cosmetic
7.3.1 Acomparisonmicroscope,consistingoftwotransmit-
treatments).
ted light microscopes joined by an optical bridge, is used for
6.2.2 Obtain at least 25 full-length hairs with roots for
microscopical hair comparisons.
examination and comparison.
7.3.2 Balance both sides of a comparison microscope for
6.2.2.1 Because the majority of pulled hairs are in the light intensity and color and check the magnification.
anagen stage, a separate combing procedure is used to obtain
7.4 Microscope Maintenance:
hairs in the telogen stage (7, 11). The regions being sampled
7.4.1 Be familiar with the instruction manual and the
are repeatedly combed or brushed over a large sheet of clean
manufacturer’s maintenance recommendations for each micro-
paper. The known pulled hairs and combed hairs should be
scope used in hair examination.
packaged separately.
7.4.2 The following maintenance procedures are performed
6.2.2.2 When applicable, pick, tape-lift with a low adhesive
on microscopes routinely used to ensure their precision,
tape (for example, sticky-note), or comb for foreign hairs or
reliability, and performance:
other trace debris (for example, fibers, paint, glass) prior to
7.4.2.1 Dust, oil, and dirt is cleaned from the optics in
combing for known hairs. accordance with the manufacturer’s recommendations.
7.4.2.2 The external surfaces are cleaned.
6.2.3 Different hairs from the same somatic region of a
7.4.2.3 The optical alignment is checked and realigned, if
person can exhibit variation in microscopic characteristics and
necessary,toestablishKöhlerormodifiedKöhlerillumination.
features.Therefore,itisimportanttoobtainasufficientnumber
7.4.2.4 When not in use, cover with a dust cover.
of hairs from the somatic region of interest in order to attempt
7.4.2.5 If the microscope cannot be cleaned or aligned,
to adequately represent the range of variation in all character-
discontinue use until the microscope is repaired.
istics present. If the variation is large (for example, color,
7.4.2.6 All service and repairs are recorded in a log;
length), it becomes necessary to obtain more hairs.
however,routinecleaningandaligningofthemicroscopeneed
6.2.3.1 Aknown head hair sample is collected from the five
not be recorded in the log.
differentareasofthehead(top,front,backincludingnape,and
both sides). Known head hairs are obtained by a combination
7.5 Calibration of the Eyepiece Reticle:
ofpullingandcombingfromthesampledregion.Inanattempt
7.5.1 Ifmeasurementsaretobemade,amicroscopewithan
toobtainthefullrangeofcharacteristicswithinanindividual’s
eyepiece reticle calibrated to a stage micrometer is used. The
head hair, it is recommended to sample at least 25 hairs from
steps for calibrating the eyepiece reticle with a stage microm-
the head (7, 11). eter are listed below.
E3316 − 22
7.5.1.1 Place a stage micrometer with a linear scale of Generally, stereomicroscopy is used to initially assess the
known dimensional divisions on the stage of the microscope. suitability of hairs for comparison, determine the presence of
7.5.1.2 Focus on the dividing lines of the stage micrometer. other trace materials, and evaluate which hairs have roots
7.5.1.3 Align the scale in the eyepiece reticle with the scale suitable for nuclear DNA analysis (5, 6).
8.2.2 Prior to mounting, some internal properties of hairs
on the micrometer.
7.5.1.4 Determine the number of reticle divisions that equal can also be examined using a transmitted-light stereomicro-
scope.
a defined increment of the stage micrometer.
7.5.1.5 Dividethenumberofstagemicrometerdivisions(in
8.3 Mounting:
microns) by the number of ocular micrometer divisions to
8.3.1 A colorless, non-yellowing, non-reactive mounting
obtain microns per ocular division.
medium with a refractive index close to that of a hair (1.54) is
7.5.1.6 Repeat this procedure for each objective used.
used to view hairs in transmitted light (5, 8, 15). The exami-
nation of surface particulates and biological material, compat-
7.6 Magnification Check:
ibilitywithDNAanalysis,easeofartifactisolation,andstorage
7.6.1 The magnification of the comparison microscope is
needs will determine the mounting medium selected.
checked using a known reference material to ensure uniform
8.3.1.1 Hairs that are to be compared are mounted in the
magnification between the left and right fields of view. If the
same type of mounting medium.
magnification is not the same, request matching objectives
8.3.2 One hair or multiple hairs from the same source are
from the manufacturer.
mounted on a glass microscope slide with a cover slip so that
7.7 Color Balance:
each mounted hair is clearly visible. Each slide is labeled as to
7.7.1 The color balance of the comparison microscope is
the source of the hair(s).
checkedtoensureuniformcolorbetweenleftandrightfieldsof
view.Ifthecolorbalanceisnotacceptable,discontinueuseand
9. Hair Characteristics and Other Determinations
correct the problem. The color balance can be checked by the
9.1 Human or Other Animal Origin:
following procedure:
9.1.1 Human hair can typically be distinguished from other
7.7.1.1 Cut a uniformly-colored semi-transparent sample
animal hair by examining the following features: scale pattern,
(that is, fibers) in half and mount on two separate slides.
shaft form, medulla width, medulla appearance/form, pigment
7.7.1.2 Place one slide on the left stage and the other slide
distribution, color banding, and root shape (5, 6, 16).
on the right stage of the comparison microscope.
7.7.1.3 Compare the color of the images.
9.2 Somatic Origin:
7.7.1.4 If the color is balanced, the sample images and the
9.2.1 Somatic origin types include head, pubic, facial, limb,
background color on both sides will appear to be the same.
trunk, and eye hairs. The somatic origin of human hair can
7.7.1.5 If the color is not balanced, correct the problem or
usually be established by considering features such as length,
contactthemicroscopemanufacturerforinstructionsonhowto
hair diameter, cross-sectional shape, shaft form, medullary
balance the microscope for color.
morphology, cortical texture, appearance of the distal end, and
appearance of the root (5, 11).
8. Evidence Handling and Sample Preparation
9.3 Characteristics of Ancestry:
8.1 Evidence Handling:
9.3.1 Features used to classify a hair as having characteris-
8.1.1 Evidence is handled in accordance with ASTM stan-
tics common among particular ancestra
...