Study guide
9.6 Serology, Bloodstain Pattern, and Tool Mark Evidence Collection
ABMDI Board Certification Fellow
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1 Chapter 1: Performance Section Mastery & Medicolegal Scene Documentation
1.1 Board Exam Architecture, Scoring, and the Performance Section 1.2 Professional Scene Narrative Drafting & Medical Terminology 1.3 Strategic Investigative Interviewing for Witnesses and Suspects 1.4 Physical, Biological, and Trace Evidence Identification & Prioritization 1.5 The Advanced Skills List: The Official 46-Task Blueprint
2 Chapter 2: Legal Framework, Jurisdiction, and Interagency Coordination
2.1 Medicolegal Systems in the United States & Statutory Authority 2.2 Jurisdictional Boundaries & Federal Interagency Liaison 2.3 Accreditation Standards (NAME, ANAB/ISO 17020, IAC4MR) & Office Operations 2.4 Health Information Privacy (HIPAA § 164.512(g)), Public Records, and Subpoenas 2.5 Reviewing a Death Certification: Cause, Manner, and the Certifier’s Role 2.6 Unique Case Problems and Soliciting Legal Opinions
3 Chapter 3: Scene Leadership, Command Structure, and Processing Protocols
3.1 Incident Command System (ICS) Integration & Scene Leadership 3.2 OSAC ASB Standard 037 Protocols & Scene Security Management 3.3 Forensic Photography, Oblique Lighting, 3D Laser Scanning & Mapping 3.4 Search Methodologies, PPE Tiers & Cross-Contamination Controls 3.5 Masqueraded and Staged Death Scenes 3.6 Media-Sensitive Deaths and Public Information Management
4 Chapter 4: Postmortem Changes, Taphonomy, and PMI Estimation
4.1 Early Postmortem Changes: Algor, Livor, and Rigor Mortis Kinetics 4.2 Decomposition Chemistry, Autolysis, Putrefaction & Specialized Taphonomy 4.3 Forensic Entomology, Insect Succession & ADH/ADD Calculations 4.4 Forensic Anthropology, Skeletal Recovery & Biological Profiling
5 Chapter 5: Mechanical Trauma: Blunt, Sharp, and Ballistic Injuries
5.1 Blunt Force Trauma: Abrasions, Contusions, Lacerations & Skeletal Fractures 5.2 Sharp Force Trauma: Incised, Stab, Chop Wounds & Defensive Patterns 5.3 Firearm Injuries, Range of Fire Determinations & Terminal Ballistics 5.4 Motor Vehicle, Pedestrian Strike & Transportation Incident Dynamics 5.5 Explosion and Blast Injury Deaths
6 Chapter 6: Asphyxial, Environmental, and Thermal Deaths
6.1 Asphyxial Deaths: Hanging, Ligature/Manual Strangulation & Positional Asphyxia 6.2 Drowning Mechanisms, Submersion Injuries & Aquatic Recovery 6.3 Fire Deaths, Inhalation Injuries, COHb/Cyanide & Heat Alterations 6.4 Environmental Extremes: Hypothermia, Hyperthermia, High/Low Voltage Electrical & Lightning Deaths 6.5 Weather-Related and Natural-Disaster Deaths
7 Chapter 7: Forensic Toxicology, Specimen Protocols, and Natural Deaths
7.1 Postmortem Specimen Collection Sites, Anticoagulants & Chain of Custody 7.2 Postmortem Redistribution (PMR), Vd & Toxicological Pharmacokinetics 7.3 Opioids, Fentanyl Analogs, Novel Psychoactive Substances & Screening Limits 7.4 Sudden Unexpected Natural Deaths: Cardiovascular, Cerebrovascular & Pulmonary 7.5 Deaths from Therapeutic Complications and Medical Misadventure 7.6 Deaths from Infectious Causes and Public Health Reporting
8 Chapter 8: Pediatric, Infant, and Vulnerable Population Investigations
8.1 Sudden Unexpected Infant Death (SUID/SUDI) Protocols & CDC Doll Re-enactment 8.2 Abusive Head Trauma (AHT), Non-Accidental Skeletal Injuries & Scalds 8.3 Elder Abuse/Neglect, In-Custody Deaths & Excited Delirium/Restraint Analysis 8.4 Deaths in Extended Care Facilities and Group Homes 8.5 Deaths Caused by Animals and Postmortem Animal Activity
9 Chapter 9: Mass Fatality Management, Ethics, and Expert Testimony
9.1 Mass Fatality Incidents, DMORT Operations & DVI Scientific Identifications 9.2 Family Assistance Centers, Antemortem Records & OSAC ASB Std 038 Next-of-Kin Notifications 9.3 ABMDI Code of Ethics, Supervisory QA Audits & Investigator Resiliency 9.4 Medicolegal Testimony & Evidence Admissibility 9.5 Leadership: Mentoring, Correcting Deficiencies, and Personal Performance Standards 9.6 Serology, Bloodstain Pattern, and Tool Mark Evidence Collection
9.6 Serology, Bloodstain Pattern, and Tool Mark Evidence Collection
Key Takeaways Explain This Topic AI
Presumptive blood tests such as Kastle-Meyer and leucomalachite green detect peroxidase activity and give false positives with plant material, some metals, and oxidizing agents, so a positive presumptive result means "possibly blood" and never "human blood".
Species and identity require confirmatory work: crystal tests confirm blood, immunochromatographic hemoglobin tests indicate human origin, and DNA establishes source.
Bloodstain evidence is collected as both a stain and a pattern: the analyst needs scaled, perpendicular, overlapping photographs with roadmapping, not only a swab.
Impact angle is calculated as the arcsine of the ratio of stain width to stain length, and directionality is indicated by the tapered or scalloped end of the stain.
A suspected tool is never fitted into a tool mark; the mark is photographed with a scale under oblique lighting and cast with a silicone casting material, and the tool is collected and submitted separately.
Last updated: September 2026
9.6 Serology, Bloodstain Pattern, and Tool Mark Evidence Collection
Every task in the Advanced Forensic Science Knowledge section of the Advanced Skills List is phrased as collect : collect data or material for serology, collect data or material on tool marks, collect trace evidence. That phrasing is the exam's guidance. The investigator is not the analyst, and items in this section test what you preserve and how , not what conclusion you draw.
- Serology at the Scene
- Presumptive testing
Presumptive tests screen for the peroxidase-like activity of hemoglobin . They are sensitive, fast, and cheap, and they are not specific.
- Test Reagent basis Positive result
- Kastle-Meyer Phenolphthalein Bright pink
- Leucomalachite green Leucomalachite green Blue-green
- Luminol Luminol, sprayed in darkness Blue chemiluminescence
- Fluorescein Fluorescein, viewed with alternate light and goggles Fluorescence
- Hemastix Tetramethylbenzidine strip Color change
False positives are the tested point. Plant peroxidases — horseradish, potato, turnip, many vegetables — react. Some metals , notably copper and iron salts, react. Oxidizing agents , especially bleach , react, which matters because bleach is exactly what someone cleaning a scene uses. Luminol in particular reacts with bleach, with some rust and metal surfaces, and with certain plant material, so a luminol-positive floor is a lead, not a finding.
A positive presumptive test supports only that the stain may be blood . It does not establish that it is blood, that it is human, or that it is the decedent's.
Confirmatory and species testing
Crystal tests — Takayama (hemochromogen) and Teichmann (hemin) — confirm the presence of blood by producing characteristic crystals under microscopy
Immunochromatographic hemoglobin tests (for example, cards that detect human hemoglobin) indicate human origin, with the known caveat of cross-reaction with some higher primates and ferrets
- DNA establishes source, and is the only method that individualizes
- Other biological fluids
Semen — acid phosphatase as a presumptive screen, p30 / prostate-specific antigen as a confirmatory human-semen indicator, and microscopic identification of spermatozoa
- Saliva — amylase-based screening
- Urine — creatinine and urea screening
Alternate light examination causes many biological stains to fluoresce, which is a locating technique, not an identification
- Collection
- Wet stains : absorb onto a sterile swab, then air dry completely before packaging
- Dry stains on a movable item : collect the whole item where possible
Dry stains on a fixed surface : double-swab technique — a swab lightly moistened with sterile water followed immediately by a dry swab over the same area
- Always collect a substrate control from an unstained area of the same surface
- Package in breathable paper — never sealed plastic, which traps moisture and degrades DNA
Change gloves between items, use single-use or cleaned instruments, and wear a mask to prevent the collector's own DNA from reaching the sample
Bloodstain Patterns: What the Investigator Documents
Bloodstain pattern analysis is a specialist discipline, and the investigator's contribution is documentation that preserves the pattern for someone else to interpret. A swab destroys the very thing the analyst needs.
Terminology
Current terminology organizes patterns by mechanism:
Passive / gravity-driven : drip stains, drip trails, flow patterns, pools, saturation stains, and transfer stains (contact stains, including wipe and swipe patterns).
Spatter : impact patterns from a force applied to a liquid blood source; cast-off patterns from blood flung from a moving object; expirated patterns from blood forced from the airway, often with bubble rings and dilution; arterial patterns with surge or spurt morphology; and drip and satellite spatter.
Altered stains : void patterns where an object blocked deposition and was subsequently removed, clotted stains, diluted stains, insect stains (which frequently mimic spatter), sequenced stains showing overlay order, and stains altered by wiping or by decomposition fluid .
Two of these carry disproportionate investigative weight. A void tells you an object or person was present and was removed — its shape may identify what stood there. Insect stains are the most common misinterpretation in an indoor scene with a decomposing decedent, and are distinguished by their irregular distribution, accompanying fly specks, and presence on surfaces such as ceilings and light fixtures.
Directionality and angle
A bloodstain's shape records its travel:
The stain is elongated in the direction of travel , and the tapered, pointed, or scalloped end points in the direction the drop was moving
The impact angle is calculated as θ = arcsin (width ÷ length) of the elliptical stain
A drop striking perpendicular to a surface produces a circular stain (90 degrees); increasingly acute angles produce increasingly elongated ellipses
Area of origin is estimated by the analyst from the convergence and the calculated angles of multiple well-formed stains
Surface texture strongly affects stain morphology, which is why the analyst needs to know the substrate and why a control photograph of the surface matters.
Documenting a bloodstain scene
Overall photographs establishing the room and the relationship of the patterns to the body and to the furniture
Roadmapping — adhesive scales or numbered labels placed adjacent to patterns, then mid-range photographs establishing each pattern's location
Close-up photographs perpendicular to the surface with a scale in the same plane as the stain , which is required for any subsequent angle measurement
- Overlapping sequential photographs across large patterns so they can be reassembled
- Sketch and measure the patterns' positions and dimensions
Photograph before applying any presumptive reagent , and document luminol results with long-exposure photography noting that reagent application alters the stain
- Only then collect samples for DNA , from the periphery of a pattern where possible so the pattern is preserved
- Tool Marks
- Class, subclass, and individual characteristics
Class characteristics are shared by all tools of a type — a screwdriver blade of a given width, a bolt cutter jaw profile
Subclass characteristics arise from the manufacturing process and are shared by a limited group of tools made in the same run; they are a well-recognized source of erroneous association
Individual characteristics are the random imperfections a specific tool acquires from manufacture, use, and damage, and are the basis for any identification claim
Mark types
Impressed (static) marks — the tool is pressed into a softer surface, as in a pry mark on a door jamb or a hammer strike
Striated (dynamic) marks — the tool moves across the surface under pressure, producing parallel striae, as when a screwdriver slips or a bolt cutter shears
The rule that gets tested
Never fit a suspected tool into a tool mark. Doing so can alter both the mark and the tool, creates the appearance of a fabricated association, and transfers material between them. Comparison is performed in the laboratory with test marks made in appropriate media on a comparison microscope.
Documentation and collection
Photograph with a scale, perpendicular to the mark , and then again with oblique lighting from several directions, since low-angle light is what makes impressed detail visible at all
Collect the whole item bearing the mark where it can be removed — a door jamb section, a padlock, a window frame member
Where the item cannot be removed, cast the mark with a silicone casting material such as a polyvinylsiloxane product, after photographing; label the cast with the case number, date, initials, and orientation before it is lifted
Package the tool separately from the marked item, and never in the same container
Protect the working surfaces of a recovered tool by wrapping or suspending it so the tip is not abraded in transport, and remember that the tool may also bear trace evidence and DNA from the surface it acted on — collect those before the tool is handled for comparison
Tool marks on bone and cartilage
Skeletal tool marks are a forensic anthropology subject with its own vocabulary. Saw marks in dismemberment cases are described by the kerf — the cut channel — with its kerf floor shape, kerf width , striae, and any breakaway spur or false start , and these features can indicate saw type and blade characteristics. Knife and chop marks on bone and cartilage may retain class-level blade features. Costal cartilage, hyoid, and sternal cartilage retain tool marks well , which is why the pathologist and anthropologist should be alerted before those structures are processed, and why the investigator's scene documentation of any recovered implement matters to that examination.
A caution on comparison claims
The scientific limits of pattern comparison disciplines have been examined closely in the last two decades, and overstated identification claims have been a recurring source of error. The Board-level investigator's defensible position is to document thoroughly, preserve completely, and leave the comparison conclusion to the qualified examiner — and, when testifying, to describe what was collected and how, rather than what it proved.