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AGR-CSI53-08 · Lesson 6 · 14 clock hours · lab

Biological Evidence: Presumptive and Confirmatory Screening

Objective. Students will perform presumptive tests for blood, semen, and saliva on prepared substrates, correctly interpret results, and document findings with appropriate limitation statements.

⚠ Pending professional review

This lesson, its worked examples and its practice questions has not yet been verified by a licensed professional in the field or by a certification or approval body. It is a study draft and may contain errors. Do not use it as instructions for work on a job site or on live equipment. Always follow your instructor, your supervising professional, and current official references and regulations.

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Biological Evidence: Presumptive and Confirmatory Screening

When a crime scene contains biological material—blood, semen, or saliva—the forensic technician must answer two questions in order: Is this substance what I think it is? and Does it match a known individual? This lesson covers the first question through presumptive testing, the critical step that guides what happens next.

What Is a Presumptive Test?

A presumptive test is a rapid screening procedure that indicates whether a particular substance may be present. A positive result narrows the field of possibilities; it does not confirm the substance's identity, its human origin, or the identity of the person who left it. Always document presumptive results with explicit limitation language—courts and reviewing scientists expect this.

Because these tests use chemical reagents and contact potentially infectious material, proper PPE is mandatory before you open any sample: nitrile gloves, safety eyewear, and a lab coat. Treat every biological sample as if it carries bloodborne pathogens (OSHA Bloodborne Pathogen Standard, 29 CFR 1910.1030 framework applies). All waste—used swabs, pipettes, reagent-contaminated materials—goes into labeled biohazard bags for proper disposal. Never recap contaminated pipettes; never eat, drink, or touch your face in the lab.

Phenolphthalein (Kastle-Meyer) Test for Blood

The Kastle-Meyer (KM) test detects the peroxidase-like activity of hemoglobin. Hemoglobin catalyzes the oxidation of reduced phenolphthalein, producing a bright pink-to-magenta color when hydrogen peroxide is added.

Procedure outline:

1. Swab a small area of the questioned stain or wet the swab with distilled water and rub the stain.

2. Add one drop of phenolphthalein reagent to the swab tip.

3. Add one drop of 3% hydrogen peroxide.

4. Observe for color change within two seconds.

A color change that occurs in two seconds is a positive presumptive result. A delayed change or no change is negative. Always run a known-blood positive control and a blank negative control alongside unknowns to validate reagent performance.

Limitations: The KM test cannot distinguish human blood from animal blood. Vegetable peroxidases (certain fruits, some vegetables) and chemical oxidants can produce false positives. A positive result must be followed by confirmatory testing—such as a species-specific immunological assay (e.g., the ABAcard HemaTrace)—before any conclusion about human origin is reported.

Acid Phosphatase Test for Semen

Seminal fluid contains high concentrations of acid phosphatase (AP), an enzyme produced by the prostate gland. The AP spot test uses a colorimetric substrate that turns purple when AP activity is present.

Procedure outline:

1. Apply the AP reagent to the swab or substrate.

2. Observe for a purple color change within one minute (many labs use a 30-second window as a strong-positive cutoff).

3. A rapid, strong purple reaction suggests the presence of semen.

Limitations: Vaginal secretions contain low levels of acid phosphatase, and some bacteria can also produce AP activity, leading to false positives. A positive AP result should be followed by confirmatory testing for spermatozoa under microscopy or prostate-specific antigen (PSA/p30) immunoassay to confirm semen of human male origin.

Amylase Testing for Saliva

Saliva contains amylase, an enzyme that breaks down starch. Commercial amylase test strips (phadebas-type or equivalent) detect elevated amylase activity above the low baseline levels found in many other body fluids. A color change on the strip indicates possible saliva. Like the other presumptive tests, a positive result requires confirmatory work—such as an immunological assay for salivary amylase isoform or RNA profiling—before a conclusion is drawn.

Controls: Why They Are Non-Negotiable

Every testing session requires:

If the positive control fails or the negative control fires, stop testing and investigate reagent integrity before proceeding. Document control results on every result form.

Documenting Results and Limitation Language

SWGDAM (Scientific Working Group for DNA Analysis Methods) guidelines and general forensic science best practices require technicians to state the limitations of every test performed. A compliant written interpretation might read:

"The phenolphthalein test yielded a positive presumptive result for blood on swab Q3. This result is consistent with the presence of blood but does not confirm human origin or exclude other substances with peroxidase-like activity. Confirmatory testing is required before a definitive conclusion can be drawn."

Using this language in your lab reports is not weakness—it is scientific integrity and professional obligation.

Biohazard Waste Disposal

All swabs, pipettes, and materials that have contacted biological samples must be placed in properly labeled biohazard bags and disposed of according to your facility's biohazardous waste protocol. Never place these items in regular trash. At the end of each lab session, the instructor will observe and confirm correct disposal—this is a graded pass/fail requirement, not optional.

Key terms10

Presumptive testA rapid screening procedure that indicates a substance may be present; a positive result supports further testing but does not confirm identity or human origin.
Confirmatory testA more specific follow-up test—often immunological or microscopic—that confirms the identity and, where relevant, the species origin of a biological substance.
Kastle-Meyer (phenolphthalein) testA color-based presumptive test for blood that detects the peroxidase-like activity of hemoglobin, producing a pink-to-magenta color in the presence of hydrogen peroxide.
Acid phosphatase (AP) testA presumptive test for semen that detects the enzyme acid phosphatase, abundant in seminal fluid; a positive result produces a purple color change.
False positiveA positive test result obtained from a substance that does not actually contain the target material, caused by interfering substances such as plant peroxidases or bacteria.
Positive controlA sample known to contain the target substance, run alongside unknowns to confirm that the reagent is functioning correctly.
Negative controlA clean sample containing none of the target substance, run to confirm the reagent does not produce spontaneous false-positive reactions.
SWGDAMScientific Working Group for DNA Analysis Methods; publishes guidelines that forensic labs follow for biological evidence testing and documentation standards.
AmylaseAn enzyme found in high concentrations in saliva that breaks down starch; its presence is used as a presumptive indicator for saliva on evidence items.
Bloodborne pathogenA microorganism present in human blood and certain other body fluids that can cause disease; forensic technicians follow OSHA standards to minimize exposure risk.
In class: Technique demonstration: instructor performs phenolphthalein (Kastle-Meyer) test for blood and acid phosphatase test for semen on positive and negative controls, then shows how to read and record results including the limitation that presumptive tests do not confirm human origin. · Blind-sample testing: each student receives five coded swabs of unknown composition (may contain blood, semen, saliva, or be blank); students apply appropriate presumptive tests, record results, and submit a written interpretation form before the instructor reveals the answers. · Limitation analysis: class reviews the blind-sample results as a group, identifies any false-positive or false-negative outcomes, discusses what additional confirmatory steps would follow in a real laboratory, and each student writes a one-paragraph limitations section appropriate for a lab report.
Materials: Phenolphthalein reagent, hydrogen peroxide, acid phosphatase reagent, amylase test strips, positive and negative control samples, coded blind swabs, swab collection tubes, disposable pipettes, timing device, PPE (gloves, eye protection, lab coat), biohazard waste bags, SWGDAM guidelines reference
Check: Student correctly identifies at least 4/5 blind samples; written results form uses proper limitation language; disposal of all biohazardous materials is observed and confirmed correct by instructor; graded pass/fail with 80% accuracy on identification and mandatory correct disposal procedure.

Practice check8

Answer each question, then check it. Nothing is recorded; this is your own practice.

Crime Scene Investigation – Evidence Documentation
1. A student performs the Kastle-Meyer test on swab Q2 and observes a bright pink color change within two seconds after adding hydrogen peroxide. What is the correct interpretation to document?
Lab Quality Assurance
2. During the acid phosphatase test for semen, the positive control fails to produce a purple color change. What should the technician do?
Crime Scene Investigation – Biological Evidence
3. Which of the following substances could produce a false-positive result on the Kastle-Meyer presumptive blood test?
Crime Scene Investigation – Biological Evidence
4. A technician is testing swab Q5 with the acid phosphatase reagent. A purple color appears after 55 seconds. The lab's strong-positive cutoff is 30 seconds. How should this result be documented?
Health and Safety
5. Why is it essential to wear nitrile gloves, safety eyewear, and a lab coat when performing presumptive biological testing?
Health and Safety
6. After completing blind-sample testing, a student places used swabs and pipettes in the regular trash can. What error has been made and what is the correct action?
Crime Scene Investigation – Biological Evidence
7. A positive amylase strip result on a questioned swab from a sexual-assault case indicates:
Crime Scene Investigation – Evidence Documentation
8. Which statement best reflects the limitation language required by SWGDAM guidelines when reporting a positive presumptive blood result in a written lab report?
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