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Blood & Bodily Fluids

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When evaluating forensic tests on suspected blood, semen, or saliva evidence (also known as serology evidence), it is important for defenders to understand first the difference between presumptive and confirmatory tests and why that distinction is so important:

Presumptive Tests

  • Also known as preliminary tests, screening tests or field tests
  • Establish the possibility that a specific bodily fluid is present
  • Do not conclusively prove the presence of a specific substance
  • Pros: Narrows possibilities, can be used on larger areas, can locate possible evidence not visible to naked eye, relatively inexpensive and quick to perform
  • Cons: Risk of false positives and may be overly sensitive
  • Uses: Provide initial information to determine what test to perform next, used in combination with confirmatory tests

Confirmatory Tests

  • Conclusively identify the identity of a biological material
  • May be one or a combination of procedures
  • Pros: Conclusively identifies a substance, smaller risk of false positives
  • Cons: May be more expensive, require additional equipment, and take longer

What Defenders Need to Look For:

  • A lab report may not reveal what type of test was done. For example, in the report below it is not clear what blood analysis tests were completed and what the meaning of “chemical indications for the presence of blood” is:
  • Testimony at trial may be misleading. To understand the meaning of phrases such as “chemical indications for the presence of blood,” see the State Crime Lab’s Body Fluid Report Format. Note that the effective date of this document is December 3, 2002 and the State Crime Lab may have an updated version. “Chemical indications for the presence” of a substance generally means that only a presumptive test gave a positive result. Counsel should be aware that a presumptive test alone establishes only the possibility that a particular substance is present. Counsel should be prepared to object to testimony or questions from the prosecutor that refer to a substance as a particular bodily fluid, such as blood, if only a presumptive test was performed.
  • Defenders must obtain the underlying notes, data, photographs and reports of the analyst.
  • Defenders must ensure that the law enforcement officer and/or analyst followed proper procedures. For example, see the State Crime Lab’s Body Fluid Casework Procedures. The NC State Bureau of Investigation Evidence Guide explains evidence submission requirements and includes a section on Forensic Biology. If you have questions about how or why evidence was collected at a crime scene or the NCSCL’s policies on what evidence the lab will examine, this guide may provide information.
  • Defenders should consider raising a challenge where:
    • Only a presumptive test was done
    • The mandated combination of tests was not done
    • Testing protocol was not followed
    • Test results were not adequately documented
    • Lab reports use improper reporting language or improper testimony is anticipated

Below are descriptions of the forensic biology presumptive and confirmatory tests that are typically used in North Carolina cases.

Blood

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Presumptive Tests

  • Phenolphthalein Test
  • Luminol Test
    • How it works: A chemical compound, known as Luminol, is used in solution or sprayed onto suspected surfaces. This compound gives a strong blue fluorescence when viewed with a UV light. The Luminol reacts with hematin, a substanced formed as bloodstains age, and produces a luminescence which is best observed in the dark. The luminescence lasts for several minutes and can be photographed. Aged bloodstains tend to give more intense and longer-lasting luminescence than fresh blood, and can be re-sprayed with Luminol to be viewed again. Source: U.S. Department of Justice, Sourcebook in Forensic Serology, Immunology, and Biochemistry, Section 6.3, page 112-114.
    • Precautions: False positives have been observed with the presence of copper salts. Most brass, bronze, and similar alloys which contain copper gave a false positive reaction, which is important to consider when dealing with locks, door handles, and other fixtures made of these materials. Source: U.S. Department of Justice, Sourcebook in Forensic Serology, Immunology, and Biochemistry, Section 6.7, page 112-114.
  • Alternative Light Sources
    • How it works: Alternative Light Sources such as the CrimeScope use ultraviolet, visible or infrared light to cause certain substances to fluoresce (glow) or absorb light (darken). Blood stains will darken rather than glow when certain light wavelengths are used. See the user’s manual for the alternative light source for more information.
    • Lab Procedure for completing this test – see the Forensic Biology portion of the ISO Procedures page of the NCSCL website.

Confirmatory Tests

  • DNA
    • DNA is not a confirmatory test for blood. For additional information about DNA evidence, click here.
  • Takayama Test
  • RSID Test for Human Blood
    • How it works: This test uses two specialized antibodies to detect the presence of human Glycophorin A which is found in red blood cell membranes. The antibodies are applied to the suspected sample by using a strip test assay. At the end of the test, certain markings will indicate whether human blood was detected or if the test failed. Source: RSID Technical Information Sheet.
    • NCSCL is using a handheld electronic reader to read the results of confirmatory tests for blood or semen. The reader records data about the type of card being read (whether it was a test for blood, semen, or prior to 1/1/2017, a test for human saliva), date of the reading, and the result rendered by the reader. The reader also creates an image of the card that it reads. The images are stored in the handheld reader (it has limited storage capacity) and can be downloaded using a cable that connects the reader to a computer. The lab is not downloading and storing this data and images.
    • Precautions: This test should be evaluated exactly 10 minutes after the addition of the sample. An appropriate sample size and dilution of the sample must be used. Kits should be stored at room temperature and buffers should be stored at 4 C.
    • Lab Procedure for completing this test – see the Forensic Biology portion of the ISO Procedures page of the NCSCL website.
  • ABAcard HemaTrace test strips
    • How it works: HemaTrace test strips are used to detect blood by indentifying the presence of human hemoglobin. The test strip contains an antihuman hemoglobin antibody. A blood sample is applied to the bottom of the test strip. If human hemoglobin is present, then a mobile antibody-antigen complex will be formed. This complex will then migrate through the test strip to a test window. This window will indicate if there is a positive result for human hemoglobin with a pink dye band. Source: NFSTC DNA Analyst Training. The State Crime Lab considers this test a back up test to examine evidence for the presence of human blood if RSID is not available. Because the test will cross react with some animal blood, it can only give a reaction consistent for the presence of blood. Source: Section 2.3 of the NC State Crime Lab Technical Procedures Manual: Body Fluid Identification.
    • Hematrace Test – NFSTC video demonstrating how the Hematrace Test is performed
    • Lab Procedure for completing this test – see the Forensic Biology portion of the ISO Procedures page of the NCSCL website.

Semen

Presumptive Tests

  • Acid Phosphatase Test
    • Also known as the Walker Test or Brentamine spot test
    • How it works: The male prostate gland produces and secrets into semen a high amount of the enzyme acid phosphatase (AP). In the presence of Alpha-Naphthyl acid phosphate and Brentamine Fast Blue, AP will produce a dark purple color in less than a minute. Source: Dr. Maher Noureddine, Forensic Tests for Semen: What You Should Know.
    • Precautions: The shade of purple color will depend on the activity of the enzyme, which can be negatively impacted by the age of the stain and the storage conditions. This test is highly presumptive because vaginal secretions and other bodily fluids contain detectable levels of this enzyme as well. Source: Dr. Maher Noureddine, Forensic Tests for Semen: What You Should Know.
    • Acid Phosphatase Test – NFSTC video demonstrating how the Acid Phosphatase Test is performed.
    • Lab Procedure for completing this test – see the Forensic Biology portion of the ISO Procedures page of the NCSCL website.
  • Alternative Light Sources
    • How it works: Under specialized lights, semen will fluoresce due to the presence of molecules such as Flavin and Choline-conjugated proteins. This color will vary from blue to yellow depending on the light equipment used. Source: Dr. Maher Noureddine, Forensic Tests for Semen: What You Should Know.
    • Precautions: This detection technique is highly presumptive because many molecules (natural and artificial) will fluoresce in a similar way as semen. Also, not all semen stains will fluoresce. Exposure to different environments, different types of fabrics, and different fabric treatments can affect this fluorescent activity. Source: Dr. Maher Noureddine, Forensic Tests for Semen: What You Should Know.
    • Lab Procedure for completing this test – see the Forensic Biology portion of the ISO Procedures page of the NCSCL website.
  • Prostate Specific Antigen
    • How it works: Test detects prostate specific antigen (PSA). PSA is produced in high amounts by male prostate gland. Source: Dr. Maher Noureddine, Forensic Tests for Semen: What You Should Know.
    • Precautions: This antigen can also be found in very small amounts of fecal material and sweat. Studies also indicate that PSA can exist in female urine and breast milk. Caution is urged when interpreting positive PSA results which are not confirmed by actual presence of sperm. Source: Dr. Maher Noureddine, Forensic Tests for Semen: What You Should Know.
    • This test is not currently performed by NC State Crime Lab.

Confirmatory Tests

  • Christmas Tree Stain
    • How it works: Positive visual identification of sperm cells using a stain. Two main reagents are used consecutively to produce this distinctive stain: Picroindigocarmine stains the neck and tail portions of the sperm in green and blue, while the Nuclear Fast Red (AKA Kernechtrot) gives the sperm heads a read color and the tip of the heads a pink color. Source: Dr. Maher Noureddine, Forensic Tests for Semen: What You Should Know.
    • Precautions: Sperm cells deteriorate quickly after ejaculation. Sperm survival will depend on the surrounding environment and type of surface. The sperm tails are the most susceptible to damage and will break down first. Therefore, the analyst must be trained to make visual distinctions between sperm heads and other types of cells in the mix. Other cells will also stain red. Source: Dr. Maher Noureddine, Forensic Tests for Semen: What You Should Know.
    • Lab Procedure for completing this test – see the Forensic Biology portion of the ISO Procedures page of the NCSCL website.
  • RSID Test for Semen
    • How it works: Identifies the presence of the seminal vesicle-specific antigen, or semonogelin. This antigen is unique to human semen; therefore, there is no cross reactivity with other bodily fluids in males and females or with semen from other mammals. This test can also identify semen even if the stain was stored in less favorable conditions. Source: Dr. Maher Noureddine, Forensic Tests for Semen: What You Should Know.
    • NCSCL is using a handheld electronic reader to read the results of confirmatory tests for blood or semen. The reader records data about the type of card being read (whether it was a test for blood, semen, or prior to 1/1/2017, a test for human saliva), date of the reading, and the result rendered by the reader. The reader also creates an image of the card that it reads. The images are stored in the handheld reader (it has limited storage capacity) and can be downloaded using a cable that connects the reader to a computer. The lab is not downloading and storing this data and images.
    • P30 Test – NFSTC video demonstrating how the P30 Test is performed.
    • Lab Procedure for completing this test – see the Forensic Biology portion of the ISO Procedures page of the NCSCL website.

Saliva

Presumptive Tests

  • Phadebas Test
    • How it works: A chemical reagent called Phadebas is used to detect the enzymatic activity of the alpha-amylase enzyme, which is found in saliva. Source: Dr. Maher Noureddine, Forensic Tests for Saliva: What You Should Know.
    • Precautions: This enzyme is found in other organisms as well. Alpha-amylases from bacteria, fungi, or chimps are very similar in structure and function to that of the human alpha-amylase. Also, in humans, there are four variants of alpha-amylase, two of which are found in saliva, and the other two are secreted by the pancreas. This test is presumptive because it will give a positive result if the alpha-amylase enzyme from any organism is present. Source: Dr. Maher Noureddine, Forensic Tests for Saliva: What You Should Know.
    • Lab Procedure for completing this test – see the Forensic Biology portion of the ISO Procedures page of the NCSCL website.

Confirmatory Tests

  • Phadebas Test and RSID Test for Human Saliva
    • How it works: The RSID Test for Human Saliva detects the alpha-amylase molecule itself, and specifically, the alpha-amylase from human saliva (in comparison to the testing for enzymatic activity as seen in the Phadebas test). Performing both of these tests is considered a confirmatory test. Source: Dr. Maher Noureddine, Forensic Tests for Saliva: What You Should Know.
    • Precautions: The RSID test has produced positive reactions in samples containing alpha-amylases from mammals such as gorillas and rats. Positive reactions were also noted in other bodily fluids, such as semen, blood, vaginal discharge, sweat, and breast milk. High reactivity of this test is observed in samples containing human feces. Reactivity was also noticed in urine samples. Improper swabbing and other factors relating to personal hygiene, personal behavior, and indirect saliva transfer from mouth to surface can result in “false” positives. Source: Dr. Maher Noureddine, Forensic Tests for Saliva: What You Should Know.
    • As of Jan. 2017, the NCSCL no longer conducts confirmatory testing for saliva.

Individualization

  • Once biological matter is confirmed to be present, lab may perform tests to determine source.
  • Current Test: DNA. However, DNA is not considered a confirmatory test for blood, semen or saliva.
  • Past Tests:
    • Ouchterlony – Species of origin test
      • How it works: The Ouchterlony test is used to determine if a blood sample is human or animal through the comparison of its reactions to specific antibodies. A sample of the unknown bloodstain is placed in a well in an agar gel. Antibodies from human and animal sources of blood are placed in other wells in the gel. Antigens from the sample and the antibodies will spread out of their respective wells and will pair up to form an immune complex if the antigen and antibody are from the same animal source. The immunne complexes can be observed as a line in the gel, thus indicating the source of the blood.
      • Precautions: A control sample consisting of a sample from the unstained area near a stained area of interest must be tested. It may take several hours up to 72 hours for the reaction to occur. This test requires expertise in interpretation.
      • This procedure is no longer performed by the NC State Crime Lab.
    • ABO typing – identifies a person’s blood type
      • How it works: ABO typing requires a multi-step procedure in which the sample is observed reacting with Anti-A and Anti-B antibodies. Next, the liquid part of the blood without cells, the serum, is mixed with blood that is known to be Type A or Type B and the reaction is observed.
      • Precautions: Appropriate negative controls must be run as false positives are possible, especially with Type B blood. Stains on denim fabric or soiled shoes may also yield false positives. Weak results may be read differently by analysts, so it is essential to have a second analyst read the results.

Reports and Publications

  • OSAC Registry Approved Standards, NIST
    Organization of Scientific Area Committees for Forensic Evidence (OSAC) is developing documentary standards for each forensic discipline. Standards under consideration as well as approved standards are available in the OSAC Registry.
  • American Academy of Forensic Sciences Standards Board Documents

    The AAFS Standards Board develops documentary standards for forensics through a consensus process, involving participation by all directly and materially affected persons. Standards are being developed for each forensic discipline. The standards that have been published are available on the ASB website.

From the Blog

  • Is there a test for saliva?, 10/9/2020
    Attorneys have asked me whether labs test for saliva, particularly in sexual assault cases. Dr. Maher “Max” Noureddine wrote the Forensic Tests for Saliva: What you should know post for this blog in 2011. His informative post explains how forensic tests for saliva work, including limitations of these tests. He discusses the RSID-Saliva test which …
  • Online tracking system for Sexual Assault Evidence Collection Kits now available, 6/28/2019
    In 2018, the NC General Assembly passed legislation (S.L. 2018-70) requiring the creation of the a statewide tracking system to track the testing of Sexual Assault Evidence Collection Kits (SAECKs) from collection to completion of forensic testing. The tracking system is now available for all stakeholders in the criminal justice system.
  • Sexual Assault Kit Tracking Now Available, 11/16/2018
    In 2018, the NC General Assembly passed legislation (S.L. 2018-70) requiring the creation of the a statewide tracking system to track the testing of Sexual Assault Evidence Collection Kits (SAECKs) from collection to completion of forensic testing. The tracking system is now available for all stakeholders in the criminal justice system.
  • Forensic Tests for Semen: What you should know, 10/19/2011
    By Maher Noureddine, Ph.D., President: ForensiGen, LLC Second in the series: Bodily Fluids and Forensics First, the biological facts about semen: Seminal fluid is a complex mixture of secretions from at least four male urogenital glands.  The seminal vesicle gland contributes approximately 60% to this mixture, the prostate gland contributes approximately 30%, and the combined …
  • Forensic Tests for Saliva: What you should know, 8/15/2011
    By Maher Noureddine, Ph.D. First in the series: Bodily Fluids and Forensics You may encounter cases in which the prosecution claims that SBI tests establish the presence of human saliva on the victim as part of its proof that an assault occurred. This type of evidence is often seen in sexual assault cases.  In cases …
  • Bodily Fluids and Forensics: Introduction to the Series, 8/14/2011
    By Maher Noureddine, Ph.D. and Sarah Rackley The field of forensic investigation continues to reap tremendous benefits from advancements made in various scientific disciplines including physics, chemistry, biology and others. The detection and analysis of biological molecules have been at the forefront of this advancement, even to the level of revolutionizing forensics as we know …
    • Click here for more blog posts on this topic

Trainings

  • Alternate Light Source Photography, Godoy Medical Forensics
    Free live webinar offered by Godoy Medical Forensics
  • Alternate Light Source Photography: Basic understanding of emerging science for use in medico-legal settings, Godoy Medical Forensics
    Free to attend live webinar offered by Godoy Medical Forensics
  • Statistical Methods for Bloodstain Pattern Analysis, CSAFE
    Presenter: Hal Stern. Offered by CSAFE, on June 14, 2021. Recording and materials are available.
  • CSAFE 2020: Bloodstain Pattern Analysis, CSAFE

    This presentation, now available to view for free, was given as a part of the Center for Statistics and Applications in Forensic Evidence’s (CSAFE) 2020 All Hands Meeting. The meeting brings together researchers, forensic partners, and interested community members to discover potential areas for collaboration, highlight the organization’s achievements, and discuss goals for the future.

    This presentation, given by Dr. Hal Stern, a professor at the University of California, Irvine in the Department of Statistics, discusses recent accomplishments in the world of bloodstain pattern analysis research including the development of a high-resolution blood spatter database, the development of an early framework for determining the region of origin of blood spatter with quantification of uncertainty, and the creation of a computer based decision process for distinguishing bloodstain patterns caused by blunt or gunshot impact. Dr. Stern also discusses the objectives for future research and details proposed future projects within this discipline.

    Materials:

    • Video of the presentation is available here
    • The slides from the presentation by themselves are available here
  • Feature-based analysis of bloodstain patterns

    The Center for Statistics and Applications in Forensic Evidence (CSAFE) invites researchers, collaborators, and members of the broader forensics and statistics communities to participate in the September Center Wide Webinar on Tuesday, September 24th from 1-2 pm EST. There will be a short introduction from CSAFE center staff followed by the presentation “Feature-based analysis of bloodstain patterns” by Dr. Hal Stern, CSAFE Co-Director.  Dr. Stern is a Professor of Statistics and the Vice Provost for Academic Planning at the University of California, Irvine.

    Presentation Description: A key task in the analysis of bloodstain pattern analysis is to identify the mechanism by which the stains were created.   Examples include impact, gunshot, castoff, and expiration.  This webinar will introduce how images of bloodstain patterns can be represented via ellipses and using algorithms to assist with characterizing complex stains.  Features defined from characteristics of the ellipses appear to have potential in distinguishing bloodstain patterns created by different mechanisms.

  • Webinar: Bloodstain Pattern Analysis Essentials

    This complimentary forensic science webinar is presented by Toby L. Wolson, retired criminalist and supervisor in the Forensic Biology Section of the Miami-Dade Police Department Forensic Services Bureau. The recorded webinar is available for on-demand viewing.

    This webinar will focus on:

    • Overview of bloodstain pattern analysis and the science behind the discipline
    • Expert training and credentials
    • Legal concerns and what attorneys should know about bloodstain pattern experts.
    • Current temperature of the field, including recent media coverage
    • Available OSAC resources

    The webinar is part of the complimentary webinar series, Crime Scene to Courtroom Forensics Training. This project was supported by Grant No. 2015-CP-BX-K006 awarded by the Bureau of Justice Assistance.

  • Presumptive and Confirmatory Tests – Virtual CLE

    For more information about presumptive and confirmatory tests, the School of Government has posted a 30 minute online program that you can view for free or for CLE credit

  • Serology Evidence (Blood, Saliva, Semen, etc.) (2011)

    NCAJ Webinar on crime scene investigation and serology evidence by Marilyn Miller.

Websites

  • Bloodstain Pattern Analysis Video Collection

    Over 500 video clips were made of blood spatter experiments that are typically performed by individuals attending a basic bloodstain pattern analysis course. The videos were made possible through a National Institute of Justice (NIJ) grant and are available to be viewed or downloaded. The principal investigators were Terry Laber, Bart Epstein, and Dr. Michael Taylor.

Cases

  • State v. Corbett & Martens, 839 S.E.2d 361 (2019)

    Expert Stuart James’s treatise on blood spatter said that suspected bloodstains should be subject to presumptive, confirmatory, and DNA testing before analysis of the spatter is conducted. The court held that blood spatter expert’s conclusions regarding untested stains on clothing did not result from reliable application of scientific methods and evidentiary errors were prejudicial. 

Motions and Briefs

Blood and Bodily Fluids Experts