Serology - Blood and other Bodily Fluids

When evaluating forensic tests on suspected blood, semen, or saliva 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, and can locate possible evidence not visible to naked eye
  • 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

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: Presumptive and Confirmatory Tests - Virtual CLE.

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:

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  • 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
    • 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.

Articles

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

  • 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.
  • DNA is not a confirmatory test for blood. See NFSTC DNA Analyst Training: "Presumptive v. Confirmatory Tests." For additional information about DNA evidence, click here.

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.