Forensic serology is the detection, identification, classification, and study of various bodily fluids, such as blood, semen, saliva, and urine, and their relationship to a crime scene. A forensic serologist may also be involved in DNADNA, or Deoxyribonucleic Acid, is the genetic material found in cells, composed of a double helix structure. It serves as the genetic blueprint for all living organisms. analysis and bloodstain pattern analysis. Serology testing begins with presumptive tests, which give the analyst an indication that a specific bodily fluid may be present but cannot wholly confirm its presence. Following the presumptive tests, confirmatory tests are done on the same sample to establish what the unknown substance is.
The detection and identification of body fluids at a crime scene can provide essential information about the events that may have occurred and the people involved. In addition, the presence of body fluids, their location, and the DNA profile obtained from them can supply police agencies with a wealth of information to aid the investigation.
Once potential biofluids are located in-situ, presumptive tests could also be used to indicate the possible identity of the stain. Following sample collection, confirmatory analysis and DNA analysis will often be conducted. Unfortunately, there’s no single method for analyzing all body fluids, and it should be necessary to employ multiple techniques depending on the biofluid type.
Types of Serology
Serology is divided into two categories of investigation:
- A presumptive test is a chemical test that indicates the presence of a body fluid by producing color or light. It is typically more sensitive and less specific than a confirmatory test.
- A confirmatory test is a test that verifies the presence of a specific compound. It is typically less sensitive than a presumptive test but more specific.
Visual Examination:
Visualization techniques may be employed at an incident scene to initially locate potential body fluids, for instance, alternative light sources (ALS). An ALS utilizes different wavelengths of light to visualize otherwise invisible or ambiguous stains, with some even having the ability to show stains that have been wiped clean or covered by paint. However, alternative light sources will not typically allow for differentiation between different body fluids.
Phenolphthalein:
The phenolphthalein test is used to test for the presence of blood presumptively. The chemicals used are ethanol, phenolphthalein, and hydrogen peroxide. Phenolphthalein testing works through a Redox reaction where the chemicals start in a reduced form. If peroxidase activity from the heme group (found on the Hemoglobin molecule in the blood) is present, the chemicals are oxidized. A color change (to bright pink) is observed if oxidation occurs. If there is no peroxidase activity, the oxidation will not happen, and no color change will be followed.
Pink color change = presumptively positive for blood*
No color change = negative for blood.
HemaTrace™:
The HemaTrace™ test determines if a bloodstain is of human origin. This test is an antigen-antibody reaction. The antibodies are present on the HemaTrace™ card. If the antigen (the heme group of the Hemoglobin molecule) is present, then an antibody-antigen reaction occurs on the card’s test site (T). The antigens and antibodies bind to each other and form a lattice (chain) which causes a pink dye band visible. The control region of the test strip should always react, and the pink dye band should be visible on both positive and negative results.
Pink line in control region + pink line in test region = presumptively positive result for human blood*.
The pink line in the control region only = a negative result for human blood.
*Note: both phenolphthalein and Hematrace™ are presumptive tests. Further DNA testing can confirm these results.
BCIP:
Suspected semen stains can be swabbed and tested using BCIP (5-Bromo-4-chloro-3-indolyl phosphate). BCIP reacts with the acid phosphatase found in seminal fluid. The reaction will cause the swabs to turn teal blue when acid phosphatase is present. BCIP testing is typically done on items of clothing or bedding. The BCIP test is a presumptive test for the presence of semen &/or seminal fluid.
p30:
p30 is a presumptive test for the presence of human prostate-specific p30 protein found in seminal fluid. The p30 test works through an antigen-antibody reaction. Antibodies are present on the p30 card, and if the antigen is present (p30 protein) in a sample, then a reaction occurs on the test site (T) of the card. The antigens and antibodies bind to each other and form a lattice (chain) which causes a pink dye band visible. The control region of the test strip should always react, and the pink dye band should be visible on both positive and negative results. The p30 test is a presumptive test for the presence of semen &/or seminal fluid.
Pink line in control region + pink line in test region = positive result for p30 protein/seminal fluid.
The pink line in the control region only = negative result for p30 protein/seminal fluid.
Sperm Hyliter™:
Sperm Hyliter is a slide staining technique used to identify sperm microscopically. Sperm Hyliter™ also works through an antigen-antibody reaction. During the staining process, the sperm heads react with an antibody found in the stain. This reaction causes the sperm heads to fluoresce a bright green color. Next, a second stain is added that stains all of the nuclei in a sample. Therefore, when sperm are present in a sample, they will fluoresce green or blue when viewed under two different contrasts (FITC & DAPI) on the microscope.
This fluorescence makes the sperm heads easily distinguish from the surrounding cells/debris. This reaction is specific to human sperm heads. Therefore, when identifying sperm heads, one looks for the correct fluorescence along with the right size, shape, and the presence of an acrosome, a small unstained part of the sperm head. Positive identification of sperm microscopically is the only confirmatory test for semen identification used at the BCA.
Christmas Tree Stain:
Christmas Tree Staining is another slide staining technique used to identify sperm microscopically. This method is much quicker than Sperm Hyliter. The sperm heads stain red, and the sperm tails and other cells stain green. When viewed under a microscope, the sperm heads are relatively easy to spot and differentiate from the surrounding cells. Similar to SpermHyliter™, when identifying sperm heads, one looks for the right color, size, shape, and the presence of an acrosome, a small unstained part of the sperm head.
Phadebas™:
The presumptive test used by the BCA to test for saliva is the Phadebas™ test. This test detects the presence of amylase in a sample, an enzyme found in high levels in saliva. A Phadebas™ tablet comprises a water-soluble starch polymer carrying a blue dye. If the amylase enzyme is present in a sample, it will degrade the starch polymer found in the Phadebas™ tablet, releasing the blue dye.
Creatinine:
The test used for urine identification is the detection of Creatinine. Creatinine is found in urine. If Creatinine is present, a color change from yellow to orange and back to yellow will be observed after adding various chemicals. A positive sample will turn yellow, orange, and back to yellow, while a negative sample will remain yellow the whole time.
