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Dr. Gabriela Roca
Research and Development Project Manager in a Forensic Biotechnology company in Germany
December 4, 2017 · 2,178 Reads

Menstrual or peripheral blood? New method permits forensic investigators to identify blood source in sexual assault cases

Authors: Claudemir R. Dias Filho and Gabriela Roca. Editor: Amanita Setari

Determining the source and nature of physical and biological evidence can be the difference between success and failure in a criminal investigation. As often depicted in movies and television series, blood is one of the most common sources of biological evidence subject to forensic analysis, making the ability to differentiate between menstrual and peripheral blood of particular importance. It is essential to determine if blood at the scene of a crime resulted from natural bleeding (i.e., menstrual blood) or from possible injury (i.e., peripheral blood). A new method has been developed to make that evidentiary distinction clear, improving the future of forensic analysis and of criminal investigation.

Sex, crime and forensics

Differentiating peripheral blood from menstrual blood is especially relevant in forensic science. In sexual assault cases,  nondescript blood evidence is often taken from an alleged victim’s undergarments. It is essential not only to confirm that a blood sample is human but also to exclude the possibility that it is menstrual. This determination is necessary to rest the common claim made by alleged aggressors that the evidence is menstrual blood, resulting naturally, as opposed to peripheral blood, resulting from sexual assault. This allows investigators to partly determine the veracity of a litigant’s allegation.

Detecting the source of blood evidence

One of the most characteristic substances comprising blood is the protein hemoglobin. As a protein, hemoglobin is composed of a unique and specific amino acid sequence that forms interlocking globular protein subunits. Among different species, the amino acid sequence that make up hemoglobin tends to be slightly different, making it possible to design antibodies (proteins capable of ‘tagging’ or binding-specific molecular sequences by design) specific to human hemoglobin.

While analyzing the amino acid sequence of hemoglobin through the use of designer antibodies is useful for determining whether a given blood sample is human or not, it is inadequate for the purpose of further determining whether blood is from a menstrual or peripheral source. In the forensic world, the final task of accomplishing this feat is to verify the presence or absence of a novel substance characteristic of menstrual blood.

Menstruation is a natural process of endometrial degradation. The endometrium is an ephemeral, organic layer of the female uterus that is rebuilt every 28 days. During menstruation, a phenomenon called “fibrinolysis” occurs to prevent blood clotting. One of the byproducts of fibrinolysis is a protein fragment called “D-dimer”. D-dimer is present in menstrual blood and is only present in peripheral blood in certain clinical conditions. It is, therefore, possible to design specific antibodies that verify the presence of D-dimer through the use of a multiplex immunochromatography assay. In fact, immunochromatography is a common method used in rapid pregnancy tests for detecting the presence of a pregnancy hormone in urine. For our purposes in forensic science, this multiplex immunochromatography assay can detect indications of menstrual blood with its D-dimer-specific antibodies.

This recently developed method of blood evidence analysis not only allows investigators to check for human hemoglobin, but also indicates the presence of D-dimer to determine if the blood is from a menstrual or peripheral source, which is often critical in sexual assault investigations.

Validation and forensic use

Validation of an assay or test gives strength to the quality of its results by indicating that the test is capable of truly measuring what it claims to measure. The paper Forensic differentiation between peripheral and menstrual blood in cases of alleged sexual assault—validating
an immunochromatographic multiplex assay for simultaneous detection of human hemoglobin and D-dimer, demonstrated the potential that D-dimer detection in immunochromatography can have in real cases involving alleged sexual assault, and validates its use in the forensic field. It has been shown, for example, that mixtures of menstrual blood and semen do not affect the results of this newly developed assay and that the material diluted in the residual buffer of the assay remains viable for subsequent DNA testing. The assay has been tested with real police cases, in ‘in vitro’ conditions, and with anthropological artifacts used for African rituals by a multi-international team.


There are possible limitations to this newly developed multiplex assay, such as false positives in cases of thrombolytic patients and blood samples from cadavers, as well as other occasions in which an increased concentration of circulating D-dimer is expected. However, this is of only limited relevance, because most blood samples observed at crime scenes can be expected to originate from a living (and, therefore actively bleeding) individual. Aside from these limitations, the test is simple to implement into forensic workflows, and it is possible to use the remaining buffer-sample solution or the sample pad for subsequent DNA analysis. The performance is not user-dependent as it is standardized. Further, no special training on the part of the analyst is needed.

Future work

Our work team had an excellent experience working together and with our international colleagues and supporters. Future work should be done to analyze potential false positives and to verify medical conditions that could affect readings. We are looking forward to, and have already planned to expand our team and embrace new scientists from abroad.

Research Article: Forensic differentiation between peripheral and menstrual blood in cases of alleged sexual assault—validating 
an immunochromatographic multiplex assay for simultaneous detection of human hemoglobin and D-dimer. International Journal of Legal Medicine, 2017.

Authors Bio

Claudemir R. Dias Filho is a crime scene analyst of the Scientific Police in São Paulo, Brazil. He is a biologist and geneticist by formation, but prefer been seen as forensic researcher, criminalistics instructor, and pop-culture junkie. Between one crime scene analysis and another, enjoys playing with his 5 dogs and drone flying.

Gabriela Roca is the Research and Development Project Manager in a Forensic Biotechnology company in Germany. She is a geneticist and has been a scientist for more than 15 years, and is especially happy to see her hard work make a live impact with real-world application. She enjoys doing in her free time swimming, scuba diving and sailing.

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