Menstrual blood is regular discharge of blood and mucosal tissue (known as menses) from the inner lining of the uterus through the vagina. This process begins at what is known as menarche (first cycle ) which happens between 12 to 15 years in girls depending on a lot of factors. The process is known as menstruation and it usually stops(menopause)at the age of 45- 55 years.
COMPOSITION OF MENSTRUAL BLOOD
Menstrual blood contains sodium, calcium, phosphate, iron, and chloride, the extent of which depends on the woman. As well as blood, the fluid consists of cervical mucus, vaginal secretions, and endometrial tissue.
Basic difference between peripheral blood and menstrual blood
Menstrual blood is not highly oxygenated; that’s why it is darker than normal blood. pH is acidic because of the vaginal secretions. The concentration of Iron, Hemoglobin and protein is found to be less in menstrual blood than in normal blood in various study.
Menstrual blood contains vaginal, cervical and decidual cells. It doesnot coagulate as it is already coagulated. Matrix mettaloproteinases are present in menstrual blood but not in blood from other sources.
Menstrual blood contains all the five isozymes of Lactate dehydrogenase , LDH-1, LDH-2, LDH-3, LDH-4 and LDH-5. Venous blood contains LDH- 1,2 and 3 where as menstrual blood contains all 5.
EXAMINATION OF MENSTRUAL BLOOD
These include microscopy, identification of the lactate dehydrogenase isoenzyme, detection of fibrinolytic products, and profiling of messenger ribonucleic acid (mRNA) and micro RNA (miRNA). Even though menstrual blood is encountered at scene of crime, a reliable routine procedure for its identification has not yet been incorporated in forensic laboratories in India .
- Physical Appearance
- The discharge is in the form of a trickling descent, it does not splash. Therefore, the nature and distribution of the suspect stain must be taken in account when making the examination.
- Colour – May vary from bright red to dark brown/ black depending on how old it is.
2. Microscopic Examination
Vaginal epithelium and residual cells will usually be present in menstrual bloodstains, along with endometrial cells.
3. Identification by Fibrin Degradation Product
Menstrual blood is discharged from the uterus in a fluid state; it contains no fibrinogen and cannot be made to clot. This situation is a result of a fibrinolysis process during which the blood clots and subsequently reliquifies prior to menstrual period. The reliquifaction process is known as fibrinolysis and defined as enzymatic digestion of fibrin clot to soluble Fibrin Degradation Products (FDP). The test is carried out in two parts:
—Assay of FDP in stain extract
—The Assay of total soluble protein in the extract.
Stain Extraction:
Extract a small piece of bloodstain (0.5 sq.cm.) on 0.3 ml of saline for one hour at room temperature. Remove the stain, centrifuge at full speed for 10 minutes. Transfer the supernatant to a small clean tube and use for fdp and total soluble protein assay.
—Assay for Fibrinogen Degradation Products:
Fibrinogen Degradation Products may be assayed by a haemagglutination inhibition method. In this technique they are quantitated by their ability to inhibit the agglutination of sheep red blood cells coated with human fibrinogen by anti- fibrinogen serum.
Materials:
Fibrinogen Standards – Human Fibrinogen Standards are diluted to a concentration of 10 µg/ml and stored at –20⁰C and should be thawed twice only.
Phosphate Citrate Buffer pH 6.4 (1.86 g disodium hydrogen phosphate, 8.95 g tri sodium citrate, 0.050 g Sodium azide, 500 ml distilled water, Citric Acid is used to bring pH to 6.4, 1.15 ml 30% Bovine albumin solution)
Store at 4⁰C.
Antiserum – Fibrinogen specific anti-sera are commercially available. Each vial of freeze-dried antiserum is dissolved in 1 ml water. The specificity of the anti-sera is to be checked with a wide variety of human serum dilution. Dilute the anti-sera with Phosphate Citrate Buffer to produce a working strength solution for the haemagglutination inhibition assay. This is usually between 1/2000 and 1/4000 dilutions of the stock antiserum depending on the batch of sensitized cells used.
Sensitized Cells– Sheep red blood cells sensitized with human fibrinogen are obtained commercially. The cells supplied usually in 50 ml aliquots of 2.5% suspension. Prior to each assay, the cells are centrifuged and resuspended in Phosphate Citrate Buffer.
Microtitre Plates – The assays are performed in Perspex Microtitre Plates. Each plate contains eight rows (A-H) with twelve V-cup shaped wells in each row.
Operating Protocol
• Using Pasteur pipette, place one drop of buffer in wells 2-10 and two drops in well 11 row A.
• Place one drop of buffer in wells 2-9 and well 12 of all subsequent rows – B, C, D, ………i.e., one row for each assay.
• Place one drop of standard fibrinogen solution in wells 1, 2 of row A. Wash pipette in water.
• Place one drop of stain extract in wells 1, 2 and 12 of rowB.
• The next sample should be placed in wells 1, 2 and 12 of row C. Wash pipette in water before application of each sample.
• Prepare doubling dilution of the fibrinogen standard and samples. Take one drop of solution from well 2 and add it to well 3. Return any remaining solution to well 2.
• Take one drop of solution from well 3 and add it to well 4. Return any remaining solution to well 3 etc.
• Discard the final drop from well 9. Wash the pipette in distilled water after dilution of each sample.
• Using a clean Pasteur pipette, place one drop of diluted antisera in wells 1-10 of row A and well 1-9 of all subsequent row.
• Mix contents of the wells by gently tapping the sides of the plates, cover and incubate at 4⁰C for an hour. Remove the plates from the refrigerator and add one drop of freshly suspended sensitized cells to wells 1-11 of row A and well 1-9 and 12 of subsequent rows.
• Mix contents of the wells by gently tapping the sides of the plates, cover the plates and incubate at 4 degree celsius for two and half hours. The results can be read after that.
Calculation and interpretation of Results
The end point of the fibrinogen standard and the samples is taken as the last well showing complete no agglutination. This appears as a compact ‘button’ at the bottom of the appropriate well. Partial or complete agglutination appears as a curtain. If the controls are different from those described then the results must be discarded.
Methods used to differentiate Menstrual Blood and Peripheral Blood
Many a times at crime scene we get menstrual blood and peripheral blood. It gets really difficult for the crime scene investigator to find out the origin of blood at the scene of crime. Depending on the case, it is important that origin of blood should be determined to best lead the investigation. There have been cases when peripheral blood was used to implicate someone and vice versa. Forensic scientists are working on developing and validating the method for differentiation of peripheral blood and menstrual blood. Below are some of the method and techniques that are used in recent times for differentiation of both the blood type.
- Teichmann test– show a negative results with menstrual blood but give a positive results with normal blood samples because hemoglobin level in menstrual blood is very low
- Raman Spectroscopy (along with chemometrics)- Raman spectroscopy is a non-destructive analytical technique that uses the inelastic scattering of light to provide information on chemical composition. The menstrual and peripheral blood samples have similar but distinct Raman spectra.
- ATR FT-IR spectroscopy – An IR beam is directed onto an optically dense crystal with a high refractive index at a certain angle. This internal reflectance creates an evanescent wave that extends beyond the surface of the crystal into the sample held in contact with the crystal. In regions of the IR spectrum where the sample absorbs energy, the evanescent wave will be attenuated. The attenuated beam returns to the crystal, then exits the opposite end of the crystal and is directed to the detector in the IR spectrometer. The detector records the attenuated IR beam as an interferogram signal, which can then be used to generate an IR spectrum. Differences in fluid composition, including proteins and small molecules, resulted in spectral differences.Blood and menstrual blood is differentiated by the peak at 1039 cm−1 attributed to phosphoric acid found in menstrual blood.
- Micro- RNA – MicroRNAs (miRNAs), a class of non-protein coding molecules that regulate gene expression at the post-transcriptional level. mRNA markers (hemoglobin alpha (HBA), matrix metalloproteinases 7 &11 (MMP7 &MMP11) as forensic markers are used for differentiation between menstrual and peripheral blood stains. It is found that MMP 7&11 are absent in peripheral blood samples. MMP7 levels is seen to be higher than that of MMP11 throughout days of menstruation.
- SERATEC PMB test– The PMB test is a duplex test combining human hemoglobin and D-dimer detection.
All the above mentioned tests are recent and lot is to be yet researched, these methods have not proven to be reliable or confirmatory, that is why these methods are not given in detail here.
Forensic significance of Menstrual Blood
This type of fluid can be found in cases of sexual assault cases, murder on the onset of menstruation of the female whether victim or suspect.
It may be relevant to establish that a bloodstain is of menstrual origin in cases of assault when grouping alone cannot differentiate between individuals or when as assailant claims that a bloodstain is menstrual.
The presence of peripheral blood indicates a traumatic cause, whereas menstrual blood points towards a natural bleeding cause. Accurate detection of menstrual blood can also help with the reconstruction or corroboration of events.
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