FRNS301 Introduction to Forensic Serology 2024/2025 PDF

C:\\Users\\Abdulmalik Isa\\Desktop\\ccuk letters\\CCUK Letterheaded.jpg **CAPITAL CITY UNIVERSITY, KANO.** **FRS 301: INTRODUCTION TO FORENSIC SEROLOGY** **BY** **ZAINAB DAUDA SULE** **2024/2025 SESSION** **INTRODUCTION** **Forensic serology** is the detection, identification, classification,...

C:\\Users\\Abdulmalik Isa\\Desktop\\ccuk letters\\CCUK Letterheaded.jpg **CAPITAL CITY UNIVERSITY, KANO.** **FRS 301: INTRODUCTION TO FORENSIC SEROLOGY** **BY** **ZAINAB DAUDA SULE** **2024/2025 SESSION** **INTRODUCTION** **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 DNA analysis and bloodstain pattern analysis. Serology testing begins with presumptive tests which gives the analyst an indication that a specific bodily fluid may be present, but cannot completely confirm its presence. Following the presumptive tests, confirmatory tests are done on the same sample to confirm what the unknown substance actually is. **Blood** is the most commonly recovered and one of the most important pieces of evidence utilized in forensic investigations. It is commonly recovered in cases of homicide, assault, and terrorist attacks, including bomb blasts. Blood as evidence holds significance in the criminal justice system as it can link a crime with a criminal or exclude an individual's involvement in a crime. Moreover, the pattern of blood stains can help in reconstruction of a crime scene by conveying information about the relative position and movement of the criminal and victim at the crime scene. At the crime scene, it is important to establish the type, origin, and other characteristics of the blood/blood stain. Preliminary investigations in this regard are to confirm whether the recovered fluid is blood, and whether its origin is human. Once the human origin of the blood/blood stain is confirmed, further analysis is done to establish other characteristics. The blood stain is initially subjected to blood typing.  **BLOOD** Blood is a specialized body fluid. It has four main components: plasma, red blood cells, white blood cells, and platelets. Blood has many different functions, including: - transporting oxygen and nutrients to the lungs and tissues - forming blood clots to prevent excess blood loss - carrying cells and antibodies that fight infection - bringing waste products to the kidneys and liver, which filter and clean the blood - regulating body temperature **The Components of Blood and Their Importance** **Plasma** The liquid component of blood is called plasma, a mixture of water, sugar, fat, protein, and salts. The main job of the plasma is to transport blood cells throughout your body along with nutrients, waste products, antibodies, clotting proteins, chemical messengers such as hormones, and proteins that help maintain the body\'s fluid balance. **Red Blood Cells (also called erythrocytes or RBCs)** Known for their bright red color, red cells are the most abundant cell in the blood, accounting for about 40 to 45 percent of its volume. The shape of a red blood cell is a biconcave disk with a flattened center - in other words, both faces of the disc have shallow bowl-like indentations (a red blood cell looks like a donut). Production of red blood cells is controlled by erythropoietin, a hormone produced primarily by the kidneys. Red blood cells start as immature cells in the bone marrow and after approximately seven days of maturation are released into the bloodstream. Unlike many other cells, red blood cells have no nucleus and can easily change shape, helping them fit through the various blood vessels in your body. However, while the lack of a nucleus makes a red blood cell more flexible, it also limits the life of the cell as it travels through the smallest blood vessels, damaging the cell\'s membranes and depleting its energy supplies. The red blood cell survives on average only 120 days. Red cells contain a special protein called hemoglobin, which helps carry oxygen from the lungs to the rest of the body and then returns carbon dioxide from the body to the lungs so it can be exhaled. Blood appears red because of the large number of red blood cells, which get their color from the hemoglobin. The percentage of whole blood volume that is made up of red blood cells is called the hematocrit and is a common measure of red blood cell levels. **White Blood Cells (also called leukocytes)** White blood cells protect the body from infection. They are much fewer in number than red blood cells, accounting for about 1 percent of your blood. The most common type of white blood cell is the **neutrophil,** which is the \"immediate response\" cell and accounts for 55 to 70 percent of the total white blood cell count. Each neutrophil lives less than a day, so your bone marrow must constantly make new neutrophils to maintain protection against infection. Transfusion of neutrophils is generally not effective since they do not remain in the body for very long. The other major type of white blood cell is a **lymphocyte**. There are two main populations of these cells. T lymphocytes help regulate the function of other immune cells and directly attack various infected cells and tumors. B lymphocytes make antibodies, which are proteins that specifically target bacteria, viruses, and other foreign materials. **Platelets (also called thrombocytes)** Unlike red and white blood cells, platelets are not actually cells but rather small fragments of cells. Platelets help the blood clotting process (or coagulation) by gathering at the site of an injury, sticking to the lining of the injured blood vessel, and forming a platform on which blood coagulation can occur. This results in the formation of a fibrin clot, which covers the wound and prevents blood from leaking out. Fibrin also forms the initial scaffolding upon which new tissue forms, thus promoting healing. A higher than normal number of platelets can cause unnecessary clotting, which can lead to strokes and heart attacks; however, thanks to advances made in antiplatelet therapies, there are treatments available to help prevent these potentially fatal events. Conversely, lower than normal counts can lead to extensive bleeding.  **\ ** **IDENTIFICATION OF BLOOD STAINS BY MICROSCOPIC METHODS** **Optical Microscopy** 1\. Morphology: Examine the shape and size of red blood cells (RBCs) to determine if they are human or animal.   Human RBCs are circular, biconcave, and lack a nucleus, with a diameter of about 6-8 microns, while animal RBCs can vary in size and shape, for example: - birds, amphibians, and reptiles have oval-shaped, nucleated RBCs. 2\. Color: Observe the color of the blood stain, which can indicate the presence of hemoglobin. 3\. Crystal formation: Look for crystals formed by the drying of blood, which can be characteristic of blood. **Polarized Light Microscopy** 1\. Birefringence: Observe the birefringence (double refraction of light in a transparent, molecularly ordered material) of RBCs, which can help identify blood. 2\. Cross-polarized light: Use cross-polarized light to enhance the visibility of RBCs. **Fluorescence Microscopy** 1\. Fluorescence: Excite the blood stain with a specific wavelength of light to produce fluorescence, which can indicate the presence of hemoglobin. 2\. Filter sets: Use specific filter sets to enhance the visibility of fluorescent signals. **Scanning Electron Microscopy (SEM)** 1\. Morphology: Examine the surface morphology of RBCs to determine if they are human or animal. 2\. Elemental analysis: Use energy-dispersive spectroscopy (EDS) to analyze the elemental composition of the blood stain. **Confocal Laser Scanning Microscopy (CLSM)** 1\. 3D imaging: Create 3D images of the blood stain to study its morphology. 2\. Fluorescence imaging: Use CLSM to study the fluorescence properties of the blood stain. **TESTS/METHODS USED IN BLOOD ANALYSIS** **CATALYTIC BLOOD TEST** The **Kastle--Meyer test** is a form of **catalytic blood test,** one of the two main classes of forensic tests commonly employed by crime labs in the chemical identification of blood. **Method** A presumed blood sample is first collected with a swab. A drop of phenolphthalein reagent is added to the sample, and after a few seconds, a drop of hydrogen peroxide is applied to the swab. If the swab turns **pink rapidly**, it is said to test presumptive **positive for blood**. Waiting for periods over 30 seconds will result in most swabs turning pink naturally as they oxidize on their own in the air. Optionally, the swab can first be treated with a drop of ethanol in order to lyse the cells present and gain increased sensitivity and specificity. This test is nondestructive to the sample, which can be kept and used in further tests at the lab; however, few labs would use the swab used for the Kastle--Meyer test in any further testing, opting instead to use a fresh swab of the original stain. **CRYSTAL TEST** The **crystal test**, also known as the Teichmann crystal test or the hemin crystal test, is a chemical test used to detect the presence of blood. **Principle:** The test is based on the reaction between hemoglobin in blood and sodium chloride (NaCl) in the presence of glacial acetic acid. This reaction forms hemin crystals, which are characteristic of blood. **Procedure** 1\. Add a few drops of the blood sample to a clean glass slide or test tube. 2\. Add a few drops of the NaCl solution to the blood sample. 3\. Carefully add a few drops of glacial acetic acid to the mixture. 4\. Observe the mixture under a microscope or with the naked eye. **Results** If blood is present, the mixture will form characteristic hemin crystals, which appear as: **- Rhombic or hexagonal crystals:** These crystals are typically yellow or brown in color. **- Needle-like crystals:** These crystals may also form, especially if the blood sample is old or degraded. **ABO GROUP** The blood groups refer to the presence on human red blood cells of certain antigens, the blood group factors. One very important group of factors present on the red blood cells is the ABO system. The ABO group of a person depends on whether his/her red blood cells contain one, both, or neither of the 2 blood group antigens A and B. There are, therefore, 4 main ABO groups: **A**, **B**, **AB** and **O**. Antibodies (agglutinins) for the antigens A and B exist in the plasma and these are termed anti-A and anti-B. The corresponding antigen and antibody are never found in the same individual since, when mixed, they form antigen-antibody complexes, effectively agglutinating the blood. **Testing for ABO Group -- Procedure** ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------ -- ------- One end of a slide is labelled Anti-A, and the other Anti-B. A drop of Anti-A test serum is added to the end marked Anti-A, and a drop of Anti-B serum is added to the end marked Anti-B. One drop of blood is added to each end of the slide, and mixed well, using separate wooden sticks.  The results are read directly from the slide. The subject is blood group A if agglutination occurred with the Anti-A test serum; group B if agglutination occurred with the Anti-B test serum; group AB if agglutination occurred with both test serums, and O if there was no agglutination in either case. **In the sample to the right, we conclude the subject has type A blood.** Examine the slides below and determine the blood type of the subject in each case **1**  **2** **3**  **4** **THE RH SYSTEM** Rh antigens, named for the rhesus monkey in which they were first discovered, are also surface antigens expressed on red blood cells. There are a few Rh antigens (common one is called D). Red cells expressing the Rh antigens are called Rh positive. Red cells which do not express this surface antigen are Rh negative (about 15% of the human population is Rh negative). Rh system becomes important when one considers the eventuality of Rh incompatibility between mother and fetus; in such a case, the antibody-mediated cytotoxicity mechanism involved threatens the well-being of the fetus. During birth, a leakage of the baby\'s red blood cells often occurs into the mother\'s circulation. If the baby is Rh positive (inheriting the trait from its father) and the mother is Rh negative, these red cells will cause the mother to manufacture antibodies against the Rh antigen. The antibodies (IgG class) do not cause problems for that first born, but can cross the placenta and attack the red cells of a subsequent Rh+ fetus. The red cells are destroyed, leading to anemia and jaundice. The disease - erythroblastosis fetalis or hemolytic disease of the newborn- may result in fetal death. **MN SYSTEM** The MNS antigen system is a human blood group system based upon two genes (glycophorin A and glycophorin B) on chromosome 4. There are currently 50 antigens in the system but the five most important are called M, N, S, s, and U. The system can be thought of as two separate groups: the M and N antigens are at one location on the ECM and S, s, and U are on a closely related location. The two groups are very closely located together on chromosome 4 and are inherited as a haplotype. **Testing Methods** 1\. Agglutination test: This is the most common method used to determine the MN blood group. The test involves mixing the blood sample with anti-M and anti-N antibodies. If the blood cells agglutinate (clump together), it indicates the presence of the corresponding antigen. 2\. Gel test: This method uses a gel matrix to separate the blood cells based on their antigenic properties. 3\. ELISA (Enzyme-Linked Immunosorbent Assay): This method uses enzymes linked to antibodies to detect the presence of the M and N antigens. **Interpretation of Results** 1**. M+ N-**: The individual has the M antigen but not the N antigen. 2\. **M- N+**: The individual has the N antigen but not the M antigen. 3\. **M+ N+**: The individual has both the M and N antigens. 4\. **M- N-**: The individual does not have either the M or N antigen. **SPECTROPHOTOMETRIC METHOD** Spectrophotometry refers to the process of analyzing samples using **electromagnetic waves**. To test a sample, spectrophotometers test how the light refracts and reflects off a particular material and analyze its color, infrared and ultraviolet properties and more. Using spectrophotometry, you can tell the difference between similar samples of hairs, fibers, drugs, bodily fluids and fingerprints.  Spectrophotometry is a gold standard of forensic analysis and is used by the FBI and the American Hazardous Material Response Unit. The spectrophotometric process is essential for telling apart two samples that are nearly indistinguishable. Most forensic analysts use UV/vis spectroscopy in forensic science to examine inks and fibers and use thin-layer chromatography as a complementary method.  Haemoglobin on treatment with acids, alkalis, reducing agents or oxidizing agents gives a variety of products which have characteristics absorption spectra. They help to identify the blood. The absorption bands are seen in length which are characteristic of blood. **CHROMATOGRAPHIC METHOD** Chromatographic methods are widely used in blood testing to separate, identify, and quantify various components of blood. Here are some common chromatographic methods used in blood testing: **Types of Chromatographic Methods** 1\. High-Performance Liquid Chromatography (HPLC): HPLC is a widely used chromatographic method in blood testing. It separates, identifies, and quantifies various components of blood, such as proteins, hormones, and metabolites. 2\. Gas Chromatography (GC): GC is commonly used to analyze volatile compounds in blood, such as ethanol, methanol, and other toxic substances. 3\. Thin-Layer Chromatography (TLC): TLC is a simple and rapid chromatographic method used to separate and identify various components of blood, such as lipids, proteins, and carbohydrates. **IMMUNOLOGICAL METHODS** Immunological methods are widely used in blood testing to detect and quantify various components of blood, such as antibodies, antigens, and proteins. Here are some common immunological methods used in blood testing: **Types of Immunological Methods** 1\. Enzyme-Linked Immunosorbent Assay (ELISA): ELISA is a widely used immunological method that detects the presence of specific antibodies or antigens in blood. 2\. Western Blot: Western blot is an immunological method that separates and identifies proteins in blood based on their size and antigenicity. 3\. Immunofluorescence: Immunofluorescence is an immunological method that uses fluorescent dyes to detect specific antibodies or antigens in blood. 4\. Immunoprecipitation: Immunoprecipitation is an immunological method that uses antibodies to precipitate specific proteins or antigens from blood. 5\. Radioimmunoassay (RIA): RIA is an immunological method that uses radioactive isotopes to detect specific antibodies or antigens in blood. **DETERMINATION OF SPECIES OF ORIGIN, RING TEST, SINGLE DIFFUSION, DOUBLE DIFFUSION, CROSSED-OVER ELECTROPHORESIS.** It is important for a forensic scientist to determine if the concerned stain is blood or not and if it is blood then whether it is of human origin or not. Supposedly, if the blood is not of human origin, then it is very necessary to do the species origin test for the concerned bloodstain so as to determine the species. Most methods in common use for determining the species of origin are immunological in nature. If an animal is injected with a protein molecule from another species, it will sometimes recognize this protein as a foreign substance (antigen) and will produce an antiserum (antibody) that respond with such protein in both vivo and in vitro. The following are the most commonly used methods for species determination in forensic laboratories: \(a) Ring Test, (b Single Diffusion, \(c) Double diffusion \(d) Crossover electrophoresis. **Ring Test** The ring test, also known as the precipitation ring test, is a serological technique used to identify the species of origin of a biological fluid, such as blood or semen. The ring test is based on the principle of immunoprecipitation, where antibodies react with antigens to form a visible precipitate.  **Single Diffusion** The single diffusion method is based on the principle of immunodiffusion, where antibodies and antigens react to form a precipitin line. **Procedure** 1\. Prepare the agar gel: Melt the agar gel and cool it to around 50°C. 2\. Add antisera: Add a small amount of species-specific antisera to the agar gel and mix well. 3\. Pour the agar gel mixture: Pour the agar gel mixture into a sterile petri dish. 4\. Create a well: Create a small well in the agar gel using a sterile pipette or a well cutter. 5\. Add the sample: Add a small amount of the sample to be identified into the well. 6\. Incubate: Incubate the petri dish at 37°C for 24-48 hours. 7\. Observe: Observe the agar gel for the formation of a precipitin line. **Interpretation** 1\. Positive reaction: A positive reaction is indicated by the formation of a precipitin line between the sample and the antisera. 2\. Negative reaction: A negative reaction is indicated by the absence of a precipitin line. 3\. Species identification: The species of the sample can be identified based on the positive reaction with the species-specific antisera. **Double Diffusion Method** This diffusion method was first described by Ouchterlony in 1949. It involves the use of agar gel plates with wells for both antibodies and antigens. The two reactants diffuse into the gel where the soluble antigens and antibodies form an insoluble complex -a precipitate. The reactants can be evaluated both qualitative and semi-quantitatively by this technique. Precipitin band formation gives the scientist considerable information regarding the identity, partial identity or non-identity of the antigen and antibody reaction. It also yields information on the diffusion coefficients and concentrations of the reactants. **Crossed-Over Electrophoresis** This method can be employed for the qualitative and quantitative determination of blood samples. In crossed-over electrophoresis, the antigen and antibody migrate towards each other under the influence of electric field thereby forming a precipitin band at the point of their interaction. Small wells about 1.5 mm in diameter are punched in an agar gel. The stain extract is placed in the cathodic well of a neighboring pair, and the antiserum in the opposite well. The antiserum travels towards the cathode (negative electrode), while the stain extract migrates anodically (toward the positive electrode). A precipitin band will form at the site of the interaction of the antibodies and the antigens. White line precipitin band means antiserum and blood match. **GROUPING OF BLOOD STAINS** Grouping of blood stains is a crucial step in forensic serology, as it helps to identify the source of the blood and link it to a suspect or victim. **Methods for Grouping of Blood Stains** 1\. ABO Blood Grouping: This method determines the ABO blood group of the blood stain by reacting it with anti-A and anti-B antibodies. 2\. Rh Blood Grouping: This method determines the Rh blood group of the blood stain by reacting it with anti-Rh antibodies. 3\. MNS Blood Grouping: This method determines the MNS blood group of the blood stain by reacting it with anti-M and anti-N antibodies. 4\. Kell Blood Grouping: This method determines the Kell blood group of the blood stain by reacting it with anti-Kell antibodies. 5\. Enzyme Typing: This method determines the enzyme type of the blood stain, such as phosphoglucomutase (PGM) or erythrocyte acid phosphatase (EAP). 6\. Protein Typing: This method determines the protein type of the blood stain, such as haptoglobin or transferrin. **Techniques Used for Grouping of Blood Stains** 1\. Agglutination: This technique involves reacting the blood stain with antibodies specific to different blood groups. 2\. Adsorption: This technique involves adsorbing the antibodies onto the surface of red blood cells. 3\. Elution: This technique involves eluting the antibodies from the surface of red blood cells. 4\. Gel Test: This technique involves using a gel matrix to separate the blood cells based on their antigenic properties. **COMPOSITION AND EXAMINATION OF BIOLOGICAL FLUIDS** **SALIVA** **Composition** Salivary fluid is an exocrine secretion consisting of approximately 99% water, containing a variety of electrolytes (sodium, potassium, calcium, chloride, magnesium, bicarbonate, phosphate) and proteins, represented by enzymes, immunoglobulins and other antimicrobial factors, mucosal glycoproteins, traces of albumin and some polypeptides and oligopeptides of importance to oral health. There are also glucose and nitrogenous products, such as urea and ammonia. The components interact and are responsible for the various functions attributed to saliva. Total or whole saliva refers to the complex mixture of fluids from the salivary glands, the gingival fold, oral mucosa transudate, in addition to mucous of the nasal cavity and pharynx, non-adherent oral bacterial, food remainders, desquamated epithelial and blood cells, as well as traces of medications or chemical products. At rest, without exogenous or pharmacological stimulation, there is a small, continuous salivary flow (SF). Whereas, stimulated saliva is produced in the face of some mechanical, gustatory, olfactory, or pharmacological stimulus, contributing to around 80% to 90% of daily salivary production. A healthy person's mean daily saliva production ranges from 1 to 1.5L. **Examination** After collection of saliva sample, we perform following test for examination of saliva sample a\) Starch- Iodine Test: Reagents Preparation: 1\) 0.5% soluble starch solution (50 mg soluble starch / 10 ml H2 O). 2\) Lugol's iodine solution. Procedure: 1\) Place 3 tubes in a rack and add the following: a\) In the first tube, place a 5 mm x 5 mm piece of sample to be tested. b\) In the second tube, place a similar sized unstained control piece. c\) In the third tube, place a 5 mm x 5 mm piece of a known saliva stain. 2\) Add 3 drops of soluble starch solution to each tube. 3\) Mix, cork and incubate the tubes for 1 hour at 37⁰C. 4\) Add 2 drops of Lugol's iodine and note the colour formed. 5\) A dark blue starch-iodine complex should be observed in the second and fourth tubes. The absence of the dark blue colour indicates that the starch has been hydrolyzed and is no longer available for complexing. Therefore, the lack of blue colour is a positive result for amylase activity, indicative of the presence of saliva. **SEMEN** **Composition** Seminal fluid is a complex minute of glandular secretion. Seminal fluid accounts for approximately 60% of the ejaculate. Seminal fluid contains the "flavin" that causes semen to fluorescence under the ultra-violet light. Prostatic fluid accounts for approximately 30% the ejaculation. This portion of the semen contains high concentration of acid phosphatase (AP) and prostate specific antigen (PSA). Both are very useful to the forensic analyst for detection of semen in case of rape, unnatural offence, hanging, etc. It is an alkaline fluid with pH=7.4. **Examination of Semen and Seminal Stains** **Physical Examination:** **Colour:** Thick, yellowish white, glairy, opalescent, secretion having a characteristic odor known as seminal odor. **Texture:** On touch, seminal stains are starchy. **Appearance:** Garments sent for forensic examination are usually dirty having variety of stains, in natural light some stains are reddish coloured, while others are brown, yellow or faint grey in colour. These are often mixed with stains of blood vaginal discharge, urine and semen, so as to restrict the investigation to seminal stains only, preliminary examination is done under filtered UV light. The fluorescence of the seminal stains is of a bluish white colour and such stains should be selected for further examination. **Confirmatory Test: Microscopic Examination** Upon obtaining a positive preliminary test for acid phosphates, the suspected stain can be extracted as follows: 1\) Cut a small portion (1 cm2 maximum) of the stain and place in a test tube. 2\) Add a few drops of acidulated (slightly acidic) water to cover the stain. 3\) Agitate the stain on a vortex, or in an ultrasonic cleaner bath or manually, by flicking the tube. This will aid in freeing the spermatozoa from the dried stain. 4\) When the solution is cloudy, withdraw the liquid with a pipette and place into a disposable 400ul plastic centrifuge tube and centrifuge in a microfuge for 30 seconds. 5\) Carefully withdraw the supernatant, which contains soluble group-specific substances, enzymes and other solutes from seminal plasma. 6\) Collect the button of cellular and other insoluble components from the tube and place on a clean-labeled microscope slide. 7\) Fix in dilute H 2SO 4 acid and dry. It is now ready for staining. **URINE** **Composition** Urine consists of water, organic and inorganic substances. Water alone forms about 95% of it and other substances form only 5%. The organic substances are mainly nitrogenous compounds but small amounts of non-nitrogenous compounds are also present. Nitrogenous organic compounds include urea, uric acid, creatinine and hippuric acid. Out of all these, urea is the principal component of human urine. Non-nitrogenous organic compounds include Vitamin C, oxalic acid, phenolic substances and traces of glucose. The inorganic substances include ammonia & mineral salts such as chlorides, sulphates and phosphates of sodium, potassium, calcium & magnesium. Sodium chloride is the principal mineral salt of the urine. Urine also contains some other substances like pigments & drugs coupled with some epithelial cells and leucocytes. **Examination** **1) Physical Examination-** A suspected urine stain may fluoresce pale yellow or pale blue, when viewed under long and short-wave Ultraviolet light. Safety glasses, which absorb UV radiation, must be worn when viewing material for fluorescence. **2) Odour test**-The characteristic odour of urine may be detected by placing a small sample of the stain in a test tube and heating it gently over flame. Avoid scorching the test material. **3) Urea Nitrate Crystal test**- An aqueous extract of stain is made and a thin film of it is made on a microscopic slide. Add one drop of concentrated. Nitric Acid (HNO3) and cover. In presence of urea, hexagonal stacked crystals of urea nitrate are formed. **4) Gee's method for detection of Urea**- A portion of the suspected stain is extracted with acetone and the acetone extract is concentrated by evaporation. The concentrated extract is filtered and evaporated to dryness. A few drops of acetone is added to the residue and stirred with a narrow glass rod. A drop of the solution thus prepared is taken on a microscopic slide and again allowed to evaporate. At this stage separation of urea crystals (long colorless 4 or 6 sided rhombic prisms) may be observed. A glass rod dipped in concentrated nitric acid is lightly run across the surface of the crystal line area. Formation of characteristic urea nitrate crystals (colorless rhombic or 6 sides tiles) may be observed under microscope indicating the presence of urea in the suspected stain. **5) Detection of Creatinine**- A concentrate of the stain is placed on a strip of chromatographic paper and it is treated with 2N sodium hydroxide solution and after it one drop of 5% picric acid is added to it. The development of red-orange color indicates the presence of creatinine in the stain. **6) Indican test**- This test though not always positive, may be undertaken for the identification of urine in addition to the above tests. 1ml of resorcinol reagent (1gm resorcinol in 20ml of ethyl alcohol) is added to a small quantity of aqueous extract of the stain followed by the addition of 1ml of cupric bromide. The mixture is extracted with amyl acetate. Red coloration of the extract indicates the presence of indican (a potassium salt). **VAGINAL FLUID** **Composition** **Chemical Composition** 1\. Water: Vaginal fluid is primarily composed of water (90-95%). 2\. Electrolytes: Sodium, potassium, calcium, and magnesium ions are present in varying concentrations. 3\. Proteins: Enzymes, such as amylase, lipase, and lysozyme, are present in vaginal fluid. 4\. Glycoproteins: Mucins, which are glycoproteins, contribute to the fluid\'s viscosity and lubricating properties. 5\. Hormones: Estrogen and progesterone are present in vaginal fluid, reflecting the hormonal fluctuations during the menstrual cycle. 6\. pH: The pH of vaginal fluid ranges from 3.8 to 4.5, which is acidic. **Cellular Composition** 1\. Epithelial cells: Squamous epithelial cells, which line the vaginal wall, are present in vaginal fluid. 2\. Leukocytes: White blood cells, such as neutrophils and lymphocytes, are present in small numbers. 3\. Bacteria: Lactobacilli, which are beneficial bacteria, are normally present in vaginal fluid. **Examination** **Physical Examination** 1\. Color and consistency: Observe the color and consistency of the vaginal fluid. Normal vaginal fluid is clear or cloudy and has a thin, watery consistency. 2\. pH: Measure the pH of the vaginal fluid using pH paper or a pH meter. Normal vaginal fluid pH ranges from 3.8 to 4.5. 3\. Odor: Note any unusual odors, which can indicate infection or contamination. **Microscopic Examination** 1\. Wet mount: Prepare a wet mount of the vaginal fluid and examine it under a microscope (400x) for: \- Epithelial cells \- Leukocytes (white blood cells) \- Bacteria (e.g., lactobacilli) \- Yeast cells or hyphae \- Spermatozoa (in cases of sexual assault) 2\. Gram stain: Perform a Gram stain on the vaginal fluid to identify bacteria and yeast. **Serological Examination** 1\. Antibody detection: Use techniques like ELISA or Western blot to detect antibodies against specific pathogens, such as HIV or HSV. 2\. Antigen detection: Use techniques like PCR or antigen capture assays to detect specific antigens, such as semenogelin or prostate-specific antigen.