Biological Evidence-Blood

Blood is a fluid type of connective tissue. It is a complex tissue which is composed of a liquid part, plasma, and cellular components. Blood is one of the most important physical and biological evidence which can be found in the crime scene in the form of blood pool, droplets, stains etc. It is a major evidence found in cases like rape, sexual assault, murder, homicide etc.

It is known as:

  • Fluid of life: Transports oxygen from the lungs to all parts of the body and carbon dioxide from the rest of the body to the lungs.
  • Fluid of growth: Transports nutrients from the digestive system and hormones from the endocrine gland to all of the body’s tissues.
  • Fluid of health: Protects the body against diseases and transports waste products and undesirable substances to excretory organs like the kidneys.


  • Colour: Blood is usually red in colour. Arterial blood contains more oxygen and hence it appears scarlet red (Oxygenated blood), but venous blood is purple red due to the presence of more carbon dioxide (Deoxygenated blood).
  • Volume: A typical adult’s blood volume is 5 litres. The volume of blood in a newborn infant is 450 ml. It increases as the child grows and reaches a maximum of 5 L at puberty. It is somewhat less in females, around 4.5 L. In a normal young healthy adult weighing around 70 kg, it accounts for roughly 8% of body weight.
  • pH and Reaction: pH is 7.4 in normal conditions and it is slightly alkaline.
  • Specific gravity: Total blood-1.052 to 1.061, Blood cells-1.092 to 1.101, Plasma-1.022 to 1.026.
  • Viscosity: Blood is 5 times more viscous than water due to the presence of red blood cells and plasma.


Blood contains 2 parts- Blood cells and a liquid portion (plasma).

The blood cells are of three main types:

>> Red blood cells (RBC or Erythrocytes): RBC’s are formed in the bone marrow. RBCs usually have a count of 4.5-5.4 million cells per mL. Hemoglobin-containing red blood corpuscles are important for transporting oxygen from the lungs to other parts of the body. Each human red cell has a diameter of around 7.5 m and a thickness of about 2 m. Agglutinogens are antigens that are found in human red blood cells.

>> White blood cells (WBC or Leukocytes): A healthy adult’s WBC count is around 4000-10,000 white blood cells per millilitre. White blood corpuscles contain antibodies that fight foreign bodies, which cause infections and disturbs the immune system. Granulocytes, also known as polymorphonuclear leukocytes, are abundantly found. Many granulocytes include neutrophilic granules (neutrophils), a small number have acid dye-staining granules (eosinophils), and a few contain basic dye-staining granules (basophils). Lymphocytes, which have big round nuclei and little cytoplasm, and monocytes, which have abundant cytoplasm and kidney-shaped nuclei, are the other two types of WBC’s.  Because DNA can be extracted from these nucleated leukocytes, it is usually used for blood genetic marker analysis.

>> Platelets (Thrombocytes): These are small, granulated bodies which are present in blood. Human blood contains around 300000 platelets per mL, each with a diameter of 2-4m.Thrombocytes are involved in blood clotting.

The liquid portion of blood is known as plasma. It is the yellowish fluid portion of blood which contains electrolytes, minerals, nutrients, vitamins and proteins. It contains 91% of water and 8% to 9% of solids.

Serum is a clear straw-colored fluid that seeps from a blood clot. Blood clots when it is shed or collected in a container. The fibrinogen is converted to fibrin in this process, and the blood cells are caught in the fibrin, producing a blood clot. Serum seeps out of the blood clot after around 45 minutes. Serum is separated from the blood by the process of centrifugation for the purpose of investigation. The only difference between plasma and serum is the absence of fibrinogen in serum as it gets converted to fibrin during clotting.


  • Nutritive substances produced from digested food, such as glucose, amino acids, lipids, and vitamins, are absorbed from the gastrointestinal tract and transported by blood to different parts of the body for growth and energy generation.
  • Blood removes waste materials generated in tissues during various metabolic activities and transports them to excretory organs such as the kidney, skin, and liver for excretion.
  • The blood is responsible for transporting respiratory gases (Oxygen and Carbon dioxide) gases. It transports oxygen from the alveoli to various tissues and carbon dioxide from tissues to the alveoli.
  • Regulation of water balance in the body.
  • Hormones secreted by ductless (endocrine) glands are discharged into the bloodstream directly. These hormones are transported to their target organs/tissues by the blood. Enzymes are also transported by blood.
  • Plasma proteins and hemoglobin act as buffers and help in the regulation of acid-base balance.
  • Blood balances between the heat loss and heat gain in the body by thermoregulatory mechanism.
  • Blood plays a crucial function in the body’s defence mechanism. This function is carried out by white blood cells. By phagocytosis, neutrophils and monocytes consume bacteria. Lymphocytes have a role in immune development. Eosinophils are responsible for foreign protein detoxification, breakdown, and elimination.


Blood examination is conducted in three different steps, which include:

  • Physical examination
  • Presumptive test or Preliminary test
  • Confirmatory test
  • Detection of species of origin-Animal/Human blood

1.) Physical examination: Blood stains appears reddish brown in natural light and if examined under UV light (230-269 nm), blood produces fluorescence.

2.) Presumptive screening test: These are preliminary tests which are done to confirm the presence of class characteristics of a biological fluid. Preliminary tests do not confirm the presence of blood as these tests react with many different biological fluids as well.

>Phenolphthalein test/ Kastel Meyer test: In a highly alkaline solution, Zn powder reduces phenolphthalein. If the reduced phenolphthalein is oxidised by oxygen released by peroxides on hydrogen per oxide (H2O2), a pink or purple colour is produced, indicating a blood stain.

>Tetra methyl Benzidine (TMB) Test: A drop of TMB solution and Hydrogen peroxide is added to the stain suspected to be blood. An immediate appearance of a blue-green colour indicated the presence of blood.

>Luminol test: Light is produced when Luminol solution interacts with blood. Both luminol (C8H7N3O2) and hydrogen peroxide are present in the luminol solution (H2O2). When hydrogen peroxide interacts with iron in the blood, oxygen is produced and this produces fluorescence.

>Leucomalachite Green test: Leucomalachite green solution is added to the suspected stain. Appearance of a green colour indicates the presence of blood.

3.) Confirmatory test: These tests confirms the presence of blood stain. It is a conclusive test for blood.

>Takayama test: This test is also known as Haemochromogen test. The assay uses a pyridine and glucose reagent to evaluate a sample. The reagent (pyridine and glucose) interacts with the heme group in blood to form hemochromogen crystals in the presence of blood. Pink needle shaped crystals of Haemochromogen (Pyridineferroprotoporphyrin) will be formed which confirms the presence of blood.

>Teichmann’s test: This test is also known as Haematin crystal acid test. The test is based on the formation of brown-colored rhombic crystals from the interaction of the heme portion of blood with NaCl and glacial acetic acid. The development of hematin chloride (ferriprotoporphyrin chloride) crystals is indicated by these prismatic-rhombic-shaped brown crystals. These rhomboid shaped crystals confirms the presence of blood.

4.) Species of origin detection: After confirming that the biological evidence is blood, it is now important to determine whether the blood is of human or animal origin. If it is not of human origin, species particular proteins in bloodstains or other bodily tissues can be recognised with the help of species specific antibodies to determine which species it belongs to. The species-specific proteins from the bloodstain or tissue are removed in a 5% ammonia solution or normal saline (8.5 g sodium chloride in one litre distilled water).

There are different methods to determine the species of origin, which include:

>>Precipitin Ring Test/Precipitin Tube Test- Place six precipitin tubes vertically in a precipitin tube stand and mark them (the number depends on the number of anti-sera used). Fill the tubes with a drop of the bloodstain / tissue extract. One drop of antiserum for the species of origin (anti-Human serum, anti-Fowl serum, anti-Dog serum, anti-Cow serum etc.) should be carefully dropped around the tube’s walls.

>>Double Diffusion Method / Ouchterlony Double Diffusion- In this approach, both the reactants, antigen and antibody, diffuse towards one other in an agar gel plate, and precipitin is produced when an antigen interacts with its particular antibody in the correct quantities. Fill the centre well with tissue extract and the periphery wells with various anti sera to determine the origin of the species. Cover the Petri dish and store the gel in a damp chamber overnight. Check for the presence of precipitin bands on the gel.

>>Crossover Electrophoresis –The stain extract (antigen) is put in the cathodic well of a buffered gel, while the anti serum is inserted in the anodic well. Electro endosmosis causes globulin antibodies to move catholically when an electric current is administered. The remaining serum proteins move in anodal directions.


The colour and nature of a blood stain can be used to estimate the age of the stain -Fresh blood stain seems to be bright red in colour, wet, and sticky. The stain acquires a reddish brown colour after 24 hours. The stain darkens to a dark brown and then black after 24 hours.


After identifying the blood’s origin, the prosecution will also look for the blood groups. For the distinction of human blood, current serological methods have split blood into three component classes: A, B, and O. The antigenic systems ABO, MN, and Rh have been proven to be useful in criminal investigations.


The test used to determine blood group is known as ABO Typing or Agglutination test.

Antibodies against type A and B blood are added into your blood sample. The sample is next examined to discover if the blood cells adhere to one another. If blood cells clump together, it implies one of the antibodies in the blood has responded.


  • Anti-A is on one end of a slide, while Anti-B is on the other. To the end marked Anti-A, a drop of Anti-A test serum is applied, and to the end marked Anti-B, a drop of Anti-B serum is added.
  • One drop of blood is put to each end of the slide and thoroughly mixed.
  • The results are directly from the slide. If agglutination occurred with the Anti-A test serum, the subject is blood group A; if agglutination occurred with the Anti-B test serum, the subject is blood group B; if agglutination occurred with both test serums, the subject is blood group AB; and if there was no agglutination in either case, the subject is blood group O.


At a crime scene, there are two forms of blood that can be collected: liquid and dried blood. Liquid blood evidence is usually gathered from blood pools, although it can also be collected using a gauze pad or a sterile cotton towel from clothes. Liquid blood can also be collected in vacutainer with a purple top (Contains EDTA).

After collecting the sample, it must be refrigerated or frozen and transported to the laboratory as soon as feasible. To begin, the sample must be completely dried at room temperature. It is critical to deliver the sample to the laboratory as soon as possible, as the sample may be rendered worthless after 48 hours. If the sample is going to be shipped, it should be thoroughly air dried beforehand. When dried blood is discovered on a tiny object, the entire thing can be packed and tagged before being submitted to the lab. When dried blood is discovered on a transportable object, an investigator should cover the stained area with paper and tape it to the object to prevent contamination. If the stained object is not transportable, an investigator might acquire the sample in a variety of methods. Cutting away the discoloured region of the big item is one method. If the part is taken off, package the sample as indicated above, but include a control sample in a separate package. The lifted stain is then to be packed and labelled and transported to the laboratory.


  1. Blood is one of the most important biological evidence found at almost every crime scenes involving a physical violence.
  2. It can link a victim to a suspect and a suspect to the crime scene.
  3. Helps in narrowing down the accused.
  4. Blood traces can help in determining the identity of individual by the process of DNA typing from blood.
  5. Bloodstain patterns can reveal the position and direction of movement during a crime. It can also reveal whether the crime was a murder or suicide to a certain extent.
Sources & References
  1. Examination of blood, Forensic Biology and serology, Paatshaala
  2. The Mcgill physiology virtual lab,
  3. Crime museum, Blood evidence: collection and preservation

Authored By:


BSc Forensic Science

Jain (Deemed-to-be-University)

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