12.1: Biological Properties

• Blood constitutes about 8% of the human body weight of a healthy individual.

• Plasma: The fluid portion of the blood.

  

Red Blood Cells

• Also known as erythrocytes.

• Their life span in humans is approximately 3–4 months.

• They do not have nuclei, and therefore lack nuclear DNA.

• They consist of hemoglobin — proteins that are responsible for the transportation of oxygen.

• Ferroprotoporphyrin: A heme molecule.

White Blood Cells

• Also called leucocytes.

• They are involved in defending the body against infection.

• They have nuclei and thus represent the main sources of nuclear DNA from the blood.

Platelets

• Also known as thrombocytes.

• They play a role in blood clotting. They aggregate at sites of vascular and blood vessel injury.

• Like erythrocytes, they lack nuclei.

12.2: Presumptive Assays for Identification

Mechanisms of Presumptive Assays

• Presumptive blood assays are designed to detect traces of blood.

• These assays are based on the basic principle of the oxidation–reduction reaction catalyzed by the heme moiety of the hemoglobin.

• Oxidation–Reduction Reactions: These involve a change in the oxidation state of a molecule.

  • The oxidation of a molecule means that the molecule has lost electrons, and the reduction of a molecule means that the molecule has gained electrons.

  • Oxidants: Chemicals that can be reduced and gain electrons from other molecules.

  • Reductants: Chemicals that can be oxidized and therefore lose electrons to other molecules.

Colorimetric Assays

• Phenolphthalin Assay

  • Phenolphthalein: A member of a class of indicators and dyes, is used in titrations of mineral and organic acids as well as most alkalis.

  • The phenolphthalin assay for blood identification is also known as the Kastle–Meyer test.

  • It presents the results of a reaction in which phenolphthalin, a colorless compound, is catalyzed by heme with hydrogen peroxide as the oxidant.

    

• Leucomalachite Green (LMG) Assay

  • Malachite green is a triphenylmethane dye.

  • The leuco base form of malachite green is colorless and can be oxidized by the catalysis of heme to produce a green color.

  • The reaction is carried out under acid conditions with hydrogen peroxide as the oxidant.

    

• Benzidine and Derivatives

  • Benzidine: Used a presumptive assay for the presence of blood after the discovery that the oxidation of benzidine can be catalyzed by heme to produce a blue to dark blue color.

  • Orthotolidine: A dimethyl derivative of benzidine. Its oxidation reaction can be catalyzed by heme to produce a blue color reaction under acidic conditions

  • Tetramethylbenzidine (TMB): A tetramethyl derivative of benzidine. Its oxidation can be catalyzed by heme to produce a green to blue-green color under acidic conditions.

• Hemastix® assay kit: TMB-based assay that utilizes a TMB-containing strip device.

Chemiluminescence and Fluorescence Assays

• In the chemiluminescence assay, light is emitted as a product of a chemical reaction.

• Fluorescence Assay requires the exposure of an oxidized product, such as fluorescein, to a particular wavelength of an excitation light source.

• Luminol

  • It is usually utilized as a chemiluminescent reagent.

  • The oxidation reaction of luminol catalyzed by heme produces light in the presence of an oxidant.

• Fluorescein: It is used to test for the presence of bloodstains at a crime scene.

  

  

  

Factors Affecting Presumptive Assay Results

• Oxidants: Chemicals that are strong oxidants may cause a false-positive reaction.

• Plant Peroxidases: It may catalyze oxidation reactions and lead to false-positive results.

• Reductants: A false-negative result can occur when a strong reductant is present in a sample.

12.3: Confirmatory Assays for Identification

Microcrystal Assays

• Microcrystal assays apply chemicals to treat bloodstains, forming crystals of heme molecules.

• Hemochromagen Crystal Assay

  • Hemochromagens are heme derivatives in which the ferrous iron of the heme forms two bonds with nitrogenous bases.

  • Takayama Crystal Assay: A bloodstain is treated with pyridine and glucose under alkaline conditions to form crystals of pyridine ferroprotoporphyrin.

• Hematin Crystal Assay

  • Also known as the Teichmann crystal assay.

  • When blood specimens are treated with glacial acetic acid and salts, and subsequently heated, hematin chloride a prismatic brown-colored crystal, is formed.

  • Hematin: A heme derivative; its iron is in the ferric state.

    

    

Other Assays

• Chromatographic and Electrophoretic Methods can identify hemoglobin by its mobility characteristics.

• Spectrophotometric methods for identifying hemoglobin are based on measurements of the characteristic light spectra, with a peak absorbance at 400–425 nm, absorbed by hemoglobin and its derivatives.

• Immunological methods utilize antihuman hemoglobin antibodies.

  • This antibody can be used to detect human hemoglobin and thus indicate the presence of human blood.

• RNA-based assays: These assays are based on the fact that certain genes are specifically expressed in certain cell types.

  • It is used in the identification of blood based on the detection of specific types of messenger RNA (mRNA) that are expressed exclusively in erythrocytes.