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Blood analysis (Forensic Serology)

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Archana Mahakalkar

This document summarizes various presumptive and confirmatory assays used for the analysis of blood. Presumptive assays like phenolphthalin, leucomalachite green, benzidine reaction, luminol and fluorescein tests identify the presence of blood through color changes. Confirmatory assays like Teichmanns test identify haemin crystals under microscope and Takayama test identifies hemoglobin through crystal formation. Immunochromatographic assays identify human hemoglobin and glycophorin A proteins. RNA-based assays can also be used to identify blood through RNA analysis.

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Analysis of Bl d Dr.Archana Mahakalkar Department of Forensic Biology Government Institute of Forensic Science, Nagpur
Blood analysis (Forensic Serology)
Haemoglobin Structure
Presumptive Assays Phenolphthalin Assay Leucomalachite Green (LMG) Assay ? Luminol (3- aminophthalhydr azide) Benzidine reaction: Fluorescin
Colorimetric Assays 1. Phenolphthalin Assay The phenolphthalin assay is also known as the Kastle-Meyer test. Oxidation¨Creduction reaction as basis for presumptive assays for blood identification. AH2 = substrate. A = oxidized substrate. Phenolphthalein(1g) dissolved in acetic acid (100ml). Sodium perborate (1.4g) is dissolved in the solution Apply on to stain
2. Leucomalachite Green reaction: The reagent is prepared by dissolving leucomalachite green (0.1g), sodium perborate (0.3g) in 65% glacial acetic acid. The reagent is applied to thestain. Intense green color indicates blood.
3.Leucomalachite Green (LMG) Assay leucomalachi te green (0.1g), sodium perborate (0.3g) 65% glacial acetic acid The reagent is applied to the stain Intense green colour indicates blood.
4. Benzidine reaction: Benzidine (0.1g) dry sodium perborate (0.1g) dissolved in glacial acetic acid (10ml) A strong blue colour indicates blood. sprayed over the stain
5. Luminol (3-aminophthalhydrazide) The oxidation products of Luminol have chemiluminescent properties. Oxidation of Luminol catalyzed by heme produces an intense light . The light emitted from a positive reaction can only be observed in a dark environment and this limits the applications of Luminol. Chemical reaction of Luminol
6. Fluorescin When oxidized and catalyzed by heme, it demonstrates fluorescent properties. Usually, fluorescin-sprayed stains are exposed to light in the range of 425 to 485 nm using an alternate light source device. In a positive reaction, the oxidized fluorescin will emit an intense yellowish-green fluorescent light and therefore indicate the presence of a blood stain. Chemical structure of fluorescein.
Confirmatory Assays for Identification TeichmannsTest Takayama Test Chromatographic Electrophoretic Confirmatory Assays
1. TeichmannsTest Take a dry crust or smear of blood on a slide Put a drop of potassium iodide, bromide or chloride (0.1gm) solution in 100ml glacial acetic acid over the blood cover it with a cover slip Warm the slide gently till it gives out bubbles Typically haemin crystals are observed under microscope Heating may have to be repeated a number of times.
2. Takayama Test The reagent is prepared from one volume of glucose solution (10%) one volume of potassium hydroxide solution (10%) two volumes of pyridine which are dissolved in six volumes of water
3. Identification of Human Hemoglobin Protein Immunochromatographic assays for the identification of Hb in human blood. (a) In a sample well, Hb in a blood sample is mixed with a labeled anti-Hb Ab. (b) The Hb binds to the labeled anti-Hb Ab to form a labeled Ab¨CHb complex. The complex diffuses toward the test zone. (c) At the test zone, the labeled Ab¨CHb complex binds to an immobilized anti-Hb Ab to form a labeled Ab¨CHb¨CAb sandwich. (d) At the control zone, the labeled anti-Hb Ab binds to an immobilized antiglobulin and is captured. Ab and Hb represent the antibody and hemoglobin, respectively. (? Richard C. Li.)
4. Human Glycophorin A protein Immunochromatographic assays for the identification of GPA in human blood. (a) In a sample well, GPA in a blood sample is mixed with a labeled anti-GPA Ab. (b) The GPA binds to the labeled anti-GPA Ab to form a labeled Ab¨CGPA complex. (c) At the test zone, the labeled Ab¨CGPA complex binds to an immobilized anti-GPA Ab to form a labeled Ab¨CGPA¨CAb sandwich. (d) At the control zone, the labeled anti-GPA Ab binds to an immobilized antiglobulin and is captured. Ab and GPA represent the antibody and GPA protein, respectively. (? Richard C. Li.)
5. RNA-based Assay
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Blood analysis (Forensic Serology)

  • 1. Analysis of Bl d Dr.Archana Mahakalkar Department of Forensic Biology Government Institute of Forensic Science, Nagpur
  • 5. Colorimetric Assays 1. Phenolphthalin Assay The phenolphthalin assay is also known as the Kastle-Meyer test. Oxidation¨Creduction reaction as basis for presumptive assays for blood identification. AH2 = substrate. A = oxidized substrate. Phenolphthalein(1g) dissolved in acetic acid (100ml). Sodium perborate (1.4g) is dissolved in the solution Apply on to stain
  • 6. 2. Leucomalachite Green reaction: The reagent is prepared by dissolving leucomalachite green (0.1g), sodium perborate (0.3g) in 65% glacial acetic acid. The reagent is applied to thestain. Intense green color indicates blood.
  • 7. 3.Leucomalachite Green (LMG) Assay leucomalachi te green (0.1g), sodium perborate (0.3g) 65% glacial acetic acid The reagent is applied to the stain Intense green colour indicates blood.
  • 8. 4. Benzidine reaction: Benzidine (0.1g) dry sodium perborate (0.1g) dissolved in glacial acetic acid (10ml) A strong blue colour indicates blood. sprayed over the stain
  • 9. 5. Luminol (3-aminophthalhydrazide) The oxidation products of Luminol have chemiluminescent properties. Oxidation of Luminol catalyzed by heme produces an intense light . The light emitted from a positive reaction can only be observed in a dark environment and this limits the applications of Luminol. Chemical reaction of Luminol
  • 10. 6. Fluorescin When oxidized and catalyzed by heme, it demonstrates fluorescent properties. Usually, fluorescin-sprayed stains are exposed to light in the range of 425 to 485 nm using an alternate light source device. In a positive reaction, the oxidized fluorescin will emit an intense yellowish-green fluorescent light and therefore indicate the presence of a blood stain. Chemical structure of fluorescein.
  • 11. Confirmatory Assays for Identification TeichmannsTest Takayama Test Chromatographic Electrophoretic Confirmatory Assays
  • 12. 1. TeichmannsTest Take a dry crust or smear of blood on a slide Put a drop of potassium iodide, bromide or chloride (0.1gm) solution in 100ml glacial acetic acid over the blood cover it with a cover slip Warm the slide gently till it gives out bubbles Typically haemin crystals are observed under microscope Heating may have to be repeated a number of times.
  • 13. 2. Takayama Test The reagent is prepared from one volume of glucose solution (10%) one volume of potassium hydroxide solution (10%) two volumes of pyridine which are dissolved in six volumes of water
  • 14. 3. Identification of Human Hemoglobin Protein Immunochromatographic assays for the identification of Hb in human blood. (a) In a sample well, Hb in a blood sample is mixed with a labeled anti-Hb Ab. (b) The Hb binds to the labeled anti-Hb Ab to form a labeled Ab¨CHb complex. The complex diffuses toward the test zone. (c) At the test zone, the labeled Ab¨CHb complex binds to an immobilized anti-Hb Ab to form a labeled Ab¨CHb¨CAb sandwich. (d) At the control zone, the labeled anti-Hb Ab binds to an immobilized antiglobulin and is captured. Ab and Hb represent the antibody and hemoglobin, respectively. (? Richard C. Li.)
  • 15. 4. Human Glycophorin A protein Immunochromatographic assays for the identification of GPA in human blood. (a) In a sample well, GPA in a blood sample is mixed with a labeled anti-GPA Ab. (b) The GPA binds to the labeled anti-GPA Ab to form a labeled Ab¨CGPA complex. (c) At the test zone, the labeled Ab¨CGPA complex binds to an immobilized anti-GPA Ab to form a labeled Ab¨CGPA¨CAb sandwich. (d) At the control zone, the labeled anti-GPA Ab binds to an immobilized antiglobulin and is captured. Ab and GPA represent the antibody and GPA protein, respectively. (? Richard C. Li.)