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    SHAKTI
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    The document describes a procedure to prepare hemin crystals from human blood. Hemin is an iron-containing porphyrin that can be extracted from hemoglobin in blood. The procedure involves pricking a finger to obtain a blood sample, spreading it on a slide, and adding Nippe's reagent which contains potassium salts in acetic acid. Upon gentle heating, the reagent ruptures red blood cells and converts hemoglobin to hemin crystals, which appear as characteristic rhomboid crystals under the microscope. Hemin crystals are used to identify blood stains in forensic investigations.

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    The document describes a procedure to prepare hemin crystals from human blood. Hemin is an iron-containing porphyrin that can be extracted from hemoglobin in blood. The procedure involves pricking a finger to obtain a blood sample, spreading it on a slide, and adding Nippe's reagent which contains potassium salts in acetic acid. Upon gentle heating, the reagent ruptures red blood cells and converts hemoglobin to hemin crystals, which appear as characteristic rhomboid crystals under the microscope. Hemin crystals are used to identify blood stains in forensic investigations.

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    336 views4 pages

    Haemin Crystal Practical Preparation

    Uploaded by

    SHAKTI
    The document describes a procedure to prepare hemin crystals from human blood. Hemin is an iron-containing porphyrin that can be extracted from hemoglobin in blood. The procedure involves pricking a finger to obtain a blood sample, spreading it on a slide, and adding Nippe's reagent which contains potassium salts in acetic acid. Upon gentle heating, the reagent ruptures red blood cells and converts hemoglobin to hemin crystals, which appear as characteristic rhomboid crystals under the microscope. Hemin crystals are used to identify blood stains in forensic investigations.

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    of 4

    Exercise 1 Preparation of Hemin Crystals

    EXERCISE 1
    PREPARATION OF HAEMIN
    CRYSTALS

    Structure
    1.1 Introduction 1.5 Observations

    Objectives 1.6 Discussion

    1.2 Materials Required 1.7 Precautions

    1.3 Principle 1.8 Terminal Questions

    1.4 Procedure

    1.1 INTRODUCTION
    Haemin or haemin (Ferric chloride heme) is an iron-containing porphyrin with
    chlorine that can be formed from a haem group, such as haem group in
    haemoglobin of human blood. The purpose of this exercise is to enable you to
    understand how can we prepare crystals from the haem part of the
    haemoglobin.

    Objectives
    After completing this exercise, you will be able to:

    ™ identify the haemin crystal, and

    ™ appreciate importance of haemin crystal test in medico-legal cases.

    1.2 MATERIALS REQUIRED


    Glass slides, Cover slips, Pricking needles, Dropper, Spirit lamp, Cotton,
    Compound microscope

    90% Alcohol, Nippe’s reagent

    Preparation of Nippe’s Reagent: Dissolve 100 mg KCl, 100 mg KBr and 100
    mg KI in 100 mL of glacial acetic acid. 5
    BZYCL-136 Physiology and Biochemistry: Laboratory
    1.3 PRINCLIPLE
    Haemoglobin is present in RBCs which imparts red colour to the blood and
    helps in the transport of gases. Haemoglobin is a conjugated chromoprotein
    which is composed of two parts- non-protein haem and protein globin. Haem is
    made up of four pyrrole rings and a central iron ion in ferrous state.

    The haemin crystals are prepared by heating of the blood with Nippe’s
    reagent. This ruptures the RBCs and the haemoglobin is released. Also, in this
    process the ferrous form of iron is converted to ferric form. The globin protein
    gets denatured by heating with acetic acid; while, heme is converted to
    oxidized heme called haematin. The haematin combines with halogens such
    as chloride ions to form insoluble haemin which appear as rhombic crystal of
    chocolate brown colour. The haemin crystals or hydrochloride of heme (Fig.
    1.1) are characteristic of blood and are thus, used for identification of blood
    stains.

    Fig. 1.1: Haemin.

    Haemin was first crystallized by Ludwik Karol Teichmann in 1853. Therefore,


    they are also referred to as ‘Teichmann Crystals’.

    1.4 PROCEDURE
    1. Sterilize the tip of finger with cotton swab soaked in 90% alcohol.

    2. Prick the finger with the sterilized pricking needle.

    3. Place a drop of blood on a clean slide and spread it to form a thin film.

    4. Add 2-3 drops of the Nippe’s reagent on the blood and place a cover
    slip.

    5. Gently heat the slide over low flame until the reagent starts boiling.

    6. Remove the slide from the flame and add few drops of Nippe’s reagent
    from the side of the coverslip. Heat it again for few more seconds.

    7. Cool the slide and observe under the microscope, first under low
    6 magnification (10x) and then under high magnification (40x).
    Exercise 1 Preparation of Hemin Crystals

    1.5 OBSERVATIONS
    Rhomboid, chocolate brown crystals as shown in Figure 1. 2. are observed.
    Porphyria is a group
    Crystals are homogeneous solids, bounded by plane faces and having a of diseases in which
    geometric shape. substances called
    porphyrins build up,
    A lyophilized form of haemin is used as a pharmacological agent in certain negatively affecting
    cases for the treatment of porphyria attacks. the nervous system.

    Fig. 1.2: Haemin Crystals of Human Blood.

    1.6 DISCUSSION
    Haemin crystals are used in medico-legal practices to distinguish fresh or
    dried blood stains from other red-coloured stains. The shape of haemin
    crystals varies in different species and thus, blood stains of human can be
    confirmed. Another advantage of the haemin test is that it can be performed
    with the dried blood stains.

    1.7 PRECAUTIONS
    • Clean the finger with alcohol before pricking.

    • Discard the first drop of blood.

    • Avoid overheating of the slide.

    • Keep the slide undisturbed at the time of cooling.

    1.8 TERMINAL QUESTIONS


    1. Write the composition of Nippe’s reagent.

    2. Write the chemistry behind formation of haemin crystals.

    3. What is the importance of haemin crystals test in medico-legal cases?

    4. Name the blood pigment involved in haemin crystal test.

    5. Differentiate between haematin and haem. 7


    BZYCL-136 Physiology and Biochemistry: Laboratory
    FURTHER READING
    1. Tortora, G.J. and Derrickson, B.H. (2009). Principles of Anatomy and
    Physiology, XII Edition, John Wiley & Sons, Inc.

    2. Victor P. Eroschenko. (2008). diFiore’s Atlas of Histology with Functional


    correlations. XII Edition. Lippincott W. & Wilkins.

    Acknowledgement of Figures
    Fig.1. 2: (a) Self-Clicked

    (b) https://www.flickr.com/photos/internetarchivebookimages
    /14780801025

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