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    Immunosero: Experiment # 1 - Preparation of Red Cell Suspension (Pg. 13-14)

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    The document discusses several immunoserology experiments including: 1. Preparing a red cell suspension with the precise dilution of normal saline solution to avoid incorrect test results. 2. Performing serial dilutions to determine dilution factors and avoid prozone phenomena. 3. Isolating peripheral blood mononuclear cells which are important for immune function studies. Functional assays using isolated PBMCs are described. 4. Performing an NBT test to diagnose chronic granulomatous disease and test neutrophil function by detecting their ability to reduce nitroblue tetrazolium dye.

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    Immunosero: Experiment # 1 - Preparation of Red Cell Suspension (Pg. 13-14)

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    swfs
    182 views8 pages
    The document discusses several immunoserology experiments including: 1. Preparing a red cell suspension with the precise dilution of normal saline solution to avoid incorrect test results. 2. Performing serial dilutions to determine dilution factors and avoid prozone phenomena. 3. Isolating peripheral blood mononuclear cells which are important for immune function studies. Functional assays using isolated PBMCs are described. 4. Performing an NBT test to diagnose chronic granulomatous disease and test neutrophil function by detecting their ability to reduce nitroblue tetrazolium dye.

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    The document discusses several immunoserology experiments including: 1. Preparing a red cell suspension with the precise dilution of normal saline solution to avoid incorrect test results. 2. Performing serial dilutions to determine dilution factors and avoid prozone phenomena. 3. Isolating peripheral blood mononuclear cells which are important for immune function studies. Functional assays using isolated PBMCs are described. 4. Performing an NBT test to diagnose chronic granulomatous disease and test neutrophil function by detecting their ability to reduce nitroblue tetrazolium dye.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    182 views8 pages

    Immunosero: Experiment # 1 - Preparation of Red Cell Suspension (Pg. 13-14)

    Uploaded by

    swfs
    The document discusses several immunoserology experiments including: 1. Preparing a red cell suspension with the precise dilution of normal saline solution to avoid incorrect test results. 2. Performing serial dilutions to determine dilution factors and avoid prozone phenomena. 3. Isolating peripheral blood mononuclear cells which are important for immune function studies. Functional assays using isolated PBMCs are described. 4. Performing an NBT test to diagnose chronic granulomatous disease and test neutrophil function by detecting their ability to reduce nitroblue tetrazolium dye.

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    of 8

    ImmunoSero

    Experiment # 1 – Preparation of Red Cell Suspension (pg. 13-14)

    204.
    205. What is the purpose of preparing a red cell suspension?
    Red cell suspension is a standard reagent used in many serological procedures. Red cell
    suspensions have the required serum to cell ratio to allow the results of tests to be interpreted.
    206. Is it permissible to make an approximation for the volume of NSS to be added to the
    washed red cells? Justify your answer.
    To avoid getting incorrect effects, one must use the precise volume or measurement of the NSS
    to be applied to the washed red cells. In order for the red cell suspension to not be too heavy or
    too diluted, it must be diluted properly. In order to avoid the phenomena or problems that are the
    prozone and postzone phenomenon in each serological test and in blood banking, it is
    necessary to do so. The equivalence would be achieved if correctly diluted and an ideal
    antigen-antibody reaction will occur.

    207. Calculate the volume of NSS to be added to a packed cell volume of 0.8mL to produce a
    2% red cell suspension
    Experiment # 2 – Serial Dilution (pg.19-20)

    208.

    208. Determine the dilution factor of tubes 2-4


    Dilution Factor:
    Tube 2: 20
    Tube 3: 40
    Tube 4: 80

    209. Cite the differences between tube dilution and microtiter plate dilution

    210. If you have a 1:10 dilution of the patient’s serum, how many parts of the sample and that of
    the diluting fluid are present in the solution?

    The solution will have 9 parts of the diluting fluid and 1 part of the sample.

    211. What is zonal phenomenon?


    ● The zonal phenomenon refers to the occurrence of prozones in antibody-antigen
    mixtures. A serial dilution is performed in the procedures for Serology-Immunology to
    avoid a zonal reaction, particularly the prozone phenomenon which refers to the
    excessive amount of antibodies.
    Experiment # 15 – Analysis of lymphocytes (pg.167-168)

    212.
    Hank’s Balanced Salt Solution​ - This is an isotonic solution that functions in regulating the
    osmolality and pH and is used to maintain, for a short period of time, the cells found outside the
    growth medium
    Ficoll-Hypaque Solution​ - This is a density gradient solution used in the purification of
    lymphocytes in high yield and purity from small and large volumes of human peripheral blood
    RPMI & McCoy Medium​ - This medium is used to support the growth of a variety of cultures. It
    is also used in the preservation of peripheral blood mononuclear cells
    10% Fetal Calf Serum​ - This serum has a functions to supplement for basal growth medium
    during cell culture
    Swing-out Bucket Rotor Without Brake​ - This enables to adjust the angle of the tube when
    performing a run.
    Nylon Wool​ - This functions as a solid phase matrix and removes monocytes and B cells

    213. Why is it important to isolate the peripheral blood mononuclear cells completely from the
    other cellular components of blood?

    A peripheral blood mononuclear cell (PBMC) is any blood cell having a round nucleus such as
    lymphocyte, monocyte, or a macrophage. These blood cells are a critical component in the
    immune system to fight infection and adapt to intruders. Peripheral blood mononuclear cells
    (PBMC) give selective responses to the immune system and are the major cells in the human
    body immunity. They contain several types of cells such as lymphocytes,monocytes or
    macrophages. Because peripheral blood is the place where exposure to chemicals occurs,
    these fundamentally important PBMCsare prone to be influenced by drugs and chemicals. This
    is why the availability of PBMCs from peripheral blood is very important for researchers studying
    toxicity of new drugs or chemical compounds.
    214. What laboratory assays can be performed to assess the efficiency of the sample
    preparation and isolation of the PBMC?
    Trypan Blue Dye Exclusion Test: this is used to assess the number of cells that are still
    viable in a cell suspension. Dead cells would turn blue, while living cells appear
    clear/transparent when viewed microscopically.

    215. Enumerate different functional assays that use isolated PBMCs.


    Human peripheral blood mononuclear cells (PBMCs) are commonly used to study immune cell
    processes, to detect biomarkers and to enhance diagnostics and therapeutics for human
    diseases. Functional assays include radiosensitivity assays on LCLs, chromosome-based
    analysis (chromosome aberration assays) for detection of genotoxicity, monitoring of autophagy
    activity, chemotaxis assays, natural killer (NK) cytotoxicity assays, leukocyte recruitment and
    adhesive interactions with endothelial cells, suppression assays, antigen-specific response
    assays such as lymphocyte proliferation assays, intracellular cytokine staining (ICS), and
    enzyme-linked immunospot (ELISPOT) assays.
    Experiment # 16 – Nitroblue Tetrazolium Reduction Test for
    Neutrophilic Function (pg. 173-174)
    216. Enumerate the purposes of performing an NBT test
    The purpose of performing an NBT test is to differentiate bacterial from non bacterial infections,
    to diagnose chronic granulomatous disease (CGD), and to test phagocytic function of
    polymorphonuclear leukocytes (PML).

    217. Why should EDTA not be used in collecting blood specimens for an NBT test?
    EDTA tubes should not be used because the anticoagulant can interfere with the results. It
    inhibits phagocytosis thus resulting in results that are falsely decreased. The preferred
    anticoagulant for NBT test is heparin.

    218. Briefly discuss chronic granulomatous disease (CGD) and how it is diagnosed.

    Chronic granulomatous disease (CGD) is a genetic disorder in which white blood cells called
    phagocytes are unable to kill certain types of bacteria and fungi.

    A diagnosis of chronic granulomatous disease is often suspected based on the presence of


    characteristic signs and symptoms. Specialized blood tests, such as the nitroblue tetrazolium
    test and/or flow cytometry with dihydrorhodamine, can then be ordered to confirm the diagnosis.

    219. What is a stimulated NBT test? Discuss its significance.


    The stimulated NBT is useful in determining the effectiveness of the phagocytic activity of
    neutrophils. A defective function is detected by a colorless dye, Nitroblue Tetrazolium (NBT),
    which is reduced to blue-black formazan deposit by the activity of the phox enzyme system. This
    test is used in the diagnosis of Chronic Granulomatous Disease (CGD) and is useful in
    differentiating bacterial from non bacterial infections.

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