Cell Biology: Degradation and Preparations
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Questions and Answers

What is ischemia primarily associated with in sample preparation?

  • Irreversible cell fixation
  • Interval phase of nutrient and oxygen depletion (correct)
  • Controlled degradation of samples
  • Bacterial attack on cells

    • Which of the following components is the fastest to degrade?

  • microRNA
  • DNA
  • Phosphorylated proteins (correct)
  • Proteins and mRNA

    • What type of anticoagulant is suitable for nucleic acid extraction?

  • Heparin (green tubes)
  • Calcium oxalate
  • EDTA (purple tubes) (correct)
  • Sodium citrate (blue tubes)

    • Which process involves halting degradation of cells and tissues irreversibly?

    <p>Fixation</p> Signup and view all the answers

    What is the significance of biomarkers in cellular analysis?

    <p>They are used as specific indicators of diseases.</p> Signup and view all the answers

    In genomic diagnostics, what blood sample type is typically used?

    <p>Whole blood or venous blood</p> Signup and view all the answers

    Which condition requires genomic analysis of bone marrow?

    <p>Myeloma</p> Signup and view all the answers

    Which statement about autolysis is correct?

    <p>It involves pH-induced destruction of cells.</p> Signup and view all the answers

    What is the primary function of chromogenic immunocytochemistry?

    <p>To produce insoluble colored products at the antigen site</p> Signup and view all the answers

    Which enzyme is NOT typically used in chromogenic detection methods?

    <p>Carbonic anhydrase</p> Signup and view all the answers

    What type of visualization does indirect immunofluorescence provide?

    <p>Bright lights on a dark background</p> Signup and view all the answers

    Which application does immunocytochemistry NOT support?

    <p>Testing for genetic mutations directly</p> Signup and view all the answers

    In the diagnosis of HBV using ICC, where can HBcAg be localized?

    <p>In both the nuclei and cytoplasm of hepatocytes</p> Signup and view all the answers

    What is the purpose of formalin fixation in tissue preparation?

    <p>To irreversibly halt degradation of the sample</p> Signup and view all the answers

    Which technique is primarily utilized for the microscopic localization of proteins within cells?

    <p>Immunocytochemistry</p> Signup and view all the answers

    What is a significant challenge in using frozen tissue preparations for microscopy?

    <p>They require samples to be immersed in isopentane</p> Signup and view all the answers

    Which of the following statements about molecular diagnostics is correct?

    <p>They can be performed on either intact cells and tissues or homogenate samples.</p> Signup and view all the answers

    Why is unmasking of epitopes important in immunocytochemistry?

    <p>It resolves issues caused by cross-linking effects of formalin fixation.</p> Signup and view all the answers

    Which of the following is not an advantage of immunocytochemistry?

    <p>Limited to morphological staining techniques</p> Signup and view all the answers

    What type of sample assessment can be performed using flow cytometry?

    <p>Intact cells and homogenate samples</p> Signup and view all the answers

    What is a fundamental challenge when applying immunocytochemistry to FFPE tissue sections?

    <p>Cross-linking effects mask epitopes</p> Signup and view all the answers

    What is the main purpose of using proteolytic-induced epitope retrieval (PIER)?

    <p>To expose hidden epitopes obscured by cross-linking</p> Signup and view all the answers

    What technique is typically employed in heat-induced epitope retrieval (HIER)?

    <p>Microwave heating with buffer solutions</p> Signup and view all the answers

    Why is blocking specific binding important prior to primary antibody incubation?

    <p>It prevents non-specific binding and reduces false positives</p> Signup and view all the answers

    Which tissue types are mentioned as expressing high amounts of endogenous peroxidase activity?

    <p>Liver and kidney</p> Signup and view all the answers

    What is the effect of treating tissues with 3-10% H2O2 prior to antibody incubation?

    <p>It quenches endogenous peroxidase activity</p> Signup and view all the answers

    What role do avidin/biotin blocking reagents play in immunohistochemistry?

    <p>They prevent non-specific staining due to endogenous biotin</p> Signup and view all the answers

    Which of these enzymes is NOT commonly used for proteolytic-induced epitope retrieval (PIER)?

    <p>Papain</p> Signup and view all the answers

    What is an important consideration when performing heat-induced epitope retrieval?

    <p>Ensuring the use of appropriate metal ions</p> Signup and view all the answers

    Study Notes

    Sample Degradation

    • Ischemia: Interval between nutrient/oxygen deprivation and preservation
    • Autolysis: Cell destruction through pH changes
    • Putrefaction: External bacterial invasion of cells
    • Components Degradation Rate: Phosphorylated proteins degrade fastest, followed by proteins and mRNA; microRNA is more stable than RNA; DNA is the most resilient

    Cell-Based Preparations

    • Standard Operating Procedures (SOPs): Detailed instructions followed without deviation
    • Cell Culture: Growing cells outside the body in a controlled environment
    • Blood Samples: Obtained from veins, used for genomic diagnostics, nucleic acid biomarkers, and genetic disorder analysis
    • Biomarkers: Cellular components indicating disease or its progression
    • Genetic Disorders: Changes in DNA, including chromosome gain/loss or single nucleotide polymorphisms (SNPs). Inheritable if germline, sporadic/somatic if originating in a dividing cell during life.
    • Anticoagulants for Molecular Diagnostics:
      • EDTA (purple tubes): Nucleic acid extraction
      • Heparin (green tubes): WBC analysis, DNA extraction, cytogenetic analysis, immunophenotyping
      • Sodium citrate (blue tubes): Genetic tests
    • Bone Marrow Analysis: Required for disorders like myeloma, leukemia, and aplastic anemia. Aspirated samples require suitable anticoagulants.
    • Cytological Analysis: Microscopic observation of individual intact cells
    • Fixation: Prevents cell and tissue degradation, preserves cytological and morphological features, and allows retention of chemical constituents for analysis

    Tissue Preparations

    • More challenging than cell preparations
    • Frozen Tissues: Degradation is halted, but special care required for some analysis.
      • Example: Frozen muscle biopsies immersed in isopentane for cytological and enzyme assessment in myopathies
    • Formalin-fixed Paraffin Embedding (FFPE): Standard for diagnostic histopathology.
      • Formalin halts degradation, paraffin provides internal and external support.

    Molecular Diagnostics

    • Can be applied to intact cells/tissues or homogenate samples

    Intact Sample Assessment

    • Immunocytochemistry (ICC): Microscopic localization of proteins within and on cell surfaces.
    • Flow Cytometry (FC): Analyzing cell populations based on their properties.
    • In situ Hybridization (ISH): Detecting specific DNA or RNA sequences within tissue sections.

    Immunocytochemistry (ICC)

    • Identifies cell types in normal and pathological samples.
    • Used diagnostically or as follow-up to initial cytological or morphological staining.
    • Provides valuable diagnostic information and contributes to disease research.
    • Early concept: Using labelled antibodies to detect specific proteins.
    • Early use involved detecting auto-antibodies in frozen sections for diagnostic purposes.
    • Challenge with FFPE sections: Cross-linking by formalin masks epitopes.
    • Solution: Unmasking epitopes to facilitate diagnosis and targeted therapy in precision medicine.

    Epitope Retrieval

    • Proteolytic-induced Epitope Retrieval (PIER): Enzymes like trypsin, pepsin, and proteinase K expose hidden epitopes. Requires careful control.
    • Heat-induced Epitope Retrieval (HIER): Superheating FFPE sections retrieves epitopes. Often involves microwave heating in buffered solutions with metal ions.

    ICC Procedure

    1. Epitope retrieval (when FFPE is used)
    2. Blocking non-specific staining
    3. Primary antibody incubation
    4. Detection of antibody/antigen interaction

    Blocking Non-Specific Binding

    • Prevents non-specific binding and false-positive results.
    • Serum Blocking: Reduces background by blocking reactive epitopes.
    • Endogenous Peroxidase Blocking: Quenches peroxidase activity in highly vascularized tissues to minimize non-specific staining.
    • Endogenous Biotin Blocking: Blocks biotin in tissues to reduce non-specific staining when using biotinylated secondary antibodies.

    Detection Systems

    • Light microscopic ICC can use two endpoints:
      • Fluorescent: Provides bright signals on a dark background and allows for multiple antigen localization.
      • Chromogenic: Uses enzymes to produce insoluble precipitates at the site of antigen/antibody reaction.

    Indirect Immunofluorescence

    • Allows for simultaneous localization of multiple antigens using different fluorophores

    Chromogenic Immunocytochemistry

    • Uses enzymes to catalyze substrates, forming insoluble colored products at the site of antigen expression.
    • Common enzymes: Horseradish peroxidase (HRP) and alkaline phosphatase.
    • Common substrates: DAB (brown) and AEC (red).

    Applications of ICC

    • Cancer diagnosis
    • Prognostic markers in cancer
    • Drug development (testing efficacy)
    • Prediction of therapy response
    • Detection of infectious agents in tissues
    • Muscle disease diagnosis

    ICC in HBV Diagnosis

    • Immunoperoxidase localization: Detects HBcAg (Hepatitis B core antigen) in the nucleus or cytoplasm of hepatocytes.

    ICC in SARS-CoV-2 Diagnosis

    • Immunoalkaline phosphatase: Detects coronavirus antigens in pneumocytes and macrophages in lung tissue.

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    Related Documents

    Biot 401 Lecture 2: Samples Preparation & Analysis (PDF)

    Description

    This quiz covers essential concepts in cell biology, including sample degradation processes and cell-based preparations. Test your knowledge on the mechanisms of ischemia, autolysis, putrefaction, and the protocols for cell culture and blood sample processing. Understand the implications of biomarkers and genetic disorders in diagnostics and research.

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