CAP Pro Course - Histology - Immunohistochemistry Part 1

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Author: Carla Shoffeitt, MSM, HT(ASCP)
Reviewer: Alexandru Casapu, MBA, MLS(ASCP)CM, PBTCM

Continuing Education Credits

Objectives

  • Discuss the basic principles of immunohistochemistry (IHC).
  • Review fixation and processing requirements related to IHC.
  • Identify and discuss methods of epitope retrieval.
  • Discuss methods and detection systems used in the visualization of antibody-antigen reactivity.
  • Outline basic IHC staining protocol and staining considerations.
  • Discuss quality control (QC) measures used in IHC, including actions taken when QC results are not acceptable.

Course Outline

  • Discuss the basic principles of immunohistochemistry (IHC).
      • Defining Immunohistochemistry (IHC)
      • Antibodies, Antigens, and Epitopes
      • Antibody-Antigen Binding
      • Monoclonal Versus Polyclonal Antibodies
      • Antibody Dilution and Titration
      • Immunohistochemistry (IHC) is a method of localizing and demonstrating specific antigens in tissues or cells based on antigen-antibody interactions. W...
      • Antibodies used in IHC staining exist as monoclonal and polyclonal antibodies. Which of the following statements MOST accurately describes the differe...
  • Review fixation and processing requirements related to IHC.
      • Fixative Considerations Based on Specimen Type
      • Formalin Fixation
      • Fixation and Processing Considerations for Breast Markers
      • Microtomy and Slide Preparation
      • Prior to performing IHC staining, blood smears, touch-preps, and cytocentrifuged cytology preparations are air-dried to allow cells to fix to the slid...
      • Which of the following statements is TRUE in regard to fixation and processing considerations for breast markers?
      • You have been assigned blocks to section for IHC staining. Which of the following should be considered when sectioning slides for these requests?
  • Identify and discuss methods of epitope retrieval.
      • Unmasking of Epitopes
      • Proteolytic-Induced Epitope Retrieval (PIER)
      • Heat-Induced Epitope Retrieval (HIER)
      • When formalin-based fixatives are utilized, cross-linkages are formed with structural proteins. As a result, antigens can become "masked." Which of th...
      • Heat-induced epitope retrieval (HIER) methods have greatly improved the quality and reproducibility of IHC staining. Which of the following represents...
  • Discuss methods and detection systems used in the visualization of antibody-antigen reactivity.
      • Direct and Indirect Methods of Immunohistochemical (IHC) Staining
      • Peroxidase Anti-Peroxidase (PAP) Method
      • Avidin-Biotin Complex (ABC) and Labeled Streptavidin-Biotin (LSAB) Methods
      • Polymer-Based Immunohistochemistry (IHC)
      • The avidin-biotin complex (ABC) method of IHC staining makes use of the essentially irreversible binding seen between avidin (an egg white protein) an...
      • In polymer-based IHC staining, what component allows for additional binding sites and ultra-sensitivity?
      • Dr. Jones has reviewed an IHC stain on a liver biopsy. He is concerned because there appears to be unexpected positive staining. He has come to you fo...
  • Outline basic IHC staining protocol and staining considerations.
      • Blocking Non-Specific Binding
      • Importance of Washing Steps
      • Incubation of Primary Antibody and Detection System Reagents
      • Chromogen and Substrates
      • Endogenous biotin is bound to enzymes and protein in a variety of tissues and is found in high concentration in brain, kidney, and liver tissue. How i...
      • Which of the following statements with regard to the function of wash buffers is TRUE?
  • Discuss quality control (QC) measures used in IHC, including actions taken when QC results are not acceptable.
      • Controls in Immunohistochemistry (IHC)
      • Validation of New Primary Antibodies
      • Validating New Lots of Antibody and Ancillary Reagents
      • Storage Considerations for Immunohistochemistry (IHC) Reagents
      • Documenting Instrument Function and Maintenance
      • Participation in Quality Assessment (QA) Programs
      • Tate is preparing an early morning staining run on the automated IHC stainer. Upon retrieval of the detection kit and needed antibodies from the refri...
      • You have received an antibody that is new to your laboratory. The antibody requires optimization and validation before being used for patient testing....
      • John receives a new lot of GATA-3 antibody. What important packaging content should John review prior to initiating validation on the new lot?
  • References
      • References

Additional Information

Level of Instruction: Intermediate
Intended Audience: This program is designed as an educational and training tool for HT, HTL, MLS, MT, MLT personnel, medical laboratory science students and interns, pathology residents, and practicing pathologists.
Author Information: Carla J. Shoffeitt, MSM, HT(ASCP) is the System Director of Anatomic Pathology for Emory Healthcare in Atlanta, Georgia. She holds a Master of Science Degree in Healthcare Management as well as a Bachelor of Science degree in Biology from Troy University, Troy, Alabama. She is certified as a Histotechnician and has 29 years of experience in the field of Histology.
Reviewer Information: Alexandru Casapu, MBA, MLS(ASCP)CM, PBTCM, has over 20 years of experience as a medical laboratory scientist, section supervisor, and laboratory manager. He is the former Director of Clinical Laboratory Technology Program at Georgia Piedmont Technical College. He is currently a Program Director at MediaLab, Inc. Alex holds BS degrees in Biology and Medical Technology from Clark Atlanta University and an MBA from the University of Georgia.