Introduction to Flow Cytometry: Blood Cell Identification

Author: Dana L. Van Laeys, MEd, MLS(ASCP)CMMBCM, CLSp(MB)
Reviewers: Dr. Linda Miller, PhD, MB(ASCP)CMSICM and Laurie Bjerklie, MA, MLS(ASCP)CM
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Continuing Education Credits

P.A.C.E.® Contact Hours: 2 hour(s)

Florida Board of Clinical Laboratory Science CE Credit Hours

Florida Board of Clinical Laboratory Science CE - General (Hematology): 1.5 hour(s)

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No other laboratory method provides as rapid and detailed analysis of cellular populations as flow cytometry, making it a valuable tool for diagnosis and management of several hematologic and immunologic diseases. Understanding this relevant methodology is important for any medical laboratory scientist.
Whether you have no previous experience with flow cytometry or just need a refresher, this course will help you to understand the basic principles, with the help of video tutorials and interactive case studies.

Basic principles include:
  • Immunophenotypic features of various types of hematologic cells
  • Labeling cellular elements with fluorochromes
  • Blood cell identification, specifically B and T lymphocyte identification and analysis
  • Cell sorting to isolate select cell population for further analysis
  • Analyzing and interpreting result reports and printouts

Objectives

  • List the appropriate specimen types used for flow cytometric analysis.
  • Describe the steps involved in analyzing samples by flow cytometry.
  • Calculate absolute cell counts from percentage values for flow cytometry analysis.
  • Identify blood cell types associated with respective specific surface markers.
  • Interpret basic flow cytometer instrument analysis printouts.
  • Examine case studies related to lymphocyte evaluation using flow cytometry while performing final interpretations.

Course Outline

  • Flow Cytometry Basics
    • Flow Cytometry Introduction
    • Table 1: Definitions
  • Flow Cytometry Principles
    • Acceptable Samples
    • Sample Preparation
    • Sample Analysis
    • Sample Analysis Animation
    • Detection of Cellular Characteristics
    • Detection of Intrinsic Cellular Characteristics: Cell Size and Granularity
    • Detection of Extrinsic Cellular Characteristics: Surface Antigens
    • True or False: In flow cytometry, cell size is indicated by forward scatter, and granularity is indicated by side scatter.
    • Staining Illustration
    • Staining: Monoclonal Antibody Binding
    • T-Helper Cell Example
    • T-Cytotoxic Cell Example
    • Flow Cytometry and Human Immunodeficiency Virus Clinical Application
    • Calculating Absolute Cell Counts
    • Calculating Absolute Cell Counts, continued
    • Which of the following types of lymphocytes express CD4?
  • Identifying and Using Cell Surface Markers
    • Identifying General Cell Types Associated with Surface Markers
  • Interpreting Instrument Printouts
    • Cytogram for Cellular Scatter: Intrinsic Characteristics
    • White Blood Cells and Cytogram for Cellular Scatter
    • Extrinsic Cell Characteristics
    • Which white blood cell populations would have the most side scatter when analyzed using flow cytometry?
    • Gating: Selecting the Population of Interest
    • Using Cell Surface Markers as Tools for Diagnosis
    • Diagnostic Process
    • True or False: When analyzing the raw data of a sample on the flow cytometer, CD marker information on the histograms represents data inside the non-gated population.
  • Review of Flow Cytometry Principles
    • Principles of Flow Cytometry
  • Case Study One
    • Case One
    • Scatter and CD45
    • What cell population is gated in this peripheral blood sample analyzed by flow cytometry?
    • CD19 and CD20
    • It has already been established that the gated cell population is lymphocytic. CD19 and CD20 cell surface antigens both appear on what type of lymphocytes?
    • CD10, HLA-DR, Kappa, and Lambda
    • If 100% of the cells in the gated lymphocyte population are positive for CD45 (i.e., they are leukocytes) and 7% are B cells (represented by CD19 and CD20 positivity), approximately what percentage of T cells would be expected?
    • T Cell Markers
    • True or False: All helper cells are T cells. Consequently, in a normal lymphocyte population, it is reasonable to expect all CD4-positive cells to be CD3-positive.
    • Final Interpretation of Case One
  • Case Study Two
    • Case Two
    • Scatter and Gating
    • T Cell Analysis
    • B Cell Analysis and Final Interpretation of Case Two
  • Conclusion
    • Conclusion
  • References
    • References

Additional Information

Level of Instruction: Intermediate
Intended Audience: Medical laboratory scientists, medical laboratory technicians, laboratory supervisors, and laboratory managers. This course is also appropriate for MLS and MLT students and pathology residents.
Author Information: Dana L. Van Laeys, MEd, MLS(ASCP)CMMBCM, is the Education Coordinator for Molecular Diagnostics and Immunology in the Clinical Laboratory Science Program at Saint Luke’s Hospital in Kansas City, Missouri. She has 14 years of experience in molecular diagnostics and flow cytometry. She has been a presenter at ASM, CLEC, and AABB conferences. Ms. Van Laeys holds a Masters in Adult Education and Distance Learning.
The author has no conflict of interest to disclose.
Reviewer Information:
Dr. Linda Miller, PhD, MB(ASCP)CMSICM, is an Assistant Editor for the Immunohematology section of Lab Medicine. She received her BS in Biology from Syracuse University and her PhD in Immunology from SUNY Upstate Medical University. She holds certifications as a Technologist in Immunology, Specialist in Immunology, and Technologist in Molecular Biology from ASCP. Dr. Miller is a professor of Clinical Laboratory Science at Upstate Medical University and the Director of the Medical Biotechnology program in the same department. There, she teaches Immunology, Human Genetics, Molecular Methods, and Medical Biotechnology courses. She has served as a member of the ASCP Immunology Exam Committee and the ASCP Lab Q Editorial Board as Associate Editor of Clinical Immunology. Dr. Miller has written numerous continuing education exercises and book chapters in clinical immunology and is co-author of the 4th edition of the textbook Clinical Immunology and Serology: A Laboratory Perspective (FA Davis).
Laurie Bjerklie, MA, MLS(ASCP)CM, is a Lead Education Developer. She earned a B.S. in Medical Laboratory Science from the University of North Dakota and an M.A. in Curriculum and Instruction from Saint Xavier University. She has over 15 years of experience in higher education and has held program director and faculty positions in both MLT and MLS programs.
Course Description: This course describes the most basic principles of flow cytometry, providing an overview and introduction to this useful tool. The focus of the course is blood cell identification, specifically B and T lymphocyte identification and analysis.