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Cytometry

Stem Cell Analysis

Overview of Stem Cell Analysis

  • Stem Cells:
    • Definition: Cells with the unique ability to self-renew and differentiate into specialized cell types
    • Types:
      • Hematopoietic Stem Cells (HSCs): Give rise to all blood cell types
      • Mesenchymal Stem Cells (MSCs): Give rise to bone, cartilage, fat, and other connective tissues
      • Embryonic Stem Cells (ESCs): Pluripotent cells derived from the inner cell mass of the blastocyst
      • Induced Pluripotent Stem Cells (iPSCs): Adult cells that have been reprogrammed to become pluripotent
  • Importance of Stem Cell Analysis:
    • Transplantation: Assessing the quality and quantity of stem cells for transplantation
    • Regenerative Medicine: Studying stem cell differentiation and function for regenerative medicine applications
    • Disease Modeling: Using stem cells to model and study diseases
    • Drug Discovery: Using stem cells to screen for new drugs

CD34 Absolute Counts

  • CD34:
    • Definition: A cell surface glycoprotein that is expressed on hematopoietic stem cells (HSCs) and progenitor cells
    • Importance: CD34 is a commonly used marker to identify and quantify HSCs
  • CD34 Absolute Count:
    • Definition: The number of CD34+ cells per unit volume of blood or bone marrow
    • Clinical Significance:
      • Predicting engraftment after stem cell transplantation
      • Monitoring stem cell mobilization
      • Diagnosing and classifying hematologic disorders
  • Methods for CD34 Absolute Counts:
    • Single-Platform Flow Cytometry:
      • Principle: Uses a single flow cytometer to simultaneously measure the number of CD34+ cells and the total white blood cell count
      • Advantages: More accurate and precise than dual-platform methods
    • Dual-Platform Flow Cytometry:
      • Principle: Uses a flow cytometer to measure the percentage of CD34+ cells and a hematology analyzer to measure the total white blood cell count
      • Advantages: Less expensive than single-platform methods
      • Disadvantages: Less accurate and precise than single-platform methods

Single-Platform Flow Cytometry for CD34 Absolute Counts

  • Principle:
    • A known volume of sample is stained with antibodies against CD34 and other cell surface markers
    • A known number of fluorescent beads is added to the sample as an internal control
    • The sample is acquired on a flow cytometer, and the number of CD34+ cells and beads is counted
    • The absolute count of CD34+ cells is calculated using the following formula:
      • CD34+ cells/μL = (Number of CD34+ cells / Number of beads) x (Number of beads added / Sample volume in μL)
  • Procedure:
    1. Sample Preparation:
      • Collect blood or bone marrow aspirate in EDTA anticoagulant
      • Lyse red blood cells (RBCs) using a lysing buffer
      • Wash the sample to remove excess lysing buffer
    2. Antibody Staining:
      • Incubate the sample with fluorochrome-conjugated antibodies against CD34 and other cell surface markers (e.g., CD45)
      • Include appropriate isotype controls
    3. Bead Addition:
      • Add a known number of fluorescent beads to the sample
    4. Flow Cytometry Acquisition:
      • Acquire the sample on a flow cytometer, following the manufacturer’s instructions
      • Collect a sufficient number of events to ensure accurate counting
    5. Data Analysis:
      • Gate on the CD45+ leukocyte population
      • Identify the CD34+ cell population within the CD45+ gate
      • Count the number of CD34+ cells and beads
      • Calculate the absolute count of CD34+ cells using the formula above
  • Reagents and supplies:
    • CD45-FITC: to gate on leukocytes
    • CD34-PE: to determine the percent of hematopoietic stem cells
    • 7-AAD: to remove any dead cells from the analysis
    • Counting beads: to determine the absolute counts

Gating Strategy for CD34 Absolute Counts

  1. Gate on the Leukocyte Population:
    • Use CD45 and side scatter (SSC) to identify the leukocyte population
    • Exclude red blood cells and debris
  2. Gate on the CD34+ Population:
    • Use CD34 and side scatter (SSC) to identify the CD34+ cell population
    • Exclude non-specific binding using isotype controls
  3. Confirm CD34+ Population:
    • Use CD34 and CD45 to identify true CD34+ cells from the background noise
  4. Bead Gate:
    • Use forward scatter (FSC) and side scatter (SSC) to identify the beads population to count how many events are being tested.

Controls for CD34 Absolute Counts

  • Isotype Controls:
    • Used to assess non-specific antibody binding
  • Negative Controls:
    • Used to set the baseline for CD34 expression
  • Positive Controls:
    • Used to validate the staining protocol and instrument performance
  • Beads Controls:
    • Used to make sure that the beads are distinguishable from any debris that may be present in the sample
  • Automated gating:
    • It is imperative that the gating is not automated to prevent the omission of a small CD34+ cell population

Clinical Applications of CD34 Absolute Counts

  • Stem Cell Transplantation:
    • Predicting engraftment after autologous or allogeneic stem cell transplantation
    • Monitoring stem cell mobilization prior to collection
    • Assessing the quality of stem cell products
  • Hematologic Disorders:
    • Diagnosing and classifying acute leukemias
    • Monitoring minimal residual disease (MRD) in acute leukemias
    • Assessing bone marrow failure syndromes
  • Other Applications:
    • Monitoring immune reconstitution after chemotherapy
    • Studying stem cell biology and differentiation

Other Stem Cell Markers

  • CD133 (Prominin-1):
    • Another marker of HSCs and progenitor cells
    • Often used in combination with CD34
  • CD90 (Thy-1):
    • A marker of mesenchymal stem cells (MSCs)
  • STRO-1:
    • Another marker of MSCs
  • Oct4, Nanog, Sox2:
    • Transcription factors that are expressed in pluripotent stem cells (ESCs and iPSCs)

Troubleshooting Stem Cell Analysis Assays

  • Low CD34+ Cell Count:
    • Possible Causes:
      • Sample degradation
      • Incorrect staining
      • Instrument malfunction
    • Troubleshooting Steps:
      • Use fresh samples
      • Verify staining protocol
      • Inspect instrument for malfunctions
  • High Background Noise:
    • Possible Causes:
      • Non-specific antibody binding
      • Autofluorescence
      • Contamination
    • Troubleshooting Steps:
      • Use blocking reagents
      • Reduce autofluorescence
      • Clean samples
  • Variable Results:
    • Possible Causes:
      • Inconsistent technique
      • Instrument instability
      • Sample heterogeneity
    • Troubleshooting Steps:
      • Standardize assay protocols
      • Calibrate instrument
      • Increase sample size