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Slide 1:

Hello, my name is Dustin Strasburg. I am a Development Technologist for the Human Cellular Therapy Laboratory at Mayo Clinic. Welcome to this Pearl of Laboratory Medicine on the “Enumeration of CD34+ Hematopoietic Progenitor Cells by Flow Cytometry.”

Slide 2:

Hematopoietic Progenitor Cells or HPCs are used to reconstitute hematopoiesis following marrow-ablative therapies. Traditional colony-forming assays take 10-14 days and are thus irrelevant when immediate assessment of HPC products, such as apheresis, is necessary. Flow cytometry is used to enumerate CD34+ HPCs to quickly determine the potency of a collected HPC product. However, these cells are rare events which may be masked by nonspecific or interfering populations. Furthermore, various methodologies led to a wide range of recommended CD34 values needed for long-term engraftment.

In 1995, the International Society of Hematotherapy and Graft Engineering (ISHAGE) developed a simple and sensitive method that utilized the CD34 antigen and several other characteristics. By using only 4 parameters and sequential gating to minimize interference, this assay can be performed by most routine clinical flow cytometry laboratories.

Slide 3:

The ISHAGE criteria for CD34+ HPCs include: expression the CD34 antigen, expression the CD45 antigen but characteristically at lower levels, low side scatter, and low to intermediate forward scatter. The scatter properties are similar to but slightly higher than small lymphocytes, which can be used as an aid in accurate gating.

Slide 4:

Furthermore, a viability marker such as 7-amino actinomycin (or 7-AAD) allows for the exclusion of dead cells. The use of fluorescent counting beads with a known concentration is used to determine an absolute concentration of CD34+ HPCs.

Slide 5:

The first gate is set around the white blood cells which are CD45+. This gate excludes CD45- negative contaminants such as platelets, platelet aggregates, non-lysed red blood cells, and other debris. It should not to exclude CD45dim events as this would exclude potential HPCs prior to further analysis. A gate is also set around the lymphocyte cluster to be used later.

Slide 6:

Using the WBC events, a gate is set around all potential CD34+ events.

Slide 7:

Looking at only the CD34+ events, the target cell population can be identified more easily. A gate is placed around the cell cluster. This cluster exhibits lower CD45 expression than typical WBCs with low side scatter, similar to lymphocytes.

Slide 8:

Utilizing the scatter properties of lymphocytes gated earlier, a linked or mirrored gate is set around the HPC population. In this example, there are a large number of HPC events; however, many times this population is more diffuse and not as easily identified. Events falling within this gate exhibit all 4 parameters to be classified as CD34+ Hematopoietic Progenitor Cells.

Slide 9:

Fluorescent beads can be assessed with any parameter. This example used the FL3 channel plotted across time to select the singlet beads. Exclusion of doublets is critical in accurate analysis of the bead concentration.

Events that meet the 4 ISHAGE parameters are then assessed for viability. Viable cells are able to exclude the dye and thus appear negative for the marker 7-AAD.

Slide 10:

To calculate the CD34+ HPC concentration, we take the ratio of viable CD34+ HPC events to the number of singlet bead events and multiply that by the CAL factor (or known bead concentration).
In this example, the sample or product would have 121.7 viable CD34 cells/μL.

Slide 11:

In summary, the ISHAGE protocol is a single-platform flow assay that determines the absolute concentration of viable CD34+ Hematopoietic Progenitor Cells in a sample or product. This value can be used with the volume of product to determine the total CD34+ HPC content and further calculated to determine the dose per patient body weight of an HPC product.

Slide 12: References

Slide 13: Disclosures

Slide 14: Thank You from

Thank you for joining me on this Pearl of Laboratory Medicine on “Enumeration of CD34+ Hematopoietic Progenitor Cells by Flow Cytometry.”