Umbilical cord blood (CB) is an important source of hematopoietic stem and progenitor cells (HSPCs) for clinical transplantation. The numbers of HSPCs in individual CB units are usually limited and can vary significantly between units. Cell processing and cryopreservation can also adversely affect the number and quality of viable stem and progenitor cells. For these reasons, it is critical to determine the HSPC content of CB units using assays to measure CB unit “potency”.Red blood cell (RBC) depletion is a critical component of sample preparation for colony-forming unit (CFU) assays of fresh blood (Cord Blood or Pereferal Blood).

The removal of red blood cells (RBCs) from samples to be tested is recommended as their presence increases background in these potency assays and may lead to an inaccurate assessment of a CB unit.

RBCs depletion minimizes background in assay plates and ensures that hematopoietic colonies can be counted accurately, whether manually using an inverted microscope or automatically using STEMvision™. RBCs must be depleted from whole fresh unprocessed blood (i.e. cord blood or peripheral blood), buffy coat fractions of fresh blood, mononuclear cells prepared by density centrifugation and blood processed using automated cell separation devices.

Typical methods for depleting RBCs from small samples, such as segments attached to the main CB unit, include immunomagnetic depletion, sedimentation, or ammonium chloride lysis.


Depletion of Red Blood Cells from Small Cord Blood Samples


As part of a complete set of tools for standardization of the CFU assay, STEMCELL Technologies has developed the ErythroClear™ Red Blood Cell Depletion Kit. This kit enables the depletion of RBCs from up to 16 small (50 - 100 μL) cord blood samples at a time, in only 2 minutes. The ErythroClear™ reagent contains immunomagnetic particles that bind to Glycophorin AB+ (GlyAB+) cells, which are then selectively depleted using the ErythroClear™ magnet.

This product is recommended for the depletion of RBCs in cord blood samples prior to downstream assays (e.g. CFU assay, ALDHbr assay).

The ErythroClear™ Red Blood Cell Depletion Kit is to be used with the ErythroClear™ magnet.


Depletion of Red Blood Cells from Fresh Blood Samples


STEMCELL Technologies Inc. has developed a simple protocol that uses HetaSep™ to remove RBCs from fresh blood samples to be tested in the CFU assay. HetaSep™ is an erythrocyte aggregation agent used to quickly separate nucleated cells from RBCs. It’s effectiveness is based on the principle that aggregated erythrocytes settle much faster than dispersed cells.

The HetaSep™ protocol requires only 50 μL of blood, takes about 15 - 20 minutes and requires a cell count only at the start of the procedure when cells are to be plated in a CFU assay. The recovery of CFUs in the RBC-depleted fraction is nearly 100% so users can be confident that the HetaSep™ procedure does not adversely affect the number or viability of hematopoietic progenitor cells. These features make it easy to incorporate HetaSep™ into an institution’s CFU assay workflow.



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