Preservation and Infusion of HSCs

Because HSCs have a short shelf life when refrigerated, cryopreservation may be necessary to prolong the storage and viability of these cells. The most commonly used cryopreservative is dimethyl sulfoxide (DMSO). This chemical will be incorporated into the cells and prevents ice crystals from forming when the cells are frozen. Some facilities use a combination of DMSO and hydroxyethyl starch (HES) which provides more viable cells than DMSO alone. After freezing, the cells can be stored in liquid nitrogen at -196° C.

Frozen HSCs are thawed in a 37° C water bath and then rapidly infused into the patient. Prior to the infusion, the patient may be given an antihistamine to prevent an allergic reaction to the DMSO. Other common side effects during infusion are fever, chills, shortness of breath, nausea, and vomiting. Some facilities remove the DMSO by washing the cells before infusion to lessen these side effects.

Successful engraftment occurs when the absolute neutrophil count is greater than 0.5 x 10⁹/L. The presence of chimerism in an allogeneic transplant is another indicator of engraftment. The rate of engraftment is dependent on the number of CD34 positive cells in the transplant, control of GVHD, and use of growth factors. Graft failure is usually the result of inadequate numbers of CD34 cells, toxic reactions to chemotherapeutic drugs, or infection with cytomegalovirus (CMV) or other opportunistic infections.

Purging is a procedure to remove unwanted cells from the peripheral blood transplant product. For allogeneic transplants. This procedure can reduce the risk of relapse from malignant cells that still may be present in the patient after radiation therapy and/or chemotherapy. For allogeneic transplants, purging is performed to reduce the number of T cells in the transplant and minimize graft versus host disease. Removal of T cells will also affect the graft versus tumor effect which requires active T cells. Patients receiving T cell depleted transplants are more likely to have a relapse than patients receiving unpurged cell products.

The absolute number of T cells in a patient's peripheral blood sample can be measured by flow cytometry. The formula to calculate the absolute T cell count is:

Absolute T lymphocyte count = Total WBC (x10⁹/L) x %lymphocytes from differential/100 x %T cells from flow cytometry/100

A typical reference range for adult T cells (CD3+) is 1.0-3.6 K/µL (1.0 -3.6 x 10⁹/L)

A number of different methods have been developed to target cells for purging. They often use monoclonal antibodies to specific targets that are conjugated with magnetic particles or are attached to a solid phase. After the antibody binds to the target cells, the remaining cells can be separated by centrifugation or other physical methods of separation.

Complete chimerism occurs when a patient’s HSC cells have been replaced by donor cells. Partial or mixed chimerism occurs when there is a mix of patient and donor hematopoietic cells. Partial chimerism may indicate a relapse and reemergence of malignant cells in the patient. These patients may benefit from chemotherapy and a second transplant of additional HSCs from the donor. Chimerism is usually determined by molecular testing using fluorescent in situ hybridization (FISH) or polymerase chain reaction (PCR) for specific target sequences of DNA. For example, if a female patient receives a transplant from a male, FISH can be used to identify and enumerate Y chromosomes in the hematopoietic cells of the patient.