PERIPHERAL BLOOD STEM CELLS(PBSC)

The presence of peripheral blood stem cells in blood samples and donor collections is monitored by measurement of CD34+ cells. CD34 is likely an intercellular adhesion molecule present on the surface of the "stem cell." After incubation with fluorescent tagged monoclonal antibodies directed toward CD34, cells bearing the CD34 antigen became detectable by flow cytomety. When the presence of CD34 is used in combination with other cell parameters such as cell size, the stem cell concentration of the sample can be determined. While not all CD34+ cells are "stem cells," stem cells are included among the CD34+ cells.

PBSC vs. ABMT
Peripheral blood stem cell transplants offer an important advantage over traditionally harvested autologous bone marrow. Peripheral blood stem cells can be harvested by an automated cell separator (leukapheresis) from a peripheral vein, or more typically, from a central venous access device. The traditional bone marrow harvest, in contrast, requires intraoperative collection using multiple iliac crest samples obtained with a large-bore needle. Of great clinical significance is the need for general anesthesia with traditional marrow harvests which has inherent risks and may be a source of anxiety for the patient. Autologous marrow collection, however, may still be necessary in those patients who do not "mobilize" stem cells due to a variety of causes including marrow injury by prior chemotherapy, radiation therapy or disease.

Patient Selection
Peripheral blood stem cell transplants, at the present time, are used in patients whose disease is "dose responsive." In general, this means that increasing the dose of the chemotherapeutic agent or agents should improve disease-free patient survival. Leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, breast carcinoma, multiple myeloma and anaplastic carcinoma of uncertain primary source are presently the diseases treated with PBSC at Yale. New applications are also emerging, including pediatric tumors and ovarian carcinoma. As a rule, patients eligible to enter the PBSC program are those with chemotherapy-responsive disease that has recurred. For a complete response, they require higher doses or more intensive chemotherapy which, in turn, causes a sublethal marrow injury. Stem cells are then reinfused to rescue the patient from complications of severely low white cell and platelet counts. The knowledge that micrometastases exist in a fair proportion of patients with certain diseases such as breast cancer has raised the prospect of using dose-intensive chemotherapy with PBSC rescue promptly after detection of the disease to prevent relapse and, possibly, to effect a cure.

Patient Preparation
Peripheral blood normally contains negligible numbers of stem cells. Therefore, it is necessary to "mobilize" them from the marrow into the blood stream. Mobilizing regimens include cytokine stimulation or chemotherapy or a combination of the two. Chemotherapeutic agents work as mobilizing agents by depleting the bone marrow. Consequently, the marrow is stimulated to replace the lost cells. In the process of rapid recovery of the marrow, the regenerating stem cells "spill" into the circulating blood. The chemotherapeutic agent most often used is cyclophoshamide (Cytoxan), a myelotoxic alkylating agent. Low dose cyclophosphamide, which may produce no therapeutic effect, is an effective "mobilizer" in many patients, but higher doses that are therapeutic as well as mobilizing are often used. Etoposide (UP-16), a topoisomerase inhibitor, is frequently used in combination with Cytoxan. Granulocyte-colony stimulating factor (G-CSF)and granulocyte monocyte-colony stimulating factor (GM-CSF) are cytokines that increase yields of stem cells. They are used either alone or in combination with chemotherapeutic agents. At Y-NHH, G-CSF is the preferred cytokine for mobilization of stem cells.

The optimal time to collect stem cells is during the rapid rise in the white blood cell count after the induced nadir. Following chemotherapy, this usually means starting leukapheresis at leukocyte counts of 3-10,000 cells/L. In the case of G-CSF alone, the rise from baseline is monitored using flow cytometry which correlates peripheral CD34+ cell numbers with expected yields. Therefore, an ordinary peripheral blood sample can be used to get a reasonably accurate indication of a patient's readiness for PBSC collection.

In addition to mobilization, the patient must have adequate access for pheresis. This almost always means the insertion of a large-bore,'hard-shelled plastic catheter into a central vein. The catheter should have a minimum bore of 10 French and must be able to withstand high draw rates. These characteristics are shared with dialysis catheters which are readily available. The insertion sites are usually subclavian or internal jugular veins or, rarely, femoral veins. Interventional Radiology performs the line placements, which can be complicated by the fact that many patients already have chemotherapy ports in place (which are not suitable for pheresis) or have had multiple prior lines with residual scar tissue.

Collection
Once the patient's PBSC are mobilized, the patient is leukapheresed in the Pheresis/Transfusion Outpatient Unit (CB-363; 785-4707) using a Baxter CS-3000 or COBE Spectra apheresis device programmed for the purpose. Pheresis machines essentially are sterile closed-loop, automated, continuous-flow centrifuges. Differences in the buoyant densities of blood cells cause the blood to fractionate into distinct layers and optical sensors facilitate collection of the specific blood fractions. A total of 12 liters of blood is processed per day over 3-4 hours and approximately 60mL of leukocyte(PBSC)-rich material is harvested. The exact harvest concentration depends on the concentration of circulating leukocytes and stem cells. The goal of collection is 2.5 x 106 CD34 positive cells/kg body weight per reinfusion cycle. The breast cancer protocol at Yale New Haven Hospital requires two reinfusion cycles and, thus 5.0 x 106 CD34+ cells/kg collected. A reinfusion cycle is defined as giving the patient myeloablative (high-dose) chemotherapy and then reinfusing the stored stem cells with subsequent hematopietic recovery.

Complications
Stem cell collections are associated with several patient complications. The central venous catheters may need to remain in place for several mobilization cycles and are at risk for thrombosis or infection. The collection necessitates the use of a citrate-based anticoagulant (formula ACD-A) which chelates calcium. The average stem cell donor will experience a 15% drop in ionized calcium level which may cause many of the complications of acute hypocalcemia, especially paresthesia and tetany. Paradoxically, the total calcium level will rise; this reflects measurement of bound citrate-calcium complexes. Fortunately, most of the problems can be averted by administration of IV calcium gluconate using a protocol developed by the Transfusion Medicine Service. Occasionally a patient will experience a near syncopal reaction with rapid onset hypotension, probably as a result of volume shifts. This is usually easily corrected by halting the procedure and replacing the volume with normal saline.

Assay and Cryopreservation
Once the PBSC material is collected, aliquots are drawn off for flow cytometry to assay the CD34+ cell count as described above and for CFU-GM, too. In addition, the laboratory performs a standard CBC with differential and a microbiology culture to monitor the collection for possible bacterial contamination. The material is then cryopreserved by addition of plasma/ dimethylsulfoxide to give a final concentration of 10% DMSO. The stem cell material is then frozen in a controlled rate freezer to -90ºC and then stored under liquid nitrogen at -196ºC.

Reinfusion
After the patient has received high-dose chemotherapy, the stem cells are reinfused. Thawing must occur at the patient's bedside because DMSO may be toxic and should not be left in contact with the cells at room temperature for very long, although the exact length of time permissible is controversial. The amount of DMSO in large volume infusions of stem cells may cause side effects - nausea, vomiting, tachycardia and hypertension are most commonly encountered. These effects, along with the characteristic "garlic" odor of the DMSO, are transient.

Engraftment Times
As previously alluded to, the intent of autologous bone marrow transplants is to hasten the recovery of hematopoiesis from marrow that has been ablated by high-dose chemotherapy. Patients will typically be at risk for infection and bleeding, with neutrophil and platelet counts near zero for several days. In addition to other advantages over the traditional aspirated bone marrow, PBSC transplants have been shown to have significantly shorter engraftment times. The data from Yale's PBSC Program show a median time to engraftment to an absolute neutrophil count over 500 PMN/uL to be 12 days post reinfusion (N=23) vs. 18 days after traditional bone marrow transplants. To obtain platelet count levels greater than 20,000/uL without transfusion has required a median of 16 days at Y-NHH.

The Future
The presence of tumor cells circulating in the peripheral blood has been well documented in breast cancer, lymphoma, and leukemia. Concern over reinfusing tumor after myeloablative therapy has been raised and the role of reinfused tumor in recurrence of disease has been demonstrated for B cell lymphomas and childhood AML. A number of approaches to increase the purity of the reinfused stem cells are being investigated, including removing tumor cells from the collected product (purging) or selectively concentrating CD 34 positive stem cells (a process called enrichment). Anti-CD34 attached to latex beads for use in elution columns or to magnetic beads for use with immunomagnetic separation are both currently being employed. In either case, CD34+ cells attach to the beads, the remaining cells are discarded and the CD34+ cells are then removed. Changes in pH, usually acidification, weaken the ionic forces holding the cell to the bead and dissociation takes place giving a more concentrated and purer stem cell product. The immunomagnetic method relies on the use of a similar antibody/antigen interaction. This time a magnetic field is used to separate the magnetically charged bead, with CD34+ cells attached, from the rest of the collected material. A recent agreement between the Baxter Healthcare Immunotherapy Division and the Transfusion Medicine Section in the Department of Laboratory Medicine has launched the Yale Stem Cell Purging and Cell Processing Program. In the near future, the laboratory will use immunomagnetic bead technology to enrich the yield of CD34 positive cells.

Ex-vivo expansion of stem cells is also being planned. This involves growing the harvested cells in culture in a "bio-reactor" so as to increase the number of cells available for reinfusion. At the same time, growth conditions can be selected which do not promote expansion of other types of cells. The intent is to harvest relatively small quantities of stem cells and then to expand them by an in vitro culture process to yield the requisite numbers of stem cells. This approach produces a twofold benefit. The first is that extensive mobilizations and repeated phereses would be avoided. The second is that a relatively pure product will be obtained, thus minimizing the problem of tumor reinfusion.

Gene Therapy
Stem cells are, theoretically, capable of unlimited renewal or regeneration and differentiation into the three blood cell lines. These characteristics make them the ideal vectors for gene insertion therapy which is under active investigation at Yale. The gene of interest could be put into the genome of a stem cell and the inborn error of metabolism corrected as the stem cell progeny repopulate the marrow. While these therapeutic modalities are limited to the research laboratory today, they may soon reach clinical utility as the field of hematopoietic stem cell therapy undergoes enormous growth.

For information on the Yale PBSC collection and cell preservation program, call Dr. Edward Snyder or Dr. Joan Judge in the Blood Bank at 785-2441.

Steve Mechanic, M.D.