Practical Aspects of Hematopoietic Stem Cell Harvesting and Mobilization

Selection of Autologous Donor

The primary selection criterion for the patient undergoing autologous HSC collection and transplantation is the diagnosis of an illness amenable to treatment with a dose-intense regimen requiring HSC support. Extensive prior treatments, especially with marrow-toxic chemotherapy regimens or with radiotherapy, may prohibit collection of adequate quantities of autologous HSCs, which would exclude a patient from this treatment option. Proper management of patients with a disease amenable to dose-intensive therapy should include provisions for HSC collection before extensive marrow-toxic agents are administered. In general, however, any serious comorbid illnesses that would preclude either marrow or PBSC collection would also disqualify the patient from treatment with dose-intensive regimens used in preparation for autologous HSC transplantation.

Selection Allogeneic Donor

The selection of the allogeneic HSC donor is more complex. The HLA major histocompatibility complex is the primary consideration in selection of a donor for allogeneic HSC transplantation, since its loci contribute significantly to host-versus-graft (leading to immunologic rejection of donor HSCs) and to graft-versus-host (leading to GVHD and graft-versus-leukemia) reactions. Donor age, gender, and parity are secondary considerations in the selection of an allogeneic HSC donor. Mismatching for killer-cell immunoglobulin-like receptor ligands may reduce the risk for posttransplant relapse of disease. More than 30% of allogeneic HSC transplants from related or unrelated donors will involve ABO-disparate donors and recipients, and donor and recipient pairs may also differ for other red blood cell antigens, with no clear evidence of deleterious effect on engraftment, survival, or GVHD. Cancer, autoimmune disorders, and genetic diseases such as the hemoglobinopathies can be transmitted to the allograft recipient; thus, donor health is an important consideration in donor selection and determination of donor eligibility.

Evaluation of Hematopoietic Stem Cell Donor Suitability and Eligibility

The immediate pre-collection evaluation of a patient or donor is intended to address the risks of the collection procedure to the donor (“donor suitability”) and the risks for transmission of disease from the donor to the recipient (“donor eligibility”). The same general health criteria apply to both bone marrow and PBSC donors. Patients undergoing autologous HSC collection and transplantation are not at risk for transmitting disease to themselves, and determination of donor eligibility is not medically required or economically justifiable, but these patients must be evaluated for their suitability for the collection procedures. All allogeneic and syngeneic HSC donors must be evaluated for donor eligibility, as well as suitability, using the same criteria currently applied to blood or other tissue donors, including a targeted history regarding behaviors exposing the donor to infection, recent or concurrent illnesses, and medication use. This evaluation of the allogeneic donor suitability and eligibility must be clearly documented in the donor medical record, with appropriate additional documentation of donor eligibility placed in the intended recipient's medical record before initiation of the transplant conditioning regimen. Older donors have a greater probability of comorbid medical conditions, which will increase the risks of the collection procedures, and the risks to the allogeneic donor with underlying health problems must be fully considered before subjecting the donor to HSC collection. Published standards describe evaluation of the donor for the risk of the donation process, as well as the risk for transmission of disease to the recipient. Evaluation by appropriate consultants may be required before autologous or allogeneic donor approval is finalized. Procedures involving donors with acute infectious illnesses should be delayed, if at all possible, because of the risk for disease transmission. Genetic disorders, such as hemoglobinopathies, will be transmitted to the recipient as a direct consequence of stem cell engraftment. Cancer can be transmitted, as illustrated by the transmission of donor leukemia not detected during initial evaluation of the donor, and donors previously treated for cancer should be evaluated for the probability of recurrent disease that could be transferred to the immunocompromised recipient.

Collection of PBSCs is generally an outpatient procedure conducted in the clinic setting. In contrast, marrow harvesting has the luxury of the intensive support capability of the operating room. PBSC collection, therefore, should never be viewed as a safer alternative to marrow harvesting for the donor with underlying health problems. Pediatric donors present different challenges, based on the smaller size and varying ages (and ability to cooperate) of the donors (see box on Evaluation of the Allogeneic or Syngeneic Marrow or Peripheral Blood Stem Cell Donor ).

Use of a donor who does not meet eligibility criteria and who poses a risk for transmission of disease requires appropriate informed consent both from the donor (for disclosure of this confidential health information to the recipient and for counseling of the recipient) and from the recipient (for use of the stem cell product). The potential conflict of interest between protecting donor confidentiality and patient needs must be recognized by the personnel caring for each person, and preferably, the donor and patient should be represented by different physicians.

Determination of Suitability for Peripheral Blood Stem Cell Donation

The PBSC donor is exposed to the risks of cytokine (and chemokine) administration and the risks related to the apheresis procedure, including the risks of central venous catheter insertion and use. No long-term health consequences have been associated with granulocyte colony-stimulating factor (G-CSF) administration, and the specific toxicities with these agents are described later. G-CSF may lead to a flare of autoimmune disorders and may increase the risk for blood clots, particularly for donors who are sedentary or who may be traveling shortly after the donation procedures. The PBSC donor must be assessed for venous access before the patient receives conditioning, and consent for use of a central venous catheter must be obtained if the venous access is deemed inadequate for the apheresis procedure.

Determination of Suitability for Bone Marrow Donation

Anesthesia and blood loss present the greatest risks for serious complications to the bone marrow donor. Most marrow harvesting is performed under general anesthesia, which requires intubation for control of the airway for a surgical procedure being performed on a prone patient. Regional (spinal or epidural) anesthesia may not be effectively established, so patients and donors who express a preference for this form of anesthesia must be counseled about the potential need for general anesthesia. The health assessment must include questioning about a history of joint disease of the cervical spine and mandible, and examination of the mouth if general anesthesia requiring intubation is chosen. Patients and donors with comorbid conditions, such as aortic stenosis sensitive to changes in blood volume and blood pressure, may require anesthesia consultation and plans for invasive monitoring during the surgical procedure. A history of marrow fibrosis, pelvic irradiation, or pelvic tumor involvement may exclude a patient from marrow harvesting, although unilateral harvesting from the posterior and iliac crests and aspiration of the sternum may achieve adequate quantities of cells for transplantation.

Suitability and Eligibility for Umbilical Cord Blood Donation

Evaluation of the donor for UCB donation begins with a history of maternal and paternal illnesses and exposures to infectious diseases. Although linkage between the infant and the product is currently maintained, an update of infant health is not obtained at the time of transplantation, which may be several years after collection. Therefore, parental medical history includes specific questions addressing the risks for transmission of hereditary or acquired blood-borne diseases. A comprehensive genetic and family history should be obtained. Testing for infectious diseases is obtained from the mother at the time of collection to minimize loss of product through such testing.

Public UCB banks set criteria for the storage of units in order to avoid the collection and storage of UCB units that would not be acceptable for transplantation. Exclusion criteria for potential donors may include multiple gestation; premature delivery; active chorioamnionitis or sepsis; mother being the recipient of an organ transplant; mother with history of cancer; mother with high-risk behaviors or previously diagnosed with human immunodeficiency virus (HIV), hepatitis, or syphilis; and mother having an active venereal disease, such as vaginal herpes simplex and delivering vaginally.

There are also private cord blood banks that usually provide service to parents that enable collection, processing, and storage of cord blood units for future use by their children when they reach adult years. This is performed as an investment in the unknown potential for cord blood to be used to treat serious illnesses in the future. In general there is less than 1% chance of any child to receive HSC transplant in their lifetime, especially if the child has no family history of leukemia or lymphoma. Therefore, the general consensus is that routine cord blood storage in healthy babies is unnecessary because it is highly unlikely even if the child needs transplant in the future, the child’s own cord blood will be suitable for the child’s transplant due to possible risk for contamination with inherently cancerous cells. Also if the indication for the transplant is a genetic condition, the child’s own cord blood will not be suitable because the cord blood will contain the same genetic problem.

In the absence of a suitable matched sibling or unrelated donor, finding a suitable UCB unit or haploidentical donor will be an important consideration. The choice between UCB and haploidentical transplant depends on the urgency of the transplantation, cell dose needed and the expertise at the transplant center. In terms of relapse rate, leukemia-free survival and transplant-related mortality, no significant difference was observed when UCB was compared with haploidentical transplant.

Bone Marrow Collection Techniques

Bone marrow is typically harvested from the posterior iliac crests using virtually the same techniques used to obtain diagnostic samples in the clinic. The primary differences between obtaining diagnostic specimens and cell quantities adequate for transplantation are the volume of blood and marrow removed, which requires attention to fluid and blood component replacement during the procedure, and the need for appropriate anesthesia. Bone marrow harvesting from healthy donors presents little risk for serious morbidity, permitting the ethical recruitment of allogeneic and syngeneic donors, including pediatric bone marrow donors and donors not related to the recipient. Multiple aspirations are performed with collection of approximately 5 mL of marrow from each puncture site. If properly spaced, no more than two or three skin-puncture sites per side usually are required. Other harvest sites, such as the anterior iliac crests or sternum, can be used, but at increased risk for complications from accidental laceration or perforation of contiguous anatomic structures. For patients with a history of radiation or tumor involvement of one pelvic crest, adequate cells can be harvested from the anterior and posterior crests of the other side.

The prescription for marrow collection will define the desired quantity of nucleated cells per kg recipient weight to be collected. Ideally, this quantity of cells will be collected in a minimal total volume and procedure duration. Although transplant registries may require physicians to be experienced in marrow harvesting, defined as the number of procedures performed, few published studies report a correlation between such experience and harvest yields or donor complications. The nucleated cell yield (cells per volume aspirated) appears greater for needles with side aspiration ports. Smaller quantities aspirated per “pull” also improves cell yield. Warming of the donor may improve cell yield. Quality-assurance management should review for each harvest team the nucleated cell yield per volume of marrow, total volume aspirated, use of blood replacement, and duration of anesthesia.

Marrow is collected in the day surgery suite using either general or regional anesthesia. With proper fluid and blood replacement, overnight hospitalization should not be required. Bone marrow harvesting necessitates placing the donor into the prone position, which has specific considerations to avoid complications directly resulting from this positioning. Donors must be supported, at a minimum, by positioning on chest rolls. For the healthy donor, the risks for serious complications from either general or regional anesthesia are minimal, although a multivariate analysis of adverse events performed by the National Marrow Donor Program for unrelated donors reported a higher risk for serious adverse events for donors receiving regional anesthesia. Use of spinal or epidural anesthesia avoids the nausea that may occur with general anesthesia, especially for younger women, but hypotension from loss of vascular tone in the lower extremities often occurs as the volume of marrow is collected. General anesthesia is preferable for the donor with comorbid disorders, such as cardiovascular or cerebral vascular disease, because of the better control of donor airway and lower risk for hypotension during the harvest procedures. Local anesthesia is acceptable only if a very limited harvest is being performed, because local anesthesia does not achieve anesthesia of the marrow space and because large quantities of lidocaine, for example, are cardiotoxic.

Both heparin and acid-citrate-dextran-A (ACD-A) can be used for anticoagulation of bone marrow products. ACD-A decreases the accumulation of lactic acid and may be preferable, especially for products that will be transported or stored for longer periods before infusion or cryopreservation.

Complications of Bone Marrow Collection

Anesthesia complications present the major health risk to the donor; marrow aspiration is generally well tolerated, although postharvest discomfort is experienced to some extent by all donors. Complications include hemorrhage and infections at skin-puncture sites. Severe hematomas and neuralgias rarely occur, and training regarding pelvic anatomy is required to decrease the risk for damage to vessels and nerves lying under or adjacent to the iliac crest harvest sites. Irritation of the sacral nerves may result from needle penetration through the pelvic bone or from blood tracking into the nerve roots, and requires several months of convalescence. Localized pain is common, may last for several days, and may require a brief period of opioid medication. In a survey of over 9000 donors for unrelated bone marrow transplantation, 82% reported collection site pain, with a median time to recovery of 3 weeks (see Figs. 102.1 and 102.2 ). Pain associated with the anesthesia procedures (throat pain, 33%; postanesthesia headache, 17%) was reported by a large proportion of the donors. Fatigue was reported by 59% of donors. Serious adverse effects were reported for 125 donors (1.35%), with 116 donors reporting serious complications considered to be a consequence of the collection procedures. Most of the serious complications ( n = 69) were mechanical injury to tissue, bone, or nerve; and a smaller number ( n = 45) were related to anesthesia. Infection and grand mal seizure were reported for one donor each. A retrospective survey of donor events reported by the European Group for Blood and Marrow Transplantation described almost 28,000 bone marrow donors, with one death from pulmonary embolism. An additional 12 donors experienced severe adverse events, including four cardiac arrests (three during anesthesia), two episodes of severe hypertension, one pulmonary embolism from heparin-induced thrombocytopenia, one episode of pulmonary edema, one donor with a subdural hematoma, and three events not otherwise specified. This retrospective survey did not include all donors and may have underreported adverse events. This report, furthermore, did not report the experiences of related and unrelated donors separately. The adverse events reported by unrelated donor registries will underestimate the risks faced by donors for related recipients, who may undergo collection despite comorbid illnesses that would preclude participation in an unrelated donor registry. Most donors are able to return to routine activities 1 to 2 days after harvesting, The total recovery time for the 67 donors reporting serious mechanical injury was a median of 10 months (range: 1 to 96 months). Toxicity was more likely to occur for less experienced harvest teams, and harvest teams must address training and maintenance of harvest skills in their quality-assurance plans. Any team associated with a second severe mechanical injury should specifically be evaluated, in light of the very low probability of these events.

The usual volume harvested from healthy donors is approximately 10 to 15 mL of marrow per kilogram of recipient bodyweight to achieve the desired nucleated cell and CD34 ⁺ cell doses. This results in a blood loss of 800 to 1000 mL for donors providing marrow for an average-sized adult recipient. The quantity of marrow harvested from autologous patients may be greater, reflecting previous chemotherapy given to these patients causing decreased marrow cellularity. Donors for pediatric recipients will lose proportionately less blood. Most patients and donors receive blood transfusions to alleviate symptoms of volume depletion. With proper pre-harvest autologous blood storage, use of homologous blood for healthy first-time allogeneic donors should be extremely rare. For a blood loss of less than 10 mL/kg of donor weight, salt solutions are acceptable for volume replacement. Colloid solutions, such as hydroxyethyl starch, can be used to avoid homologous blood transfusion for blood losses between 10 and 20 mL/kg donor weight. Blood transfusion will be required for larger blood losses (>20 mL/kg) or for patients with comorbid illnesses. Homologous blood transfusions must be irradiated to prevent transfusion-associated GVHD in the transplant recipient caused by “passenger lymphocytes” from the third-party blood donor. Donors undergoing a second harvest shortly after the first harvest are more likely to require homologous blood. Oral iron supplements should be considered for healthy donors, particularly for female donors or donors from whom a proportionately large blood volume is to be harvested.

Collection of Umbilical Cord Blood Stem Cells

Advantages of this source of HSCs include the ability of public cord blood banks to target collections from ethnic-minority populations not well represented in the various unrelated donor registries and the relative immaturity of the donor immune system allowing transplantation of HLA-mismatched units without overwhelming GVHD. The primary obstacle to the widespread use of cord blood cells is the limited quantity of HSCs collected, and one public UCB bank predicted that from less than 5% to less than 38% (depending on the cell dose criterion for transplantation) of the units stored in that bank would be acceptable for transplantation of an 80-kg adult patient. The speed and success of engraftment are predicted by the total nucleated cell dose and, more important, by the quantity of CD34 ⁺ cells or infused colony-forming units (CFUs).

CB Collection Techniques

UCB is collected from the placental vein after delivery of the infant and transection of the cord, either before delivery of the placenta by the obstetrician or by laboratory personnel after delivery of the placenta. Published reports conflict regarding the volume of UCB collected and the likelihood of obtaining a product inadequate for storage with either in utero or ex utero collection techniques. The timing of cord clamping after delivery of the infant is associated with the volume of cord blood collected, and greater volumes are collected with earlier clamping. Greater cell quantities were found for infants with greater birth weight, but no difference was found based on gender or gestational age. Ethnic background appears to predict the cell quantities, with smaller quantities of cells collected from persons from other ethnic populations compared with Whites. UCB is usually collected by cannulation of the umbilical cord veins with aspiration of the blood into a collection bag. Collection of cord blood into open containers results in an unacceptable rate of bacterial contamination. Perfusion of the placenta with salt solutions may increase the cell number collected, but this technique has not been widely adopted. Many cord blood banks reduce the volume of the product by red cell and plasma depletion to minimize storage space and to reduce possible infusion-related toxicities from mature blood cells contained in unfractionated cord blood units. Bacterial contamination of UCB products is of concern, especially in the collection of products for related donor transplantation by obstetricians with limited or no experience in HSC collection and processing. The identification and evaluation of the donor and the collection techniques used should be viewed as the first steps in a manufacturing process with adequately validated procedures, personnel training, quality control, and performance improvement oversight.

Mobilization

The self-renewal and differentiation of HSCs is controlled by the surrounding microenvironment of the stem cell niche(s) in which the HSCs reside. These niches are composed of a complex three-dimensional architecture of a variety of cell types, including sinusoidal endothelial cells, sympathetic nerve fibers, cells of the osteoblastic lineage, macrophages, and mesenchymal stem cells that are responsible for controlling the balance between HSC quiescence, self-renewal, and differentiation. A number of pathways with mutually recognized cellular adhesion molecules and their respective ligands responsible for the spontaneous migration of HSCs from the stem cell niche, as well as the multistep process of homing back into the niche, have been identified. The mechanisms by which G-CSF and other cytokines promote mobilization of HSCs are being elucidated and appear to be an indirect effect (HSCs do not express receptors for G-CSF) on the CXC-chemokine receptor 4 (CXCR4)/stromal cell-derived factor-1 (SDF-1) axis mediated by monocytes and the sympathetic nervous system. The mechanisms of mobilization by chemotherapy and CXCR4 antagonists such as plerixafor are also being determined, and the elucidation of the mechanisms of mobilization and homing may result in more effective harvesting and transplantation techniques.