HEMATOPOIETIC STEM CELL (HSC) TRANSPLANTATION (HSCT / HCT)

Table of contents :

Sources of HSCs :

• bone marrow transplantation (BMT) (BMSC)

History : in 1939 Osgood unsuccessfully infused a few milliliters of marrow into patients with aplastic anemia. In 1949, Jacobson discovered that shielding the spleen protected mice from lethal irradiation. Lorenz performed allogenic BMT in animals protected by total irradiation. His findings, and research by Barnes and colleagues, demonstrated that a cellular rather than a humoral factor played a role in graft survival. Vanbekkum, DeVries, and other scientists performed important experiments to study immunological reactions in transplantation recipients. They demonstrated that successfully grafted allogeneic marrow cells could later produce an immunological reaction against host tissues; this phenomenon is now known as graft versus host disease (GVHD). Experimental canine studies during the 1960s resulted in important discoveries that were applicable to human transplantation. Total body irradiation did not seem to prevent GVHD; mismatched canine leukocyte antigen always resulted in graft rejection or GVHD. Methotrexate was also found to be an immunosuppressive agent that could prevent or ameliorate the graft versus host reaction. Although initial attempts at allogeneic BMT in the 1950s and 1960s were unsuccessful, renewed attempts occurred after additional knowledge of the HLA system was obtained. Getty performed a successful allogeneic marrow graft in a patient with severe combined immunologic deficiency using a sibling donor with identical HLA. 2 similar successes were reported around the same time. These patients did not require immunosuppressive therapy and survived for > 25 years. In the 1970s and 1980s, clinical BMT required matched HLA haplotypes between the donor and recipient. Although earlier BMT procedures occurred between siblings, current molecular techniques have enabled the precise characterization of MHC genes and have allowed the possibility of matching unrelated individuals. In the 1970s, researchers performed a large number of BMT procedures using HLA-matched sibling donors for patients with end-stage leukemia or aplastic anemia. These endeavors resulted in mixed success because BMT was only considered for patients with advanced leukemia after conventional therapy failed. In the 1980s and 1990s, researchers noted that BMT performed at first remission or after first relapse resulted in significantly prolonged survival. BMT procedures for treating other disorders, such as thalassemia major and sickle cell disease, were also successful when the procedure was performed at an earlier stage. Because stem cells have been shown to be present in peripheral blood, these cells have now been used for autologous grafting. Extensive studies reviewed by Korngold and Brent have established the role of both T cells and histocompatibility systems in the GVHD reaction. Even with HLA-matched sibling donors, GVHD occurs half of all patients. Although corticosteroids and methotrexate partially ameliorated GVHD in early studies, cyclosporine in combination with other drugs have proven superior in subsequent studies. Following major efforts to determine the effectiveness of autologous BMT in the 1970s and 1980s, autologous BMT has become the therapy of choice for patients with malignant lymphoma and for selected patients with acute myelocytic leukemia and other malignancies. Recent research has also shown that a large number of stem cells from peripheral blood could be harvested following cytotoxic chemotherapy and specifically after growth factor administration. Following high-dose chemotherapy, PBSCT results in more rapid recovery of platelets than BMT. In the future, stem cell transplantation may allow many patients to attain a state of minimal residual disease so that other treatments, such as chemotherapy maintenance, monoclonal antibodies, vaccines, nonspecific immune modulators, and dendritic cell infusions, may be performed^{ref1, ref2, ref3}
Removing RBCs from BM buffy coat products :
• inverted spin procedure removes a median value of 81.2% of the RBCs, while still recovering a median of 94.3% of the mononuclear cells (median: 0.35 x 10⁸/kg; range: 0.17-0.9 x 10⁸/kg). The median volume of RBCs remaining in the product is 15.0 mL (range: 7.3-21.9 mL). The CD34⁺ cell dose of the final product ranged from 1.0 x 10⁶/kg to 4.8 x 10⁶ cells/kg (median: 1.9 x 10⁶/kg). Granulocyte recovery (defined as ANC count >or=500/mL for a period of 3 consecutive days) ranged from 18-30 days post-infusion of the allograft (median: 24.0 days)^ref.
Only 2-3 ml of blood should be sampled per bone marrow aspiration : if 5-10 ml are samples, the number of T lymphocytes increases
• peripheral blood stem cell transplantation (PBSCT) (peripheral blood progenitor cell (PBPC)) : easier accessibility and availability, faster engraftment of neutrophil and platelets when compared to standard bone marrow transplant^ref. PBPC collection in children weighing </=25 kg is hampered by technical and clinical problems related to vascular access, low total blood volume, anticoagulation, side effects, and psychological impact but large-volume leukapheresis (LVL) is a safe and effective procedure^ref

Mobilization of HSCs into PB : recruitment of hematopoietic stem and progenitor cells to the blood after administration of treatment.
• cytokine-mobilized PBSCs (expecially in elderlies):
• G-CSF mobilization (30-fold increase) coincides in vivo with a decrease in BM SDF-1 concentration related to SDF-1 cleavage by marrow serine proteases^{ref1, ref2} and dipeptidylpeptidase IV (CD26)^{ref1, ref2}. Furthermore, treating HSPCs by pertussis toxin (PT), which causes an interruption of Ga_i-protein signaling, did not affect their BM engraftment^ref but induced their release in the peripheral blood^ref, suggesting that a Ga_i-protein signaling identical to that mediated by CXCR4 receptor or other chemokine receptors must be required for HSPC retention in BM microenvironment^{ref1, ref2, ref3}. Consistent with the hypothesis that the CXCR4/SDF-1 axis plays a major role in the retention of hematopoietic cells, it has been shown that elevated numbers of granulocytic precursors were present in the peripheral blood of mice reconstituted with CXCR4^–/– FL cells^{ref1, ref2}. The ribosomal protein SA (RPSA) / 67 kDa laminin receptor (67LR) is a non-integrin cell-surface receptor with high affinity for laminin which plays a key role in tumor invasion and metastasis. The role of 67LR in G-CSF-induced mobilization of CD34⁺ HSCs from 35 healthy donors was investigated. G-CSF-mobilized HSCs, including CD34⁺/CD38^-- cells, showed increased 67LR expression, as compared with unstimulated marrow HSCs; noteworthy, the level of 67LR expression in G-CSF-mobilized HSCs significantly correlated with mobilization efficiency. During G-CSF- induced HSC mobilization, the expression of laminin receptors switched from 6 integrins, which mediated laminin-dependent adhesion of steady-state human marrow HSCs, to 67LR, responsible for G-CSF-mobilized HSC adhesion and migration toward laminin. In vitro G-CSF treatment, alone or combined with exposure to marrow-derived endothelial cells, induced 67LR up-regulation in marrow HSCs; moreover, anti-67LR antibodies significantly inhibited transendothelial migration of G-CSF-stimulated marrow HSCs. Finally, G- CSF-induced mobilization in mice was associated with 67LR up-regulation both in circulating and marrow CD34⁺ cells, and anti-67LR antibodies significantly reduced HSC mobilization, providing the first in vivo evidence for 67LR involvement in stem cell egress from bone marrow after G-CSF administration. In conclusion, 67LR upregulation in G-CSF-mobilized HSCs correlates with their successful mobilization and reflects its increase in marrow HSCs, which contributes to the egress from bone marrow by mediating laminin-dependent cell adhesion and transendothelial migration^ref
• G-CSF + SCF in poor mobilisers
• GM-CSF
• FLT3L
• CXCR4 antagonists^{ref1, ref2}
• induction of CXCL12 / SDF-1 plasma elevation^{ref1, ref2}
• combination mobilization (200-fold increase) : PBSC peak at day +12 (collect when CD34⁺ cells > 20/ml)
• low-dose cyclophosphamide (1.5 g/m²) plus lenograstim is a safe and effective mobilizing regimen^ref
• ifosfamide (1.8 g/m² per day for 5 days) + carboplatin (400 mg/m² per day for 2 days) + etoposide (100 mg/m² per day for 5 days) (ICE) /IL-11 (50-100 mg/kg per day) + G-CSF (5 mg/kg per day) is successful in mobilizing large numbers of CD34⁺ PBSC cells with a limited number (one) of apheresis collections in patients that have previously been heavily pretreated with chemotherapy/radiotherapy
• high doses of cyclophosphamide (600 mg/m² twice daily for 10 doses) and etoposide (200 mg/m² twice daily for 10 doses (continuous; n=57) or 2 g/m² over 10 hours on day 5 of etoposide (bolus; n=22)) + filgrastim (5 mg/kg/d beginning day 14)  to mobilize autologous hematopoietic progenitor cells in 79 patients with high-risk or relapsed/primary refractory NHL, MM, and Waldenstrom macroglobulinemia. 59% of patients achieved the primary end point (a CD34 cell dose of 5 million/kg with a single leukapheresis). More bolus etoposide patients achieved the primary end point (86%) compared with continuous etoposide patients (47%; P<.0001). The CD34 cell dose collected was greater in bolus etoposide patients (44 million/kg) than in continuous etoposide patients (10.9 million per kilogram; P<.0001). Patients took 3 weeks to recover >500/ml neutrophils and >20000/ml platelets after cyclophosphamide and etoposide. The overall response rate was 69% for NHL patients and 71% for MM/WM patients. The TR; was 2.5%. 16% of surviving patients experienced grade>or=3 nonhematologic toxicity. Patients receiving bolus etoposide had significantly less grade>or=2 oral mucositis, less use of TPN, and less need for red blood cell and platelet transfusions. 64 patients (81%) underwent autologous HSCT, with prompt engraftment. 4 patients (5%) did not undergo autologous HSCT because of toxicity from high-dose cyclophosphamide and etoposide^ref
This process mimics enhancement of the physiological release of stem cells and progenitors from the bone marrow (BM) reservoir in response to stress signals during injury and inflammation. Currently, mobilized cells are the preferable and major source of stem and progenitor cells harvested for autologous and allogeneic transplantations because of the higher yield of these cells, leading to faster engraftment and decreased procedural risks compared with harvested BM cells. The emerging picture of stem cell mobilization involves interfering with the physiological interplay between mesenchymal stromal and hematopoietic cells regulating both bone and BM remodeling processes, which also mediate stem/progenitor cell proliferation and migration. The mobilization process is initiated by stress-induced activation of neutrophils and osteoclasts by chemotherapy and repeated stimulation with cytokines such as G-CSF, resulting in shedding and release of membrane-bound SCF, proliferation of progenitor cells, as well as activation and/or degradation of adhesion molecules such as VLA-4 and P/E selectins. The dynamic secretion and inactivation of the chemokines SDF-1/CXCL12 and IL-8/CXCL8, and multiple cycles of inactivation and degradation of BM components by proteolytic enzymes, such as elastase, cathepsin G, proteinase 3, CD26, and various MMPs, are implicated as major players in progenitor mobilization. These mechanisms will be reviewed in this section. The road to stem cell mobilization began in the 1960s, with publication of a few reports documenting the presence of hematopoietic stem cells in the peripheral blood of mice, dogs, and monkeys, followed by reports revealing low levels of progenitors in the human circulation during steady-state homeostasis. However, steady-state progenitors in the circulation of mice were shown to be inferior to BM as a source of repopulating stem cells. More important, although sporadic clinical transplantations documented successful hematopoietic recovery, in most cases graft failure was detected in autologous, syngeneic, and fully matched related allogeneic transplantations, using steady-state peripheral blood cells. Moreover, the first successful human blood leukocyte transplantations were not better than BM as a source of repopulating stem cells (reviewed in Korbling and Fliender (Korbling M, Fliender TM. History of blood stem cell transplants. Blood stem cell transplants. In: Gale RP, Juttner CA, Henon P eds. Peripheral blood stem cell autographts. New York: Cambridge University Press; 1994:9) and To et al^ref). While the levels of human progenitors in the circulation during steady-state homeostasis are low, they are significantly elevated in patients with myeloproliferative disorders^ref. Similarly, a transient increase in circulating progenitors was documented in dogs treated with dextran sulfate and humans treated with endotoxin or other stress-inducing mobilization agents. These encouraging results were followed by preliminary reports in the late 1970s documenting increased levels of progenitors in the circulation of patients after chemotherapy treatment with cyclophosphamide and other drugs. Technical improvements including in vitro colony assays and the availability of large-scale harvesting of human progenitors by continuous-flow leukopheresis enabled closer examination of chemotherapy-/druginduced mobilization in treated patients^ref (Korbling M, Fliender TM. History of blood stem cell transplants. Blood stem cell transplants. In: Gale RP, Juttner CA, Henon P eds. Peripheral blood stem cell autographts. New York: Cambridge University Press; 1994:9). In the early 1980s, To and colleagues reported high levels of progenitors in the circulation of leukemia patients in early remission^ref. Clinical studies by this group were the first to document the beneficial faster repopulation in patients transplanted with autologous mobilized peripheral blood (PBL). Initially, the mobilization protocols were based on chemotherapy alone. However, after the discovery of human G-CSF by Welte et al^ref, mobilization protocols began to include G-CSF, which today is the standard mobilizing agent^ref.  During late embryonic development, both human and murine stem cells migrate via the blood circulation from the fetal liver to the BM and repopulate this tissue with high levels of immature and maturing cells. These in turn are released into the circulation, while a small pool of undifferentiated stem and progenitor cells within the BM is maintained throughout life. Prior to their localization in the murine fetal liver, hematopoietic stem cells can be isolated from the aorta-gonad-mesonephros (AGM) region and the yolk sac even before completion of blood circulation development^ref. However, some of these stem cells are not definitive, since they cannot rescue and repopulate lethally irradiated recipients because of their inability to migrate in the host circulation and home to and repopulate the BM. These predefinitive stem cells isolated from the murine yolk sac can still engraft the liver of newborn mice preconditioned as fetuses with chemotherapy if the predefinitive stem cells are directly injected into this organ^ref. Furthermore, an additional maturation step provided to predefinitive progenitors isolated from the AGM region, in the form of in vitro incubation on stromal cells and stimulation with cytokines, shifts these cells into definitive stem cells with functional migration, homing, and repopulation potential^ref. Murine embryos that lack the chemokine SDF-1 or its receptor CXCR4 have multiple lethal defects, including impaired seeding of the fetal BM^ref. The essential roles of SDF-1/CXCR4 interactions has been shown in a functional, preclinical model for human stem cells using non-obese diabetes severe combined immune deficient (NOD/SCID) mice as recipients. Homing and repopulation in both primary and serially transplanted mice with human CD34 stem cells was dependent on CXCR4 signaling^{ref1, ref2}, which is dynamically expressed by human progenitors^ref. Moreover, SDF-1-mediated migration of human CD34 cells in vitro correlated with their in vivo repopulation potential in transplanted patients^ref. In conclusion, stem cell motility (migration, homing, and release) is essential for BM repopulation and for the development of the hematopoietic system. The role of circulating stem cells in steady-state homeostasis is currently unknown. While some progenitors are needed for seeding of the thymus, which requires migration of lymphocyte precursors from the BM, this process is markedly reduced in adult life^ref. Stem cells migrate also to nonhematopoietic organs such as the liver, especially during liver injury/inflammation, creating an alarm situation and transmitting stress signals that mobilize and recruit stem cells as part of organ repair^ref. Ramshaw et al suggest that circulating stem cells can reengraft the BM, documenting successful engraftment of unconditioned murine recipients transplanted with high doses of BM cells^ref. In support of this approach, Wright et al elegantly demonstrated rapid clearance of intravenous transplanted mouse stem cells from the murine circulation within a few minutes. By using genetically marked parabiotic mice with a shared circulation, they showed that the low levels of circulating stem cells can durably reengraft the BM, suggesting continuous release and reengraftment of the BM as sequential events taking part in physiological processes^ref. In parallel, Abkowitz et al, using the same parabiotic model, revealed only low levels of reengrafted stem cells with secondary recipient repopulation potential (1–2.5%) in the partner BM as opposed to high levels of progenitors in the spleen. These investigators suggest the release of stem cells into the circulation functions also as an apoptotic pathway for the BM to regulate excess amounts of progenitors because of limited numbers of stem cell niches^ref. Of interest, these investigators also applied G-CSF- and SCF-induced mobilization to the parabiotic mice pairs, documenting a dramatic 4- to 10-fold increase (from 1–2.5% to 10.1%) in the levels of reengrafted stem cells with secondary repopulation potential in the partner BM. These results demonstrate that inducing the release of stem cells in response to stress signals also increases their homing back and reengraftment of the stimulated/stressed BM, confirming the repopulation potential of mobilized progenitors harvested for clinical protocols. These results also support the hypothesis that tissue repair of damaged BM can be supported by intensified homing and reengraftment of circulating hematopoietic stem cells, in response to stress signals, which is similar to migration of mature leukocytes to sites of inflammation as part of the immune system host defense. The concept of BM niches occupied by stationary, quiescent stem cells may be an oversimplification of a more dynamic situation. Higher proliferative and migratory turnover of definitive, repopulating stem cells involves their release into the circulation, migration, homing, and reengraftment of the BM as part of their physiological development. Bradford et al revealed that even the most primitive murine stem cells in the BM must cycle at least once every 30 days^ref. Proliferation and release of hematopoietic stem and progenitor cells require dynamic cycles of BM destruction/restructuring, which seem to be linked to bone remodeling by osteoclast/osteoblast interactions. Moreover, both processes are likely to be orchestrated by the same factors. For example, continuous G-CSF treatment to prevent neutropenia has also been shown to induce osteoporosis in some juvenile patients with severe congenital neutropenia^ref and in transgenic mice that overexpress this cytokine^ref. More important, there is a dramatic increase in the number of murine osteoclasts in response to repetitive G-CSF stimulation, leading to osteoclast-mediated bone resorption during stem cell mobilization and calcium release, which can induce detachment of hematopoietic progenitors from fibronectin in vitro^ref. Unexpectedly, treatment of mice with pamidronate, an inhibitor of osteoclast-mediated bone resorption, together with G-CSF increased the levels of mobilized progenitors in the murine circulation^ref. Osteoclasts secrete high levels of IL-8, a mobilization-inducing chemokine^{ref1, ref2}. Another chemokine, SDF-1, which also participates in G-CSF–mediated mobilization, induces osteoclasts to secrete the metalloproteinase MMP-9^ref. This enzyme is involved in stem cell mobilization by shedding the membrane-bound cytokine SCF within the BM and releasing this cytokine into the circulation^ref. MMP-9 also induces migration of osteoclasts to the cortical bone, adjacent to the SDF-1 and stem cell–rich endosteum region, to form resorption pits^{ref1, ref2}. These results suggest that the role of osteoclasts in mobilization could be related to the rapid increase in their numbers and to the secretion of chemokines and proteolytic enzymes. Activation and secretion of proteolytic enzymes lead to cleavage and degradation of the BM extracellular matrix, adhesion molecules, cytokines, and chemokines and therefore facilitate transendothelial migration and stem cell release from the BM.7 These processes mediate both peripheral bone and medullar BM remodeling as well as stem cell proliferation and release by inactivating the BM microenvironment. Subsequently, stem cells migrate via the circulation, home back to the BM, and repopulate the damaged/destructured sites in this organ as part of tissue repair and the continuous replenishment of the blood with new immature and maturing hematopoietic cells of all lineages, while maintaining a small pool of undifferentiated stem and progenitor cells within their renewed BM niches. In support of this hypothesis, by applying complementary DNA array technology, 2 recent studies revealed high expression of the proteolytic enzyme proteinase 3 and low expression of a proteinase 3 inhibitor in isolated human BM CD34 cells compared with enriched mobilized CD34 cells isolated from G-CSF-treated healthy donors^{ref1, ref2}. These results suggest that proteinase 3, with its ability to cleave connective tissue in the BM, is needed for maintaining a niche for hematopoietic stem and progenitor cells. Recent findings document that proteinase 3 also cleaves the cell cycle checkpoint p21, inducing cells to enter the cell cycle^ref. This could explain the higher levels of cycling BM CD34 cells compared with the more quiescent mobilized PBL CD34 cells^{ref1, ref2}. Immature mesenchymal bone-forming osteoblasts lining the stem cell–rich endosteum region secrete high levels of SDF-1^ref. In response to stimulation with this ligand, hematopoietic stem cell–derived osteoclasts secrete MMP-9^{ref1, ref2}. Thus, peripheral periosteal bone and medullar BM destruction and remodeling are sequential events mediated by the same factors, i.e. MMP-9, G-CSF, SCF, IL-8, and SDF-1, which regulate both new bone formation and stem cell self-renewal, migration, and development. Applying single or multiple stress signals interferes with steady-state homeostasis, creating an alarm situation leading to increased release of immature and maturing hematopoietic cells from the BM into the blood circulation, which occurs naturally during inflammation and injury. For example, increased levels of G-CSF and other cytokines secreted by long-distance runners during marathons cause blood leukocytosis, in particular higher levels of CD34/CD38⁺ committed progenitor cells, but not more primitive CD38^- stem cells compared with control individuals, reflecting adaptation of BM activity to stress induced by extensive exercise^ref. Increased cell release from the BM reservoir is part of the immune system host defense during inflammation as a result of infection- or injury-mediated release of stress signals. This release is induced clinically or in animal models by a wide range of molecules and/or treatments: DNA damaging, single chemotherapeutic drugs such as cyclophosphamide, or combined chemotherapy regimens such as iphosphamide, carboplatin and etoposide (ICE) and etoposide, methylprednisolone, ara-c and cisplatin (ESHAP); cytokines such as G-CSF, GM-CSF, SCF, and flt-3 ligand; and chemokines such as IL-8, Mip-1, Groß, and SDF-1^ref. These molecules differ in their mode of administration, the time frame needed to achieve mobilization, the type of cells mobilized, and the efficiency. G-CSF, the most commonly used mobilization agent, is usually administered daily at a dose of 5–10 µg/kg for 5–10 days, alone or after chemotherapy^ref. However, a substantial number of patients, especially patients having extensive chemotherapy that leads to BM aplasia, older patients, and a minority of healthy individuals, are poor mobilizers^ref. For example, increasing age and longer intensive chemotherapy treatment in old multiple myeloma patients inversely correlate with successful mobilization^ref. These patients are usually treated with higher doses of G-CSF, GM-CSF followed by G-CSF, or a combination of G-CSF and SCF. Recently, it was reported that some chronic myeloid leukemia (CML) patients treated with high doses of imatinib (Glivec, STI 571) suffer hematological toxicity and can develop drug-induced neutropenia^ref. In addition, some treated patients failed to mobilize well, requiring discontinuation of the drug during the mobilization process and the addition of SCF to G-CSF, to successfully overcome the poor mobilization^ref. However, while SCF stimulation increases the levels of mobilized CD34⁺ progenitors, this treatment also has side effects such as allergic reaction due to activation of mast cells. These studies stress the need to identify and characterize the mechanisms of stem cell mobilization in order to develop better strategies for poor mobilizers. Chemotherapy followed by repetitive G-CSF–induced mobilization is a multistep process. Anchorage of stem and progenitor cells to the BM microenvironment by activation of adhesion interactions, which are facilitated by membrane-bound SCF or SDF-1, needs to be disrupted^ref. Papayannopoulou elegantly demonstrated that the integrin VLA-4 plays a critical role in stem cell mobilization since anti-VLA-4 antibodies prevent adhesion molecules expressed on the surface of hematopoietic progenitors from binding their ligand VCAM-1 expressed by stromal cells. This induces rapid mobilization of primate and murine stem cells, which also involves signaling via the c-kit receptor. Moreover, as a result of inactivation during the mobilization process, mobilized CD34⁺ cells have lower levels of VLA-4 and c-kit expression compared with BM progenitors^{ref1, ref2}. In addition, sulfated polysaccharides such as Fucoidan lead to rapid mobilization of progenitors^ref since they compete for adhesion interactions with selectins and for SDF-1 binding to the BM endothelium via its nonsignaling C-terminus, inducing inactivation of selectin interactions as well as release of SDF-1 into the circulation. Levesque et al revealed increased release of elastase and cathepsin G within murine BM during Cy- and G-CSF–induced mobilization; peak levels were reached during stem cell egress^ref. These enzymes cleaved VCAM-1 expressed by stromal cells, preventing adhesion of progenitors via VLA-4 as part of the mobilization process. In addition to their distinctive adhesive properties, mobilized CD34 progenitors differ from their BM counterparts in several ways. Besides reduced VLA-4, c-kit, and CXCR4 expression, a significantly higher percentage of mobilized progenitors are noncycling quiescent cells^{ref1, ref2}. Prior to mobilization, hematopoietic progenitors proliferate within the BM, and this feature could involve proteinase 3–mediated cleavage of p21 in response to neutrophil stimulation^ref. Mobilized progenitors have higher levels of the proapoptotic genes caspase 3, 4, and 8 and reduction in inhibitors of apoptosis such as antiproteinase 2, compared with human BM CD34 cells^ref, supporting the hypothesis of Abkowitz et al that release into the circulation may also serve as an apoptotic pathway for stem cells^ref. Enhanced repopulation documented with mobilized progenitors is due to significantly higher cell doses, in particular committed progenitors, which lead to faster neutrophil and platelet recovery^ref. However, better leukemia-free survival and overall survival were recently documented in acute myeloid leukemia (AML) patients transplanted with increased cell doses of matched BM cells compared with lower doses of BM cells and more important, also compared with high cell doses of matched mobilized PBL, suggesting BM as a superior source of stem cells for HLA-identical allogeneic transplantations^ref. New protocols aimed at increasing the levels of long-term repopulating stem cells as the major source while maintaining short-term repopulating cells need to be developed. Mobilized human CD34 progenitors express reduced levels of the SDF-1 receptor CXCR4, which correlates with improved mobilization^{ref1, ref2}, suggesting involvement of SDF-1/CXCR4 interactions in the mobilization process. Overexpression of SDF-1 in the murine circulation leads to stem cell mobilization^ref. In functional preclinical animal models for human stem cells such as preimmune sheep and immune-deficient NOD/SCID mice, mobilized CD34⁺ cells are inferior in their repopulating potential compared with similar cell doses of CD34 cells obtained from human BM. This is most probably due to their reduced levels of surface CXCR4, which is needed for SDF-1–mediated directional homing and repopulation in transplanted mice and increased levels of short-term repopulating cells^ref. These results should be interpreted with caution, since short-term stimulation with human SCF upregulated surface CXCR4 expression on human CD34⁺ cells and murine SCF is much less potent in stimulating human progenitors^{ref1, ref2}. In addition, other factors such as complement C3a improve SDF-1–mediated directional migration of human progenitors and the in vivo homing of murine progenitors. However, some of these factors can be species-specific or absent in immune-deficient mice^ref. Of interest, priming of mobilized human progenitors by several inflammatory molecules improves their chemotactic responses to SDF-1 (Wysoczynski M, Reca R, Kucia M, et al. Mobilized peripheral blood stem/progenitor cells are primed by several inflammatory molecules for their chemotactic responses to sdf-1: a molecular explanation as to why mobilized peripheral blood cells engraft faster than bone marrow cells after transplantation. Paris, France -July 5–8, 2003; 32nd Annual Scientific Meeting of the International Society for Experimental Hematology (ISEH). abst 277. 2003). We have demonstrated increased levels of SDF-1 production in the murine BM in response to DNA-damaging chemotherapy drugs, including Cy and 5-fluorouracil (5-FU), most probably to prevent cell death since this chemokine is also a survival factor for stem cells^{ref1, ref2, ref3}. Rafii and Hattori et al demonstrated that the increased levels of SDF-1 in response to stress-mediated 5-FU mobilization in mice induce release of MMP-9, which cleaves and releases membrane-bound SCF, leading quiescent stem cells to proliferate in the BM prior to their mobilization into the circulation^ref. SDF-1 mediates secretion of MMP-2 and MMP-9 from human CD34⁺ cells^ref, and these proteolytic enzymes inactivate SDF-1 by cleaving a few amino acids in the N terminus^ref. We demonstrated that each injection of G-CSF stimulates mesenchymal cells such as immature osteoblasts to secrete SDF-1, leading to a transient increase in SDF-1 levels within the BM. This oscillating increase is followed by a profound decrease due to inactivation by proteolytic enzymes, mostly by neutrophil elastase, with the lowest levels of this ligand released during cell mobilization^ref. Levesque et al reported that in addition to inactivation of BM SDF-1 by elastase and cathepsin G, these proteolytic enzymes can also cleave part of the CXCR4 receptor N terminus in the BM^ref, partially inactivating SDF-1 signaling and directional migration. Of interest, neutrophils secrete the proteolytic enzymes elastase, cathepsin G, and proteinase 3 in response to G-CSF, and neutrophil elastase also inactivates G-CSF^ref, which is essential for regulating release of neutrophils from the BM. However, expression of G-CSF receptors by neutrophils is not required because of an indirect mechanism that involves proteolytic reduction of SDF-1 within the murine BM^ref. Of interest, the central role of elastase in regulating release of BM leukocytes is suggested by inherited genetic mutations in the elastase gene, leading to 2 forms of neutropenia in juvenile patients (severe congenital neutropenia, or Kostmann disease, and cyclic neutropenia) that require continuous treatment with G-CSF^ref. CD26, another proteolytic enzyme that inactivates SDF-1, is expressed by human CD34 progenitors and is involved in G-CSF–induced mobilization in mice^ref. A role for CXCR4 signaling in cell egress from the BM to the circulation emerges from several studies. In parallel to gradual reduction of BM SDF-1 during G-SCF administration, CXCR4 expression is elevated within the human and murine BM, reaching peak levels at the time of mobilization^ref. While some CXCR4 inhibitors also lead to release of human progenitors^ref, and treatment with pertussis toxin, which blocks Gi-mediated signaling in 7 transmembrane G coupled receptors such as CXCR4, induced mobilization in mice^ref, SDF-1/CXCR4 interactions are also needed for cell egress. Treatment of mice with G-CSF and neutralizing anti-CXCR4, anti-SDF-1 Ab, or inhibitors of CD26 reduced the mobilization levels^{ref1, ref2}. Finally, warts, hypogammaglobulinemia, immunodeficiency and myelokathexis (WHIM) syndrome, a rare inherited immunodeficiency disease associated with mutations in CXCR4, is characterized by neutropenia and B-cell lymphopenia. In one case, an affected individual was born with cardiac malformation, resembling the murine model in which knocking out CXCR4 or SDF-1 results in a lethal phenotype associated with lack of BM seeding, B-cell development, and cardiac septum formation^ref. Thus, SDF-1/CXCR4 interactions may have a role in the regulation of the routine and active egress of progenitor and maturing cells from the BM into the circulation. Taken together, these results decipher key mechanistic insights into stress-induced mobilization that mimic and amplify naturally occurring recruitment of progenitors during alarm situations. We suggest the BM as a reservoir for immature and maturing hematopoietic cells to be released into the circulation upon stress signals, to migrate to injured sites, and to contribute to host defense and tissue repair. The steady-state balance in the BM is disrupted, leading to transient increased production of SDF-1, and proliferation and activation of neutrophils and osteoclasts. Release of proteolytic enzymes is followed by shedding of membrane-bound SCF, proliferation of hematopoietic progenitors, increasing surface CXCR4 expression and inactivation of SDF-1, G-CSF, the BM adhesion machinery, and extra cellular matrix (ECM). These events are intensified in each cycle of stimulation by G-CSF, eventually leading to release of progenitors into the circulation. A better understanding of the process by which progenitors egress from the BM will eventually lead to the development of improved mobilization protocols, in particular for patients who are poor mobilizers.
• mobilizing the older patient with multiple myeloma
• individually optimized collection of HPCs
• enumeration of HPCs

Variables having an impact on the ability to collect hematopoietic progenitor cells (HPCs) may be donor related or procedure related. Donor-related variables include age, previous chemotherapy, mobilization regimen, and platelet count at the time of mobilization. Procedure-related variables include central access devices as well as variables inherent to the different cell separation devices used. Nonetheless, at present, the basis for optimizing HPC collection is the ability to determine when to start collection, based on the ability to enumerate CD34⁺ cells in the peripheral blood of a donor/patient on a daily basis. It has been demonstrated repeatedly that the best currently available predictor of an adequate collection is the number of CD34⁺ cell/µL in the blood on the morning of collection, both for good mobilizers and for poor mobilizers^{ref1, ref2, ref3, ref4, ref5}. A number of authors have recommended starting collection when a particular number of CD34⁺ cells/µL is present (usually a number between 8 and 20) in order to increase the likelihood of collecting at least 2–4 x 10⁶ CD34⁺ cells/kg in a single apheresis, i.e., an acceptable number of HPCs for either one or two autologous transplants or a single allogeneic transplant (Weaver C, Birch R, Schwartzberg L, et al. CD34+ content of peripheral blood progenitor cells is the single most powerful predictor of recovery kinetics in patients receiving myeloablative high-dose chemotherapy and PBPC infusion. Blood. 1994;84:1388a)^ref. In the past it was important that each center determine a number of its own as the starting point for apheresis, given the historic difficulty in comparing flow cytometry results from center to center^ref. At present, however, there are 2 commercially available single platform tests available for measuring CD34⁺ cells in blood (ProCount from Becton-Dickinson, Mt View, CA, and StemKit from Beckman-Coulter, Fullerton, CA), so that any center using the same technology should have a reasonable chance of success using the same numbers. More importantly, it is possible to use the number of CD34⁺ cells/µL obtained using single platform technology in a predictive formula which allows the clinician to know when to start apheresis in order to optimize collection and how many liters of blood need to be processed in order to collect a given number of CD34⁺ cells (O’Connell B, Ng Y, Rapoport A, Fassas A, Tricot G, Cottler-Fox M. Maximizing collection of human peripheral blood progenitor cells while minimizing the number of aphereses. Blood. 1999;94:4749a) using the formula in Figure 3. While results of collection using this formula have only been reported for the COBE Spectra cell separators, it seems likely that it would work with any continuous flow apheresis device if the appropriate value for machine collection efficiency is used.
Predictive formula that allows the clinician to know when to start apheresis in order to optimize collection and how many liters of blood need to be processed in order to collect a given number of CD34⁺ cells : # of L of blood processed = # CD34⁺ cells desired / [CD34⁺ cells/ml blood x machine collection efficiency)/patient weight in kg]
Sadly, despite the consensus that daily CD34⁺ cell numbers in the blood are the best currently available predictor for achieving an acceptable HPC collection,8 the practice has not been universally adopted because it is both time consuming and relatively expensive. Alternatives that have been reported to be useful include measuring the number of CD34⁺ cells in the blood the day before starting collection, with or without the total white cell count, or change in white cell count from the day before apheresis to the day of apheresis^ref, and the rapidity of rise in white cell count and platelet count. More recently, the Food and Drug Administration (FDA) has approved the use of an HPC window on an automated cell counter (Sysmex, Kobe, Japan) and this number may be used to predict when to start apheresis^ref. The HPC number measured by the Sysmex does not correlate well with CD34⁺ cell number in the blood (nor does CD133, an antigen expressed by the more primitive CD34⁺ cells^ref, but since it is now recognized that not all HPCs express measurable CD34^ref it has been presumed that the HPCs measured by the Sysmex include some that are CD34^-. Thus, the HPCs measured by Sysmex are available rapidly and less expensively, and may serve as a guide for starting apheresis, but they are difficult to use in the above predictive formula.
HPCs expressing high levels of the enzyme aldehyde dehydrogenase (ALDH) are believed to be pluripotent and generally express CD34 on their surface^ref. A commercial assay has now been developed for this intracellular enzyme (Aldecount, Stemco Biomedical, Durham, NC). As this assay detects both CD34⁺ and CD34^- cells, and appears to differentiate viable from non-viable cells (Balber A, Gentry C, Pritchard C, Baucom C, Deibert E, Smith C. ABC-mediated efflux of ALDH reaction product: implications for enumeratioin and isolation of blood progenitor cells. Biol Blood Marrow Transplant. 2003;9:231a), its use may eventually lead to a major change in how HPCs are enumerated in transplant grafts.
Timing of apheresis : there is a fall in circulating leukocytes immediately after G-CSF has been given, with the peak of CD34⁺ cell mobilization approximately 3–6 hours after each dose is given subcutaneously, paralleling its half-life^{ref1, ref2, ref3}. To optimize collection, it seems reasonable therefore, to wait at least 1 hour after giving the injection, trying to time the duration of the collection for the period which includes the rise in leukocytes and as much of the peak as possible. For example, at least one center routinely waits for 2 hours after the injection, since their average collection lasts about 2–3 hours^ref. It is possible that the apparent ability of large volume leukapheresis lasting 4–5 hours to "recruit" CD34⁺ cells into the blood, reported by several groups but not others^{ref1, ref2}, in fact simply reflects the timing of the apheresis collection relative to the dose of G-CSF. With the arrival of pegylated filgrastim (Neulasta, Amgen, Thousand Oaks, CA) it is possible that the relationship of collection yields relative to the time of dosage will cease to be a factor in collection.
Predicting who will mobilize poorly : the effect of age has been shown to be a continuous, incremental variable in the myeloma population, with no threshold past which there is an accelerated decline in mobilization of CD34⁺ cells^ref. That is, the older the patient, the fewer stem cells are likely to be collected, but there is no fixed age past which it is impossible to collect HPCs. Over and above age, however, 2 other variables are statistically significant: number of months of previous chemotherapy and platelet count at the time of mobilization^ref. In 85% of myeloma patients over age 70 who had < 12 months of therapy and a platelet count > 200 x 10⁹/L, it was possible to obtain  4 x 10⁶ CD34⁺ cells/kg in a single apheresis using individually optimized collection conditions after combined chemotherapy and growth factors. Those patient over 70 years of age with > 12 months of prior therapy and platelets < 200 x 10⁹/L, however, were poor mobilizers.
Choosing a mobilization regimen : mobilization with chemotherapy and growth factors has been shown in a number of settings to be significantly more effective than growth factors alone. Nonetheless, it is often the case that the toxicity of chemotherapy makes this combination less attractive in the fragile older patient. It is therefore important to know that in the subgroup of myeloma patients over the age of 70 who were predicted to be poor mobilizers (> 12 months prior therapy and platelets < 200 x 10⁹/L), as many stem cells were collected with growth factors alone as with chemotherapy and growth factors together. Thus, for this group of patients it seems worthwhile trying to mobilize with growth factors alone in order to avoid toxicity, assuming the disease itself does not require chemotherapy at the time of attempted mobilization.
• current standard agents :
• filgrastim has become the standard against which all other mobilization agents are measured. This is because it has been shown to both mobilize more CD34⁺ cells and have less toxicity than any other single agent against which it has been tested to date. It is not completely without toxicity, however, given that there have been deaths attributed to thrombosis (acute myocardial infarction and stroke) in sibling donors^ref, possibly related to receptors on platelets for G-CSF^ref. Also, a recent study of serial ultrasounds in donors receiving G-CSF prompted by 4 reports of splenic rupture related to G-CSF demonstrated universal enlargement of the spleen during mobilization, with regression of size after discontinuing the growth factor^{ref1, ref2}. Other effects of G-CSF which are shared with GM-CSF include pain, nausea, vomiting, diarrhea, insomnia, chills, fevers, and nightsweats^{ref1, ref2}
• sargramostim as a single agent is used less often today for mobilization than G-CSF, because it mobilizes somewhat less well than G-CSF^ref and because of a relatively higher incidence of both mild and severe side effects^ref. However, the fact that it can be more cost effective, and that there are reports of improved immune reconstitution with GM-CSF relative to G-CSF^{ref1, ref2, ref3, ref4}, has caused some clinicians to reconsider its use. Further, for the patient, or normal donor who has failed to mobilize adequately on G-CSF alone, the combination of GM-CSF with or followed by G-CSF has been shown to be efficacious^{ref1, ref2, ref3}. (Law P, Young D, Peterson S, Lane T, Ho A. Mobilization and collection of peripheral blood progenitor cells from normal subjects treated sequentially with GM-CSF and G-CSF. Blood. 1996;88:1578a)
• erythropoietin, now commonly used among cancer patients undergoing chemotherapy to maintain hemoglobin in the near normal range, also has some ability to mobilize CD34⁺ cells^ref. In the hard to mobilize patient, its use may therefore be doubly beneficial.
• stem cell factor (SCF) has been shown to be an excellent mobilizing agent, particularly when used in combination with G-CSF^ref. Unfortunately, the high incidence of allergic reactions to this agent and the need for observation after it is given have made it difficult to move into standard clinical practice. It is not currently available commercially in the US.
• pegfilgrastim and darbopoietin are now available and are in clinical trials as mobilizing agents. They have the benefit of very long half-lives and so add an important measure of patient convenience and the probability that timing of collection may be more flexible without sacrificing optimal collections.
• a new factor (AMD3100, AnorMed, Vancouver, Canada), which is a reversible inhibitor of the binding of stromal derived factor (SDF-1a) to its cognate receptor CXCR4, is currently in clinical trials as a mobilizing agent. It is the first agent to be tried for mobilization based on a rational understanding of its mechanism of action relative to HPC-stromal cell interactions. While it mobilizes CD34⁺ cells adequately on its own, it significantly improves the mobilization capacity of G-CSF when used in combination with G-CSF in mice. Clinical trials in humans with various diseases are in progress, including myeloma.
Remobilization : for the patient who fails to mobilize the necessary number of cells for transplant on the first attempt, but for whom it is clear that transplant is the best option, two decisions need to be made simultaneously: when to remobilize, and with what? When to remobilize is still a subject of debate, and depends to some extent on whether the failed mobilization was with chemotherapy plus growth factor or growth factor alone. Although at least one set of authors recommend immediate remobilization with growth factors for a patient who has not mobilized adequately after chemotherapy plus growth factor (Miclea J, Makki J, Lefrere F, et al. Successful PBPC harvesting with G-CSF alone a short time after previous mobilizataion failure by both chemotherapy and hematopoietic growth factors. Blood. 1999;94:1465a), others feel that at least 2–3 weeks off growth factor prior to remobilization offers the best chance of success.40–42 If adequate cells are given for an autologous transplant, it is even possible to collect enough for a second transplant during the period of white blood cell recovery following the first transplant (Schenkein D, O’Connor C, Morelli J, et al. A randomized trial of two different priming methods for stem cell mobilization in patients with relapsed Hodgkin’s disease and Non-Hodgkin’s lymphoma. Blood. 1999;94:1460a). It is also possible to collect HPCs at least 1 year after a prior transplant: of 38 myeloma patients who attempted such a collection, 36 achieved an adequate number of cells for transplant (Cottler-Fox et al, in preparation). What to use for a repeat mobilization attempt for a specific patient and disease may be a complex decision. However, some general guidelines may be found in the literature. First, mobilization with chemotherapy plus growth factor will generally yield more CD34⁺ cells than growth factor alone (for the single exception see #2 in the Algorithm for Mobilizing Myeloma Patients below)^{ref1, ref2}. Second, chemotherapy plus G-CSF and SCF is more effective than chemotherapy + G-CSF alone^ref. Further, chemotherapy with either sequential or concurrent GM-CSF plus G-CSF may be more effective than G-CSF alone^ref, as erythropoietin may improve the response to G-CSF^ref. Finally, retrospective analysis of data in myeloma patients from a single institution has shown that for patients who have undergone as many as 4 attempts at mobilization, only 1 of the 4 may yield an adequate collection. It is possible that AMD3100 will find a niche in this hard to mobilize population: in an ongoing trial at the University of Arkansas for Medical Sciences in myeloma patients who have previously failed to collect at least 5 x 10⁶ CD34⁺ cells/kg, it provides adequate mobilization in a significant number of patients. Although many variables may affect the decision of how and when to mobilize a myeloma patient, the following is a generally useful approach :
• attempt first collection relatively early, i.e., with < 12 months prior therapy.
• attempt collection first with chemotherapy plus growth factor (except for patients over 70 with > 12 months prior therapy and platelets < 200 x 10⁹/L, for whom growth factors alone may be tried first). The choice of growth factor (G-CSF versus GM-CSF) may depend on data under development regarding the importance of early immune reconstitution on time to progression and long-term disease-free survival. Pegylated filgrastim may replace standard G-CSF if studies show it to be equivalent or better than standard G-CSF.
• if mobilization is inadequate and
• a. patient is in CR or near-CR: wait at least 3 weeks, then remobilize with combination growth factors. In the future, AMD3100 may be a possibility.
• b. patient is not in CR or near-CR
: give planned chemotherapy and combine it with sequential GM-CSF and G-CSF. Consider adding erythropoietin. In the future, AMD3100 may be a possibility.
• if patient is not progressing, the procedure above may be repeated until adequate cells are collected.
• if patient is progressing, and adequate cells are available for autologous transplant (>= 3 x 10⁶ CD34⁺ cells/kg), monitor CD34⁺ cells in the blood at the time of leukocyte recovery, and consider collecting HPCs if there is adequate mobilization.
• if patient achieves a CR with transplant but does not mobilize adequately to collect during the leukocyte recovery phase post-transplant, consider attempting collection with combination growth factors at least 1 year after transplant.
• mobilization of autologous HSCs :  although early attempts to use unmobilized peripheral blood stem cells (PBSCs) for autologous stem cell transplantation were problematic, the use of cytokines such as G-CSF to enhance the peripheralization of CD34⁺ cells and the collection of these stem cells using leukapheresis procedures has become the standard for autologous stem cell transplantation around the world^ref. IBMTR and EBMT data suggest that over 80–90% of all autologous stem cell transplants in the world are performed using cytokine or chemotherapy/cytokine mobilized PBSCs as a source of stem cells^ref. In addition to reducing patient morbidity, the use of mobilized PBSCs has resulted in higher CD34 content of grafts, shorter hospital stays, and reduced engraftment times for both neutrophils and platelets as well as improved lymphocyte recovery resulting in enhanced immunologic reconstitution when compared to patients receiving autologous BM^{ref1, ref2, ref3, ref4}. These beneficial effects of mobilized PBSCs as a source of stem cells for autologous stem cell transplantation have been confirmed in a number of randomized trials^{ref1, ref2, ref3, ref4, ref5}.
• mobilization of allogeneic HSCs : based on the sustained success of using mobilized PBSCs for autologous stem cell transplantation, investigators began to pilot the use of PBSCs for allogeneic stem cell transplantation. Initial concerns focused on the possibility of increased risk of acute and chronic GVHD due to the presence of 10- to 50-fold increased numbers of T cells present in mobilized PBSC products. It was not clear if the function of mobilized allogeneic T cells might be qualitatively altered resulting in even greater risk of GVHD or relapse. In addition, the risk of infusing increased numbers of CMV⁺ granulocytes, dendritic cells, and monocytes into both CMV^- and CMV⁺ recipients remained unknown and potentially posed an increased risk to the recipient. On the other hand, mobilized PBSCs contain 3- to 4-fold more CD34⁺ cells, which might result in faster engraftment and more efficient transformation to complete donor chimersim. Early Phase 2 studies demonstrated that G-CSF had a generalized effect on the peripheralization of many different types of allogeneic peripheral blood cells, not just CD34⁺ cells. These data are consistent with the notion that G-CSF has a generalized effect on remodeling the BM microenvironment, which results in the egress of many types of cells including T cells and monocytes. Activation of neutrophils by cytokines such as G-CSF results in the release of proteases that facilitate the egress of HSCs from the BM microenvironment. Interruption of the G-CSF signal through genetically "knocking out" the G-CSF receptor results in not only the expected elimination of G-CSF-induced mobilization of HSCs but also IL-8- and chemotherapy-induced HSC mobilization in these G-CSF receptor knock-out mice^{ref1, ref2}. Although it is not completely clear which is the most important tether binding HSCs to the microenvironment, LFA-1, VLA-4, CXCR4, and c-kit have all been implicated as critical stem cell adhesion molecules^{ref1, ref2, ref3, ref4}. Likewise, a number of neutrophil-specific enzymes have been implicated in mediating critical cleavages that result in stem cell egress from the microenvironment. These include neutrophil elastase, cathepsin G, proteinase 3, gelatinase B (MMP-9), and other metalloproteinases^{ref1, ref2, ref3, ref4}. Recent evidence has strongly implicated CD26, a CD34-associated protease, as the prime protease that may cleave SDF-1 off the marrow microenvironment resulting in the release of CD34⁺ HSCs into the periphery^ref. Of interest, a collaborative effort of the Link, Simmons and Levesque laboratories have shown that mice deficient in MMPq, neturophil elastase and cathepsin-G and mice deficient in dipeptidyl peptidase I (CD26) all mobilized hematopietic precursors in response to G-CSF normally. These data question the role of neutrophil specific proteins in stem cell egress
• effects of G-CSF mobilization on allograft content : although a number of cytokines and cytokine combinations have been used to mobilize autologous HSCs, only filgrastim and sargramostim have been approved by the Food and Durg Administration (FDA) for use as autologous stem cell mobilizing agents. Thus, these have been the only cytokines used to mobilize allogeneic PBSCs. The majority of the initial Phase 2 studies using mobilized PBSCs in an allogeneic setting utilized G-CSF (10–16 µg/kg/day for 5 days). Leukapheresis was performed on day 4 or day 5 after G-CSF treatment. Fischmeister et al^ref followed CD34⁺ in the peripheral blood after either G-CSF or GM-CSF treatment and showed that CD34⁺ cells peaked in the blood between days +4 and +5 for G-CSF and days +5 and +6 after GM-CSF treatment. Data from Seattle suggested that larger doses of G-CSF (16 µg/kg/day) may result in even higher CD34 yields at the time of pheresis on day 5^ref. We have assessed the effect of 5 days of of G-CSF (10 µg/kg) on the numbers of leukocyte subsets in the peripheral blood of 100 consecutive normal allogeneic PBSC donors. It is clear from these data that G-CSF has a pleiotropic effect of increasing the numbers of circulating neutrophils and monocytes (WBC) as well as T cells (both CD4 and CD8), NK cells and B cells. Korbling and Anderlini compared the allograft content after G-CSF mobilization to cellular contents of BM harvests. These data suggest a 3- to 4-fold enhancement of CD34⁺ cells and a 10- to 20-fold increase in the number of CD3+ T cells in PBSC products compared to BM harvests^ref

It is well known that certain factors may help predict those autologous stem cell recipients who might be expected to be "poor mobilizers." These include extent of previous treatment, treatment with certain drugs such as nitrosoureas and certain diseases such as Hodgkin’s disease, non-Hodgkin’s lymphomas and preleukemic syndromes. No such data exist for allogeneic donors. We have examined stem cell mobilization from over 400 HLA-matched sibling donors since 1995. Using G-CSF as the sole mobilizing agent, only 2.0% of normal donors mobilized with G-CSF (10 µg/kg/day; 20 L exchange on day 5) did not achieve > 2 x 10⁶ CD34/kg and 25% did not achieve > 5 x 10⁶ CD34/kg after a single collection. These data have been recapitulated by other groups. We have studied the few normal donors who did not achieve > 1 x 10⁶ CD34/kg after 3 collections (1.0% of all normal donors in our data set). All of these donors underwent BM harvests as well but these all yielded < 1.0 x 10⁶ CD34 cells consistent with the notion that these poor allogeneic PBSC mobilizers were not defective in cytokine induced mobization per se but had low levels of BM stem cell reserves. Brown et al26 correlated premobilization PB CD34/mL with G-CSF-induced mobilization. None of the normal allogeneic donors who had < 2000 CD34/mL prior to mobilization yielded > 5 x 10⁶ CD34⁺ cells/kg while 95% of those normal donors with > 4000 CD34/mL PB yielded > 5 x 10⁶ CD34/kg after G-CSF mobilization. Although provocative, this has not been widely accepted as a method of identifying poor autologous or allogeneic PBSC donors. In the mid- and late-1990s, a large number of small Phase 2 studies were performed using mobilized PBSCs as a source of HSCs for allogeneic stem cell transplantation. All of these studies yielded similar results. Although neutrophil and platelet recovery was enhanced using cytokine mobilized allogeneic PBSCs, rates of acute GVHD were similar or less than that documented for BM as a source of allogeneic HSCs. The majority, but not all of these early studies, demonstrated increased actuarial rates of limited and extensive^{ref1, ref2, ref3} chronic GVHD (cGVHD). Cost and hospitalization appeared to be reduced compared to patients transplanted using allogeneic BM in these small Phase 2 studies. Similar to many autologous PBSC studies, the number of allogeneic CD34 cells infused correlated well with both neutrophil and platelet engraftment. Brown demonstrated that those allogeneic PBSC recipients who had > 5 x 10⁶ CD34/kg infused had a 95% chance of both neutrophil and platelet engraftment by day +15^ref. In this study, no correlation could be found between GVHD or survival and the number of CD3⁺ cells infused. In one retrospective study by the MD Anderson group, infusion of > 8 x 10⁶/kg CD34 resulted in decreased survival presumably due to increased rates of cGVHD associated complications^ref. These data have not been corroborated by other groups. Therefore, the infusion of high numbers (> 8 x 10⁶ CD34/kg) of allogeneic stem cells remains a controversial negative predictor for outcomes after allogeneic PBSC transplantation.
A major question is whether allogeneic stem cell harvesting results in less morbidity than BM harvesting for allogeneic stem cell donors. Anderlini et al^ref reviewed 1448 mobilized allogeneic PBSC collections from the IBMTR and EBMT registries. G-CSF was used in > 99% of donors. Similar to multiple Phase 2 studies, 20% of normal donors required placement of a central line for apheresis. 11% required > 2 leukapheresis procedures and "serious complications" occurred in 1.1% of allogeneic PBSC collection versus 0.5% after BM harvest. Rowley et al^ref utilized an 11-point scale (0 = minimum and 10 = maximum) for 23 different symptoms occurring during the first 14 days after either BM or PBSC collection. There were no statistical differences between the PBSC and BM groups for any of the symptom complexes or for the severity of any symptoms.
Many of the Phase 2 studies assessing the role of mobilized allogeneic PBSCs on GVHD, relapse and overall survival are limited by design (Phase 2), paucity of patients, short-term follow-up, and heterogeneity of diseases for which allogeneic PBSC transplantation was performed. Several trials have provided insight into the relative effect of PBSC versus BM on GVHD, relapse, and survival. These include randomized studies, case control retrospective studies, and meta-analyses. Table 1 summarizes the results of all randomized trials comparing BM and PB as a source of stem cells for allogeneic stem cell transplantation^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}. Half of these trials suffer from low numbers of patients. The 3 largest studies^{ref1, ref2, ref3} demonstrate no significant difference in overall survival when peripheral blood is compared to BM as a source of allogeneic stem cells. These studies, similar to the smaller randomized trials, did demonstrate a significant enhancement in both neutrophil and platelet recovery consistent with the significantly increased numbers of CD34 cells that are harvested in mobilized allogeneic PBSC products compared with BM. Randomized trials comparing allogeneic peripheral blood to bone marrow :

study	n	absolute neutrophil count (ANC) (engraftment (days) ANC > 500/mm3)		PLT (engraftment (days) Plt > 25000/mm3)		TRM		aGvHD		cGVHD		OS
		PB	BM	PB	BM	PB, %	BM, %	PB, %	BM, %	PB, %	BM, %	PB, %	BM, %
Vigoritoref	37	16	18	12	17	78	63	27	19	100	50	47	51
Blaiseref	101	15	21	13	21	23	21	44	42	50	28	61	61
Powlesref	39	17.5	23	11	18	31	35	68	58	44	40	70	68
Heldalref	61	17	23	13	21	17	10	21	10	56	27	80	73
Schmitzref	350	12	15	15	20	ND	ND	52	39	74	53	ND	ND
Coubanref	228	19	22	16	22	7.5	16	40	40	71	55	68	55
Bensingerref	172	16	21	13	19	21	30	64	57	46	35	66	54

In one of the largest and best-designed studies, Bensinger et al^ref, using identical conditioning regimens, GVHD prophylaxis (cyclosporine and methotrexate), and post-transplant growth factor support (no G-CSF), found a slight advantage in both disease-free and overall survival in those patients receiving mobilized allogeneic stem cells versus BM (P = .03 and P = .06, respectively). Although there was no difference in probability of 2-year overall survival in the subgroup of patients with less advanced disease (75% for PB and 72% for BM), those patients with more advanced disease demonstrated a significantly enhanced overall survival when mobilized allogeneic PBSCs were used as a source of stem cells (57% for PB and 33% for BM; P = .04). With a median follow-up for all surviving patients of 26 months (9–47 months), the cumulative incidence of grade III–IV acute GVHD at 100 days was 64% in the PB group and 57% in the BM group (P = .35). The cumulative incidence of grade II–IV acute GVHD was 15% in the PB group and 12% in the BM group (P = NS). Although the follow-up was relatively short, the cumulative incidence of extensive cGVHD was 46% in the PB group and 35% in the BM group (P = .54). These results were inconsistent with many of the other smaller Phase 2 and Phase 3 studies, which all showed no difference in rates of acute GVHD and increased rates of cGVHD in recipients of allogeneic PBSC.
A recent meta-analysis was performed by Cutler et al^ref summarizing 15 Phase 2 and Phase 3 trials assessing the risk of GVHD in recipients of allogeneic PBSC and BM. This analysis demonstrated a modest increased relative risk of acute GVHD (RR = 1.2) and a significant increased risk of developing cGVHD (RR = 1.8) in recipients of allogeneic PBSCs. It also demonstrated a modest reduction in relative risk of relapse in recipients of allogeneic PBSCs compared to BM (RR = 0.8). Champlin et al and the IBMTR40 performed a retrospective case controlled study comparing the outcomes of recipients of allogeneic PBSCs and BM. Median follow-up was 1 year, and this study focused on 1-year outcomes. A total of 288 HLA-identical sibling PBSC recipients was compared with 536 case control allogeneic BM recipients. All patients received T-replete stem cell products. There was no significant difference in the incidence of grades II–IV acute GVHD (40% for PB and 35% for BM; P = NS) or grades III–IV acute GVHD (13% for PB and 19% for BM; P = NS). There was less variability in recovery times of both platelets and neutrophils after allogeneic PBSC compared to BM and statistically faster neutrophil and platelet recovery after allogeneic PBSC compared to allogeneic BM. Treatment-related mortality (TRM), disease-free survival, and overall survival were similar for patients with acute leukemia in first remission. In contrast, acute leukemia patients in second remission and patients with CML in accelerated phase experienced lower TRM, improved DFS and overall survival when allogeneic PBSCs were used as a source of stem cells. There was no apparent difference in the risk of relapse after allogeneic PBSC versus BM transplantation. There was a trend toward lower relapse rates in patients with high-risk leukemia (acute leukemia in second remission and CML in accelerated phase). The relative risk of both limited and extensive cGVHD was increased in recipients of allogeneic PBSCs (RR = 1.3). Mohty et al^ref have performed the only long-term follow-up of allogeneic PBSC and BM recipients focusing specifically on the rates of cGVHD. At a median follow-up of 45 months (range 31–57 months), the 3-year cumulative incidence of cGVHD was 65% in the PBSC group (n = 53) and 36% in the BM group (n = 48) (P = .004). Extensive chronic GVHD was also more frequent in the PBSC group (44% versus 17%; P = .004). These data suggest that although hematopoietic recovery is increased in recipients of allogeneic PBSC, there appears to be no increased risk of acute GVHD and a modest increase risk of cGVHD including extensive cGVHD. Overall survival in recipients of allogeneic PBSCs may be improved modestly but only in those patients with more advanced hematologic malignancies. Although no study has correlated rates of either acute or chronic GVHD with the number of CD3 cells/kg in the stem cell products, several studies have suggested that CD34 cells in excess of 8 x 10⁶/kg found in allogeneic PBSC products are associated with a greater risk of both acute GVHD and GVHD.
Impact of G-CSF mobilization on graft content and immune reconstitution : very little data exist on the relative impact of allogeneic PBSCs versus BM on immunologic reconstitution after allogeneic stem cell transplantation. Storek et al^ref analyzed the incidence of documented and suspected infections after transplantation of mobilized allogeneic PBSCs and BM in the randomized trial carried out by Bensinger et al^ref. The cumulative incidence of infections was higher in the allogeneic BM group (120 versus 90 at 1 year). Since rates of acute GVHD after allogeneic PBSC infusions are similar to BM in spite of these products having 10- to 50-fold increased CD3 cells/kg over allogeneic BM products, a number of investigators have tried to understand the reason for this. To date, no studies have clearly shown a difference in either B-cell or T-cell recovery after allogeneic PBSC transplantation compared to BM transplantation. A number of reports have emphasized the role of cytokines as mediators of GVHD. Cytokines produced by both CD4 and CD8 T cells can be segregated into two patterns: type I cytokines such as interferon- and IL-2 and type 2 cytokines, such as IL-4 and IL-10. Type 1 cytokines are proinflammatory and type 2 cytokines are considered anti-inflammatory. Multiple studies have shown that T cells that elaborate T_h1 cytokines  mediate GVHD whereas those T cells that elaborate type 2 cytokines (T_h2 cells) inhibit GVHD. Pan et al^ref demonstrated that splenocytes from mice mobilized with G-CSF were polarized toward the T_h2 phenotype. Those mice who received splenocytes from G-CSF-mobilized donor mice demonstrated significantly longer survival and less GVHD that those allogeneic transplant recipient mice who were infused with splenocytes from naïve unmobilized donor mice. T cells from G-CSF treated mice showed a significant increase in IL-4 production with a simultaneous decrease in IL-2 and interferon- production. This polarization persisted in secondary mixed lymphocyte reactions (MLR) despite the absence of G-CSF during in vitro MLR. Arpinati et al hypothesized that G-CSF-mobilized PBSC contained antigen-presenting cells which prime T cells to produce T_h2 cytokines^ref. 2 distinct lineages of dendritic cells (DC) have been described in humans. DC1 cells or myeloid DCs express HLA-DR, CD11c, CD13, and CD33 and require GM-CSF for their survival. These cells are negative for both myeloid and lymphoid specific markers (Lin-), produce high levels of IL-12 when stimulated with tumor necrosis factor (TNF) or CD40 ligand and drive the differentiation of naïve T cells into the T_h1 phenotype. DC2 or lymphoid DC are HLA-DR⁺/CD11c^-/CD4⁺/IL-3Ra⁺ express high levels of T-cell receptor  chain and depend on IL-3, and not GM-CSF, for their survival and differentiation. After appropriate activation, they can induce T-cell differentiation into T_h2 cells. These investigators studied the effects of G-CSF mobilization (10–16 µg/kg/day for 5 days) on DC content in the peripheral blood in these allogeneic donors. G-CSF treatment was found to mobilize DC2 but not DC1. Although the numbers of donors and controls studied were very small, the median number of DC1 per liter in the G-CSF group was not different (11 versus 10 x 10⁶/liter; P = .52) than in the control premobilization group. In contrast, the numbers of DC2 were significantly increased in the G-CSF mobilization group compared with control (median 24.8 versus 4.9 x 10⁶/liter; P = .0009). As expected, a proliferative response of naïve allogeneic T cells could be detected in vitro to fresh DC1 but not to fresh DC2. Activation of DC2 in vitro with TNF, GM-CSF, and IL-3 resulted in the rapid maturation of these precursors to mature DC2 cells overexpressing costimulatory molecules such as CD80 and CD86 restoring their ability to induce a proliferative response to naïve CD4⁺/CD45RA allogeneic T cells. Incubation of naïve allogeneic T cells with DC1 resulted in polarization of these T cells toward the T_h1 phenotype as measured by restimulation of these T cells with PMA and ionomycin and detecting primarily IL-2 and interferon- as the major intracellular cytokines produced after restimulation. In contrast, incubation of naïve T cells with DC2 cells results in the polarization of these T cells toward the T_h2 phenotype as noted by the intracellular accumulation of IL-4 and IL-10 after restimulation in vitro with PMA and ionomycin. In conclusion, G-CSF mobilization results in stem cell products with 10- to 50-fold more T cells and 4- to 6-fold more DC2 cells. The increased numbers of DC2 in G-mobilized products may reduce the relative risk of acute GVHD as seen in preclinical murine allogeneic transplant studies described above and observed in the initial Phase 2 and 3 clinical trials in humans comparing BM versus mobilized PBSCs as sources of allogeneic stem cells. It is of interest that cord blood stem cell products, which are associated with a low risk of severe acute GVHD, contain primarily DC2 and negligible DC1^ref. Consistent with this notion, Waller found a strong inverse correlation with the number of precursor DC2 infused in allogeneic bone marrow and the incidence of both cGVHD and, more importantly, relapse^ref.
Alternative allogeneic PBSC mobilization regimens : although other cytokines, in addition to G-CSF, have been used to mobilized autologous PBSCs from humans including GM-CSF, Flt-3 ligand, stem cell factor (SCF), Daniplestim (IL-3 agonist), thrombopoietin agonists, chimeric cytokines including Leridistim (IL-3 agonist-G-CSF chimeric molecule) and Progenipoietin-1 (Flt-3 ligand-G-CSF chimeric molecule), peg-filgrastim (NeulastaTM) and SDF-1 antagonist (AMD 3100), only G-CSF and GM-CSF have been approved by the FDA and only G-CSF has been studied extensively for the mobilization of allogeneic PBSCs in humans. We have performed several sequential nonrandomized trials to determine the comparative effects of allogeneic PBSCs mobilized with G-CSF at 10 µg/kg/day (n = 96), G-CSF (10 µg/kg/day) combined with GM-CSF at 5 µg/kg/day (n = 102) or GM-CSF alone (10 or 15 µg/kg/day (n = 32). All donors underwent leukapheresis (20 liter) on the fifth day of cytokine administration. The target CD34⁺ content was 5.0 x 106 CD34/kg with a minimum of 2 x 10⁶/kg. PBSCs mobilized with G/GM resulted in collection of grafts with similar CD34 content compared with G alone. Grafts obtained following mobilization with GM alone contained significantly fewer CD34⁺ cells, but sufficient numbers for rapid engraftment. Grafts mobilized with GM or G/GM contained significantly fewer T and NK cells. There were no obvious differences in donor toxicities including bone pain. All recipients of GM mobilized cells engrafted with kinetics similar to recipients of G and G/GM, although neutrophil recovery was delayed about 1 day. Rates of neutrophil and platelet recovery and cGVHD for the G and GM groups are shown in Table 3. In 30 evaluable recipients of peripheral blood grafts mobilized with GM alone, the actuarial risk of grades 2–4 acute GVHD was only 0.13 ± 0.05 and 0.00 (0/31) for grades 3–4 acute GVHD. In a multivariate analysis including patient and donor age, sex mismatching, conditioning regimen received, CD3⁺ cell dose and CD34⁺ cell dose, only the receipt of PBSCs mobilized with GM-CSF alone correlated with a lower risk of grades 2–4 acute GVHD. These data suggest that altering the mobilization regimen and cytokines used may alter the functional aspects of the graft thereby modifying outcomes such as GVHD. Randomized Phase 3 studies will need to be performed to more accurately dissect the phenotypic differences in grafts mobilized by different cytokines/chemokines or combinations and to more accurately assess the impact on important endpoints such as multilineage engraftment, disease-free survival, overall survival, GVHD, and relapse. Comparison of G versus G/GM versus GM mobilization on allograft content :

parameter	G alone	G/GM	GM alone	P value
# donors	96	102	32
Total # LP procedures required	1.26 ± 0.5	1.20 ± 0.5	1.6 ± 0.7	G vs G/GM: .5; G vs GM: .002
CD34 (x 106/kg)	9.8 ± 7.2	10.9 ± 7.9	3.7 ± 2.0	G vs G/GM: .28; G vs GM: < .001
CD3 (x 108/kg)	3.5 ± 2.4	1.8 ± 1.5	1.8 ± 1.8	G vs G/GM: < .0001; G vs GM: < .001
CD4 (x 108/kg)	2.3 ± 1.4	1.3 ± 1.0	0.9 ± 0.8	G vs G/GM: < .0001; G vs GM: < .001
CD8 (x 108/kg)	1.1 ± 0.7	0.4 ± 0.4	0.3 ± 0.26	G vs G/GM: .56; G vs GM: .002
CD19 (x 108/kg)	0.97 ± 0.9	0.89 ± 0.63	0.45 ± 0.4	G vs G/GM: .56; G vs GM: .002
CD16/56 (x 107/kg)	4.63 ± 3.7	2.98 ± 2.36	1.66 ± 1.18	G vs G/GM: < .001; G vs GM: < .0001

Effect of cytokine mobilization regimen on graft-versus-host disease (GVHD)/survival :

	G-CSF, % n = 40	G-CSF and GM-CSF, % n = 66	GM-CSF, % n = 30	P Value
acute grade 2–4	51	65	13	G vs GM: .003 GM vs G/GM: < .0001 G vs G/GM: .11
acute grade 3–4	11	13	0	.07
chronic GVHD	86	88	65	NS
survival (2 years)	62 high 38 low 88	56 high 28 low 5	59 high 35 low 69	NS

Chemokines such as IL-8 have been used to induce the egress of HSCs into the peripheral blood of mice and nonhuman primates. This effect is rapid (30 minutes-4 hours) and may result from the ability of these chemokines to induce the release of a protease from mature myeloid cells resulting in a decrease in the intramedullary concentration of SDF-1, the ligand for the receptor CXCR4, which is expressed in many cells including hematopoietic stem cells^{ref1, ref2}. Increasing evidence points to the critical role of the CXCR4/SDF-1 axis in both murine and human stem cell mobilization. The bicyclam molecular AMD 3100 was first clinically developed for its potent and selective inhibition of HIV type 1 and 2 replication through binding to the chemokine receptor, CXCR4. Initial clinical trials in AIDS patients demonstrated that AMD-3100 induced a rapid (within 1 hour) increase in both WBC and circulating progenitor cells. Broxmeyer and colleagues demonstrated a 40-fold increase in the mobilization of hematopoietic progenitors within 1 hour of AMD-3100 injection in mice^ref (Broxmeyer HE, Hangoc G, Cooper S, Bridger G. Interference of the SDF-1/CXCR4 axis in mice with AMD3100 induces rapid high level mobilization of hematopoietic progenitor cells and AMD3100 acts synergistically with G-CSF and MIP-1 alpha to mobilize progenitors. Blood. 2001;96:3371a). Studies in human volunteers and the first Phase 1 and 2 clinical trials in patients undergoing autologous stem cell transplantation are currently underway (Devine S, Flomenberg N, Vesole D, et al. Safety and effect on WBC count and CD34 cell mobilization following administration of AMD3100 to patients with multiple myeloma or non-Hodgkins lymphoma. 8th Congress of the European Hematology Association, Abstract 0406, 2003). These studies have shown a consistent and rapid impact on stem cell mobilization when given alone and a synergistic effect on CD34⁺ mobilization when coadministered with G-CSF. Since CXCR4 is expressed on different types of cells including CD34⁺ progenitor and T cells, the impact of AMD-3100 on both multilineage engraftment and GVHD will need to be appropriately explored in preclinical animal models before it can be safely used as a rapid mobilizing agent for allogeneic PBSC transplantation in humans.
• umbilical cord blood stem cell transplantation (UCBSCT / UCBT)^{ref1, ref2}
• in 1989 Gluckman first successfully transplanted cord-blood stem cells, which are immature and less likely to cause GVHD. It was observed that the numbers of progenitor cells present in the low-density (< 1.077 gm/ml) fraction after Ficoll/Hypaque separation typically fell within the range that has been reported for successful engraftment by bone marrow cells. Another observation of practical importance is that procedures to remove erythrocytes or granulocytes prior to freezing, and washing of thawed cells before plating, entailed large losses of progenitor cells, the yield of unwashed progenitor cells from unfractionated cord blood being many times greater. The provisional inference is that human umbilical cord blood from a single individual is typically a sufficient source of cells for autologous (syngeneic) and for MHC-matched allogeneic hematopoietic reconstitution^ref.
• cord blood banks (CBBs) are not always closely located to a GMP-licensed microbiology laboratory, resulting in time delays for transport of specimens prior to microbiological testing : inoculation of culture bottles as soon as possible after sample preparation is preferable. Bacteria can maintain viability in BacT/ALERT bottles inoculated and held at RT for up to 7 days prior to automated culture testing. Bacteria can be successfully recovered from cryopreserved UCB^ref
• liquid storage of cord blood for 24 h due to delays prior to RBC depletion and cryopreservation does not adversely influence the post-thaw cell recovery but the post-thaw mononuclear cell recovery and viability of cord blood stored for 72 h was significantly lower. The use of dilution with a storage solution (STM-Sav with 1:1 ratio) enhances the retention of colony-forming capabilities post-thaw^ref
• a small proportion of  high proliferative potential (HPP)-Q cells recovered from 7-day expansion cultures of of UCB HSCs in the presence of TPO, SCF, flt-3 ligand (FL) and IL-6 retain their multilineage potential: longer culturing of these cells results in the loss of multilineage potential while they maintain quiescent behavior and high proliferative potential^ref.
• as a source of HSCs, UCB has the advantages of speed of availability, tolerance of 1-2 antigen HLA mismatch, and a low incidence of severe GVHD. Thus, UCB represents a highly convenient HSC source that may significantly extend the HSC donor pool. UCBT has become a standard practice in the treatment of pediatric malignancies. Now, UCBT is being investigated in adults using both conventional and non-myeloablative preparative regimens. As graft cell dose is the major factor determining hematopoietic recovery and survival in URD UCBT and cord blood has a limited number of stem cells (each sample is < 200 mL (10-20-folds less than a conventional bone marrow sample) and is unlikely to contain enough stem cells for an efficient transplant (usually 1 x 10⁹ nucleated cells = 0.5 x 10⁶ CD34⁺ cells/kg), its use has largely been restricted to children : methods to increase cell dose such as multiple-unit transplant and ex vivo expansion are being pursued^ref. CD26/dipeptidylpeptidase IV (DPPIV) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N terminus of polypeptide chains. The N terminus of chemokines is known to interact with the extracellular portion of chemokine receptors, and removal of these amino acids in many instances results in significant changes in functional activity. CD26/DPPIV has the ability to cleave the chemokine CXCL12 / SDF-1a at its position Pro². CXCL12/SDF-1a induces migration of HSCs and progenitor cells, and it is thought that CXCL12 plays a crucial role in homing/mobilization of these cells to/from the bone marrow^ref. In mice upon treating mice bone marrow HSCs for 15 minutes with a CD26 inhibitor (Diprotin A), a 9-fold increase in homing efficiency is measured. Similarly, cells in which CD26 had been deleted (CD26^-/-) showed a 11-fold increase. The treatment of low-density bone marrow donor cells, which more accurately resemble stem cells used in a clinical setting, also increased homing efficiency. Furthermore, CD26^-/- cells made a more significant contribution to long-term donor HSCs engraftment in lethally irradiated mice, with fewer CD26^-/- HSCs being required for mouse survival compared with normal HSCs^ref.
Indications :
• Arteriocyte (Cleveland, Ohio) : allogeneic cord stem cell transplant for ischemic disease (preclinical studies)
• Chemokine Therapeutics (Vancouver, Canada) : CTCE-0214 (chemokine agonist for ex vivo cord-blood stem-cell expansion) for blood disorders (phase 1 trial)
• Gamida-Cell (Jerusalem) : StemEx (allogeneic ex vivo expanded cord-blood stem cells combined with unexpanded cells Blood cancers Phase 1/2
• Histostem (Seoul, Korea) : developing technologies for expanding cord-blood stem-cells & for mixing cord blood for transplant Multiple indications including spinal cord injury, type 2 diabetes, liver cirrhosis (phase 1/2 trial)
• Saneron CCEL Therapeutics (Tampa, Florida) U-Cord-Cell: umbilical cord-blood stem-cell platform for myocardial fraction (preclinical studies)
• ViaCell (Cambridge, Massachusetts) : CB001: allogeneic ex vivo expanded cord-blood stem cells combined with unexpanded cells for blood cancers (phase 1/2 trial)
UCB cells coinfused through the jugular vein with a blood-brain barrier (BBB) permeabilizer (mannitol) into a stroke animal model in the first hours and days following a stroke, decrease stroke size by 40% and resulting disability is significantly reduced (source : Saneron CCEL Therapeutics)^ref
The first known attempt at UCBT occurred in 1970 when a young 16-year-old boy with acute lymphoblastic leukemia received cord blood units from 8 different donors. He was receiving 6-mercaptopurine and prednisone and did not receive a preparatory regimen. One unit engrafted as demonstrated by red cell antigens and the graft lasted for 38 days^ref. However, it was the success of the allogeneic UCBT approximately 16 years ago in a child with Fanconi anemia that opened a new source of allogeneic hematopoietic stem cells^ref. UCB is now accepted as an alternative source for hematopoietic stem cells for transplantation in children.
Advantages :
• the relative ease of procurement and availability (given the ability to store fully tested and HLA-typed cord blood available for immediate use)
• the absence of risk for mothers and donors
• the reduced likelihood of transmitting infections, particularly cytomegalovirus
• potential reduced risk of GVHD
• less stringent criteria for HLA matching for donor-recipient selection (with the potential of finding donors for minority populations)
• absence of donor attrition.
Contraindications to UCB stem cell collection :
• preterm delivery
• dystocic delivery
• newborn malformations of hereditary diseases
• previous maternal HCV, HBV, HIV or syphilis infection
• fever of unknown origin in the mother
• vaginal sampling positive for Streptococcus
• rupture of amniotic bag for > 24 hrs
UCB banks have been established for related and unrelated UCBT with about 100,000 units currently available^{ref1, ref2, ref3, ref4}. While the clinical data are encouraging for pediatric patients, the limited number of hematopoietic stem cells in UCB has been a cause for caution for its use in adult patients. Moreover, the use of myeloablative preparatory regimens with their known toxicities together with the known delays in engraftment of all cell lineages and the enrollment of high-risk patients with advanced disease all contributed to this caution. Recently, older allogeneic recipients have been treated successfully following a variety of less intense nonmyeloablative conditioning regimens. On the basis of these encouraging observations, it has been hypothesized that a reduced-intensity preparative regimen would allow engraftment of the UCB stem cells. While the total numbers of mononuclear cells are limited, the progenitor content and the proliferative potential of cord blood cells are high. This nonmyeloablative approach could diminish some of those concerns raised with the use of ablative regimens
• myeloablative preparative regimens : in comparison to the published studies on pediatric patients, the clinical data on the use of UCBT in adult patients are relatively limited, although growing^{ref1, ref2, ref3, ref4, ref5} (Rocha V, Arcese W, Sanz G, et al. Prognostic factors of outcome after unrelated cord blood transplant (UCBT) in adults with haematologic malignancies [abstract]. Blood. 2000;96:587a; Goldberg SL, Chedid S, Jennis AA, Preti RA. Unrelated cord blood transplantation in adults: a single institution experience [abstract]. Blood. 2000;96:208a; Cornetta K, Laughlin M, Carter S, et al. Umbilical cord blood transplantation in adults: results of a prospective, multi-institutional, NHBLI sponsored trial [abstract]. Blood. 2002;100:42a). As can be gleaned from the composite data, these patients received standard total body irradiation or busulfan-based myeloablative regimens. GVHD prophylaxis consisted of cyclosporine alone or in combination with steroids or methotrexate. The degree of HLA disparity was not associated with outcome in any of these studies, although the numbers in each were small. However, in a recent review update with 861 unrelated UCBT recipients from the Placental Blood program of the New York Blood Center, which includes 181 (21%) patients with age  18 years and 170 patients (20%) weighing  60 kg, Rubinstein et al (Rubinstein P, Carrier C, Carpenter C, et al. Graft selection in unrelated placental/umbilical cord blood (PCB) transplantation: influence and weight of HLA match and cell dose on engraftment and survival [abstract]. Blood. 2000;96:588a) have demonstrated in a multivariate analysis that HLA match is an independent predictor of event-free survival. Studies of unrelated umbilical cord blood (UCB) transplantation in adults :

		Laughlin et alref	Sanz et alref	Rocha et alref (Rocha V, Arcese W, Sanz G, et al. Prognostic factors of outcome after unrelated cord blood transplant (UCBT) in adults with haematologic malignancies [abstract]. Blood. 2000;96:587a) (Eurocord)	Goldberg et al (Goldberg SL, Chedid S, Jennis AA, Preti RA. Unrelated cord blood transplantation in adults: a single institution experience [abstract]. Blood. 2000;96:208a)	Cornetta et al (Cornetta K, Laughlin M, Carter S, et al. Umbilical cord blood transplantation in adults: results of a prospective, multi-institutional, NHBLI sponsored trial [abstract]. Blood. 2002;100:42a)  (COBLT)	Ooi et alref	Ooi et alref13	Long et alref14
number of patients		68	22	108	19	34	18	13	57 (19 of the 57 patients were included in the study published by Laughlin et al.)
median age (range, years)		31.4 (17.6–58.1)	29 (18–46)	26 (15–53)	48 (20–59)	34.5 (18.2–55)	43 (21–52)	40 (20–51)	31 (18–58)
median weight (range, kg)		69.2 (40.9–115.5)	70 (41–85)	60 (35–110)	69 (52–126)	NA	55.2 (NA)	51 (43–68)	70 (46–110)
diseases (n)	hematological malignancies	54	21	96	18	32	18	13	50
	bone marrow failure syndrome/MDS	13	1	12	1	2			6
number of HLA loci disparity (n)	0	2	1	6	4	1	0	0	2
	1	18	13	38	8	10	4	4	8
	>= 2	48	8	64	7	23	14	9	47
preparative regimen (n)		TBI-based/ATG  (n = 51)  Bu-based/ATG  (n = 14)  Others/ATG (n = 4)	Thio/Bu/Cy/ATG  (n = 21)  Thio/Flu/ATG  (n = 1)	NA	Cy/TBI/ATG  (n = 13)  Mel/TBI/ATG  (n = 4)  Bu/Cy (n = 2)	Cy/TBI  (n = 27)  Bu/Mel  (n = 7)	TBI/Ara-C/Cy  (n = 17)  TBI/Ara-C/Flu  (n = 1)	Cy/TBI/Ara-C  (n = 13)	Cy or Mel with  TBI or BU/  ATG n = 41  thoraco-abdominal radiation (TAI)/Cy/ATG (n = 1)
GVHD prophylaxis		CSA/Pred (n = NA)  CSA (n = NA)	CSA/Pred (n = 22)	CSA/Pred (n = 77)  Others (n = 31)	CSA/Pred (n = 13)  FK506/Pred (n = 6)	NA  CSA/MTX (n = 16)	CSA (n = 2)	CSA/MTX (n = 13)	CSA (n = 1)  CSA/Pred (n = 56)
median time to engraftment (range, days)	ANC > 500/µL	27 (13–59)	22 (13–52)	32 (13–60)	28 (NA)	28.5 (13–55)	23 (16–41)	22.5 (19–35)	26 (12–55)
	Platelet > 20,000/µL	58 (35–142)	69 (49–153)	129 (26–176)	56 (NA)	NA	49 (31–263) (platelet > 50,000/µL)	49 (30–164) (platelet > 50,000/µL)	84 (35–167)
probability of myeloid engraftment		90% by 42 days	100% at 60 days	81% by day 60	75% by day 60	72% at day 60	94%	92%	80% at day 50
GVHD (n) (probability, %)	acute grade II–IV	33 (60%)	16 (NA)	44 (38)	NA	11 (38%)	10 (55%)	9 (9 of 12 evaluable patients had acute acute graft-versus-host disease)	17 (41%)
	acute grade III–IV	11 (20%)	7 (NA)	27 (NA)	NA	6 (21%)	1 (6%)	-	9 (22%)
	chronic/patients at risk	12/33	9/10	15/58	NA	NA	14/18	8/11	8/25
median cell dose (range)	N.C. infused (x 107/kg)	1.6 (0.6–4)	1.71 (1.01–4.96)	1.71(0.2–6)	1.8 (0.4–5.3)	1.73 (1.11–3.75	2.51 (1.16–5.29)	2.43 (2.09–4.06)	1.5 (0.54–2.78)
	CD34+ cells infused (x 105/kg)	1.2 (0.2–16.7)	0.79 (0.27–2.60)	NA	NA	NA	NA	NA	1.37 (0.02–12.45)
therapy-related mortality (%)		50% at 100 day	43% at 100 day	54% at 100 day	NA	NA	6%	0%	56%
survival (%)		NA	NA	27% at 1 year	20% for good risk  and 21% for poor risk  at 1 year	30% at 180 days	76% at 2 years	76.2% at 2 years	19% at 3 years
event-free survival (%)		26% at 40 months (EFS is better in patients receiving graft with CD34+ cells >1.2 x 105/kg)	53% at 1year (patients under the age of 30 had significantly better survival)	21% at 1 year (100 day TRM is lower in patients with disease in chronic phase or remission, N.C.dose infused  2.0 x 107/kg and transplant performed after January 1998)	NA (10 patients received ex vivo expanded UCBT)	NA (the high mortality and relapse reflects the poor risk patients enrolled)	77% at 2 years (all de novo AML)	NA (all except 1 patients received UCB transplant as an upfront treatment and all patients received > 2 x 107 N.C. per weight, perhaps due to smaller size of patients (median weight 51 kg))	15% at 3 years (CD34+ cell dose infused correlated with rate of platelet recovery and age > 31 years was significant predictor of poorer event-free survival)

The median age varied considerably from a low of 29 to a high of 48 years with a range of 15 to 58 years. Age is an important factor in the outcome, with the younger patients generally doing better than the older patients. The median weight for these studies also varied from a low of 51 to 70 kg. As the cell dose of the UCB is given per recipient weight, the patients with lower body weights would have a higher chance of receiving a higher cell dose per cord blood unit. The range of mononuclear cells/kg recipient weight ranged from 1.5 to 2.43 x 10⁷/kg. The total numbers of CD34⁺ cells has been correlated with both engraftment and the speed of engraftment. The diseases for which these patients were transplanted consisted primarily of hematological malignancies. The differences in outcome can be partially attributed to the proportion with more favorable disease status at transplantation. For example, most of the data from Sanz et al^ref or Ooi et al^ref included recipients with chronic myeloid leukemia (CML) in first chronic phase or acute leukemia in first remission or de novoacute myeloid leukemia or untreated myelodysplastic syndrome. In contrast, the less favorable survival in the other studies reflects the poor-risk subjects enrolled into the study. The probability of myeloid engraftment ranged from 72% to 100% by day +60; however, some patients were censored to this event if they died prior to the set date for the definition of engraftment. The median times to engraftment of both neutrophils and platelets varied as well with approximately 1 month to reach 500 neutrophils/mL and 2–4 months to reach platelet counts > 20,000/µL. Again, the pace of recovery is likely to reflect the UCB stem cell dose as well as the amount of prior therapies a patient could have received. As compared to the pediatric patients, the incidence of severe grade III-IV acute GVHD was low (approximately 20%) given that the majority of the patients received HLA mismatched unrelated grafts. Of note, the HLA matching in general was done serologically for HLA-A and-B and molecularly for HLA-DR. Thus it is likely that the mismatching would have been greater than reported if molecular matching had been performed. Therapy-related mortality (TRM) was high, reaching approximately 50% in most series. The overall survival again varied considerably, reflecting the risk factors outlined above. Comparative studies of UCB transplantation versus unrelated bone marrow have been performed from two registry datasets. These two studies have demonstrated that if there is a cord blood unit that is at least a 4/6 HLA match with sufficient cells, the outcomes are similar following UCB transplantation to matched or one antigen mismatched unrelated donor bone marrow grafts^ref (Laughlin M, Rubinstein P, Stevens C, et al. Comparison of unrelated cord blood and unrelated bone marrow transplants for leukemia in adults: a collaborative study of the International Bone Marrow Transplant Registry and the New York Blood Center. Blood. 2003;103:857). Thus, UCB contains sufficient numbers of hematopoietic stem cells to achieve engraftment in adult patients with lower than anticipated risk of severe acute GVHD, even when HLA-disparate grafts are infused. The use of UCB allows allografting to be offered to a greater proportion of patients, many of whom do not have a matched sibling or unrelated donor to allow allogeneic therapy as the only chance to cure the underlying disease. The results thus far suggest that UCBT can result in long-term disease-free survival in many of these patients. Similar to the pediatric series, clinical experience in the adult patients has also documented the importance of graft cell dose in determining engraftment and survival. The critical "threshold dose," below which engraftment and survival become significantly inferior, remains to be defined in a larger study with a longer follow-up. Based on current results, it appears that a UCB graft that contains at least 2 x 10⁷ nucleated cells/kg and 1.7 x 10⁵ CD34⁺ cells/kg is acceptable for adult recipients^{ref1, ref2, ref3}. It is hoped that the advantage of a lower GVHD without any apparent increase in relapse will offset the adverse impact of reduced cell dose on survival. As in the pediatric setting, TRM remains the main obstacle for success in adult recipients, although several investigators have demonstrated that the TRM is not different from that seen with matched unrelated donors. With the profound influence of UCB cell dose (both nucleated cell dose and CD34⁺ cell dose) on engraftment, survival and probably TRM in the adult setting, future research should also focus on increasing the cell dose of the UCB graft. Possible methods to increase cell dose include ex vivo expansion, infusion of multiple cord blood units or use of possible additional cells such as mesenchymal stem cells or improving HLA matching.
• nonmyeloablative regimens : the nonmyeloablative stem cell transplantation (NST) regimen was proposed initially based on the rationale that the therapeutic benefit of an allogeneic transplantation is partially related to the crucial immune-mediated graft-versus-malignancy effect. The concept of graft-versus-malignancy as the pivotal therapeutic component of allogeneic transplantation is supported by the observation from clinical studies that :
• patients with acute and chronic GVHD have a reduced risk of relapse
• patients with syngeneic BMT and after T cell–depleted allotransplant have a higher incidence of relapse compared with other allogeneic donors
• patients with a relapsed malignancy after allogeneic transplantation can be re-induced into complete remission without any chemotherapy by donor lymphocyte infusion (DLI).
NST, with its reduced-intensity preparative regimen, makes allogeneic transplantation applicable to patients with relative contraindications to myeloablative regimens given its lower rate of TRM with fewer infections and less GVHD. However, the preparative regimen does not contribute significantly to the antimalignancy effect; thus, the risk of disease progression after transplantation remains higher compared with the myeloablative approach. NST using UCB provides an opportunity for immunotherapy for older patients, sicker patients and also patients without suitable donors who are not eligible for this potentially curative approach. However, there is increased concern about graft rejection using this approach given that there are, on average, 2 logs fewer cells infused than would be considered for a standard matched sibling or unrelated donor transplant. The clinical outcome of 2 patients with malignant lymphoma using this novel approach was first reported by investigators at Duke University^{ref1, ref2}. In that study, 2 patients with relapsed lymphoma who had no matched siblings, partially matched family members, or matched unrelated donors successfully underwent nonmyeloablative conditioning therapy followed by infusion of 4 and 6 matched, unrelated donor UCB cells, respectively, at the nucleated cell dose of 2.9 and 6.5 x 10⁷/kg, respectively. The conditioning regimens consisted of fludarabine 30mg/m² and cyclophosphamide 500 mg/m² daily for 4 days with antithymocyte globulin 30 mg/kg per day for 3 days. Cyclosporine and prednisolone were given for acute GVHD prophylaxis. Both patients had 100% donor engraftment by the third month of transplant and remained in remission 6 and 12 months following transplantation. Extension of these data to 11 other patients confirms the low morbidity and the potential for robust engraftment using this approach. The favorable outcome demonstrates the feasibility of the mismatched unrelated UCB cells, even with the NST regimens. Summary of results of nonmyeloablative unrelated umbilical cord blood transplantation in adult patients :

investigator	Barker et alref	McSweeney PA, Bearman SI, Jones RB, et al. Nonmyeloablative hematopoietic cell transplant using cord blood [abstract]. Blood 2001;98:666a	Chao et alref
number of patients	43	5	13
age, years: median (range)	49.5 (22–65)	64.5 (25–78)	49 (19–62)
diagnosis	HM	HD; AML; CLL; NHL	NHL; MDS; AML; melanoma; ALL
preparative regimen	F + Bu + TB I (200cGy (n = 21)  F + C + TBI (200cGy) (n = 22)	F + TBI (200cGy)	F + C + ATG (n = 10)  F + C + ATG + TBI (200cGy) (n = 3)
GvHD prophylaxis	CsA + MMF	CsA + MMF	CsA + Pred (n = 8)  CsA + MMF (n = 5)
cell dose
nucleated cell (x 107/kg): median (range)	2.6 (1.6–3.8)  3.2 (1.1–5.1)	1.1 (1.75–1.3)	2.07 (1.07–5.53)
CD34 (x 106/kg): median (range)	3.7 (1.1–8.1)  4.3 (1.1–10.3)	NA (0.01–0.04)	1.3 (0.5–9.6)
CD3 (x 106/kg): median (range)	0.06 (0.02–0.15)  0.05 (0.02–0.12)	NA (1.4–3.3)	4.6 (2.02–22.82)
engraftment (n)	C.I. = 76%  C.I. = 94%	2	12
median days to ANC  > 500/µL (range)	26 (12–30)  9.5 (5–28)	NA	12 (6–24)
median days to platelet  > 20,000/µL (range)		NA	14 (6–61)
grade II–IV aGVHD / cGVHD (n)	C.I. for aGVHD = 44%  C.I. for cGVHD = 21%	1/NA	1/1
outcome	O.S. : 39% at 1 year  DFS.: 31% at 1 year	Alive: n = 3  PR: n = 1	O.S.: 43% at 1 year  DFS: 43% at 1 year

The largest experience with transplantation using a reduced-intensity regimen was evaluated by investigators from the University of Minnesota on a cohort of high-risk patients with hematological malignancies^ref. In their study, unrelated UCB graft with a median nucleated cell dose of 3.7 x 10⁷ per kg (range, 1.6–6.0 x 10⁷/kg) was infused into 43 patients (median age 49.5 years; range, 22–65 years), after receiving 2 types of conditioning regimens: fludarabine 200 mg/m², total body irradiation (TBI) 200 cGy and busulfan 8 mg/kg (Flu/Bu/TBI) for the initial 21 subjects; fludarabine 200 mg/m², TBI 200 cGy and cyclophosphamide 50 mg/kg (Flu/Cy/TBI) for the subsequent 22 subjects. All patients received GVHD prophylaxis with cyclosporine and mycophenolate mofetil. The median time to neutrophil recovery of more than 0.5 x 109/L was 26 days (range, 12–30 days) for the Flu/Bu/TBI recipients, but only 9.5 days (range, 5–28 days) for the Flu/Cy/TBI recipients. The cumulative incidence of engraftment for Flu/Bu/TBI and Flu/Cy/TBI recipients was 76% and 94%, respectively. Despite the use of 1–2 HLA-antigen mismatched graft in 93% of the recipients, the cumulative incidence of grade II–IV GVHD and grade III–IV GVHD for the entire cohort of patients were 44% and 9%, respectively. The disease-free survival of these high-risk subjects was also favorable: 24% at 1 year for Flu/Bu/TBI recipients and 41% at 1 year for Flu/Cy/TBI recipients. A similar approach and data have also been reported by investigators in University of Colorado Health Sciences Center (McSweeney PA, Bearman SI, Jones RB, et al. Nonmyeloablative hematopoietic cell transplant using cord blood [abstract]. Blood 2001;98:666a). Immune recovery in 5 recipients of UCB transplant following the NST regimen were compared to recovery in adult recipients of UCB following a myeloablative regimen by investigators at Duke University^ref. Recipients of NST regimens had a more rapid and robust recovery of myeloid and lymphoid cells. The T cell repertoire in UCB recipients treated with the NST regimen was markedly more diverse and robust compared with the repertoire in those receiving the myeloablative regimen at similar time points. T cell receptor excision circles (TRECs), which are generated within the thymus and identify new thymic emigrants and those that have not divided, were detected 12 months after transplantation in the NST recipients. This compared favorably to the delayed detection of TRECs at 18–24 months among recipients of myeloablative regimens. Thus, in adults receiving an NST preparatory regimen, the quantitative and qualitative recovery of T cells occurs through rapid peripheral expansion. The ability of patients receiving NST transplantation to recover within a few months suggests that the peripheral "niches" in which T cells can proliferate are preserved in these patients compared to those receiving myeloablative regimens. Moreover, the presence of TREC-positive cells within 1 year suggests that thymic recovery is likewise accelerated in NST compared with recipients of myeloablative regimens. The favorable results of T cell recovery following the NST suggest that it may be possible to have an excellent outcome with an unrelated mismatched UCB transplantation in adult patients. Patients have a rapid recovery of T cells with a complex diversity. The primary difference between the recipients of ablative and NST regimens was the extent of physiologic damage caused by the preparatory regimen. When the damage is relatively mild, as in nonmyeloablative regimens, the donor T cells are able to expand effectively in the periphery, and the development of new T cells through the thymus is also accelerated compared to the rate of development in those receiving ablative regimens. Alternatively, the lower incidence of GVHD in NST may also play an important role in the preservation of the peripheral and central niches for T cell development.
Future directions for UCB transplantation in adults : while the use of NST signifies an advancement in the field of transplantation and immunobiolology, several unresolved questions remain:
• what is the optimal UCB cell dose required for adult patients and can we increase it?
• what is the optimal NST conditioning regimen and GVHD prophylaxis?
• is the incidence of GVHD similar in ablative and NST regimens?
• does the incidence of infection differ in ablative versus NST regimens and can one improve immune recovery?
• what is the difference in overall efficacy between myeloablative and NST UCBT?
UCB is a viable alternative to bone marrow and peripheral blood as a source of stem cells capable of hematopoietic reconstitution for adults, when related or unrelated marrow donor is not available. UCBT following NST preparative regimen is an exciting new approach that provides an option for patients who are otherwise excluded from conventional hematopoietic stem cell transplantation, including elderly or medically infirm patients with no matched sibling donor. Preliminary results have shown that such an approach can be associated with timely engraftment with full donor chimerism. Comparison between myeloablative and NST approaches will be needed before this therapy can be considered for younger patients eligible for myeloablative transplant. At the moment, the use of NST UCBT cannot be encouraged outside of a clinical trials or selected patients. The future challenge will be to develop strategies to optimize the chance of early and durable engraftment, as well as to minimize the risk of GVHD and transplant-related death.
Expansion of UCBC progenitors and stem cells : the close correlation between preclinical assays and in vivo clinical biology that is observed with bone marrow stem cells and with hematopoietic growth factors such as Epo, G-CSF and GM-CSF has been very reassuring and has accelerated the development and improvement of clinical hematology practice. Clinical experience with UCB transplants, in contrast, has proved much more paradoxical and problematic. Early laboratory measurements showed that the density of both CD34⁺ cells and clonogenic progenitor cells was extremely high in UCB^ref. Similarly, the proliferative capacity of progenitor cell differentiation in laboratory assays and of UCB stem cells transplanted into immunodeficient mice was measurably higher than for progenitors and stem cells from bone marrow (BM) or peripheral blood (PB)^{ref1, ref2}. But when comparable numbers of UCB progenitors (1–5 x 10⁶/kg) and stem cells are transplanted as are delivered to patients with standard BM or PB grafts, engraftment is far slower, and graft failure is more likely^ref. Given that opportunistic infection and organ failure rise exponentially with time to engraftment, and that overall long-term survival correlates closely with platelet engraftment, this delay has proved to be a major barrier to adoption of what would otherwise be an overwhelmingly attractive stem cell source. One of the striking lessons of BM and peripheral stem cell (PSC) transplant is that time to engraftment correlates inversely with progenitor and stem cell dose, with most graphed comparisons showing hyperbolic curves with asymptotic limits at 7–8 days for neutrophil engraftment and 10–12 days for platelet engraftments, both achieved with PSC stem and progenitor cell doses of > 5 x 10⁶ CD34⁺ cells per kg. Providing lower doses of early hematopoietic cells results in increasingly longer nadirs, but providing higher doses does not further accelerate hematopoietic recovery. Based on these well-documented observations, the question has been raised whether one could significantly shorten UCB transplant nadirs by providing increased numbers of stem and progenitor cells to patients by expanding the numbers of hematopoietic stem cells (HSCs) and progenitor cells, in the laboratory, prior to transplant. This approach should succeed if the delay in engraftment is due to a paucity of HSC and progenitor numbers, but would fail if the delay were due to an uncharacterized-but-insurmountable qualitative defect in UCB stem and progenitor cells. Attempts to characterize UCB expansion protocols have relied directly on the assays used to measure the cell populations that are targets for expansion. Over the past 20 years, these assays have become more sophisticated, and clinical investigators have progressively adjusted their clinical trial designs based on more recent, better assays. Pluripotent hematopoietic stem cells are very rare, representing only 1 cell in 2–10 x 10⁶ BM cells, perhaps slightly more frequent in UCB. There is no phenotypic assay, to date, that directly identifies and measures these rare cells. Rather, all efforts to measure HSCs rely on functional assays. The easiest and most popular assays have been colony-forming cell (CFC) assays, which measure cells that are partially differentiated between HSCs and mature blood cells. These cells, which correlate closely but inexactly with CD34⁺ cell numbers, produce easily detectable blood cell colonies in 2 weeks in the laboratory. But careful cell fractionation studies have shown that the intermediate-maturity cells detected in these assays contribute only very slightly, and temporarily, to hematopoietic recovery after transplantation, since they don’t produce enough cells that survive long enough after transplantation; it is only the immature pluripotent stem cell population that contributes to sustained engraftment^{ref1, ref2}. So, although CD34⁺ cell numbers in an unmanipulated graft may correlate with clinical time to engraftment after transplantation, that is likely because the ratio of intermediate progenitor cells to very primitive stem cells is fairly constant between clinical samples, and therefore CD34⁺ cell numbers indirectly reflect primitive stem cell numbers transplanted. This assumption, however, may not be true following many ex vivo expansion procedures, in which progenitors may be greatly expanded but primitive stem cells may not^ref. Better functional assays for primitive stem cells, developed in the late 1980s and 1990s, include the long-term culture initiating cell (LTCIC), cobblestone area forming cells (CAFC), and best of all the human to immunodeficient NOD/SCID mouse (NOD/SRC) repopulating cell assay^ref. In evaluating UCB expansion protocols, it is important to determine what populations were measured and specifically whether LTCIC, CAFC or NOD/SRC were identified as being expanded.
• UCB expansion ex vivo: cytokine cocktails : when UCB progenitors were first detected, investigators noted that the progenitor cells formed colonies in agar more rapidly than did BM or PSC progenitors, and that a higher fraction of UCB colonies could be replated to form secondary colonies. These data on UCB progenitors, together with their much higher frequency in the UCB mononuclear cell fraction, suggested that they were more proliferative than their adult counterparts. In the early 1990s Moore and Hoskins applied the techniques of PB CD34⁺ cell isolation followed by culture in high concentrations of multiple hematopoietic growth factors first pioneered by Haylock et al to UCB, with prima facie very promising results. After removal of more mature CD34^– cells, clonogenic progenitors could be expanded 50- to 100-fold, while slightly more primitive  cells could be multiplied 10–20X, all in 2 weeks^ref. Subsequently, many investigators confirmed that UCB progenitor populations could be readily expanded, and some showed that LTCIC could also be expanded 10X, particularly if combinations of low-dose cytokines were combined with perfusion conditions that simultaneously removed the secreted products of maturing blood cells and supplied continuous fluxes of cytokines^ref. Of note, however, no assays for primitive NOD/SRC repopulating cells were measured in these studies, so it is not known to what extent, if any, true HSC were expanded in these protocols. When Lewis et al assayed UCB NOD/SRC repopulating cells in parallel with CFC and LTCIC during ex vivo cytokine expansion cultures, they found that while CFC were increased 20–25X, LTCIC were only increased 40%, and NOD/SCR were maintained but not increased at all^ref. True UCB NOD/SRC stem cell expansion may be achievable by very judicious combinations of HSC purification and cytokine selection. SDF-1 treatment increases the engraftment of UCB HSCs, but whether this effect reflects quantitative increases in HSCs or a qualitative improvement in their behavior is not certain^ref. This may be the basis of the reported enhancement of UCB engraftment achieved by supplementing UCB grafts with mesenchymal stem cells. UCB HSCs are selectively responsive to IL-6 stimulation^ref, and recent studies suggest that combining UCB CD34⁺ cell selection with IL-6/soluble IL-6R/SCF/Flt-3l/Tpo stimulation induces NOD/SRC expansion 4–5X. Overall, it appears that UCB stem cell expansion may be possible with ex vivo cytokine cocktails, but conditions that can generate such HSC expansions, as opposed to the expansion of more mature progenitor populations, may have not yet been used in clinical trial settings.
• stem cell expansion: Hox gene products : individual cytokines are known to trigger many intracellular signal transduction pathways, which presumably activate multiple gene transcription pathways. Thus, in retrospect, combinations of cytokines might well be expected to have multiple effects on HSCs that might not permit UCB stem cell expansion. An alternative, more direct, approach to expanding stem cells might focus on the specific genetic pathways known or believed to support primitive HSC proliferation. Over the past decade several such pathways and transcription factors have been proposed, including Notch-1 receptor activation, Wnt/LEF-1 pathway induction and telomerase. One of the most promising intracellular targets for HSC expansion are Homeobox (Hox) gene products. These transcription factors, which were first identified as master switch transcriptional regulators of early development, were discovered a decade ago to be expressed in the most primitive HSCs. When cloned and overexpressed in mouse HSCs, several of these Hox genes induce HSC proliferation, in addition to more or less disruption in differentiation. One of these Hox genes, HoxB4, has proven to be the most interesting to date. HoxB4 is highly expressed in primitive HSCs, and its expression declines with differentiation. Overexpression of HoxB4 mRNA by retroviral infection results in HSCs that expand over 100X if transplanted in very low numbers (analogous to the clinical HSC scenario)^ref. Of even more interest, recent experiments suggest that the same effect can be achieved by treating purified HSCs with a modified form of soluble HoxB4 protein (TAT-HoxB4), which increases intracellular HoxB4 protein levels for several hours only^ref. While this approach has thus far only been applied to murine HSCs, and not to human BM or UCB HSC expansion, the application to human UCB expansion could be very direct. More recently, Zhu et al have discovered that the protein NF-Y is a normal transcriptional activator for multiple Hox genes, as well as telomerase, Notch-1 and LEF-1^ref. When overexpressed in murine HSCs by as little as twofold, NF-Ya increases HSC numbers by 20X following transplantation. Application of TAT-NF-Y protein transduction to UCB HSC expansion thus offers the attractive possibility of activating multiple HSC expansion pathways simultaneously. The absolute and relative efficiencies of these protein treatments for expanding human UCB HSCs will need to be evaluated directly in NOD/SCID mouse xenotransplants, and then in appropriate Phase I clinical trials. Similar approaches using other soluble, reversible, biochemical stimulators of stem cell transcription programs, including perhaps copper chelation, have also been proposed and are under preclinical and clinical trial.
• clinical experience with ex vivo expanded HSCs : clinical trials of ex vivo culture UCB cells have begun to be reported, but their interpretation should be tempered by the knowledge that the conditions of culture used to date have not been shown to support USC amplification, only CFU expansion. At least three trials evaluating the combination of unmanipulated with ex vivo expanded UCB grafts have been published to date; in all three studies expanded cells were added on day 10–12 to supplement unmanipulated cells. One study involved the use of a static culture system in defined media while the other two used a perfusion culture expansion system. Although no toxicities were seen in these studies, no significant acceleration of neutrophil or platelet engraftment was seen^{ref1, ref2, ref3}. Neutrophil engraftment to ANC > 500 was seen on day 24–33, and platelet engraftment to > 50,000 at approximately day 90. These results, while disappointing, highlight the importance of building clinical trial design based upon the most precise and appropriate preclinical data. The cytokine cocktails employed in the three published studies included some but not all of the cytokines required to achieve UCB HSC expansion in vitro, as measured by our best NOD/SRC assays: One trial used SCF and a Tpo analogue, but not Flt3-l, and the other 2 used Flt3-l, but not Tpo or SCF, and neither included IL-6 or soluble IL-6R. In addition, a lesser but possibly contributing variable was that in all three trials the ex vivo expanded cells were added 10–12 days later, thus mitigating any accelerating effect they might have otherwise have achieved. It must be pointed out that these trial design limitations were known to the clinical investigators, but the lack of availability of clinical grade cytokines, and possibly also HSC selection devices, prevented the optimal cocktails and culture conditions from being employed. These difficulties would be surmounted if simpler expansion conditions, requiring few added reagents and/or devices while still supporting HSC expansion, could be devised and employed.What if UCB HSC Numbers Are Not Rate Limiting? Exploring the Null Hypothesis Another possibility that must be considered is that increasing UCB numbers will not, by itself, accelerate engraftment. Perhaps there is a qualitative "immaturity" defect that prevents rapid engraftment. If so, then is there a way to circumvent this defect: providing a maturation effect through some combination of in vivo cytokine treatment, or perhaps ex vivo cell activation targeted at maturation? Viewed through this perspective, one can envision manipulations of the non-HSC components of the UCB graft, including the mature hematopoietic cells, the immune cells, antigen-presenting cells, and mesenchymal stromal cell components of the graft. Interactions between HSC and non-HSCs could form the basis for potentially improved engraftment in mixed graft settings as well^ref. Each of these manipulations could be evaluated by comparing engraftment in NOD/SCID mice, prior to clinical transplantation trials. In summary, experience with hematopoietic cell expansion to date supports the fundamental principle of experimental hematology, that the best clinical results follow from the most careful, comprehensive and creative preclinical trials in the proper in vitro and in vivo models. For the future, this experience suggests concentrating on ex vivo manipulations that can be shown to directly increase HSC numbers following NOD/SCID engraftment and/or accelerate the pace of engraftment in these same models.
Immune reconstitution after UCBSCT : one of the events necessary for the success of HSCT, regardless of the source of stem cells, is the development of a functional immune system from donor-derived cells. The production of adequate granulocytes, platelets, and red blood cells usually occurs rapidly after HSCT. In contrast, the ability to produce lymphocytes, especially T lymphocytes, is delayed. As a result, serious infection in the first 1–2 years after HSCT occurs in about 50% of uncomplicated transplants from histocompatible sibling donors, and up to 80–90% of recipients of matched unrelated donor (MUD) marrow transplants or histocompatible recipients who developed GVHD^ref. Infections attributable to poor lymphocyte function include viral pathogens such as the herpes group (CMV, HSV, VZV, HHV-6 and HHV-8), respiratory viruses (RSV, parainfluenza viruses), adenovirus, and enteroviruses (Coxsackie and ECHO viruses), as well as EBV-associated lymphoproliferative disorders. Other infections that may result from inadequate cellular immunity include fungal infections such as Pneumocystis carinii, Candida, and Cryptococcus. Increased susceptibility to Aspergillus infection may reflect defects in innate immunity. Susceptibility to some bacterial infections, notably those caused by encapsulated organisms (Staphylococcus pneumoniae, Haemophilus influenzae), is determined by the inability to produce antipolysaccharide antibodies. The emphasis of this review will be the relationship between stem cell source and the development of T lymphocyte functions required for protection of the HSCT recipient from infection. In particular, the unique aspects of UCBT, compared to either BMT or mobilized peripheral blood stem cell transplantation (PBSCT), will be described. Factors influencing the development of adequate immune function after HSCT include those related to the donor progenitor cell populations, pre-formed lymphocytes in the donor product, the host microenvironment, pharmacological interventions, and antigenic exposure. All clinically relevant stem cell sources include both progenitor cells and preformed lymphocytes. Prethymic progenitors may include HSC, common lymphoid progenitors (CLP), committed T progenitors (CTP), whose T lymphoid progeny depend on the host thymus for their development^ref. 2 pathways for generation of T lymphocytes after hematopoietic stem cell transplantation (HSCT) :

Shown at top is the adoptive transfer of post-thymic T cells which then undergo expansion in the host, resulting in a limited repertoire of mature T lymphocytes that can rapidly confer antigen-specific responses, including graft-versus-host disease (GVHD). Below is shown the pathway for differentiation of pre-thymic progenitors by migration into the recipient thymus, resulting in the slow generation of a diverse repertoire that is host-tolerant.
• development of thymic progenitors : upon entry to the thymus, progenitor cells undergo expansion; differentiation events including V(D)J recombination to generate functionally rearranged TCR genes; and both positive and negative selection events that cull potential T lymphocytes that would either be unresponsive to antigenic peptides presented by self-major histocompatibility complex (MHC) antigens or overly responsive to either MHC or self antigens. The ultimate fate of negatively selected cells is apoptosis. Since most thymocytes are fated to die, maintenance of thymic output depends on continual expansion and differentiation of the immature prothymocytes, which lack expression of the TCR invariant CD3 complex, and the co-receptors CD4 and CD8 ("triple negative," TN). The major signals for expansion of immature prothymocytes are 2 cytokines, IL-7 and c-kit ligand (KL), which are produced by thymic epithelial cells (TEC). Murine studies have demonstrated that pre-HSCT radiotherapy and chemotherapy kill TEC, resulting in defective intrathymic IL-7 production. Early studies demonstrated that the thymic microenvironment is a target of GVHD, and more recent studies have shown that TEC organization and function is abnormal in experimental GVHD models. In non-HSCT studies, aging also results in TEC damage and decreased intrathymic IL-7 production. Thus, thymic function may be adversely affected by several phenomena commonly associated with clinical HSCT-cytotoxic therapies given pre-HSCT, GVHD, and recipient aging. Clinical studies have demonstrated that decreased capacity for production of new T lymphocytes is associated with GVHD and increasing recipient age^{ref1, ref2}. The effects of pre-transplant conditioning on thymopoiesis has been more difficult to assess because of the complexity of the problem of dose intensity as well as the effects of previous therapy for malignancies. Any assessment of the effect of stem cell source, e.g., UCB, on immune reconstitution must take into account the function of the thymic microenvironment. The biology of prethymic progenitors is also highly relevant to the outcome of HSCT. The lack of pre-existing TCR rearrangements means that donor-derived prethymic progenitors are unlikely to cause GVHD, as long as the recipient thymus maintains the ability to select out undesirable T cells. However, the development of an immune repertoire from prethymic progenitors may be delayed by ontogenic factors such as commitment to the T lymphoid lineage and the time normally required for development of large numbers of diverse T cells. Between 7 to 9 weeks of gestation, hematopoietically derived cells enter the thymic anlage, which forms from mesenchyme and endoderm of the III-IV pharyngeal pouches. Alloreactive T cells are present by week 14–15 and antigen-specific T lymphocytes appear later in the second trimester. Recapitulation of fetal ontogeny provides a framework for understanding the development of lymphoid cells after HSCT. The generation of T lymphocytes post-HSCT requires events similar to those observed in normal immune development, e.g., lymphoid commitment, thymic migration and entry, and thymic differentiation. It is likely that the time period required for such development is similar to the time normally required for prenatal T cell development. For example, in patients with severe combined immune deficiency (SCID) receiving CD34⁺ haploidentical HSCT, the appearance of functional mature T lymphocytes in the peripheral blood is 90–120 days after transplant, a time period similar to that required for normal prenatal immune ontogeny.
• post-thymic T lymphocytes : in contrast to prethymic progenitors, post-thymic T cells have already undergone maturation and have antigenic and functional specificities that were conferred by the donor’s thymus. Donor-derived post-thymic T cells express a fixed TCR and thereby are programmed to respond to a set of antigenic determinants. Although adoptive transfer of mature T lymphocytes had long been thought to be a relatively static process, recent studies have indicated that mature T lymphocyte populations are dynamic and regulated by interactions with other T lymphocytes as well as the host microenvironment. Mature T lymphocytes may undergo activation if they encounter a stimulating antigen and the proper set of co-stimulatory signals. Activation of T lymphocytes leads to acquisition of functional properties such as cytolytic capacity or cytokine secretion. Activated T cells may proliferate and mature into memory cells, undergo apoptosis (activation-induced cell death [AICD]), or be anergized. Expansion of activated T lymphocytes is dependent on production of IL-2 by helper T lymphocytes and expression of high affinity IL-2R by the responsive helper and cytotoxic cells. Recent studies have demonstrated that naive T lymphocytes in a lymphopenic environment can undergo proliferation while maintaining a mainly naive phenotype^ref. Such homeostatic proliferation differs from activation-induced proliferation in which naive T cells recognize foreign antigens and proliferate in response to IL-2. In homeostatic proliferation, the stimuli recognized by the TCR are the same self antigens that normally induce positive selection in the thymus. Like thymopoiesis, homeostatic proliferation also requires IL-7, although the source is probably extrathymic, not TEC. Homeostatic proliferation has also been described for memory T lymphocytes with evidence that IL-15 is critical for the expansion of these cells. The appearance of T lymphocytes after HSCT is conventionally seen as evidence of de novo generation of T lymphocytes from progenitor cells. However, as previously noted, transplantation of stem cell products that have been extensively depleted of mature T lymphocytes in order to prevent GVHD results in significant delays in immune reconstitution. Therefore, it is likely that much of what has been attributed to T cell generation in the first few months after transplant is the result of adoptive transfer of post-thymic cells. Such cells may undergo homeostatic proliferation in the lymphopenic environment of the host, or IL-2 driven proliferation if activated by exposure to alloantigens or nominal antigens. Analyses of the length of time required for de novo generation of T lymphocytes after HSCT are best performed in patients receiving highly purified progenitors are depleted of all post-thymic T lymphocytes.
• UCBC HSC : HSC derived from UCBC differ from those of adult marrow or PBSCT in several ways. The most notable difference is quantitative—the cell dose in UCBT is significantly less than the HSC dose in adult sources such as PBSC or marrow. While low cell dose has been associated with failure of hematopoietic engraftment, HSC dose may also influence immune reconstitution. In a murine congenic transplant model, increasing the dose of phenotypic HSC administered resulted in greater thymic cellularity post-transplant, as well as greater numbers of mature T lymphocytes derived from the transplanted cells^ref. Thus, one of the limiting factors for T lymphocyte reconstitution after HSCT, besides microenvironmental defects, may be the number of progenitor cells entering the thymus and contributing to thymopoiesis. Limited numbers of HSC in UCBC compared to marrow or PBSC products may delay or decrease thymic regeneration. There are also qualitative differences between UCBC and other HSC sources. Comparisons of CD34⁺ CD38^– phenotypic HSC derived from UCBC versus adult marrow have shown that UCBC contain a higher frequency of LTCICs and a higher percentage of cycling cells^ref. UCBC-derived HSC also had greater cloning efficiency and generative capacity. Thus, the HSC in UCBC may be both more primitive and more capable of regenerating hematopoiesis in the recipient. The contrasting biological properties may represent intrinsic or environmental differences in the UCBC and adult marrow HSC. The most likely intrinsic difference may be the age of the HSC—a few months for UCBC versus decades for adult HSC. There have been some studies of the aging process in hematopoietic progenitors, but there is little direct evidence that HSC from adult donors have decreased capacity to contribute to lymphocyte development compared to other progenitors. Properties of umbilical cord blood (UCB) cell populations relevant to immune responsiveness :

hematopoietic stem cells	dendritic cells	T lymphocytes
decreased number increased cell cycling increased generative capacity more immature?	decreased immune activity decreased co-stimulation decreased IL-12 production	decreased number naive phenotype resistance to activation decreased cytokine expression skewing to Th2 responses (decreased IL-2, INF-g production)

The placenta as unique microenvironment that influences UCBT : besides intrinsic differences, HSC in UCBC have had a different set of microenvironmental exposures compared to those of adult marrow or PBSC. All HSC sources are influenced by the microenvironment from which they are derived. An example of differences between sources are some of the observed changes in HSC cell cycle status, gene expression and adhesive and invasive properties induced by mobilization procedures used to generate PBSC, e.g., G-CSF. The placenta is a complex organ that regulates maternal-fetal interactions. Many cytokines that can influence lymphohematopoietic development, e.g., G-CSF, c-kit ligand (SCF), GM-CSF, IL-15, and others, are produced by the placenta. Production of G-CSF by the placenta may be especially relevant to UCBT. G-CSF is produced both by the maternal decidua and the fetal chorionic villi8 and enters the fetal circulation by a process that does not require a functional G-CSF receptor. G-CSF from the mother probably does not enter the fetal circulation as administration of recombinant human G-CSF (rhG-CSF) to pregnant macaques did not result in detectable rhG-CSF in the fetuses. The function of placental G-CSF production is unknown; however, it may serve as an immunoregulator that protects the mother and fetus from each other’s allogeneic immune systems. G-CSF inhibits the ability of placental mononuclear cells to mediate cytotoxicity against allogeneic targets including choriocarcinoma cells. The production of G-CSF and other cytokines capable of HSC mobilization, e.g., SCF, is likely to explain the high percentage of HSC in UCBC and their rapid disappearance from the neonate’s circulation after birth. In contrast to mobilization induced by a short period of monotherapy with G-CSF before PBSCT, the HSC in UCBC have been exposed continually to a complex mixture of cytokines that likely affect their behavior, e.g., induce a high rate of cell cycling^ref, or alter the homing and invasive properties needed for entry of progenitors into the thymus. Placental G-CSF production affects umbilical cord blood cells (UCBC). G-CSF produced in the placenta may affect both fetal HSC and dendritic cells. Placental G-CSF may induce mobilization of the fetal HSC, resulting in significant numbers of circulating HSC among UCBC, which declines rapidly after delivery. G-CSF may also suppress the immunological potency of fetal dendritic cells by interfering with IL-12 production and co-stimulatory activity. As a result, the immune responsiveness of fetal T lymphocytes is attenuated and skewed away from T_h1 responses.

UCBC and lymphoid progenitors : little is known about the numbers and functional properties of circulating lymphoid progenitors present in UCBC compared to other sources. Commitment to lymphoid differentiation by donor HSC could be a rate-limiting step in the ability to repopulate the recipient immune system after HSCT. At present, there is some controversy regarding the nature of intermediate lymphoid progenitors. The murine common lymphoid progenitor (CLP) has been described as a phenotypic population in the marrow that can contribute to T, NK, and B lymphopoiesis, but it is not clear if CLP generate all thymocytes. There is some evidence that early thymic progenitors (ETP) in the thymus can arise in a CLP-independent manner. At present, CLP are the only lymphoid progenitor that are relevant to transplantation; besides the logistical problem of transplanting the intrathymic ETP population, it is unclear if such cells could home and engraft after transplantation. Murine CLP can be transplanted into irradiated hosts and contribute to post-transplant lymphopoiesis. Co-transplantation of CLP with HSC is able to more rapidly restore immune function than transplantation of HSC alone, and can result in increased protection of mice from experimental challenge with murine CMV^ref. Although human cells with CLP-like features can be found abundantly in UCBC, rigorous comparison of the numbers and functional properties of CLP in UCBC versus marrow or PBSC still need to be performed.
• UCBC post-thymic T cells : studies over the last 20 years have demonstrated differences between neonatal T lymphocytes and those of adults. The major notable difference between the T lymphocytes in UCBC and those derived from adult marrow or mobilized PBSC is the maturational status. Because the fetus is exposed to few foreign antigens, the T lymphocytes in UCBC are almost exclusively naive. Naive T lymphocytes express a phenotype that is identifiable as CD45RA⁺ CD45R0^– and CD62L⁺. As individuals age, there is an increase in the frequency of T cells that have differentiated into a memory phenotype, CD45RA^– CD45R0⁺ and CD62L^–/low as a result of antigenic exposure. Memory cells are more readily activated by antigen than naive T cells and may be able to respond to weaker co-stimulatory signals through the B7-CD28 pathway. The predominant naive phenotype of T lymphocytes in UCBC may contribute to the reduced alloreactivity observed after UCBT. Investigations of neonatal T cell function have been driven by interest in the increased susceptibility to severe viral infections in neonates. For example, newborns infected with HSV have higher rates of dissemination (sepsis, meningoencephalitis) than adults with primary infection, and increased severity of some viral infections such as enteroviruses is observed until at least several months of age. Investigations of neonatal helper T lymphocytes have demonstrated decreased production of T_h1 cytokines needed for cytolytic and anti-viral responses, compared to adult T lymphocytes^ref. Specifically, the activation of the IFN-g gene is markedly reduced in neonatal T lymphocytes, because of decreased induction of transcription factors, e.g., NF-AT, which are required for activation of the IFN- gene after TCR stimulation. A central question is whether such differences are intrinsic to UCBC versus adult T lymphocytes. Interpretation of the differences has been confused by comparison of signaling properties of UCBC T lymphocytes to unfractionated adult T lymphocytes. Since adult T lymphocytes are comprised of both naive and memory T cells, while UCBC are almost exclusively naive T cells, ascribed differences between them may simply reflect differences between the stringent activation requirements of naive helper T lymphocytes and the more easily activated memory cells. Only comparisons between UCBC and purified naive T lymphocytes from adult blood can be used to evaluate whether UCBC T cells have unique properties different from adult peripheral blood naive T lymphocytes. Regardless of the reason, T lymphocytes from UCBC appear to be less capable of mediating T_h1 cytotoxic responses than those derived from adult sources. The decreased capacity for T_h1 responses has both positive and negative implications for HSCT. The decreased absolute numbers and decreased responsiveness of UCBC T lymphocytes contribute to the decreased alloreactivity and greater tolerance for MHC disparity observed in clinical UBCBT. However, the decreased responsiveness probably means that there is also less adoptive immunity derived from UCBC T lymphocytes. There are fewer T cells overall, and the number of memory cells capable of mediating recall responses to antigen is extremely limited. Any adoptive immunity must be derived from the naive T lymphocytes present in the UCBC product. As discussed above, naive T lymphocytes are difficult to activate, rendering them less capable of responses to pathogens. These properties of the mature T lymphocytes in UCBC would predict that antiviral and fungal immunity may be decreased after UCBT than after BMT or PBSCT.
• UCBC dendritic cells : besides intrinsic differences in T lymphocytes, there may be differences in antigen presenting cells (APC) derived from UCBC versus adult sources. Analyses of dendritic cells (DC) in UCBC have shown that both so-called myeloid and plasmacytoid DC are present, although there may be a decreased proportion of myeloid DC compared to adult peripheral blood. The myeloid dendritic cells are thought to promote T_h1 responses by producing IL-12. Studies of the DC derived from UCBC have indicated that there may be intrinsic defects in production of IL-12 as well as other functions of DC. Recent data on DC generation after HSCT suggests that G-CSF administration significantly decreases the ability to produce IL-12^ref. The decrease in IL-12 production after post-HSCT G-CSF administration may be relevant to UCBT. As discussed above, cells in the fetal circulation are probably constantly exposed to placental G-CSF. Furthermore, many clinical UCBT protocols have used post-transplant G-CSF administration to accelerate the development of granulocytes. The routine administration of G-CSF, combined with intrinsic defects in DC function, may decrease the generation of T_h1 responses after UCBT. The expected consequences of such an effect would be decreased capacity to control opportunistic viral and fungal infections.
Anticipated and empiric properties of UCBT : based on the progenitor and immunological properties of UCBC, certain predictions can be made regarding the expected immune reconstitution after UCBT. Compared to the detailed information available about the recovery of marrow function and the decreased risks of severe GVHD, there are few systematic data on lymphoid recovery, and even less on such properties as antigenic repertoire and effector function. As discussed above, immune function after HSCT can be derived from either the adoptive transfer of preformed mature T lymphocytes in the stem cell product or from the development in the host of donor-derived lymphocytes from the donor HSC or other prethymic populations, e.g., CLP^ref. The adoptively transferred T cells would be expected to represent most of the lymphocytes seen in the first few months after transplant, but are more likely to be both impermanent and narrow in repertoire. Adoptively transferred T cells after UCBT are largely naive cells which will be less responsive to specific antigen than the memory cells in adult sources and probably less able to mediate T_h1 effector functions than marrow or PBSC. The HSC and pre-thymic progenitors are expected to produce a long-lasting, broad immune repertoire but would be slow to develop. The increased generative capacity of UCBC HSC might predict more rapid engraftment kinetics, but would be expected to be offset by the decreased dose of transplanted HSC in the average UCBT. Analyses of UCBT recipients have demonstrated slow de novo generation of T lymphocytes in the first year after transplant; however, by 2 years post-UCBT recipients have evidence of both a broad repertoire and thymic function^{ref1, ref2, ref3}. It is unclear if there are differences in the kinetics of post-transplant lymphopoiesis in UCBT versus BMT. More rapid recovery in children versus adults and nonmyeloablative than myeloablative conditioning suggests that damage to the thymic microenvironment as a result of aging or cytotoxic therapies may be important determinants of post-HSCT immune reconstitution^ref. Although several studies have demonstrated the importance of UCBC CD34⁺ cell dose for likelihood of hematopoietic engraftment and engraftment kinetics, analyses of the engraftment kinetics of lymphoid populations are more limited. In a study from Case Western University, unrelated donor UCBT was compared to the matched unrelated donor marrow transplantation^ref. UCBT led to decreased rate of appearance of lymphoid populations in the first 3 months after transplantation. In the subsequent year, the absolute lymphocyte count of the UCBT recipients exceeded that of the MUD recipients. Indeed, the mean ALC in the UCBT group was normal by day 200 post-transplant but remained abnormally low in the MUD recipients during the entire first year. Since the T lymphocytes seen in the first 100 days post-transplant are likely to be derived from adoptively transferred mature T cells, the decreased lymphocyte count in the first 3 months after UCBT would be expected as a result of lower amount of such cells in UCBC products. The increased numbers of lymphocytes observed later could reflect greater capacity of the UCBC progenitors to produce lymphocytes de novo. However, this is uncertain: an alternative explanation could be that the increased GVHD observed in MUD recipients led to more severe lymphopenia than that seen after UCBT. The differences in UCBC T cells from adult sources would be expected to translate into UCBT recipients having less GVHD but also less protection from viral infections such as CMV or EBV-LPD. Overall, the risk of infectious death in studies of UCBT may be higher than that of transplant from other sources, but large, multi-institutional, randomized studies have not been done, and there is a bias toward use of UCBT in sicker patients who cannot await a MUD BMT^{ref1, ref2, ref3, ref4, ref5}. In a non-randomized study of children with ALL, the incidence of viral infections was greater after UCBT than after unrelated BMT^ref. Complicating the analysis of actual infectious risk for some infections like herpes viruses is the difference in potential source of virus between UCBT and either BMT or PBSCT. CMV- or EBV-infected B cells can be derived from either the donor or recipient in BMT or PBSCT. In UCBT, CMV- or EBV-infected cells would be expected to exclusively arise from the recipient. Assuming that CMV-negative blood products are administered to CMV-negative recipients, then only CMV-positive recipients of UCBT are at significant risk of CMV disease, while CMV-negative BMT or PBSCT recipients could be infected through their seropositive donor. Therefore, comparisons of herpes virus infections after UCBT versus BMT or PBSCT need to analyze at risk populations, not the total number of recipients. For several infections, notably CMV and HHV-6, there is evidence that at-risk recipients may have greater rate of progression to symptomatic infection, with decreased evidence of viral control, although this is not a universal finding^{ref1, ref2, ref3}. In the comparison of UCBT with MUD transplants at Case Western University, the overall risk of infection, including bacterial infection, correlated with the degree of lymphopenia^ref. The risk of early bacterial infection in UCBT recipients may reflect differences in duration of neutropenia, not just lymphocyte numbers or function^ref. The available information about immune reconstitution after clinical UCBT is quite limited. In order to better understand both the kinetics and quality of immune reconstitution after UCBT, analyses of specific lymphoid populations, immunological repertoire, mitogen and antigen-driven proliferation and function, and responses to test neo-antigens, e.g., keyhole limpet hemocyanin, are needed. Ideally, such studies would be multi-institutional but use similar degrees of matching, CD34⁺ cell dose, pretransplant conditioning, supportive care and post-transplant immune suppression to control for the variables of GVHD, conditioning related microenvironmental damage and progenitor cell dose that likely have an impact on immune reconstitution. At this time, the lack of direct comparisons of immune reconstitution hinders the ability to make decisions regarding the optimal source of HSC for transplantation. In a patient with an active viral infection, e.g., CMV or a history of Epstein-Barr virus lymphoproliferative disease (EBV-LPD), unrelated marrow from an immune donor may be better than UCBT. However, potential HSCT recipients may have limited choices regarding potential HSC sources, and frequently the ability to rapidly perform transplant may trump any immunological disadvantages of UCBT.
Strategies to overcome the problems of immune reconstitution after UCBT : a paradox of UCBT is that some of the associated immunological problems are causally related to the properties that make UCBT attractive in the first place. For example, the decreased alloreactivity that has been clinically observed in UCBT reflects the quantitative and functional abnormalities that make adoptive transfer of antiviral immunity so unlikely immediately after UCBT. Efforts to improve immune reconstitution after any form of HSCT have been directed at two somewhat different questions—accelerating the rate of development of functional immunity, especially that required to control certain early infections, e.g., CMV; or altering the long-term qualitative and functional defects such as hypogammaglobulinemia that contribute to morbidity and mortality over the first decade after transplant. The first goal has been more pressing because of the high impact of opportunistic infection on transplant-related mortality. Strategies in BMT or PBSCT designed to increase the adoptive transfer of antigen-specific T cells directed against important pathogens such as CMV or EBV have used co-transplantation of cloned T cells that have been expanded in vitro. The application of such strategies to UCBT would require either cloned cells that are derived from the host or a third party, or techniques for in vitro priming of the naive T cells in the UCBC graft. Nonspecific activation of the T cells in the UCBC product would likely increase the risk of GVHD. Besides the manipulation of the preformed T cells in a graft, other strategies that have been proposed for accelerating immune reconstitution have been aimed at increasing the rate at which prethymic cells contribute to the mature T lymphocyte compartment, either by increasing the number of transplanted lymphoid progenitors or by accelerating their development in vivo. One potential strategy that has been demonstrated in murine models is the co-transplantation of committed progenitors, such as CLP^ref. Administration of 3000 CLP in addition to 500 HSC in lethally irradiated mice provided significant protection from experimental infection with murine CMV, compared to HSC alone. Impressively, small numbers of CLP provide levels of protection from MCMV that are comparable to that of millions of post-thymic cells. The technical difficulty for UCBT would be obtaining sufficient CLP to affect reconstitution, e.g., via ex vivo expansion. The use of ligands for the Notch receptors that regulate both proliferation and commitment to lymphoid lineages might be useful for in vitro generation of prethymic progenitors that could be used to augment those present in the UCBC product^ref. Based on the data of Chen et al, expansion techniques to increase the number of transplanted HSC might be expected to increase thymic output^ref. Other strategies have focused on ways to increase the in vivo development of T lymphocytes in the recipient after transplantation. These studies have been driven by the hypothesis that the thymic microenvironment of HSCT recipients lacks sufficient capacity to efficiently produce new T lymphocytes from prethymic progenitors. Our group has emphasized that defects in TEC numbers or function caused by aging, cytotoxic therapies, or GVHD lead to a relative lack of intrathymic IL-7 needed for thymopoiesis. Systemic administration of IL-7 in murine models can dramatically improve thymic output after transplantation, but the effects have not been reproducibly observed in larger animal models and IL-7 has additional effects on mature T cells that could limit its usefulness in allogeneic HSCT. Besides promoting the development of thymocytes, IL-7 treatment also increases the expansion of mature T lymphocytes by promoting their proliferation and survival. These effects have led to increased GVHD in some preclinical experiments but have not been observed by others. Experimental usage of IL-7 in clinical allogeneic transplants should probably be initially restricted to T cell–depleted grafts, not UCBT, until more is known about both efficacy and promotion of GVHD. Another strategy to address the problem of microenvironmental damage has been to decrease the toxicity of the conditioning regimen. Besides decreasing the intensity of conditioning, another approach is the administration of a biological response modifier, recombinant keratinocyte growth factor (KGF). KGF is a mesenchymally derived member of the fibroblastic growth factor family which interacts with an epithelial-specific receptor. Administration of KGF in both preclinical experimental models and clinical trials has demonstrated decreased regimen-related toxicities such as oral mucositis, but also less GVHD. KGF protects TEC in murine transplant and aging models, thereby increasing thymopoietic capacity and improving both the immediate rate of lymphocyte recovery and medium-term (3–4 months) immune reconstitution. The clinical relevance of protection of murine thymopoiesis by KGF needs to be determined.
Immune reconstitution after UCBT is promoted by the increased generative capacity of UCBC HSC, and the decreased GVHD, but is hampered by fewer HSC, decreased adoptively transferred antigen-specific T cells, and functional defects in the T cells. Clinical studies to understand the relative importance of each of these biological properties to outcome are needed. At this time, it is difficult to use relative differences in immune reconstitution between HSC sources as a basis for choosing the mode of HSCT, although BMT might be preferential to UCBT for a recipient with an active viral infection. Some but not all strategies aimed at improving immune reconstitution after HSCT in general may be relevant to addressing the immunological and infectious problems observed after UCBT. Currently available assays of hematopoietic progenitor and stem cells in umbilical cord blood :

assay	ease and rapidity for clinical application	correlation with stable engraftment
colony assays	+/–	indirect, unmanipulated grafts only
CD34+	+++	indirect, unmanipulated grafts only
long-term culture-initiating cell (LTCIC)	-	possibly direct
human to immundeficient NOD/SCID mouse repopulating cell assay (NOD/SRC)	-	likely direct

Ex vivo approaches to progenitor and stem cell expansion :

	evidence for HSC expansion	application to clinical trials
CD34+ selected cells, high-dose cytokines	+	limited trials, without best combinations due to cytokine availability. No nadir reduction
low-dose cytokines, continuous perfusion	?	in trials, without best cytokine combinations due to cytokine availability. No nadir reduction.
direct transcriptional activation of HSC cycling: HOXB4, telomerase, NF-Y	+++	future

One of the criticisms of UCBT, compared with other unrelated sources of hematopoietic stem cells, has been that donor anonymity precludes augmenting the GVL effect by infusions of donor-derived lymphocytes^{ref1, ref2, ref3}. The ability to generate B-ALL–specific T cells from UCB by enforcing expression of a chimeric immunoreceptor provides a potential solution to this limitation. Furthermore, the setting of UCBT may be particularly well suited to adoptive immunotherapy, since the slow pace of hematopoietic recovery provides a period of relative lymphopenia in which adoptively transferred T cells might expand using homeostatic mechanisms inherent to recovering the lymphocyte pool, and the relatively low incidence of severe GVHD means that recipients will be less likely to be taking immunosuppressive medications that might impair the function and persistence of the adoptively transferred T cells.
• placental stem cell. Unlike other sites where blood stem cells are found during embryonic development, such as the liver, the stem cells in the placenta can increase in number without giving rise to mature, specialized cells.
• fetal liver : experimental

• in vivo expansion :
• recombinant cytokines
• gene transfer of transcription factors : HOXB4 homeoprotein protein is of particular interests as it promotes the expansion of mouse HSCs without inducing the development of leukemia. Direct delivery of the HOXB4 homeoprotein by passive translocation through cell membranes (alone or as recombinant HIV Tat-HOXB4 protein eliminates any deleterious effects that might be associated with stable HOXB4 gene transfer
• in vitro expansion :
• a 3D, tantalum-coated porous biomaterial (Cytomatrix) supports the maintenance and expansion of human BM pluripotent HPCs (with multi-lineage engraftment capability) in the absence of cytokines and animal-derived serum. The number of CD45⁺ and CD34⁺ cells both increase 3-fold. Trends demonstrate an increase in the frequency of CD34⁺C38^- cells, and an increase in both CD34⁺C33⁺ cells and CD34⁺C61⁺ cells. No expansion of T or B lymphocytes is observed^ref

• cytofluorimetry
• standardization on the lyse-no-wash and single platform (SP) method reduces variation of results^ref

• syngeneic HSCT
• syngeneic BMT
• syngeneic PBSCT
• autologous HSCT (ASCT)
• autologous PBSCT (90-95%) : 4-8 ^. 10⁶ CD34⁺ cells/kg is the minimal dose to ensure a safe engraftment = 8 x 10⁴ CFU-GM
• autologous BMT (ABMT) (for bad mobilizers only) : 2 ^. 10⁸ CD34⁺ cells/kg is the minimal dose to ensure a safe engraftment
Purging :
• positive selection :
• anti-CD34 antibodies
• immunomagnetic CD34⁺ cell selection (ICS) is a widely employed technology in autotransplant and allotransplant settings. When an haploidentical transplant is performed, a high dose of purified CD34⁺ cells together with efficient T and B cell depletion are required to minimize the risks of GVHD and EBV-related lymphoma. To ameliorate the performances of the CliniMACS (Miltenyi Biotec) ICS device, 73 ICS performed following the manufacturer's recommended platelet depletion were compared versus 48 performed after adjunctive centrifugations to increase platelet depletion of the leukapheresis (LKF) product. A total of 121 ICS (from single or fractioned LKF products) were carried out on 93 LKF collected from 47 related healthy donors. A statistical significance in terms of CD34⁺ cell recovery (81.8% vs. 71.2%) was found in favor of the modified ICS procedure (p=0.0049) with a comparable stem cell purity and viability. The modification of the standard manufacturer's technique for increasing platelet depletion can further improve the recovery of stem cells with no influence on T and B cell depletion. These results demonstrate the negative influence exerted on CD34⁺ cell recovery by LKF platelet contamination^ref
• immunoadsorption on avidin-biotin columns
• panning techniques (obsolete)
• FACS
• anti-CD133 antibodies
• negative selection :
• pharmacological methods
• cyclophosphamide derivatives (mafosfamide and 4-hydroxyperoxycyclophosphamide)
• etoposide
• methylprednisolone
• vincristine
• alkyllysophospholipids
• dibutylphthalate
• eilatine
• photoactive substances (merocyanine, hematoporphyrin derivatives)
• immunological methods :
• monoclonal antibodies and complement-mediated lysis
• in vivo
• in vitro
• immunotoxin
• immunomagnetic beads
• biological methods
• in vitro culture
• IL-2, IL-3, IL-6
• antisense oligonucleotides
• TNF-a
• combinations :
• drug combinations
• drug and mAb combinations
• in contrast to leukaemia-initiating cells, HSCs were therefore unable to maintain themselves without Pten. These effects were mostly mediated by mTOR as they were inhibited by rapamycin. Rapamycin not only depleted leukaemia-initiating cells but also restored normal HSC function. Mechanistic differences between normal stem cells and cancer stem cells can thus be targeted to deplete cancer stem cells without damaging normal stem cells^ref.
Indications :
• diseases with bone marrow involvement : following results in animal models^{ref1, ref2} and clinical observations in patients with malignancies and concomitant MS^{ref1, ref2, ref3}, HSCT was first proposed for the treatment of autoimmune diseases^{ref1, ref2}. Debulking of autoreactive clones followed by restoring of self-tolerance during the immunologic recovery has been postulated^ref :
• AML
• CML
• ALL
• CLL
• lymphomas
• autoimmune diseases :
• rheumatoid arthritis
• systemic lupus erythematosus
• multiple sclerosis : autologous HSCT was first adopted for the therapy of MS in the late 1990s^{ref1, ref2, ref3}. Data on the MS cases treated with autologous HSCT have been analyzed and published as single-center studies^{ref1, ref2, ref3}, multicenter trials^{ref1, ref2}, and as a collective European Group for Blood and Marrow Transplantation (EBMT) retrospective study^ref. It is significant that 74% of 85 MS patients reviewed are progression free at 3 years from transplantation^ref. However, due to the different clinical forms of MS treated as well as the multiplicity of the employed immunoablative regimens, these results should be interpreted with caution. A phase II trial showed that HSCT is able to induce a prolonged clinical stabilization in severe progressive MS patients, resulting in both sustained treatment-free periods and quality of life improvement^ref.
• systemic sclerosis
• diseases without bone marrow involvement :
• lymphomas
• solid chemo- and radiosensitive tumors
• CHF : intracoronary infusion of G-CSF mobilised PBSC improves left ventricular systolic function but causes in-stent restenosis after coronary stenting in myocardial infarction (MAGIC cell randomised clinical trial^ref). Some researchers have argued that any human trials are premature because animal experiments have not yet shown exactly how the stem cells repair the heart. They may generate new heart muscle, encourage new blood vessels to grow or secrete molecules that boost tissue survival. Ultimately, blood stem cell injections might be unnecessary if doctors can persuade stem cells dwelling within the heart to start dividing and make new tissue
• BM mononuclear and side population (SP) cells generated progeny in some compartments of the lung (mean 45%, range 4-70% : primarily monocytes and macrophages, but also fibroblasts and type I, but not type II, alveolar cells and rare cells in the bronchial epithelium) and liver (mean 4%, range 0.4-8.3% : Kupffer cells, inflammatory cells and small clusters of hepatocytes, but not bile duct cells), but only in 7-9.5 Gy TBI recipient mice. Stem cells in BM can contribute to repair of tissue injury in some compartments, but not to the same extent in the lung and liver^ref.
• in elderly patients with non-Hodgkin's lymphoma (NHL). In 1994-2004 altogether 88 NHL patients > 60 years old received ASCT in six Finnish transplant centres. There were 57 male and 31 female patients with a median age of 63 years (range 60-70 years); 17 patients were>65 years. The histology included diffuse large B cell (n = 29), mantle cell (n = 27), follicular (n = 15), peripheral T cell (n = 12) and other (n = 5). Disease status at ASCT was I complete remission/partial remission (CR/PR) in 53 patients, II CR/PR in 30 patients and other in five patients. The conditioning regimens included BEAC (n = 49), BEAM (n = 34), TBI-CY (n = 4) and other (n = 1). 84 patients received PB grafts. The medians to reach neutrophils > 0.5 and platelets > 20 were 10 and 14 days, respectively. The early treatment-related mortality (TRM) (<100 days) was 11%. With a median follow-up of 21 months for all patients, 45 patients (51%) are alive. A relapse or progression after ASCT has been observed in 32 patients (36%). ASCT is feasible in selected elderly patients with NHL, but the early TRM seems to be higher than in younger patients^ref
Hematopoietic regeneration after HCT is associated with increased proliferation and differentiation of primitive progenitors. Increased proliferative stress on stem cells bearing genotoxic damage could contribute to the pathogenesis of t-MDS/AML. Extended follow-up of a larger number of patients is required to confirm whether alterations in progenitor and telomere recovery predict for increased risk of t-MDS/AML^ref.
• tandem autologous transplantation : used for ...
• multiple myeloma
• primary progressive or refractory Hodgkin's disease (HD) or aggressive non-Hodgkin's lymphoma (NHL) : tandem HDCT followed by autologous stem cell transplantation (ASCT) offers a chance of cure in these poor prognostic patients, but is associated with risks^ref
Conditioning regimens :
• busulphan-melphalan
• idarubicin-intensified busulphan-melphalan (ida-bu-mel)^ref
• BEAM
• BVAC
• CBV
• cyclophosphamide 60 mg/kg i.v. for 2 days + total body irradiation (TBI) (12 Gy in 4 fractions over 4 days)
Reconstitution :
• T-cell CD3⁺ repopulation typically occurs by 2 to 4 months. Low CD4/CD8 ratios, related to both low numbers of CD4⁺ and elevated CD8⁺ with low levels of naive CD4⁺ CD45RA cells may exist during the first 6 months but return to baseline by 1 year. T-cell function, as measured by mitogen studies and mixed lymphocyte reactions, returns to baseline by 1 year^ref. Even the introduction of CD34⁺ selection techniques to reduce potential tumor contamination does not greatly influence long-term immunologic reconstitution after PBSC infusions, with no significant differences in the kinetics of CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺ cells^{ref1, ref2}.
• CD19⁺ B-cell numbers return to baseline within 3 months, but in vivo function measured by immunoglobulin levels typically requires > 6 months after both selected and unselected infusions. Preliminary studies have suggested delayed immune reconstitution with the combination of rituximab and CD34-selected transplantation. Among recipients of CD34-selected, B-cell antibody-purged stem cell infusions receiving posttransplantation rituximab, decreased IgG levels were noted in 10 of 20 patients at 6 months and 6 of 11 at 1 year. T-cell recovery was also impaired with CD4 counts less than 200/µL observed in 12 of 20 patients at 6 months and in 4 of 11 at 1 year (Horwitz SM, Breslin S, Negrin RS, et al. Adjuvant rituximab after autologous peripheral blood stem cell transplant results in delayed immune reconstitution with increase in infectious complications [abstract]. Blood. 2000;96:707a). A second study of CD34-selected autographs with pretransplantation rituximab noted 5 of 13 patients with T_h < 200/µL at 6 months, and 8 of 8 CMV-seropositive patients developing reactivation CMV-DNA in blood at least once after transplantation (Flohr T, Hess GR, Kreiter S, Meyer RG, Huber C, Derigs G. Immune reconstitution after autologous CD34-positive selected peripheral blood stem cell transplantation combined with rituximab for refractory B-cell non-Hodgkin's lymphoma [abstract]. Blood. 2000;96:709a). Cases of PML and CMV disease suggest that the addition of peritransplantation rituximab, which results in delayed T-cell reconstitution after autografting, may have serious consequences for late clinical infectious disease^ref
• dendritic cell (DC) content has been identified as a predictor of relapse and EFS after autologous HSCT^ref.
Complications :
• mucositis : amifostine protects against mucositis and other toxicities in patients with advanced, refractory, or recurrent hematologic malignancies undergoing HDT and TBI. 35 patients (20 with NHL, 12 with HL, and 3 with AML) who underwent autologous stem cell transplantation were conditioned with TBI 2 Gy twice daily on days -8 through -6; cyclophosphamide 6 g/m², etoposide 1.8 g/m², and carboplatin 1 g/m² on days -5 through -3; and amifostine 500 mg/m² on days -8 through -2. Prior institutional experience in patients treated without amifostine was used as a historical comparison (no-amifostine group). Severe mucositis occurred in 14 (40%) of 35 patients in the amifostine group, compared with 33 (94%) of 35 in the no-amifostine group (P < .0001). TPN was used by 4 (11%) of 35 amifostine-treated patients and 34 (97%) of 35 no-amifostine patients (P < .0001). The median duration of narcotic use decreased from 15.5 days with no amifostine to 11 days with amifostine (P = .002). Granulocyte and platelet engraftment times were similar. Prospective trials with innovative designs and clearly defined stopping rules are warranted to confirm whether amifostine reduces the toxicities of a myelosuppressive conditioning regimen before autologous stem cell transplantation without compromising therapeutic response^ref.
• reactivation of latent viruses :
• HHV-1 / HSV-1
• HHV-3 / VZV
• HHV-4 / EBV
• HHV-5 / CMV : although viral reactivation occurs with similar frequencies after allogeneic and autologous transplantation, clinical disease occurs in only 2% to 8% of autologous marrow recipients^{ref1, ref2}. A comparative study noted an increased odds ratio of 17.0 of CMV disease among recipients of CD34-selected peripheral blood stem cells (7 of 31 patients) versus recipients of unselected peripheral blood stem cell support (10 of 237 patients)^ref. Clinical disease after HSCT typically involves a pattern of early (first 3 months) pneumonitis and enteritis (including all 17 patients in the above study) rather than retinitis that is more common among patients with HIV disease and severe T-cell deficiencies^ref
• HHV-8 / KHSV^ref
• HBV and HCV
• JCV
• allogeneic HSCT : 2-4 ^. 10⁸ CD34⁺ cells/kg is the minimal dose to ensure a safe engraftment. If RBC incompatibility exists, RBCs can be removed by chemical lysis of gradient centrifugation. See HLA and HLA typing

Sources :
• allogeneic PBSCT : PBSCT provides 10 times more T cells than BMT, but G-CSF converts granulocytes into low-density granulocytes (LDG), which undergo apoptosis after 48 h in culture, able to inhibit T-cell function by H₂O₂ production => estimated numbers of T_h1 and T_h2 cells infused in BMT and PBSCT do not differ significantly^ref => the incidence and severity of acute GvHD is similar among recipients of both types of transplants. Nephrotic syndrome^ref
• allogeneic BMT : usually performed in patients under 50 years of age and having a HLA identical sibling
• related donor
• HLA-matched related donor (MRD) : In 1959, Thiomas and his colleagues reported that a patient with end-stage leukemia who was treated with total-body irradiation, followed by infusion of her identical twin's marrow, had a 3-month remission^ref. Transplantation of bone marrow from an HLA-matched child to his immunodeficient sibling was successful because the recipient could not reject the allograft^ref. In the 1970s, Thomas and colleagues cured some patients who had end-stage leukemia by using marrow from their HLA-identical siblings after ablating the host marrow with total-body irradiation combined with cyclophosphamide^ref. Transplantation during the first remission of the leukemia was successful in > 50% the patients^ref. The occurrence of GVHD reduced the incidence of leukemic relapse^ref, which suggested that donor lymphocytes can eradicate tumor cells that survive preparative regimens.
• prognostic significance of NOD2/CARD15 variants in HLA-identical sibling HSCT : effect on long-term outcome is confirmed in 2 independent cohorts and may be modulated by the type of gastrointestinal decontamination^ref
• HLA-partially matched related donor (PMRD)
• haploidentical BMT : transplantation of HSCs from mismatched related donors makes a potential donor available for every child in need of stem cell transplantation. 3 different graft manipulation methods were compared in patients with leukemias and lymphomas: positive selection of stem cells with either CD34 (n = 39) or CD133-coated magnetic microbeads (n = 14) and a new strategy which depletes T- and B-cells through the use of CD3- and CD19-coated microbeads (n = 11). Median purity of stem cells was comparable after CD34⁺-selection and CD133⁺-selection, whereas stem cells were only slightly enriched after CD3⁺/CD19⁺-depletion (97.5 %, 93.4 % and 1.02 %). Indirect depletion of T-cells by positive selection resulted in 1 x 10⁴ (median) residual CD3⁺-cells/kg (0.7-3 x 10⁴). Patients with CD3/CD19-depleted grafts received 3.2 x 10⁴ (median) (0.7-16 x 10⁴) residual T-cells/kg. Those grafts also comprised NK-cells (median number: 86 x 10⁶/kg), dendritic cells and monocytes/granulocytes. Primary engraftment of the stem cell products was comparable after CD34- and CD133-selection (85 and 72 %). In the CD3/CD19 group, 91 % had a primary engraftment. After reconditioning, all patients (64/64) were finally engrafted. Patients with CD34-selected or CD133-selected grafts had similar incidences of a GvHD II-IV (3 and 7 %), whereas a GvHD was slightly increased in patients receiving CD3/CD19-depleted cells (27%). Reconstitution of CD3⁺ T-cells was faster in the CD3/CD19 group than in the CD34 or CD133 group. These preliminary results indicate, that CD3/CD19-selected grafts may be advantageous regarding engraftment and immunoreconstitution. Since effector cell with potential antileukemic activity are cotransfused, such grafts may be suited in particular for patients with insufficient remission^ref
• HLA-matched unrelated donor (MUD) / voluntary unrelated donor (VUD)
• age < 10 years : congenital anomalies
• age < 25 years : severe bone marrow aplasia
• age < 30 years : acute leukemias even in second CR
• age < 40 : CML, MDS, MM
• unrelated CB transplantation (UCBT) : unrelated HLA partially matched UCBT in adults is associated with delayed neutrophil and T- and B-lymphocyte recovery, T-cell subsets recovery, platelet recovery, higher rates of bacteraemia, deficiencies of CB mononuclear cells (MNC) in producing cytokines (including G-CSF, GM-CSF, M-CSF, IL-12, and IL-15) at early time points after transplantation compared with MUD grafting. While phenotypically similar, CB and APB DC have differential potency in allogeneic MLR, which may account for the difference in GvHD and infection incidence and severity between UCBT and allogeneic stem cell transplantation^ref. The "naive" nature of UCB lymphocytes may explain the lower incidence and severity of GvHD encountered in UCBT compared to the allogeneic transplant setting. Furthermore, UCB is rich in primitive CD16^-56⁺⁺ NK cells, which possess significant proliferative and cytotoxic capacities and can be expanded using IL-12 or IL-15, so as to mount a substantial GvL effect. The major disadvantage of UCB is the low yield of HSCs, resulting in higher graft failure rates and slower time to engraftment compared to bone marrow transplantation. A rational approach thus involves ex vivo expansion of UCB-derived HSCs^ref.
After allogeneic transplantation MSC from the donor can engraft in bone marrow. Moreover, since the stem cells were obtained from peripheral blood, it can be concluded that MSC circulate among mobilized peripheral blood stem cells and can engraft in bone marrow after allogeneic transplantation^ref.
Conditioning regimens :
• myeloablative or conventional stem cell transplantation (CST) : mortality = 10%
• cyclophosphamide^ref
• cyclophosphamide (100-120 mg/kg for 2-4 days)/total body irradiation (TBI) (CY/TBI) 5-10 Gy has been the standard preparation since the 1980s. In one study, higher-than-standard doses of TBI reduced the rate of relapse but did not improve survival, because transplantation-related mortality increased^ref.
• busulphan/cyclophosphamide (BU/CY)
• cyclophosphamide, BCNU / carmustine, etoposide (CBV)
• carmustine, etoposide, cytarabine, melphalan (BEAM)
• melphalan 140 mg/m² + etoposide 60 mg/kg + total body irradiation 500 cGy
• TBI 12 Gy/etoposide/cyclophosphamide
• selective radiation of leukemias involves the delivery of radiolabeled monoclonal antibodies against antigens on marrow cells^ref
• newer methods of selective radiation promise increased specificity^ref
Blood group incompatibility does not appear to affect the overall outcome in patients undergoing myeloablative conditioning before allogeneic BMT. Older age has been linked to increased transplant-related mortality from GvHD, but depletion of T cells from stem cell grafts seems to protect from complications of GvHD particularly in older patients. Myeloablative conditioning is well tolerated up to the age of 59 in patients receiving T-cell-depleted grafts^ref.
• reduced-intensity conditioning (RIC) HSCT (RIST / RI-HSCT) / minitransplant / mini-allotransplant / nonmyeloablative stem cell transplantation (NMSCT) : submyeloablative, immunosuppressive conditioning regimen without bone marrow destruction, which reduces early transplant-related mortality (TRM) in older patients.
• fludarabine-based RIC regimen :
• fludarabine (30 mg/m²/day on days -6, -5, and -4), cyclophosphamide 60 mg/m² on days -6, -5, and -4
• fludarabine (30 mg/m²/day on days -6, -5, and -4), cyclophosphamide 60 mg/m² on days -6, -5, and -4, rituximab 375 mg/m² on day -6 and 1,000 mg/m² on days +1, +8, and +15^ref
• fludarabine, low-dose busulfan, and antithymocyte globulin (ATG). In 37 heavily pretreated patients >/=55 years, aGVHD grade III-IV and cGVHD developed in 15.6% and 44.4% of cases, respectively. With a median follow-up period of 22 (range 3-113) months, the 1-year OS and DFS were 55% and 53%, respectively, while the overall non-relapse mortality was 35%^ref.
• fludarabine (30 mg/m²/d x 6 days) or cladribine (0.11 mg/kg/d x 6) with busulfan (BU, 4 mg/kg/d orally x 2), with or without antithymocyte globulin (ATG)
• fludarabine (30 mg/m²/d x 4 days) with melphalan 140 mg/m² as a single dose or
• fludarabine 25 mg/m² for 5 days with melphalan 90 mg/m² daily for 2 days.
• fludarabine 200 mg/m², busulfan 8 mg/kg, and total body irradiation 200 cGy
• fludarabine, BCNU / carmustine and melphalan (FBM)^ref reduced intensity conditioning results in reduced, if any, stromal damage as compared to standard myeloablative treatment. The novel, donor-derived, hematopoiesis in FBM patients after allogeneic transplantation is supported and maintained by a host-derived BM stromal microenvironment^ref
• cyclophosphamide 100 mg/kg i.v. on days -5 and -4; thiotepa 10 mg/kg
• cyclophosphamide 60 mg/kg i.v. on days -5 and -4; 15 mg/kg equine antithymocyte globulin (ATG) IV on days -1, +1, and +3; and thymic irradiation (700 cGy) on day -1^ref
• low-dose total body irradiation
• pentostatin/extracorporeal photopheresis (ECP)/total body irradiation have been shown to be well tolerated and associated with early full donor engraftment with a predominance of donor DC2 cells and a low incidence of aGVHD^ref
• busulfan alone, 4 mg/kg/day for 2, 3, or 4 consecutive days : it is the most commonly used myeloablative alkylating agent, but is considered a poor anti-lymphocyte agent. Engraftment of allogeneic stem cells depends not only on adequate immunosuppression but also on successful hematopoietic competition : residual lymphocytes of host origin may play a beneficial role in preventing GVHD. No additional pre- or post-transplant immunosuppressive agents were used in order to exploit the capacity of donor lymphocytes to induce graft-versus-malignancy (GVM) effects. Conditioning was well tolerated, trilineage engraftment was documented in all patients and none exhibited immune-mediated rejection. Time to recovery of absolute neutrophil count >0.5x10⁹/L and 1.0x10⁹/L was 12 - 38 (median 15) days and 12 - 41 (median 15) days, respectively. The time to platelet recovery >or=20 and >or=50x10⁹/L ranged from 0 to 26 (median 11) days, and from 0 to 83 (median 14) days, respectively. Acute GVHD (<or=grade I) occurred in 13/15 patients. Three patients benefited from long-term survival. Using busulfan alone for the preparation of patients for SCT may be sufficient for engraftment, in very high-risk heavily pre-treated patients^ref.
RIC compared to standard conditioning preserves the in vitro growth capacity of bone marrow stroma, which remains of host origin^ref. It is used to exploit graft-versus-tumor reaction in myeloproliferative disorders (MPD), myelodysplastic syndromes (MDS), and leukemias and lymphomas.
The safety and efficacy of RIC allo-HSCT using either unrelated umbilical cord blood (UCB) donors or matched-sibling donors (MSDs) was assessed in 21 adults at high risk with advanced HL (UCB, n = 9; MSD, n = 12). Both groups were comparable except for younger age in the UCB cohort (median, 28 vs 42 years; P = .02). Neutrophil recovery occurred earlier in the MSD group (median, 7 vs 10 days; P = .02). All patients had sustained donor engraftment by day 60. Cumulative incidence of acute severe graft-versus-host-disease (33% vs 33%; P = .99), chronic graft-versus-host-disease (11% vs 33%; P = .24), and 100-day treatment-related mortality (11% vs 17%; P = .80) were comparable. With median follow-up periods of 17 and 24 months, the 2-year progression-free survival rates were 25% (95% confidence interval [95% CI], 0%-55%) for UCB and 20% (95% CI, 0%-44%) for MSD allo-SCT (P = .67). These results suggest comparable outcomes for RIC allo-SCT using UCB or MSD in adults at high risk with advanced Hodgkin lymphoma^ref.
Immunosuppression to avoid GvHD :
• FK506 since day -2 0.03 mg/kg i.v. continuous infusion (maintaining morning levels 5-15 ng/ml and cheching dose adjustements after 72 hrs. Reduce since day +60 and discontinue at day +90 if MRD is present, otherwise continue until month 6) + methotrexate 5 mg/m² at days +1, +3, and +6
• cyclosporine and methotrexate^ref
• increased mycophenolic acid (MPA), the active metabolite of MMF, concentrations steady state (Css) predict higher degrees of donor T-cell chimerism after unrelated donor nonmyeloablative HCT and suggest that targeting MPA Css's > 2.5 mg/mL could prevent graft rejection^ref.
Patients with ABO mismatch have problems with engraftment, including graft rejection, pure RBC aplasia, and mixed-lineage chimerism. RBC transfusions are significantly higher in the ABO-mismatched recipients. GVHD may play a role in engraftment, possibly by facilitating the disappearance of native ABO antibodies via graft-versus-plasma cell effect.
Immune reconstitution : G-CSF 5 mg/kg/day since day 0 until ANC > 10⁹/ml
Chimerism :
• full donor chimerism : donor CD3⁺ T cells in bone marrow > 95%
• mixed hematopoietic chimerism : donor CD3⁺ T cells in bone marrow = 1-95%
• graft rejection : lack of donor cells in bone marrow at day +56 after donor cells were > 50% at day +28
• graft failure : lack of donor cells in bone marrow at day +56 after donor cells were 1-50% at day +28
Complications :
• infections : in 62 patients undergoing NMSCT with low-dose TBI +/- fludarabine and postgrafting CsA and MMF, the proportion of patients with any infection was 77%, but the majority of infectious events occurred beyond day 30. Donor other than sibling, older age, early disease and male gender were significant risk factors. The incidence of bacteremia was 55% at 1 year and the number of bacteremic episodes was 0.9 per patient (0.08 before day 30). The risk of bacteremia increased with older age and the use of a donor other than an HLA-identical sibling, but not with neutropenia. The incidence of infections other than bacteremia correlated with the use of corticosteroids. The risk of CMV infection increased with high-risk CMV serology, and risk of CMV disease with high-risk CMV serology, older age, first transplantation and a diagnosis of lymphoma^ref
• deep vein thrombosis (DVT) might be a significant problem associated with RIST in patients with solid tumours and anticoagulation therapy might be needed.
• monoclonal antibodies in conditioning :
• alemtuzumab : TCD (reduced GvHD, increased graft rejection (20%), relapse (expecially CML, not related to lymphomas), and delayed immune recovery)
• Campath-1G in vivo + Campath-1M in vitro reduce both rejection and GvHD
• Campath-1H 50-100 mg days -8 to -4 or -6 to -2 (dosable until day + 10, so with greater anti-rejection activity) or -3 to -1 (dosable until day +40, so with greater anti-GvHD activity). Still dosable after 2 months, T-cell recovery after 1 year
• Campath in the bag : (CD52 binding sites per lymphocyte = 5x 10⁵) x (lymphocytes per transplant = 3 x 10¹⁰) x (molecular weight of IgG = 1.5 x 10⁵) x 10³ / NA (= 6 x 10²³) = 3.75 mg per bag
• ATG^ref : reduces cGvHD to 4% => indicated in PBSCT^ref.
Indications :
• malignancies :
• leukemias and lymphomas : allo-SCT for relapsed or refractory aggressive histology (DLBCL, PTCL, transformed B cell) NHL results in long-term EFS and OS of 40-50%. Patients with chemorefractory disease can have a durable remission post-transplant. TRM was 25% at 1 year. Grade III-IV aGVHD was the only significant variable affecting OS and EFS, and had a negative impact. cGVHD did not influence survival. Lymphoma relapse <12 months after initial therapy predicted for increased risk of relapse post-transplant^ref
• myeloproliferative disorders (MPD)
• selected advanced solid tumors (see situation in Europe^ref and USA^ref), including nonmyeloablative conditioning regimens^{ref1, ref2, ref3}
• metastatic breast cancer^{ref1, ref2}, including nonmyeloablative conditioning regimen^ref
• renal cell carcinoma, including nonmyeloablative conditioning regimen^ref
• colorectal carcinoma, including nonmyeloablative conditioning regimen^ref
Among patients who have relapsed after allogeneic HSCT, an immunologically based GVT effect may be obtained :
• simply by transfusing T cells obtained by leukopheresis of the original bone marrow donor (donor leukocyte infusion (DLI)) : this technique has been used to obtain complete remissions in relapsed acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), multiple myeloma, non-Hodgkin's lymphoma, myelodysplastic syndrome (MDS), and chronic myeloid leukemia (CML)
• combination of nonmyeloablative BMT followed by immunotherapy with DLI.
• tumor vaccines in conjunction with autologous transplantation offer a promising method for eliminating tumor.
Patients undergoing autologous HSCT may have bone marrow and peripheral blood that has been contaminated with tumor (Minimal residual cancer detection in hematopoietic stem cell products and its prognostic significance in patients with breast cancer, lymphoma, or multiple myeloma by Thomas J. Moss, MD), which places them at a higher risk of relapse :
• purging bone marrow with either 4-hydroperoxycyclophosphamide, mafosfamide or with mAb have proven feasible although inducing prolonged myelosuppression. Selection of CD34⁺ progenitors appears to be a promising alternative with a 2.5-4.5 log depletion of plasma cells.
• PBSCT have possibly lower tumor contamination than BMT, despite sensitive immunofluorescence studies or PCR based techniques have demostrated that virtually al PBPC harvests are contaminated with malignant cells. Several pilot studies have confirmed the feasibility of autologous transplants with CD34⁺ selected PBPC in MM
Prior exposure to chemotherapy, specially to alkylating agents, significantly impact stem cell collection and hematopoietic recovery : thus PBSC should be collected prior to ASCT.
• nonmalignant conditions :
• paroxysmal nocturnal hemoglobinuria
• congenital immune deficiencies
• Glanzman thromboasthenia
• thalassemia
• Diamond-Blackfan anemia (DBA)
• sickle cell anemia
• osteopetrosis
• inborn errors of metabolism (IEM) :
• childhood cerebral form of X-linked adrenoleukodystrophy (X-ALD) is a rapidly progressive demyelinating condition affecting the cerebral white matter, which rapidly leads to total disability and death. The only known curative treatment for this condition is allogeneic HSCT. Procedure-related toxicity is assumed to be the cause of death of patients with X-ALD. 3 cases of ALD successfully transplanted with the use of non-myeloablative fludarabine based conditioning are described. Patients showed smooth peri-HSCT course with fast and stable engraftment. In the 3- to 5 yr follow-up period, patients showed no deterioration in their clinical and neurological condition. Levels of very long chain fatty acids were very variable and had a tendency to decrease in at least 1 of the 3 patients. In another patient, an improvement of MRI changes was found. Non-myeloablative HSCT should be considered as an early treatment for X-ALD^ref
• aplastic anemia
• myeloablation resulting from anti-proliferative chemotherapy or radiotherapy (sometimes to increase the MTD)
• RI-HSCT provides a good alternative to myeloablative HSCT for children with non-malignant disorders, except for haemoglobinopathies, in which engraftment is poor : even patients with MUD-HSCT have adequate engraftment with acceptable toxicities^ref.
• spinal cord inury (SCI) : Hwang Mi-Soon, 37-yo, received UBSCT in injured spinal cord and stood up her wheelchair after 20 yrs of paralysis caused by an accident. Stem cells transplanted after SCI mostly generate astrocytes, which can promote aberrant sprouting of sensory neurons, leading to allodynia. Making cells produce more oligodendrocytes reduces allodynia and improves functional recovery^ref.
Contraindications :
• creatinine clearance < 40 mL/min because of concerns regarding excessive toxicity of HDT and pharmacokinetics of melphalan in patients with renal failure, despite renal failure has no impact on the quality of stem cell collection and does not effect engraftment
• performance status > 2
Pathogenesis : myeloablative total body irradiation following semi-allogeneic (parental) BMT in mice resulted in a gradual reduction of splenic CD3⁺, CD4⁺ and CD8⁺cells until day 45 post-BMT. Concomitantly, there was an increase in monocytes and CD4⁺/CD8⁺ (double positive) cells, accompanied by a persistent elevation in the percentage of B lymphocytes. The total thymic and splenic T cell populations were reduced until day +30. The cellular reduction correlated with the poor proliferative response of the thymic and splenic cells. A decrease occurred in IL-2 mRNA expression in thymic cells during days 15-20 post-transplant, corresponding with the low level of IL-2 secretion in the spleen and thymus of the transplanted mice. In conclusion, following semi-allogeneic BMT, there was an overall immune down-regulation in the cells, gene and protein levels. Reduced immunological responsiveness following BMT reinforces the need for improving the immune dysfunction by immunotherapy post-BMT^ref.
Reconstitution : engraftment within 2 weeks
• ANC > 500/ml at week 3
• slower for PLT and RBC
• long periods of post-transplant immune deficiency (4 months to 3 years). Peripheral T-cell reconstitution is particularly slow : in mice 3 populations of donor T cells are found in allogenic hosts after transplantation : fast proliferating alloreactive cells (that downregulate IL-7R, respond to host antigens and cause GvHD), slow proliferating non-alloreactive cells and non-proliferating non-alloreactive cells. IL-7 administration could improve the immunocompetence of patients after allogeneic HSCT driving the clonal expansion of non-alloreactive T-cells without inducing GVHD^ref. Allograft dendritic cell (DC) content has been identified as a predictor of relapse and event-free survival after allogeneic bone marrow transplantation. The immune recovery of 66 patients undergoing allogeneic HSCT with either conventional or non-myeloablative conditioning regimen was studied. Infections post-transplant were enumerated and quantitative immunoglobuilins (IgG, IgA, IgM) and lymphocyte sub-sets 3, 6 and 12 months post-transplant were measured. A significant difference was found in the immunologic recovery of non-myeloablative and conventional ASCT in the patient population. The T-helper cell reconstitution was significantly faster after NMA than conventional transplantation and the recovery of B cells was faster after conventional transplantation. Regarding immunoglobulin levels, a faster recovery of IgM levels after NMA-ASCT and a delayed recovery of IgA levels was observed in both groups. These were accompanied by a significant difference in the frequency and severity of infectious episodes^ref.
Complications :

• graft rejection :
• HLA-matched related donor : 2%
• MUD : 12%
• 7% in HLA-matched
• 9% in 1-HLA mismatched
• 21% in 2-HLA mismatched
• graft-versus-host disease (GvHD) (> 70%)
• acute GvHD (70%)
• chronic GvHD (30%) : the incidence of cGVHD in 584 individuals who had undergone allogeneic HCT between 1976 and 1999 and had survived > 2.5 years was 54%, of whom 46% reported active cGVHD. In multivariable analyses, subjects with active cGVHD were more likely to report adverse general health, mental health, functional impairments, activity limitation, and pain compared to those with no history of cGVHD. However, health status did not differ between those with resolved cGVHD and those who never had cGVHD^ref
• fatal immune-mediated pancytopenia and a TRALI-like syndrome associated with high titers of recipient-type antibodies against donor-derived peripheral blood cells after allogeneic bone marrow transplantation following dose reduced conditioning^ref
• infections :
• invasive fungal infections (IFIs) (3%; 6.9% in MUD alloHSCT, 4.5% in MRD alloHSCT, 0.9% in autoHSCT; 3-mo OS after IFI : 38%) : Candida glabrata, Aspergillus fumigatus, Aspergillus ustus, Cryptococcus neoformans var. neoformans, Fusarium spp., zygomycosis, Pneumocystis carinii
• reactivation of latent viruses :
• HHV-1 / HSV-1 (70-80%)
• HHV-3 / VZV (50-60%)
• HHV-4 / EBV (60%)
• HHV-5 / CMV (70%) : although viral reactivation occurs with similar frequencies after allogeneic and autologous transplantation, clinical disease occurs in only 2-8% of autologous marrow recipients^{ref1, ref2}. A comparative study noted an increased odds ratio of 17.0 of CMV disease among recipients of CD34-selected peripheral blood stem cells (7 of 31 patients) versus recipients of unselected peripheral blood stem cell support (10 of 237 patients)^ref. Clinical disease after HSCT typically involves a pattern of early (first 3 months) pneumonitis and enteritis (including all 17 patients in the above study) rather than retinitis that is more common among patients with HIV disease and severe T-cell deficiencies^ref. A murine model of a liver-adapted B-cell lymphoma has previously shown a survival benefit and tumor growth inhibition by nonlethal subcutaneous infection with murine CMV. Virus replication proved to be required for antitumoral immunity, since inactivated virions or the highly attenuated enhancerless mutant mCMV-DMIEenh did not impact the lymphoma in the liver. Surprisingly, the dissemination-deficient mutant mCMV-DM36 inhibited tumor growth, even though this virus fails to infect the liver. On the other hand, various strains of HSV consistently failed to control the lymphoma, even though they infect the liver. A quantitative analysis of the tumor growth kinetics identified a transient tumor remission by apoptosis as the antitumoral effector mechanism. Tumor cell colonies with cells surviving the CMV-induced "apoptotic crisis" lead to tumor relapse even in the presence of full-blown tissue infection. Serial transfer of surviving tumor cells did not indicate a selection of apoptosis-resistant genetic variants. NK cell activity of CD49b-expressing cells failed to control the lymphoma upon adoptive transfer. An innate antitumoral mechanism is triggered by CMV infection and involves an apoptotic signal effective at a distant site of tumor growth^ref. While donor CMV seropositivity had no effect on overall survival, having a CMV-seropositive donor was associated with a significantly lower risk for relapse (20% vs. 52%, P = 0.02). The beneficial effect of a CMV-seropositive donor, however, was offset by a higher TRM (42% vs. 19% in patients with a CMV-seronegative donor, P = 0.2). Donor CMV serostatus had no effect on the incidence of CMV infection/disease, and acute or chronic graft-vs.-host disease. Overall, 27 patients died during the observation period. Causes of death in patients with a CMV-seropositive donor were primarily transplant-related (11 vs. 4 in patients with CMV-seronegative donors), whereas leukemic relapse was the leading cause of death in patients with a CMV-seronegative donor (8 vs. 4 in patients with a CMV-seropositive donor, P = 0.038, chi-squared test). Multivariate Cox regression analysis showed risk category by the underlying disease and donor CMV seropositivity to be significant predictors of leukemic relapse following reduced-intensity transplants. These preliminary results suggest a possible role of donor CMV serostatus on the risk of relapse following reduced-intensity allogeneic SCT^ref
• HHV-8 / KHSV^ref (in RIC^ref)
• HBV and HCV
• JCV (6.7%)
• BKV (55%)
Prevention : evidence-based rating system used in the Hematopoietic Stem Cell Transplantation (HSCT) Guidelines.
• strength of the recommendation : category and definition
• A : both strong evidence for efficacy and substantial clinical benefit support recommendation for use. Should always be offered.
• B : moderate evidence for efficacy—or strong evidence for efficacy, but only limited clinical benefit—supports recommendation for use. Should generally be offered.
• C : evidence for efficacy is insufficient to support a recommendation for or against use, or evidence for efficacy might not outweigh adverse consequences, (e.g., drug toxicity, drug interactions), or cost of the chemoprophylaxis or alternative approaches. Optional.
• D : moderate evidence for lack of efficacy or for adverse outcome supports a recommendation against use. Should generally not be offered.
• E : good evidence for lack of efficacy or for adverse outcome supports arecommendation against use. Should never be offered.
• quality of evidence supporting the recommendation : category and definition
• I : evidence from at least one properly randomized, controlled trial.
• II : evidence from at least one well-designed clinical trial without randomization, from cohort or case-controlled analytic studies (preferably from more than one center), or from multiple time-series or dramatic results from uncontrolled experiments.
• III : evidence from opinions of respected authorities based on clinical experience, descriptive
• AI recommendations :
• 1. All persons should wash their hands before entering and after leaving the rooms of HSCT recipients and candidates undergoing conditioning therapy, or before any direct contact with patients regardless of whether they were soiled from the patient, environment or objects.
• 2. All health care workers with diseases transmissible by air, droplet, and direct contact (e.g. varicella zoster virus, infectious gastroenteritis, herpes simplex lesions of lips or fingers and upper respiratory tract infections) should be restricted from patient contact and temporarily reassigned to other duties.
• 3. When a case of laboratory confirmed legionellosis is identified in a person who was in the impatient HSCT center during all or part of the 2-10 days before illness onset, or if two or more cases of laboratory-confirmed Legionnaire's disease occur among patients who had visited an outpatient HSCT center, hospital personnel in consultation with the hospital infection control team should perform a thorough epidemiologic and environmental investigation or determine the likely environmental source(s) of Legionella species (e.g. showers, tap water faucets, cooling towers and hot water tanks.
• 4. To control VRE exposure, strict adherence to standard infection control measures is necessary, as outlined in the text.
• 5. All HCWs who anticipate contact with a Clostridium difficile-infected patient or the patient's environment or possessions should put on gloves before entering the patient's room and before handling the patient's secretions and excretions.
• 6. HSCT candidates with a recently positive tuberculin skin test or a history of a positive skin test and no prior preventive therapy should be administered a chest radiograph and evaluated for active TB.
•  AII recommendations :
• 1. HCST centers should prevent birds from gaining access to hospital air-intake ducts.
• 2. Appropriate gloves should be used by all persons when handling potentially contaminated biological materials.
• 3. Work exclusion policies should be designed to encourage HCWs to report their illnesses or exposures.
• 4. Visitors who might have communicable infectious diseases (e.g. upper respiratory tract infections, flu-like illnesses, recent exposure to communicable diseases, an active shingles rash whether covered or not, a VZV-like rash within 6 weeks of receiving a live attenuated varicella vaccine, or a history of receiving an oral polio vaccine within the previous 3-6 weeks) should not be allowed in the HSCT center or have direct contact with HSCT recipients or candidates undergoing conditioning therapy.
• 5. If Legionella species are detected in the water supplying an HSCT center, the water supply should be decontaminated and eradication of Legionella should be verified.
• 6. HSCT centers should follow basic infection control practices for control of MRSA infection and colonization, including hand washing between patients and use of barrier precautions, including wearing gloves whenever entering the MRSA-infected or MRSA-colonized patient's room.
• 7. HSCT personnel should institute prudent use of all antibiotics, particularly vancomycin, to prevent the emergence of staphylococci with reduced susceptibility to vancomycin.
• 8. Use of intravenous vancomycin is associated with the emergence of VRE; vancomycin and all other antibiotics, particularly antianaerobic agents, should be used judiciously.
• 9. All patients with Clostridium difficile disease should be placed under contact precautions for the duration of the illness.
• 10. When caring for an HCST recipient or candidate undergoing conditioning therapy with upper or lower respiratory tract infection, HCWs and visitors should change gloves and wash hands in circumstances outlined in the text.
• 11. Visitors and HCWs with infectious conjunctivitis should be restricted from direct patient contact until the drainage resolves and the ophthalmology consultant concurs that the infection and inflammation have resolved to avoid possible transmission of adenovirus to HSCT recipients.
• 12. For patients with suspected or proven pulmonary or laryngeal TB, HSCT personnel should follow guidelines regarding the control of TB in health care facilities.
• EII recommendations : Bacillus of Calmette and Guerin (BCG) vaccination is contraindicated among HSCT candidates and recipients because of its potential to cause disseminated or fatal disease among immunocompromised persons.
Practices :
• room ventilation : staff in HSCT centers should prevent access of birds to hospital air intake ducts (AII), as studies have linked such exposures to the subsequent development of invasive pulmonary respiratory tract infections in HSCT recipients^{ref1, ref2}. Although well designed clinical trials have not validated this approach, it is recommended that all allogeneic recipients be placed in rooms with > 12 air exchanges per hour and point-of-use HEPA filters that are capable of removing particles  0.3 mm in diameter (Streifel AJ. Chapter 80. Design and maintenance of hospital ventilation systems and the prevention of airborne nosocomial infections. In Mayhall CG, ed. Hospital Epidemiology and Infection Control. Second edition. Philadelphia PA: Lippincott Williams & Wilkins; 1999:1211–1221) (AIII). The utility of HEPA filtration has not been established in autologous transplant recipients, but the guidelines suggest consideration of its use in high risk autologous recipients with prolonged neutropenia who are at risk of invasive aspergillosis (CIII). Although data from the early 1980s suggested the benefit of laminar air flow (LAF) rooms in allogeneic transplant recipients with aplastic anemia who received grafts from HLA-identical siblings^ref, more recent studies suggest no survival benefit from routine LAF use in all HSCT recipients^ref. Survival of patients with aplastic anemia patients undergoing HSCT has improved in recent years, and no follow-up studies have been performed to evaluate the utility of LAF use in this population. The use of LAF rooms, even if available, is therefore deemed optional (CII).
• construction, renovation, and building cleaning : this portion of the guidelines contains no AI, AII, or E level recommendations. There are five AIII level guidelines. To minimize the risk of nosocomial aspergillosis, individuals responsible for HSCT construction or renovation are advised to consult published recommendations regarding environmental controls during construction^ref. In addition, when construction or renovation is anticipated, staff in HSCT centers should intensify aspergillosis-control measures (AIII), as outlined in a series of BIII recommendations. HSCT recipients, health care workers and visitors should avoid construction and renovation areas to the extent possible (AIII). N95 respirators have been proposed for HSCT recipients during transport near construction or renovation areas (CIII), but fit-testing and training are necessary for maximal benefit; standard surgical masks afford little protection against inhalation of fungal spores and are not recommended (DIII). Newly constructed areas should be cleaned before patients are permitted to enter them (AIII)^ref as outlined in several BIII guidelines. HSCT patients should not be exposed to activities such as vacuuming of floors or carpets, which are apt to result in the aerosolization of Aspergillus spores (AIII)^ref
• isolation and barrier precautions : this section of the guidelines contains no AI, AII, or E recommendations. Members of HSCT centers are advised to follow published guidelines for hospital isolation practices, including guidelines for the prevention of nosocomial infections (AIII)^{ref1, ref2, ref3}. The utility of specific isolation and barrier precautions in the prevention of nosocomial infections in HSCT recipients has not been studied, however, and these recommendations are extrapolated from other settings.
• hand hygiene : the importance of good handwashing practices in the care of HSCT patients cannot be over-emphasized. All persons, especially HCWs, are advised to wash hands before entering and after leaving the rooms of HSCT patients and of those receiving conditioning therapy. In addition, handwashing should be performed before and after direct contact with patients irrespective of whether soiling from the patient or from objects in the environment occurs (AI)^ref. Handwashing produces a reduction in the carriage of potential pathogens on the hands and has been shown to reduce morbidity and mortality from nosocomial infections in numerous studies^ref. Handwashing may be performed with an antimicrobial soap and water, or with hygienic hand rubs (AIII). Gloves should be worn by all persons when handling potentially infected biologic material (AII). Gloves should be changed between patients or prior to touching a clean area when soiled (AIII). Items worn on the hands by HCWs such as rings, artificial nails, and adhesive bandages should be avoided, as they may serve as a nidus for pathogenic microorganisms (BII).
• equipment : this section contains no AI-II or E level recommendations. HSCT centers are advised to adhere to established guidelines for the sterilization, disinfection and maintenance of devices and equipment utilizing only EPA-registered agents (AIII)^{ref1, ref2, ref3, ref4}.
• health care workers : all HCWs with diseases transmissible by direct contact, droplet or airborne transmission should be restricted from direct patient contact and temporarily reassigned to other tasks (AI) and should follow published guidelines regarding the duration of work restriction (BIII)^ref. These include individuals with illnesses such as varicella zoster virus (VZV) infection, infectious gastroenteritis, herpes simplex infections involving the fingers or lips, and upper respiratory tract infections. Staff at HSCT centers should formulate work exclusion policies to ensure that HCWs report illnesses or potential exposures to communicable pathogens (AII). HSCT institutions should have a written policy regarding HCW immunizations that meets guidelines recommended by the CDC, the Advisory Committee on Immunization Practices, and the Healthcare Infection Control Practices Advisory Committee (BIII)^ref.
• visitors to transplant centers : visitors with potentially communicable illnesses should be barred from the HSCT center and should not have direct contact with HSCT recipients or with candidates receiving preparative therapy (AII). Such illnesses include upper respiratory tract infection, flu-like illnesses, varicella zoster infection, a VZV-like rash developing within 6 weeks of a live attenuated VZV vaccine, or a history of receiving oral polio vaccine within the preceding 6 weeks. All visitors to HSCT centers should be capable of practicing recommended handwashing guidelines irrespective of age (AIII). HSCT centers should have written policies regarding screening of visitors for potentially communicable diseases (BII).
• patient skin and oral care : this section of the guidelines contains no AI, AII or E guidelines. 6 AIII level recommendations are offered. Education regarding dental hygiene is recommended for HSCT recipients and their caregivers concerning the importance of good dental hygiene following transplantation, so as to minimize the likelihood of oral and dental infection (AIII). In addition, all HSCT candidates should be evaluated for dental disease (AIII) and likely sources of infection should be eliminated (AIII) (Schubert MM, Peterson DE, Lloid ME. Oral complications. In : Thomas ED, Blume KG, Forman SJ, eds. Hematopoietic Stem Cell Transplantation, 2nd edition. Oxford, England: Blackwell Sciences, Inc.; 1999:751–763) 10 to 14 days should elapse between completion of dental therapy and initiation of a preparative regimen (AIII). Oral hygiene should be maintained in HSCT recipients or candidates receiving conditioning therapy who have oral mucositis through the use of 4-6 oral rinses per day with normal saline, sterile water or sodium bicarbonate solutions^ref. Following urination or defecation, female HSCT recipients should wipe the perineum from front to back to prevent urethral contamination and subsequent urinary tract infection (AIII).
• prevention of intravascular catheter-related bacterial infections : this section of the guidelines contains no AI-II or E level recommendations. HSCT centers should follow published guidelines for preventing intravascular device-related infections (AIII)^ref
• control of specific nosocomial and community-acquired pathogens :
• Legionella species : legionellosis should be considered in the differential diagnosis of pneumonia in HSCT recipients (AIII). The incubation period for Legionnaires' disease (LD) is 2-10 days. Therefore, patients with laboratory-confirmed LD who are continuously hospitalized for  10 days prior to symptom onset have definite nosocomial legionellosis. Those with proven LD who have been in hospital 2–9 days prior to illness onset have possible nosocomial acquisition. If LD is confirmed in a patient hospitalized on the HSCT unit for all or part of the 2-10 days before illness onset, or if 2 or more cases of proven LD occur in patients visiting an outpatient HSCT clinic, a thorough epidemiologic and environmental investigation should be conducted in conjunction with the infection control team to determine the environmental source(s) of the outbreak (AI)^ref.20 This should include assessment of cooling towers, hot water tanks, showers and tap water faucets. Once identified, the environmental source should be removed or decontaminated (AIII). If Legionella species are found in the HSCT center water supply, it should be decontaminated (AII) and patients should not take showers (DIII) but should receive sponge baths utilizing Legionella-free water (BIII). In addition, water from faucets contaminated with Legionella species should not be used in the HSCT unit so as to prevent the creation of infectious aerosols (CIII). Only sterile water should be used for rinsing nebulation devices and other respiratory care equipment after cleaning, and for filling reservoirs of these devices (BII)^ref
• methicillin-resistant staphylococci : personnel at HSCT centers should adhere to standard infection control practices in order to optimize the control of methicillin-resistant Staphylococcus aureus (MRSA) (AII)^ref. These practices include handwashing between patient encounters, use of appropriate barrier precautions, and wearing gloves when entering the room of an MRSA-infected or colonized patient. Intravascular devices infected or colonized with MRSA should be removed (AIII).
• Staphylococci with reduced susceptibility to vancomycin : at present, staphylococci with reduced susceptibility to vancomycin are fortunately rare^ref. HSCT centers should have laboratory facilities capable of identifying and performing antimicrobial susceptibility testing on all staphylococci (AIII). In addition, routine surveillance should be performed for staphylococci with reduced susceptibility to vancomycin (AIII), defined as a vancomycin minimal inhibitory concentration (MIC)  4 µg/mL for Staphylococcus aureus and  8 µg/mL for coagulase negative staphylococci. If such isolates are identified and confirmed, the laboratory should contact hospital infection control personnel, the HSCT center, the patient's physician, the local or state health department, and the CDC's Hospital Infection Program so that a prompt epidemiologic investigation may be performed (AIII). Hospitalized patients who are infected or colonized with these organisms should be cohorted (AIII). Prudent antibiotic use by HSCT centers, especially of vancomycin, is essential to prevent the emergence of resistant staphylococci and is recommended (AII). In addition, intravascular devices colonized or infected with staphylococci with reduced susceptibility to vancomycin should be removed (AIII).
• Vancomycin resistant enterococci (VRE) : the emergence of VRE is associated with the use of vancomycin and antianaerobic agents, and these drugs should be used judiciously (AII)^ref. To limit exposure of HSCT recipients and patients receiving conditioning therapy to VRE, adherence to standard infection control measures is recommended as summarized in the following guidelines (AI). These measures include compliance with handwashing guidelines outlined above and the cohorting of VRE-colonized or infected patients. In addition, patient rooms and equipment should be disinfected with an FDA- or EPA-registered disinfectant, including surfaces of the hospital environment such as walls, floors, bed frames, doors and bathrooms. In pediatric areas, a nontoxic disinfectant should be used (BIII). Patients with VRE should be placed under contact precautions until cultures are negative and antibiotics are discontinued (BIII). Gloves should be worn in the room of VRE-infected or colonized patients and discarded prior to leaving the room
• Clostridium difficile : patients with Clostridium difficile disease should be placed under contact precautions for the duration of their illness^ref. Gloves should be worn by HCWs who anticipate contact with HSCT patients who are colonized or infected with C. difficile or with their possessions or environment; gloves should be put on before entering the patient's room (AI). Standard published guidelines for the prevention and control of C. difficile infection should be followed, including the judicious use of antibiotics (AIII)^ref. The prophylactic use of lyophilized Saccharomyces boulardii to prevent diarrhea among HSCT recipients receiving antibiotics is contraindicated, as it has no salutary effect and has been associated with the development of S. boulardii fungemia (DII)^ref
• community-acquired respiratory virus infections : community-acquired respiratory viruses (CRVs) may produce significant morbidity and mortality in HSCT recipients^ref, and may produce nosocomial infection if introduced into the HSCT center. Measures to prevent the introduction and spread of CRVs on the HSCT unit are therefore recommended. When caring for a patient at the HSCT center with upper or lower respiratory tract infection, HCWs and visitors should change gloves and wash hands after contact with a patient, after handling respiratory secretions or objects contaminated with respiratory secretions, and between contact with a contaminated body site and the respiratory tract of or respiratory device used on the same patient (AII). This practice is important because most CRVs are transmitted by contact, especially from hand to nose and eye Appropriate infection control measures to prevent nosocomial pneumonia among HSCT patients should be followed, especially during outbreaks of community or nosocomial CRVs (AIII)^ref. It is crucial to identify and place under contact precautions HSCT recipients with respiratory syncytial virus (RSV) infection so as to prevent nosocomial transmission (AIII). To minimize the risk of CRV spread on the HSCT unit, HCWs and visitors with symptoms of upper respiratory infection should be restricted from contact with HSCT patients and individuals undergoing conditioning therapy (AIII). Because of the risk of adenovirus transmission to HSCT recipients, HCWs and visitors with infectious conjunctivitis should be prohibited from contact with patients until ocular drainage resolves and an ophthalmologist agrees that inflammation and infection have subsided (AII). Finally, when suctioning patients with upper or lower respiratory tract infections, HCWs should wear surgical masks, eye protection and gowns to avoid splashes with the patient's secretions. In addition, protective clothing should be donned when entering the patient's room, removed and discarded prior to leaving, and always changed between patient rooms (AIII)
• tuberculosis : because HSCT recipients may reactivate tuberculosis in the face of immunosuppression, candidates for HSCT should be screened by history and chart review for a history of prior exposure to tuberculosis (AIII). A tuberculin skin test utilizing 5 tuberculin units of either the Tubersol or Aplisol formulations may be administered by the Mantoux method, but may be unreliable because of the patient's baseline immunosuppression. For immunocompromised individuals, a positive skin test is defined as  5 mm of induration^ref. Individuals with a recently positive skin test or a previously positive skin test and no prior preventive therapy should have a chest radiograph and an evaluation for active tuberculosis (AI). Staff at HSCT centers should follow published guidelines concerning the control of tuberculosis in health care facilities, which include the institution of airborne precautions and the use of negative pressure rooms for patients with suspected or confirmed laryngeal or pulmonary tuberculosis (AII)^ref (CDC. Guidelines for preventing the transmission of Mycobacterium tuberculosis in health care facilities, 1994. MMWR. 1994;43 (No. RR-13):1–132). With such patients, HCWs should wear N95 respirators, even in isolation rooms, when exposed to potentially infected patients (AIII). For maximal efficacy of N95 respirators, HCWs should be fit-tested and trained in their use (AIII). Immunization with the Bacillus of Calmette and Guerin (BCG) vaccine is contraindicated in HSCT candidates and recipients because of its potential to produce disseminated and fatal infection in immunocompromised hosts (EII)^ref
• Infection Control Surveillance : this section of the guidelines contains no A or E level recommendations. HSCT centers are advised to refrain from performing routine bacterial or fungal surveillance cultures of asymptomatic HSCT recipients (DII)^ref or, in the absence of clusters of infections, of the environment or of equipment or devices used for pulmonary function testing, delivery of inhalation anesthesia, or respiratory care (DIII). In addition, in the absence of an outbreak of nosocomial fungal infection, personnel at HSCT centers should not perform routine fungal surveillance cultures of devices or dust in patient rooms (DIII). Several studies of relevance to infection control practices on HSCT units have been published in the past year. Weinstock et al reported an outbreak of nosocomial influenza A in January 1998 occurring on a 30 bed HSCT unit, and described enhanced interventions implemented to terminate the outbreak and to prevent nosocomial influenza the following season^ref. These interventions included strict isolation of patients with influenza, reverse isolation of all other patients on the HSCT unit during the outbreak, more rapid laboratory turnaround of diagnostic tests for influenza, restriction of visitors, cohorting of HCWs assigned to the HSCT unit, universal rimantadine prophylaxis of all patients and HCWs on the unit during the outbreak, and an enhanced influenza vaccine campaign for HCWs the following season.
• Mayfield et al evaluated the role of environmental disinfectants in reducing the incidence of nosocomial Clostridium difficile-associated diarrhea (CDAD)^ref. In a before and after intervention study, the incidence rate of CDAD among HSCT patients fell from 8.6 to 3.3 cases per 1000 patient days when the environmental disinfectant was switched from quaternary ammonium to 1:10 hypochlorite solutions in the rooms of patients with CDAD. When quaternary ammonium solution was re-instituted, the CDAD rate among HSCT recipients again increased to 8.1 cases per 1000 patient days.
• Laura et al performed a randomized multicenter study in HSCT patients comparing two different time interval protocols for central venous catheter (CVC) dressing changes^ref. 399 patients with tunneled (230, Group A) or non-tunneled (169, Group B) CVCs were randomized to receive dressing changes with transparent impermeable polyurethane dressings. Patients in Group A had dressings changed at 5 versus 10 days, compared with 2 versus 5 days in Group B. There was no significant increase in local infections in those individuals randomized to longer dressing change intervals, and longer intervals were associated with lower costs and less patient discomfort.
• The relative paucity of level AI, AII, BI, and E level recommendations in the infection control guidelines is striking. Among the AI and AII recommendations, most are more global and generic recommendations, derived from and applicable to other clinical settings. More evidence-based studies are necessary to define optimal infection control practices in these high-risk patients undergoing hematopoietic cell transplantation.
• prevention of viral infections : viral infections are common and potentially fatal complications after HSCT. A key aspect of prevention of viral infections is risk assessment based on viral and host factors as well as the time patterns of infections after transplant. This time pattern has changed over the past two decades due to the introduction of standard viral prevention strategies. Several important changes in transplantation techniques have occurred over the past decade, such as use of different sources of stem cells (i.e. peripheral blood or cord blood instead of marrow), graft manipulation (e.g. CD34 selection or T cell depletion), and the use of novel and less toxic conditioning regimens (i.e. non-myeloablative or regimens with reduced toxicity). All these factors may affect the posttransplant risk and thus the prevention regimens. For some of the more recent changes, little information is available on their impact of infectious risk following transplantation. Ultimately, the choice of the prevention strategy is determined by the host immune status as well as the efficacy, toxicity, feasibility, cost and ratio of treated versus untreated individuals (i.e. ‘number needed to treat’) of the intervention. All evidence ratings in the paragraphs "preventing exposure" and "preventing disease and disease recurrence" are identical with those in the published guidelines^ref. Ratings in the paragraph "update since publication of the guidelines" are assigned by the author of this chapter and have not been authorized by CDC or any of the underwriting organizations. If not otherwise noted, recommendations apply for both adult and pediatric HSCT candidates and recipients (although dosing may be different in children). For specific dosing information, the reader is referred to the original guidelines^ref. This chapter only addresses infection prophylaxis in recipients of myeloablative conditioning regimens. Infection prophylaxis in non-myeloablative conditioning regimens is subject of ongoing studies.
• HHV-5 / CMV
• preventing exposure : determination of cytomegalovirus (CMV) IgG serostatus is recommended in all transplant candidates and their prospective donors (AIII). Seronegative HSCT recipients should receive seronegative or leukocyte-depleted blood products (AI). Seronegative autograft recipients are susceptible for primary CMV infection via blood products. Thus, seronegative or leukocyte-depleted blood products may be used to prevent transfusion-transmitted CMV infection (CIII). Seronegative candidates and HSCT recipients who are sexually active and not in long-term monogamous relationships should use latex condoms during sexual contacts (AII). Handling or changing diapers or wiping oral secretions from toddlers also represents a potential risk for CMV transmission to seronegative individuals and should therefore be avoided (AII). Summary of major recommendations for prevention of cytomegalovirus (CMV).
• preventing disease or disease recurrence : 1 of 2 strategies should be used to prevent CMV disease in allogeneic HSCT recipients between engraftment and day 100 after transplant (AI). The first strategy is to give ganciclovir prophylaxis from engraftment until day 100^ref. An alternative strategy is to use virologic surveillance and administer ganciclovir for early evidence of subclinical CMV infection^{ref1, ref2}. Tests used for this purpose include CMV blood cultures, the pp65 antigenemia assay, and DNA detection methods^ref. Due to lower sensitivity, blood culture methods are generally not recommended. Limited data exist on other detection methods such as detection of pp67 mRNA and DNA by the hybrid capture method^ref and no comparative study has been reported for preemptive therapy using these assays. Centers without access to highly sensitive CMV detection methods should use prophylaxis (BII). In seronegative recipients with a positive donor, the incidence of CMV infection is only 15-20% when seronegative or leukocyte-reduced blood products are given. Thus, preemptive therapy with ganciclovir rather than prophylaxis is preferred in these patients (BII). Preemptive therapy is started on the basis of any positive antigenemia assay or two positive DNA PCR test results (BII). Ganciclovir is continued for a minimum of 3 weeks with one week of induction dosing or until day 100, whichever is longer (AI). Other regimens use 2 weeks of induction dosing and maintenance until PCR negativity^ref (BIII). Ganciclovir recipients should have absolute neutrophil counts (ANC) checked at least twice a week during the first 100 days after transplant (BIII). Management of ganciclovir-related neutropenia (ANC < 1000/mm³) includes discontinuations of ganciclovir (CIII) and/or use of G-CSF (CIII). Neutropenic patients with ongoing CMV detection in blood should be treated with foscarnet (CIII). Late CMV disease is an emerging problem in HSCT recipients given ganciclovir prophylaxis or preemptive therapy^{ref1, ref2, ref3}. Risk factors for late CMV disease include chronic graft-versus-host disease (GVHD), low CD4 counts (< 50/mm³), and CMV infection before day 100. If there is continued evidence of CMV in blood by day 100, ganciclovir should be continued until CMV is no longer detectable (AI). CMV surveillance should be continued until one year after transplant in these high-risk HSCT recipients. Ganciclovir should be administered for antigenemia, viremia, or PCR test positivity (e.g. 3 weeks or until CMV is no longer detectable) (BIII).
• High-dose acyclovir given until day 30 or until engraftment has limited efficacy in preventing CMV disease, especially when antigenemia-guided preemptive therapy is given^ref; however, survival was improved in one randomized trial with extended use of high-dose acyclovir^ref. High-dose acyclovir given until day 30 does not seem to be effective in preventing CMV infection and disease in seropositive autograft recipients and is therefore not recommended (DII). Intravenous immunoglobulin is not recommended for prevention of CMV disease (DI).
• Autologous transplant recipients generally have a lower risk of CMV disease than allograft recipients. However, among recipients of CD34-selected autografts the risk may be similar to that seen after allografting^ref. Therefore, CMV prevention strategies in CD34-selected autologous transplant recipients should be similar to those applied in allogeneic transplant recipients (BII). In recipients of unmodified autologous transplants, CMV surveillance and preemptive therapy is recommended in patients with hematologic malignancies or in those who have recently received fludarabine (CIII). Quantitative rather than qualitative CMV detection methods are preferred in autograft recipients (BII). In seropositive nonmodified autograft recipients a treatment course of 3 weeks is recommended if antigenemia levels of > 5 cells per slide are detected (BII).
• New information indicates that the in vivo replication time of CMV is short and influenced by the host immune status^ref. This information can be used to optimize the duration of induction dosing. For example, viral load may continue to rise despite start of ganciclovir in transplant recipients, especially in those receiving high-dose corticosteroids or anti-thymocyte globulin (ATG)^{ref1, ref2}. In such a situation, induction dosing should be continued until a decline of viral load is documented by quantitative detection methods such as DNA PCR or the pp65 antigenemia assay. Conversely, in HSCT recipients with immediate decline of viral load, shorter induction courses may be possible. Increases of CMV loads early during preemptive therapy (first 4 weeks) are only very rarely due to antiviral resistance in ganciclovir-naïve transplant recipients^ref. However, genotypic ganciclovir resistance has been described early after transplant in T cell depleted pediatric HSCT recipients^ref. More information is now available on the optimal duration of ganciclovir preemptive therapy. In an uncontrolled series of 84 patients there was no early rebound CMV disease after discontinuation of ganciclovir following a short course of ganciclovir with 2 weeks of induction and subsequent maintenance treatment until polymerase chain reaction (PCR)-negativity^ref. Thus, discontinuation of ganciclovir based on negative PCR testing is an alternative to treatment until day 100. Continuation of surveillance and preemptive therapy as well as long-term secondary prophylaxis have been reported in uncontrolled trials^{ref1, ref2}. A randomized trial is ongoing to assess the relative benefits of these two strategies for prevention of late CMV disease. Foscarnet has been shown to be as effective as ganciclovir for preemptive therapy in a prospective randomized study. Cidofovir has been associated with moderate renal toxicity similar to that reported for foscarnet in a retrospective analysis of the clinical experience of European centers^ref; however, no recommendations about its use can be made at this time. Only limited data exist on oral ganciclovir in allogeneic HSCT recipients^ref (Spielberger R, Zaia J. Use or oral ganciclovir for the preemptive treatment of CMV following allogeneic HCT: safety and feasibility results. Blood. 2000;96:586a. (abstract)). To date, no data exist on valganciclovir in HSCT. High-dose valacyclovir (8 g per day) moderately reduced the incidence of CMV infection (and therefore the need for preemptive therapy) compared to high-dose acyclovir in a randomized placebo-controlled double-blind study and showed similar efficacy as ganciclovir when given prophylactically in another randomized trial. The incidence of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome was not increased compared to acyclovir (Ljungman P, Camara R, Milpied N, Volin L, Reilly L, Crisp A, Group VIBMTS. A randomized study of valacyclovir as prophylaxis against CMV infection and disease in BMT recipients. 25th Annual Meeting European Group for Blood and Marrow Transplantation. Hamburg, 1999). A CMV-specific monoclonal antibody (MSL-109) has not effective in preventing CMV infection^ref. The type of posttransplant immunosuppression seems to determine, and in some cases limit, the efficacy of preemptive treatment strategy. One non-randomized study showed a high CMV disease rate in cohort of allogeneic HSCT patients who received high-dose steroid prophylaxis, mycophenolate mofetil, and ATG with preemptive PCR-guided therapy based on 2 consecutive positive results two weeks apart and discontinuation based on negative PCR^ref. Thus, earlier intervention, more frequent monitoring or ganciclovir prophylaxis may be required in patients who are heavily immunosuppressed. Analysis of the impact of pretransplant CMV serostatus on survival indicates that seronegative recipients of a seropositive donor have a higher mortality than seronegative recipients of a seronegative donors (Nichols WG, Corey L, Gooley T, Davis C. The role of CMV serostatus on mortality after stem cell transplantation in the era of pre-emptive ganciclovir therapy. 8th International CMV Conference. Monterey, CA, 2001, p 64). Thus, a CMV seronegative donor should be used if possible.
• HHV-4 / EBV
• preventing exposure : serologic testing is generally not recommended; however, it may be useful in recipients of T cell depleted or pediatric HSCT. Known Epstein-Barr virus (EBV) seronegative transplant candidates should be advised of behavior that could decrease the likelihood of EBV transmission (AII), including safe hygiene practices (e.g. handwashing [BIII]), and should avoid contact with potentially infected respiratory secretions (AII).
• preventing disease and disease recurrence : data are emerging that donor-derived EBV-specific cytotoxic T lymphocytes (CTL) can be used effectively for prophylaxis in high-risk patients, i.e. T cell depleted transplant recipients. The strategy has potential but has not been rated by CDC. Posttransplant surveillance with quantitative PCR for EBV DNA and preemptive therapy with rituximab, an anti-CD20 monoclonal antibody, have been reported to be effective in reducing viral load in a small number of high-risk patients for EBV posttransplant lymphoproliferative disease^ref. However, additional studies are needed and no firm recommendations can be made at this time. Additional data have been presented on prophylactic use of donor derived EBV-specific T cells (Bollard CM, Rooney CM, Huls MH, et al. Long term follow-up of patients who received EBV specific CTLs for the prevention or treatment of EBV lymphoma. Blood2000;96 (Suppl):478a. (abstract 2057)). Although this strategy is complex it may be considered in high-risk patients.
• HHV-1 / HSV-1
• preventing exposure : determination of herpes simplex virus (HSV) IgG serostatus is recommended in all transplant candidates (AIII). Testing of donor serostatus is currently not routine in most centers since HSV transmission by marrow or stem cell products has not been documented. HSV seronegative recipients should be informed about the mode of transmission of HSV and be advised of behavior that will decrease the likelihood of exposure, e.g. avoiding potentially infectious secretions such as cervical secretions and saliva (AII). Summary of major recommendations for prevention of herpes simplex virus (HSV).
• preventing disease or and disease recurrence : acyclovir prophylaxis is recommended for all HSV seropositive allograft recipients (AI) (Table 5). Most centers administer acyclovir from the onset of conditioning until engraftment and resolution of mucositis (BIII). This may occur before day 30 after transplant, which is the time period that was used in the original randomized studies of acyclovir prophylaxis. Routine extension of HSV prophylaxis beyond day 30 is not recommended in the absence of randomized trials (DIII); however, patients with frequent recurrences might benefit from extended suppressive therapy (CIII). Although the original studies were performed with intravenous (IV) acyclovir, oral administration is often used in individuals who can take oral medication; however, adequate dosing seems important^ref. Patients receiving ganciclovir (CIII), foscarnet, or cidofovir for CMV prevention or treatment do not require concomitant acyclovir prophylaxis due to the in vitro activity of these compounds against HSV. Routine acyclovir prophylaxis in HSV seronegative recipients is not indicated (D III), even if the donor is seropositive. Valacyclovir has been used instead of oral acyclovir for prevention of HSV in HSCT (CIII). There are no data on the use of famciclovir for HSV prophylaxis in HSCT recipients; however, the drug has in vitro activity as well. Foscarnet should not be used for routine HSV prophylaxis due to substantial renal toxicity (DIII).
• HHV-3 / VZV
• preventing exposure : determination of VZV IgG serostatus is recommended in all transplant candidates (AIII). Testing of donor serostatus is currently not routinely conducted by most centers since VZV transmission by marrow or stem cell products has not been documented. VZV is highly contagious and vaccination of family members, household contacts, and prospective visitors who are known seronegative for VZV or have no history of VZV infection should be performed (AII). This is usually done at the time of first contact with the transplant center to allow for sufficient time to complete the vaccination (ideally > 4 weeks prior to start of conditioning) (BIII). Seronegative transplant candidates and recipients should be informed about VZV transmission and advised of strategies on how to avoid exposure (AII). Transplant recipients and candidates should also avoid vaccine recipients who experience a rash after vaccination (BIII). Such rash may occur between 5 and 35 day after vaccination (median 22 days)^ref.1 Summary of major recommendations for prevention of varicella-zoster virus infection.

All patients with VZV disease should be placed under airborne and contact isolation (AII). Isolation should be continued until all lesions have crusted. Airborne precautions should be instituted 10 days after an exposure of a susceptible transplant recipient to VZV and continued until 21 days after the last exposure or 28 days postexposure if the patient received varicella-zoster immunoglobulin (VZIG) (AI). Note that individuals infected with VZV can be contagious before the rash appears.
• preventing disease or and disease recurrence : VZIG should be administered to a seronegative recipient at risk for complications of VZV (i.e. < 24 months after transplant, or  24 months if on immunosuppressive therapy and/or having chronic GVHD) as soon as possible after exposure (< 96 hours) (AII) (Table 6). Seronegative recipients or candidates undergoing conditioning therapy should also receive VZIG if they are exposed to a VZV vaccinee having a varicella-like rash (BIII); these recommendations may be extended to seropositive recipients (CIII). If a second exposure occurs less than 21 days after a dose of VZIG in a susceptible individual (see above), a second dose of VZIG should be administered. Any HSCT recipient or candidate undergoing conditioning therapy who experiences a VZV-like rash should be started immediately on preemptive intravenous acyclovir until  2 days after all lesions are crusted (BIII). Virologic confirmation may be useful in situations where the appearance of the rash is atypical. A rash following exposure to a VZV vaccinee should be treated similarly (BII). Long-term acyclovir prophylaxis for 6 months to prevent VZV infection is generally not recommended (DIII); however, such prophylaxis could be considered in HSCT recipients with severe, long-term immunodeficiency (CIII). VZV vaccine use is contraindicated for individuals < 24 months after HSCT (EIII). Use of VZV vaccine among HSCT recipients is restricted to research protocols for individuals > 24 months after HSCT who are presumed immunocompetent (i.e., off immunosuppressive therapy and free of chronic GVHD). Long-term suppressive prophylaxis with acyclovir with doses higher (800 mg twice daily) and of longer duration (one year) than previously reported appears to be successful in preventing VZV reactivation (Bowden RA, Rogers KS, Meyers JD. Oral acyclovir for the long-term suppression of varicella zoster virus infection after marrow transplantation. 29th Interscience Conference on Antimicrobial Agents and Chemotherapy. Anaheim, CA, 1989, p abstract 62). Valacylovir (500 mg twice daily for prophylaxis, 1000 mg 3 times daily for recipients with stable non-disseminated disease) is being increasingly used due to its higher bioavailability. Initial concerns of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome which had been observed in HIV-infected individuals, have not been observed in two large randomized trials at much higher doses than those used for VZV (Ljungman P, Camara R, Milpied N, Volin L, Reilly L, Crisp A, Group VIBMTS. A randomized study of valacyclovir as prophylaxis against CMV infection and disease in BMT recipients. 25th Annual Meeting European Group for Blood and Marrow Transplantation. Hamburg, 1999; Winston DJ, Yeager AM, Chandrasekar PH, et al. Randomized comparison of oral valaciclovir and intravenous ganciclovir for prophylaxis of cytomegalovirus disease and complications after allogeneic bone marrow transplantation. 2nd International Conference on Transplant Infectious Diseases. Stockhom, Sweden, 2000) Whether seropositive transplant recipients can be reinfected with a second strain of virus is poorly defined. However, this may occur in the context of severe immunosuppression such T cell depletion, high-dose steroid therapy, or use of ATG. Thus, exposure should also be avoided in highly immmunosuppressed seropositive recipients, and postexposure prophylaxis with VZIG or acyclovir^ref or valacyclovir (1000 mg three times daily) given from day 2-22 of exposure may be considered. As VZV vaccination is more commonly used in the general population, an increased frequency of previously vaccinated transplant candidates can be expected. The risk of reactivation of vaccine strains after transplantation is currently unknown. Also, whether transplant recipients who have been vaccinated prior to transplant are more susceptible to infections with wild-type VZV than those who have been naturally infected with VZV is unknown. Because the vaccine is not 100% protective even in immunocompetent individuals^ref, an exposure to wild-type VZV in a HSCT recipient is likely to carry a high risk and should be treated similar to exposures in seronegative HSCT recipients or candidates, especially when intense immunosuppression is administered (e.g. T cell depletion, high-dose corticosteroids, or ATG).
• respiratory virus infections
• preventing exposure : respiratory viruses (respiratory syncytial virus [RSV], parainfluenza viruses, influenza viruses, adenovirus) are highly contagious. Strict infection control practices are advised (AIII). A targeted surveillance system that evaluates HSCT recipients with upper respiratory symptoms for the presence of respiratory viruses should be in place (AIII). Isolation should be instituted in all symptomatic patients, even before virologic confirmation (AIII) (Table 7). Summary of major recommendations for prevention of community respiratory virus infection. Influenza vaccination is strongly advised for all staff members, family members and close household contacts during each influenza season before HSCT and at least 24 months after HSCT (AI). During a nosocomial influenza A outbreak situation of strains that are not covered by the vaccine health care workers, family members, and close household contact should be offered chemoprophylaxis with amantidine or rimantidine (for influenza A) (BIII) or with neuraminidase inhibitors (zanamavir, oseltemavir) if amantidine/rimantidine is not tolerated, the outbreak strain is resistant to these agents, or the outbreak is due to influenza B (BI). Neuraminidase inhibitors have not been evaluated in children.
• preventing disease or diseases recurrence : respiratory secretions of HSCT candidates and recipients with upper respiratory symptoms should be evaluated for the presence of respiratory viruses (BIII). Several strategies have been proposed to prevent progression from upper to lower tract RSV infection, including aerosolized ribavirin alone and combination with RSV-specific antibodies or palivizumab, an RSV-specific monoclonal antibody. Controlled clinical trials are currently underway to establish the efficacy of these strategies, thus, no firm recommendations can be made. Intravenous ribavirin is not recommended for preemptive therapy (DIII). Amantidine or rimantidine chemoprophylaxis is recommended in influenza A outbreak situations for all HSCT recipients < 6 months after transplant (BIII). After 6 months, vaccination should be resumed (BIII). Chemoprophylaxis for all HSCT recipients < 24 months or > 24 months if treated for chronic GVHD has also been recommended during outbreak situations due to the poor immunologic response to the vaccine. However, no formal recommendation regarding such prophylaxis can be made because of lack of data. Influenza vaccinations during the first 6 months after transplant are unlikely to be effective (DII). Neuraminidase inhibitors are now available for treatment and prevention of influenza infection in immunocompetent subjects. However, no formal evaluation has been performed in immunocompromised patients. Thus, no recommendations can be made. Palivizumab, a RSV-specific monoclonal antibody, has been licensed for prevention in high-risk immunocompetent children; however, no prevention studies have been performed in HSCT recipients. A study of palivizumab for treatment of RSV infection showed no significant toxicity (Boeckh M, Berrey MM, Bowden RA, Crawford SW, Balsley J, Corey L. Phase I evaluation of the RSV-specific humanized monoclonal antibody palivizumab (MEDI-493) in hematopoietic stem cell transplant recipients. J Infect Dis 2001). Some centers use palivizumab for prophylaxis in pediatric (age < 2 years) HSCT recipients and candidates throughout the respiratory virus season. Whether rhinovirus is a significant pathogen in HSCT recipients remains controversial^ref (Boeckh M, Hayden F, Corey L, Kaiser L. Detection of rhinovirus RNA in bronchoalveolar lavage in hematopoietic stem cell transplants recipients with pneumonia. 40th Interscience Conference on Antimicrobial Agents and Chemotherapy. Toronto, 2000, p 262 (abstract 190)).
• prevention of fungal infection : a primary goal in the practice of transplant medicine is the prevention of infection. This is particularly true when one considers fungal infections in HSCT recipients. Fungal infection in this patient population is difficult to diagnose and treat, and, not surprisingly, is associated with a prohibitively high mortality. Factors contributing to this mortality include the following^ref:
• diagnosis is difficult, as conventional microbiologic approaches are both insensitive and often nonspecific. Invasive sampling of tissues is often necessary for diagnosis, or, alternatively, an empiric therapeutic approach is employed.
• because of the impaired inflammatory response present during the neutropenic phase preengraftment and during the treatment of GVHD postengraftment, clinical signs and symptoms of invasive fungal infection can be greatly blunted until disease is relatively far advanced. As a result, the microbial burden is usually far greater at the time of diagnosis than in nonimmunocompromised patients, a potentially important prognostic factor in determining the outcome of therapy.
• outcome is directly related to how early the therapy is instituted, with the previously listed two factors making early diagnosis difficult.
• currently licensed antifungal therapies are not ideal, having limited spectra of activity (e.g., fluconazole), a propensity for selecting resistance (e.g., flucytosine), or a particular ability to induce systemic (e.g., infusion toxicity resulting in a cytokine release syndrome with amphotericin) or organ toxicity (e.g., renal toxicity from amphotericin and hepatic toxicity from certain azoles).
• the treatment of invasive fungal infection needs to be more prolonged than that needed in the management of invasive bacterial infection.
Fungal infections of major concern can be divided into three general categories: invasive infection due to Candida and Aspergillus species, which account for > 90% of the fungal infections occurring in this patient population; infection due to the geographically restricted systemic mycoses (e.g., Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma capsulatum); and invasive infection due to the so-called "newly emerging fungi" (e.g., Fusarium, Paecilomyces, the zygomycetes, and such dematiaceous fungi as Scedosporium, Scopulariopsis, and Dactylaria)^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8}
• risk factors in the pathogenesis of fungal infection : the occurrence of invasive fungal infection in the HSCT patient is largely determined by the interaction of three factors: the presence of technical/anatomic abnormalities that compromise or attenuate the functioning of the primary mucocutaneous barrier to infection; excessive environmental exposures to potential fungal pathogens; and the net state of immunosuppression. The major technical/anatomic abnormalities that occur in the HSCT patient are those that arise from the placement of a central venous catheter for blood sampling and fluid administration and post-chemotherapy mucositis- both of which providing a portal of entry for Candida isolates. In addition, serious injury to the skin (e.g., water immersion injury, dermal or subcutaneous injury due to extravasation of noxious chemicals into soft tissues because of malfunction of vascular access devices) will commonly become secondarily infected by Candida, Aspergillus, and even zygomycetes species. Invasion of the skin and blood vessels can then occur, followed by secondary dissemination from the cutaneous portal of entry. The creation of devitalized tissue, the occurrence of undrained fluid collections, and the development of significant tissue injury (especially of the lungs) increases the risk of secondary fungal invasion^ref (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). In the hospital setting, two epidemiologic patterns of exposure are observed: domiciliary and nondomiciliary. The term domiciliary refers to exposures occurring where the patient resides in the hospital. There is often clustering of cases in time and space. Although such exposures are usually due to a contaminated air supply, with molds such as Aspergillus being the major concern, person-to-person spread (usually on the hands of medical personnel) of Candida species, often azole resistant strains, can also occur^ref. Nondomiciliary exposures can occur anywhere within the hospital as the patient travels for an essential procedure (the operating room, the radiology or endoscopy suite, or the halls of the hospital where construction is taking place). Nondomiciliary exposures are probably more common than domiciliary, but are often more difficult to define because of the lack of clustering of cases. Indeed, the leading clue to the presence of such an environmental problem is the occurrence of a case of invasive fungal infection at a point in time when the net state of immunosuppression should not be great enough for such an infection to occur without a particularly intense exposure^{ref1, ref2} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press; Rubin RH. Infection in solid organ transplant recipients. In Rubin RH YL, ed. Clinical Approach to Infection in the Compromised Host. New York: Plenum Press, 2001:in press) Particularly notable contributors to the net state of immunosuppression for the HSCT recipient include neutropenia, GVHD and its treatment, and immunomodulating viral infection. In addition, such metabolic factors as protein-calorie malnutrition, uremia, and, perhaps, hyperglycemia contribute to the net state of immunosuppression. The patient receiving an allograft is far more immunosuppressed than the autologous HSCT recipient since the duration of neutropenia is greater and GVHD has a sustained effect in the allogeneic recipient. Reflecting this difference, the incidence of invasive fungal infection is far higher among the allogeneic population. Not unexpectedly, preventative strategies that are shown to be effective in the allogeneic HSCT recipient can be assumed to be effective in the autologous recipient; unfortunately, the converse is not necessarily true (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press)
• prevention of fungal infection in the HSCT recipient : as delineated in 2 recent publications^{ref1, ref2}, there is a dearth of well designed, randomized, concurrently controlled clinical trials, not because of a lack of will on the part of investigators, but rather because of the inherent challenges in approaching fungal infection in this patient population: confounding factors (e.g., duration of neutropenia and other markers of the severity of the underlying disease) can obscure the efficacy of one or another antifungal regimen. In addition, the underlying disease for which the HSCT has been performed can be the cause of mortality, rather than failure of the antifungal program (other endpoints such as fever can also be problematic). The diagnosis of invasive fungal infection remains a problematic endpoint as well. Thus, the evidence relies less on randomized clinical trials and more on comparative studies that employ historical controls and careful clinical observation. Studies carried out by Benson^ref and Sacks^ref and their colleagues are reassuring in demonstrating that in other therapeutic areas, studies that employed historical controls yielded comparable results to those obtained in concurrently controlled clinical trials in at least 75% of instances.
• prevention of exposure to potentially invasive fungal species : aerosols containing excessive numbers of the geographically restricted systemic mycoses, as well as such molds as Aspergillus species, constitute a significant risk. It is generally recommended that HSCT recipients be educated to avoid circumstances in which aerosols of fungal spores might be encountered (BIII): areas of high dust exposure, urban renewal and other construction projects, chicken coops, bat caves, avocational (e.g., gardening) and vocational activities that require digging up soil, marijuana smoking, and the preparation and handling of foods that contain molds (e.g., blue cheese). Similarly, HSCT recipients should avoid exposures to naturopathic medicines, which can be contaminated with molds (CIII)^{ref1, ref2, ref3, ref4, ref5, ref6}. Although invasive candidal infection in the HSCT patient is almost always of endogenous origin, the endogenous candidal flora is modified within the hospital. A particular issue is the person-to-person transfer of Candida species (which are frequently azole-resistant) from the hands of medical personnel. Appropriate handwashing procedures by medical is strongly recommended to prevent the introduction of more difficult to treat Candida species into the "endogenous" flora (AIII)^{ref1, ref2} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). Of even greater importance are exposures to air contaminated with fungal spores, which result in invasive infection with such molds as Aspergillus species and such newly emerging species as Fusarium and the Mucorales. The portals of entry of these infections are the lungs and nasal sinuses. Special effort is required to protect HSCT recipients from contact with air containing excessive fungal spore counts (BIII)^{ref1, ref2, ref3} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press)
• high efficiency (> 90%) particulate air (HEPA) filtration in patient rooms.
• controlled air pressures, such that the air pressure in patients' rooms, are positive in relation to the corridor, and that air flows from the rooms into the corridors.
• correctly sealed rooms, including correctly sealed windows and electrical outlets.
• high rates of room air exchange (i.e., > 12 air changes/hour).
• the construction of effective barriers between patient care and renovation or construction areas (e.g., sealed plastic) that prevent dust from entering patient care areas and that are impermeable to Aspergillus species.
• careful attention to travel routes within the hospital to avoid areas of renovation and construction. Ideally, patients leaving their rooms should be wearing highly effective masks as a further barrier to the inhalation of fungal spores.
Although HEPA filters and air pressure systems are essential and effective cleaning procedures to minimize fungal spores are important, routine surveillance cultures of the air or of the patient are not recommended (DIII)^ref
• minimization of deficits in host defenses against IFI : the first principle of practice is the eradication of all treatable infection prior to transplant. In patients with hematologic malignancies, it is not uncommon that previous attempts to induce remission with cytotoxic chemotherapy were complicated by invasive fungal infections due to Candida species or molds, especially Aspergillus. Such patients are still candidates for HSCT provided certain requirements are fulfilled (BIII): the patient has had a complete response clinically, microbiologically, and radiologically to effective antifungal chemotherapy; the patient can be continued on such therapy at least until engraftment occurs post-HSCT; and any residual anatomic abnormality (e.g., an infarcted area of lung due to invasive Aspergillus infection) or devitalized tissue has been considered for surgical resection (CIII)^{ref1, ref2, ref3, ref4} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). The first host defense deficit usually encountered by HSCT recipients is neutropenia secondary to the preparative regimen. Growth factors (e.g., GM-CSF and G-CSF) reduce the duration of neutropenia, although there is currently no evidence that this shortened effect has translated into a lower incidence of invasive fungal infection. Therefore, these agents cannot be recommended for the routine prevention of invasive fungal infection (DIII)^{ref1, ref2} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). CMV infection is an important cause of morbidity and mortality with both direct and indirect effects. The direct infectious disease effects of CMV in this population include life-threatening pneumonia and a systemic multisystem disease. CMV infection also contributes to the net state of immunosuppression, with an increased susceptibility to both yeast and mold infection being noted during active CMV disease. Accordingly, control of CMV replication is recommended, not only for its direct benefits but also to decrease the incidence of invasive fungal infection (BIII)^{ref1, ref2} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). Effective control of GVHD can reduce occurrence of invasive fungal infection^{ref1, ref2, ref3} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). A major lesson from the solid organ transplant experience is the need for so-called steroid sparing therapy; that is, the use of additional drugs such as cyclosporine +/- mycophenolate permit a decrease in prednisone dose and lower the risk of opportunistic infection, including that caused by fungi.10 In addition, the correction of metabolic abnormalities such as protein-calorie malnutrition, uremia, and hyperglycemia should decrease the net state of immunosuppression and the subsequent risk of invasive fungal disease (Rubin RH. Infection in solid organ transplant recipients. In Rubin RH YL, ed. Clinical Approach to Infection in the Compromised Host. New York: Plenum Press, 2001:in press)
• antifungal therapy in the prevention of invasive fungal infection : there are 3 different modes in which antifungal drugs can be administered to prevent invasive fungal infection: a prophylactic mode, an empiric mode, and a preemptive mode.28 In HSCT recipients the administration of topical (non-absorbable) antifungal agents (e.g., clotrimazole, nystatin, amphotericin B) orally or to the skin can be shown to decrease the colonizing fungal burden, particularly with yeasts, when applied to specific skin sites or the gastrointestinal tract. In addition, mucosal manifestations of superficial yeast infection (e.g., oral thrush, pharyngeal, and esophageal candidiasis) are probably decreased by topical therapy (CIII). However, such topical therapy does not reduce the incidence of either invasive candidiasis or invasive mold infection in HSCT patients and cannot be recommended for these purposes (DII)^ref (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press)
• prophylaxis of invasive yeast infection : the efficacy of oral fluconazole, a well absorbed triazole antifungal agent with particular efficacy against C. albicans and C. tropicalis, appears to be dose dependent. At a dose of 100-200 mg/day orally, fluconazole has variable effects in the prevention of invasive candidal infection. Indeed, this regimen has been associated with the emergence of infection due to such invasive species of Candida as C. krusei and C. glabrata, which are fluconazole resistant. Therefore, this regimen is not recommended for the prevention of invasive yeast infection (nor, since molds are universally resistant to fluconazole, for invasive mold infection) in HSCT recipients (DII). In contrast, at a dose of 400 mg/day there are convincing data that fluconazole administered orally or intravenously, beginning at the time of HSCT and continuing until at least the time of engraftment, provides clinically important protection against invasive yeast infection (AII). When breakthrough candidal infection does occur despite such a regimen, it should be assumed that the isolate is fluconazole resistant. The increasing incidence of C. krusei, C. glabrata, and other non-albicans strains is in large part related to the widespread use of fluconazole prophylaxis. Overall, however, the incidence of invasive candidiasis appears to be significantly decreased by fluconazole prophylaxis (at a dose of 400 mg/day) in allogeneic HSCT recipients.1,11,29,30 In general, the risk of invasive yeast infection in recipients of autologous HSCT is low enough not to require fluconazole prophylaxis (DIII). However, certain autograft patients are at higher risk and do merit prophylaxis employed for allogeneic recipients (BIII): autologous patients with underlying hematologic malignancies who will have prolonged neutropenia and mucosal damage from intense conditioning regimens; and those who have been receiving fludarabine or 2-CDA before the transplant^ref
• antifungal prophylaxis of invasive mold infection : no regimen has been shown to be clearly effective in the prevention of invasive mold infection, whether the species of mold be Aspergillus or one of the newly emerging molds such as Fusarium; hence, the importance of the previously described environmental protection strategies. Regimens studied thus far include moderate dose (0.5 mg/kg/day) amphotericin B, low dose amphotericin B (0.1–0.25 mg/kg/day), intranasal amphotericin B spray, lipid formulations of amphotericin B, aerosolized amphotericin B, and itraconazole. Similarly, there is no convincing evidence that these strategies prevent the newly emerging molds (DIII)^ref (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press) As mentioned above, increasing success is being achieved in patients with previous invasive Aspergillus infection, which has been brought under control with an amphotericin preparation. HSCT, both allogeneic and autologous, has been successfully carried out in these individuals under the cover of antifungal prophylaxis, usually with an amphotericin preparation^{ref1, ref2, ref3, ref4} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press)
• antifungal prophylaxis of the geographically restricted systemic mycoses : although case reports or small series of HSCT patients infected with one of the endemic mycoses (particularly coccidioidomycosis and histoplasmosis) have been reported, this has not been a significant clinical problem. Indeed, prophylactic antifungal therapy against the endemic mycoses is not recommended (DIII)^{ref1, ref2, ref3, ref4}.
• empiric therapy of fungal infection : the importance of neutropenia, particularly profound (absolute neutrophil count < 100/mm³) and prolonged (> 7 days) in the pathogenesis of invasive fungal infection has long been recognized. Because of the previously discussed difficulty in establishing the diagnosis of invasive fungal infection and the need for early initiation of therapy, empiric therapy of the febrile, neutropenic HSCT has become the standard of care. Such empiric therapy is defined as the initiation of systemic antifungal therapy to neutropenic patients who remain febrile after four days of empiric broad spectrum antibacterial therapy, and whose clinical and laboratory evaluation (including multiple blood cultures) remain negative. Amphotericin B was established over the past two decades as the therapy of choice for this purpose, with a significantly lower incidence of invasive fungal infection observed among patients receiving amphotericin B^{ref1, ref2, ref3, ref4, ref5}. More recently, liposomal amphotericin B has been shown to have equal efficacy to conventional amphotericin B, with significantly less infusion related toxicity (fever and chills) and dose related nephrotoxicity, but with significantly greater cost (BI)^{ref1, ref2}. Perhaps of greatest interest is the report of a multicenter, randomized trial in which standard liposomal amphotericin therapy at a dose of 3 mg/kg/day was compared to voriconazole at a dose of 3 mg/kg twice daily intravenously (or 200 mg orally) following two loading doses of 6 mg/kg 12 hours apart in a standard febrile neutropenia protocol. Voriconazole, a new broad-spectrum triazole agent available in oral and intravenous formulations, was equal or superior (fewer breakthrough cases of invasive fungal infection) to liposomal amphotericin. The major side effect from the voriconazole was transient visual disturbances, whereas febrile reactions and nephrotoxicity were significantly more common among the recipients of liposomal amphotericin (Walsh TJ, Finberg, R., Arndt, C., et al. Voriconazole versus liposomal amphotericin B for empirical antifungal therapy in persistantly febriele neutropenic patients: a prosepective, randomized, multicenter, international trial, 40th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, ON, Canada, 2000). Thus, empiric therapy of the febrile, neutropenic HSCT patient has been accepted as standard care. Amphotericin B, liposomal amphotericin B, and voriconazole have comparable efficacy. However, voriconazole appears to have less toxicity and the added convenience of an oral formulation.
• preemptive therapy of fungal infection : preemptive therapy is defined as the administration of an antimicrobial regimen to a patient population felt to be at high risk of life threatening infection before the onset of clinically recognizable disease^ref. In the case of invasive fungal infection in the HSCT patient, three opportunities for preemptive therapy have been defined:
• Colonization of the respiratory tract with Aspergillus species as demonstrated by positive cultures of respiratory secretions (sputum or bronchoalveolar lavage specimen) carries a > 50% risk of subsequent or concurrent invasive disease and is an indication for the initiation of preemptive therapy to eradicate such colonization^ref (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). In contrast, colonization with Candida species does not correlate with the risk of invasive disease and is not an indication for antifungal therapy. There are currently two problems with this approach: the absence of such early colonization in a significant percentage of patients with invasive pulmonary aspergillosis and the lack of clear-cut information as to which is the best regimen for accomplishing preemptive therapy—amphotericin B, liposomal amphotericin, or voriconazole. Itraconazole appears to be less desirable for this task given less reliable pharmacokinetics and penetration into respiratory secretions^ref
• Protocol high resolution computerized tomographic scans of the lungs at regular intervals in HSCT patients during periods of high risk for invasive aspergillosis can result in earlier diagnosis of disease, and the opportunity for preemptive therapy. A particular morphology of the lung lesion, the so-called "halo sign," is particularly suggestive of invasive aspergillosis (although Aspergillus infection is the most common cause of a halo sign, such other opportunistic infections as nocardiosis, mucormycosis, and others can also cause a halo sign). Antifungal therapy with an amphotericin preparation or one of the newer drugs (see below) is indicated in this circumstance^ref (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press)
• Detection of fungal antigens (e.g., Aspergillus galactomannan) or fungal metabolites (e.g., arabinitol) could give early evidence of invasive infection at a time when therapy would be most effective. Of particular interest are PCR based assays that will detect both yeast and mold infection. Linkage of such results to optimized antifungal strategies appears to be the wave of the future once these assays achieve adequate sensitivity and specificity^{ref1, ref2, ref3, ref4} (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press)
• new antifungal drugs in the prevention of IFI : the licensure of the lipid associated amphotericin preparations has provided drugs with efficacy comparable to conventional amphotericin, but with considerably less toxicity, although cost issues remain of concern^ref (Boeckh M, Marr KA. Infection in hematopoietic stem cell transplantation. In Rubin RH YL, ed. Clinical approach to infection in the compromised host. New York: Plenum Press, 2001:in press). Also of great interest are two drugs that are in the late stages of clinical development. One of these, caspofungin acetate, the first of the echinocandins to be released for human use, has been approved for the salvage therapy of invasive aspergillosis. However, the preclinical data demonstrating broad-spectrum activity against yeasts and molds, and its potential utility in combination with other antifungal agents, suggest further benefits of this new agent^{ref1, ref2}. Particularly appealing is its side effect profile, both in terms of infusion related and organ specific toxicity. A potential disadvantage is its availability only in a parenteral formulation. Voriconazole, a triazole with fungicidal activity against Aspergillus species and potent fungistatic activity against a wide variety of yeasts and molds, including several of the newly emerging fungi resistant to presently available agents, appears especially well suited to the management and prevention of fungal infection in the HSCT patient^{ref1, ref2, ref3}. Particularly noteworthy is a large randomized study of invasive aspergillosis in immunocompromised patients, a large percentage of whom were HSCT recipients. In this study, voriconazole was significantly more efficacious than amphotericin, with significantly less toxicity (Herbrecht R, Denning D.W. , Patterson, T.F., et al. Open, randomized comparison of voriconazole and amphotericin B followed by other licensed antifungal therapy for primary therapy of invasive aspergillosis. 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2001). Table 8 provides a summary of major recommendations for prevention of fungal infection after hematopoietic cell transplantation. Summary of major recommendations for prevention of invasive fungal infection.

The prevention of invasive fungal infection in the HSCT recipient remains an important goal of transplant medicine. Accomplishing this task has been made difficult by the lack of sensitivity of diagnostic techniques and the lack of an ideal antifungal agent. Three different strategies have emerged to accomplish the task of antifungal prevention: prophylactic, empiric, and preemptive. Prophylactic fluconazole at a dose of 400 mg/day has been shown to decrease both the rate of invasive yeast infection and mortality. Thus far, no prophylactic strategy has been shown to decrease the incidence of invasive mold infection, and the emphasis is on environmental protection. Empiric therapy in the febrile neutropenic patient has been established as a standard of care, with amphotericin B, lipid associated amphotericin, and voriconazole appearing to offer comparable efficacy. Preemptive therapy based on microbiologic markers, particularly non-culture based microbiologic assays, appears to be an approach for the future.
• prevention of bacterial infections : bacterial infections may complicate HSCT at any time but pose a particular risk during the neutropenia produced by cytoreduction. The main sources of infection include central venous catheters, mouth flora, and gut flora via bacterial translocation. As with all patient care, attention to proper handwashing is strongly recommended to minimize risk of a bacterial infection (AIII) (Table 9)^ref

Summary of major recommendations for prevention of bacterial infections. In general, many decisions regarding use of prophylactic agents for bacterial infections must weigh the potential immediate benefit (prevention of infection) against the longer-term consequence (development of antibiotic resistance). Because of the potential severity of antibiotic-resistant infections, particularly among HSCT recipients, every effort should be made to use preventative antibacterials with restraint. The widespread development of VRE in most US hospitals is a recent reminder of how devastating drug-resistant bacteria may be.
• neutropenia : the neutropenia from cytoreduction results in risk for predictable organisms from bowel and mouth flora. Gut flora decontamination is not recommended (DIII) prior to HSCT given the lack of clear efficacy and its potential for fostering development of resistant organisms^{ref1, ref2, ref3, ref4}. Because of limited data, no recommendation can be made regarding routine use of antibiotics for bacterial prophylaxis in afebrile and asymptomatic neutropenic patients. In particular, vancomycin should not be used in this setting (DIII), since overuse of this drug may encourage development of VRE^ref. Routine use of growth factors may shorten neutropenia but does not appear to change the rate of serious bacterial infection and so is not recommended to prevent bacterial infections^ref
• central venous catheters : guidelines for care of central venous catheters recently have been promulgated to reduce infectious and other complications^{ref1, ref2}. Handwashing is recommended prior to changing dressings or manipulation at the site. Use of silver or antibiotic-impregnated catheters may result in lower rates of infection although no studies have been performed on indwelling or implantable devices in HSCT^{ref1, ref2}. Similarly, chlorhexidine-impregnated gauze dressings may reduce infection rates, although no studies of this approach have been completed in HSCT^ref. Antibiotic ointments at the exit site are not recommended^ref. Catheters should be removed as soon as is practical; for patients with transfusion requirements, however, the central venous catheter may be required for months.
• chronic graft-versus-host disease : patients with chronic GVHD are at risk for invasive infection from encapsulated organisms, particularly S. pneumoniae, although infection with Haemophilus influenzae and Neisseria meningitis may also occur. Appropriate prophylactic antibiotics directed at these pathogens should be selected based on local resistance patterns. These should be used until resolution of chronic GVHD. Intravenous immunoglobulin (IVIG) is not recommended (DII), unless the patient is among the 20-25% of HSCT recipients with unrelated marrow grafts who experience severe hypogammaglobulinemia in the first 100 days post-HSCT (CIII)^{ref1, ref2}
• specific organisms
• Streptococcus pneumoniae infection is a particular risk for HSCT recipients with chronic GVHD or those with profound hypogammaglobulinemia^{ref1, ref2}. 3 distinct strategies can help prevent invasive S. pneumoniae infection in specific groups: In patients with chronic GVHD, antibiotic prophylaxis is recommended for as long as GVHD therapy is administered (BIII)^{ref1, ref2}. Penicillin is the drug of choice in communities where S. pneumoniae remains likely to be penicillin-susceptible^ref. Trimethoprim-sulfamethoxazole, if given daily for pneumocystis prophylaxis, is adequate to prevent S. pneumoniae as well. However, no data were found to support its use for preventing S. pneumoniae disease. Vaccination with the currently available 23-valent vaccine may be of benefit to any HSCT patient and should be administered 12 and 24 months post transplant (BIII)^ref. The role of the new conjugate 7- valent vaccine has not been determined for HSCT recipients. Among those profound hypogammaglobulinemia, use of IVIG may be effective (CIII)^{ref1, ref2}.
• Streptococcus viridans : mucositis may facilitate translocation of S. viridans from the mouth into the blood. A full dental evaluation should therefore be conducted for all patients prior to initiating HSCT conditioning (AIII). Routine use of prophylaxis directed at S. viridans is not recommended (DIII) as such a strategy has not been shown to be effective and may promote the emergence of antibiotic-resistant organisms. At certain institutions, frequent cases of overwhelming shock due to S. viridans have been described^ref. In such a circumstance, prophylaxis might be considered (CIII). Selection of the best agent for prophylaxis should be made based on the hospital antibiotic susceptibility profile.
• Haemophilus influenzae : pediatric household contacts of HSCT recipients should be current with H. influenzae type B (Hib) vaccine (AII). Exposure to infected household contacts and persons in the house who themselves had been exposed to an active case of invasive H. influenzae should be minimized. Hib conjugate vaccine should be administered to all HSCT recipients at 12, 14, and 24 months post-HSCT (BII) because HSCT recipients have low levels of H. influenza capsular antibody post-transplant.16 This approach is particularly important among persons with chronic GVHD, whose risk for invasive H. influenzae is increased.
• prevention of protozoal infections
• Pneumocystis carinii pneumonia (PCP) : the potential risk of nosocomial PCP remains controversial. Although some hospitals place patients with proven PCP into contact precaution isolation, the value of this strategy has not been established. When possible, HSCT recipients should avoid contact with persons with PCP (CIII). Prophylaxis against PCP should be given to all allogeneic recipients from the time of engraftment until 6 months after HSCT (AII). Prophylaxis should be extended for those with chronic GVHD or those receiving immunosuppressive therapy for other indications until resolution of the underlying problem (BII)^{ref1, ref2}. In some centers, prophylaxis is given for 1-2 weeks prior to transplant. However, because no studies have been performed on this approach, no recommendations can be made. Summary of major recommendations for prevention of protozoal infections. Trimethoprim-sulfamethoxazole is the preferred prophylactic agent (AII). For those unable to receive trimethoprim-sulfamethoxazole, alternative agents include dapsone 100 mg daily^ref; atovaquone 1500 mg daily^ref, or aerosol pentamidine^ref (BIII). Although these second-line agents have not been formally compared, daily dapsone appears the preferred agent. Dapsone given three times weekly should not be used^ref. Leucovorin (folinic acid) is often administered with trimethoprim-sulfamethoxazole but no studies support the use of this costly medication^ref. PCP among autologous transplant recipients is rare but does occur^ref. Patients for whom prophylaxis might be considered are those who have received fludarabine or 2-CDA, those with underlying lymphoma or leukemia, or those receiving intense conditioning regimens or graft manipulation (BIII). No recommendation can be given for other patients. In determining the best approach for a given transplant center, the risk of an adverse event related to trimethoprim-sulfamethoxazole, such as fever and rash or myelosuppression, should be weighed against the potential benefit of preventing this rare but sometimes fatal pneumonia.
• Toxoplasma gondii : all HSCT recipients should be advised regarding the risk of acquiring toxoplasmosis via ingestion of undercooked meat or through exposure to cat feces (BIII). Toxoplasmosis is a substantial risk (8.5% incidence) for T-cell depleted graft recipients if the host is seropositive for T. gondii pre-transplant^ref.24 Importantly, however, cases of toxoplasmosis post-HSCT have been reported when both host and donor are seronegative for T. gondii, reflecting the limited sensitivity of the serologic test^ref.24 Disseminated toxoplasmosis does occur, although less commonly, in other transplant setting^ref. Prophylaxis might be considered for those with a history of invasive toxoplasmosis, particularly chorioretinitis, and for T. gondii seropositive patients with acute GVHD, but data demonstrating efficacy are limited (CIII). Trimethoprim-sulfamethoxazole is recommended for prophylaxis (BII). For those in whom trimethoprim-sulfamethoxazole cannot be given, clindamycin-pyrimethamine or pyrimethamine/sulfadoxine (Fansidar)^ref should be effective (CIII). Duration of prophylaxis should parallel that of PCP prophylaxis (CIII). Secondary prophylaxis should be given for the duration of immunosuppression (BIII). Autologous transplant recipients do not have discernible risk and should not receive prophylaxis (DIII) nor should they be screened for serologic evidence of previous infection (DIII).
• Strongyloides stercoralis : HSCT recipients should avoid situations, such as exposure to potentially infected soil that might result in acquisition of S. stercoralis (AIII). A thorough travel and residence history should be obtained for all HSCT candidates (BIII)^ref. Areas with risk for S. stercoralis include the Southeastern US, the tropics, the sub-tropics, and Europe. Any potential HSCT recipient with unexplained peripheral eosinophilia or those with travel or residence to endemic areas should be screened by serologic test for asymptomatic S. stercoralis (BIII). If the serologic screen is unavailable, examination of three stool specimens can be performed, although this approach is less sensitive (BIII). Patients with a positive serology, unexplained eosinophilia despite a negative serology, or a compelling history, should receive prophylaxis. Ivermectin is the preferred drug; if it is unavailable, thiabendazole may be given (BIII). No recommendation can be made for secondary prophylaxis. All infected patients should have  3 negative stools after completion of treatment, prior to beginning HSCT (AIII).
• Trypanosoma cruzii, which causes Chagas' disease, can be transmitted by blood transfusion and possibly via HSCT. It may also be transmitted congenitally. Because the infection is difficult to treat, potential donors should be screened for IgG against T. cruzii if they were born in, received a blood transfusion in, or ever lived for  6 months in Chagas' disease endemic areas, such as parts of Mexico, Central and South America (BIII)^ref (Altclas J, Sinagra A, Jaimovich G, et al. Reactivation of chronic Chagas' disease following allogeneic bone marrow transplantation and successful pre-emptive therapy with benznidazole. Transplant Infectious Dis. 1999;1:135–137). Persons with active diseases should not serve as donors (DIII).
• guidelines for active and passive immunizations for HSCT : for the vast majority of individuals, immunizations are safe, effective and inexpensive means to prevent infectious disease^{ref1, ref2} (Centers for Disease Control and Prevention. General recommendations on immunization. Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1994;43(RR-1):1–38). While it is difficult to overestimate the benefit of vaccines on world health, misconceptions and missed opportunities may abrogate this protective advantage^{ref1, ref2} (Plotkin SL, Plotkin SA. A short history of vaccination. In Plotkin SA, Mortimer EA Jr, eds. Vaccines. W B Saunders: Philadelphia; 1988). This appears especially true among individuals undergoing myeloablative HSCT, where care by multiple providers, faulty assumptions regarding immunocompetence or persistence of protective immunity and lack of immunization schedules contribute to underutilization or variability in best practices^{ref1, ref2, ref3}
• immunization of the immunocompromised host : myeloablative conditioning before HSCT impairs immune function, fosters opportunistic infection and dampens immunogenic responsiveness to vaccination^{ref1, ref2}. With passage of time, most patients recover adequate immune function, although the tempo of recovery can be delayed by older age, T-cell depletion, GVHD, anti-thymocyte globulin or other immunosuppressive agents^{ref1, ref2, ref3} (Lum LG. The kinetics of immune reconstitution after human marrow transplantation. Blood). Persisting immunodeficiency contributes to serious late infections, especially with encapsulated organisms^{ref1, ref2, ref3}. Since vaccine efficacy is lessened during the immunocompromised period but the tempo of immune recovery varies widely, it is understandable that standardized data on antibody response to vaccination following HSCT are limited. Moreover, since the number of HSCT patients enrolled in controlled vaccination trials has been small, published efficacy measuring the frequency of late infections has been scanty. Thus, guidelines of level II and III recommendations are based on either surrogate markers of protective antibody titers or clinical experience extrapolated from other patient groups^{ref1, ref2} (Centers for Disease Control and Prevention. Use of vaccines and immune globulins in person with altered immunocompetence. Recommendation of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1993;42(RR-4):10–18). Antibody titers persisting from childhood immunizations decrease after HSCT to barely protective or unprotective levels. This decline has been shown in allogeneic and autologous recipients of blood and marrow grafts^{ref1, ref2, ref3, ref4, ref5}. In general, HSCT recipients respond better to vaccinations if several are given at and after 1 year posttransplant^{ref1, ref2, ref3, ref4, ref5}. Unfortunately, responses are poorer for unconjugated pneumococcal polysaccharide vaccine (PPV) and unconjugatged Haemophilus influenzae type b (Hib) vaccine^{ref1, ref2, ref3, ref4}. Booster immunizations result in anamnestic antibody responses, but this benefit may be lost in those with chronic GVHD^{ref1, ref2, ref3}
• active immunization for HSCT recipients : as shown in Table 11, guidelines are provided for allogeneic and autologous recipients given myeloablative conditioning (Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients: recommendations of CDC, the Infectious Diseases Society of America, and the American Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2000;6:705–706). Major recommendations for vaccinations for hematopoietic stem cell transplant (HSCT) recipients, including both allogeneic and autologous recipients.For these guidelines, HSCT recipients are presumed immunocompetent at  24 months after HSCT if they are not on immunosuppressive therapy and do not have graft-versus-host-disease (GHVD).
• Diphtheria, tetanus and pertussis : vaccinations at 12, 14 and 24 months posttransplant are recommended for diphtheria and tetanus toxoid (BII) and pertussis in children < age 7 years (BIII)^{ref1, ref2, ref3, ref4}. Patients should be reimmunized for tetanus-diphtheria toxoids every 10 years (Centers for Disease Control and Prevention. Use of vaccines and immune globulins in person with altered immunocompetence. Recommendation of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1993;42(RR-4):10–18)
• Haemophilus influenzae type b. Hib conjugate vaccine is given 3 times beginning one year after HSCT (BII)^{ref1, ref2, ref3}. This is recommended for patients of any age (Centers for Disease Control and Prevention. Use of vaccines and immune globulins in person with altered immunocompetence. Recommendation of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1993;42(RR-4):10–18)
• Hepatitis B : immunization with the recombinant vaccine is recommended for those who are otherwise susceptible: children < age 18 or adults with risk factors for hepatitis B (BIII)^ref (Nagler A, Klan Y, Adler R, et al. Successful immunization of autologous bone marrow transplantation recipients against hepatitis B virus by active vaccination. Bone Marrow Transplant. 1995;15: 4750478). Persons who fail to exhibit a response should receive a second 3-dose series.
• Pneumococcal : the 23-valent PPV had been recommended at 12 and 24 months after HSCT along with chemoprophylaxis in individuals with chronic GVHD (BIII)^ref. Recently, a 7-valent conjugate vaccine has been licensed. No data are currently available in HSCT recipients, but one strategy could be to give one or more does of the 7-valent conjugate vaccine with subsequent broader coverage with the 23-valent product^ref.
• Influenza : beginning before HSCT and resuming > 6 months after transplant, individuals should receive life-long seasonal immunization (BII)^ref (Centers for Disease Control and Prevention. General recommendations on immunization. Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1994;43(RR-1):1–38; Centers for Disease Control and Prevention. Use of vaccines and immune globulins in person with altered immunocompetence. Recommendation of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1993;42(RR-4):10–18). Children < age 9 receiving vaccine for the first time require two doses. Those < 12 years old should receive only split-virus vaccines^ref. For optimal protection, immunization and chemoprophylaxis can be combined.
• polio : inactivated polio vaccine is recommended following allo and auto transplant (BII)^{ref1, ref2}. More information is needed regarding optimal schedules of vaccination.
• measles, mumps, rubella : MMR is a live-attenuated viral product and is indicated only in immunocompetent recipients at least 24 months posttransplant who are not receiving immunosuppressive drugs and are free of GVHD (BIII)^{ref1, ref2, ref3} (Centers for Disease Control and Prevention. Use of vaccines and immune globulins in person with altered immunocompetence. Recommendation of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1993;42(RR-4):10–18). No data are available for use before 24 months posttransplant.
• varicella : insufficient data are available to recommend this vaccine (EIII)^ref
• others : vaccinations for HSCT patients travelling to endemic areas are either contraindicated (live-attenuated vaccines) or not rated due to limited experience (Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients: recommendations of CDC, the Infectious Diseases Society of America, and the American Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2000;6:705–706)
• active immunization for family members and healthcare workers : as shown in Table 12, a number of recommendations apply to close contacts of the patient in order to prevent spread of infectious disease (Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients: recommendations of CDC, the Infectious Diseases Society of America, and the American Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2000;6:705–706) The published evidence supporting these recommendations is provided by the Centers for Disease Control and Prevention^ref. Major recommendations for vaccinations for family, close contacts, and health-care workers (HCWs) of hematopoietic stem cell (HSCT) recipients.
• passive immunization for HSCT recipients (Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients: recommendations of CDC, the Infectious Diseases Society of America, and the American Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2000;6:705–706) : CMV immunoglobulin is not indicated (DI) but VZIG is recommended (AII)^ref. Intramuscular immunoglobulin may be of benefit for those who travel to areas endemic for hepatitis A or were exposed to measles but not vaccinated after HSCT (BIII)^ref (Centers for Disease Control and Prevention. Prevention of Hepatitis A through active or passive immunization. Recommendation of the Advisory Committee on Immunization Practices (ACIP). MMWR. 1999;48 (RR-12): 1–37). Intravenous immunoglobulin can be administered to patients with persisting hypogammaglobulinemia (Ig < 400 mg/dl) and recurrent bacterial infection (CIII)^{ref1, ref2, ref3, ref4, ref5}. Major recommendations for use of passive immunization for hematopoietic stem cell transplant (HSCT) recipients exposed to vaccine-preventable diseases.
• secondary cancers : 17 cases of squamous cell carcinoma owing to oral GVHD in patients undergoing allogeneic HSCT^ref
• osteoporosis
There are no significant immunological or alterations in clinical benefit of post-transplant G-CSF administration in T-repleted allotransplant recipient^ref.
Whereas active HBV infection was non significantly (P=0.13) higher (11%) in the BMT patients in comparison to 3% in untransplanted patients, 53% of the HHV-5 / CMV-seropositive recipients developed CMV reactivation compared to 11% of the CMV-seronegative recipients who developed CMV seroconversion. In addition, 38% of the HBV-seropositive recipients developed HBV reactivation in comparison to 0% of the HBV-seronegative recipients. Moreover, 39% of the HCV-seropositive recipients developed HCV reactivation in comparison to 6% of the HCV-seronegative recipients who developed HCV seroconversion. Previous exposure to CMV, HBV, and HCV infections in the recipients of BMT patients influences the risk of developing those viral infections^ref.

• International Bone Marrow Transplant Registry (IBMTR)/Autologous Blood and Marrow Transplant Registry (ABMTR)
• Bone Marrow Donors Worldwide (BMDW) (updated every 4 months)
• Anthony Nolan Bone Marrow Trust (ANBMT)
• National Marrow Donor Program (NMDP)
• World Marrow Donor Association (WMDA)
• ABO Mismatch Reporting Center
• European Group for Blood and Marrow Transplantation (EBMT)
• Total Body Irridation (TBI) Registry
• British Society of Blood and Marrow Transplantation (BSBMT)
• American Society for Blood and Marrow Transplantation (ASBMT)
• Canadian Blood and Marrow Transplant Group (CBMTG)
• European Council for Accreditation in Hematology (ECAH)
• HAPLO.ORG - Haploidentical Stem Cell Transplantation
• International NetCord Organisation (NETCORD)
• International Society for Hemotherapy and Graft Engineering (ISHAGE)
• BMT Net: "a central resource of all blood and marrow transplantation -related topics"
• BMT Information
• journals :
• Biology of Blood and Marrow Transplantation
• Bone Marrow Transplantation
• Marrow Transplantation Reviews: Issues in Hematology, Oncology and Immunology

• E. Donnall Thomas (Editor), Karl G. Blume (Editor), Stephen J. Forman (Editor), Frederick R. Appelbaum (Editor), Stephen J. Jorman : Thomas' Hematopoietic cell transplantation ; 3rd edition (March 2004)
• E, Donnall Thomas (Editor), Karl G. Blume (Editor), Stephen J. Forman (Editor) : Hematopoietic Cell Transplantation; 2nd edition (January 2004)

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