In vitro induction of neoantigen-specific T cells in myelodysplastic syndrome, a disease with low mutational burden.

Therapies that utilize immune checkpoint inhibition work by leveraging mutation-derived neoantigens and have shown greater clinical efficacy in tumors with higher mutational burden. Whether tumors with a low mutational burden are susceptible to neoantigen-targeted therapy has not been fully addressed. To examine the feasibility of neoantigen-specific adoptive T-cell therapy, the authors studied the T-cell response against somatic variants in five patients with myelodysplastic syndrome (MDS), a malignancy with a very low tumor mutational burden. DNA and RNA from tumor (CD34+) and normal (CD3+) cells isolated from the patients' blood were sequenced to predict patient-specific MDS neopeptides. Neopeptides representing the somatic variants were used to induce and expand autologous T cells ex vivo, and these were systematically tested in killing assays to determine the proportion of neopeptides yielding neoantigen-specific T cells. The authors identified a total of 32 somatic variants (four to eight per patient) and found that 21 (66%) induced a peptide-specific T-cell response and 19 (59%) induced a T-cell response capable of killing autologous tumor cells. Of the 32 somatic variants, 11 (34%) induced a CD4+ response and 11 (34%) induced a CD8+ response that killed the tumor. These results indicate that in vitro induction of neoantigen-specific T cells is feasible for tumors with very low mutational burden and that this approach warrants investigation as a therapeutic option for such patients.

Adoptive transfer of neoantigen-specific T-cell therapy is feasible in older patients with higher-risk myelodysplastic syndrome.

BACKGROUND: Myelodysplastic syndromes (MDS) represent the most common type of acquired bone marrow failure in adults and is characterized by ineffective maturation of myeloid precursor cells and peripheral cytopenias associated with higher rates of infection, bleeding and transfusion dependence. In higher-risk patients with MDS who relapse or do not respond after standard hypomethylating agent (HMA) therapy, the 2-year survival rate is 15%. METHODS: Here the authors report the feasibility and safety of a novel experimental T-cell therapy called personalized adoptive cell therapy, which selects, immunizes and expands T cells against MDS-specific mutations and is targeted to patient-specific tumor cell neoantigens. Somatic mutations serve as the pathogenic drivers of cancer, including MDS, as these transformative genetic mutations may generate novel immunogenic proteins (i.e., neopeptides and possible neoantigens) that may be targeted therapeutically. RESULTS: The authors demonstrate that the adaptive immune system can be trained ex vivo to recognize neopeptides as neoantigens and that the infusion of culture-expanded, neoantigen-immunized autologous T cells has been feasible and safe in the three patients treated to date. DISCUSSION: The authors report on early results from their first-in-human phase 1 clinical trial that aims to assess the safety and tolerability of this novel form of adoptive T-cell immunotherapy for HMA-refractory patients with higher-risk MDS.

Augmentation of autologous T cell reactivity with acute myeloid leukemia (AML) blasts by Toll-like receptor (TLR) agonists.

This study investigated whether TNF-α, Toll-like receptors (TLRs) 7/8 agonist resiquimod (R848), the TLR4 agonist lipopolysaccharide (LPS) and their combinations can enhance autologous AML-reactive T cell generation in an in vitro culture. AML peripheral blood or bone marrow mononuclear cells were cultured in medium supplemented with GM-CSF/IL-4 to induce dendritic cell (DC) differentiation of AML blasts (AML-DC). The impact of TNF-α, LPS, R848 and their combinations on AML-DC cultures was analyzed. Significantly enhanced CD80, CD40, CD83, CD54, HLA-DR and CD86 expression of AML cells was observed by addition of TNF-α, LPS, R848 alone or combinations. Induced CD80 expression of AML cells was significantly higher through the combination of TNF-α, LPS and R848 (T + L + R) than that by T alone. CTL induced from T + L + R, T + R, T + L, L + R and R, but not T, L alone stimulated cultures showed significantly higher IFN-γ release than the medium control in response to autologous AML cells. IFN-γ release by T + L + R was significantly higher than T or L alone, and T + R was significantly higher than T alone. CTL generated from T + L + R, T + L, T + R, L + R and L alone exerted significantly higher AML cell killing than medium control. AML cell killing by T + L + R and T + R was significantly higher than T or R alone. These results indicate that the combination of T + L + R induces a significantly enhanced antigen presentation effect of AML-DC. We speculate that the complementary effects of reagent combinations may better address the heterogeneity of responses to any single agent in AML cells from different patients.

A mild acute hemolytic transfusion reaction in a patient with alloanti-Ge3: a case report and review of the literature.

BACKGROUND: The clinical significance of the Gerbich antibodies has been described as variable and there are no well-documented reports of hemolytic transfusion reactions (HTRs). CASE REPORT: We present the case of a woman with a long history of documented anti-Ge3 alloantibody who received multiple units of Ge+ red blood cells (RBCs) uneventfully. During the first admission to our hospital she was transfused 8 units of Ge+ RBCs and had a negative monocyte monolayer assay (MMA) before receiving the units. Within 2 weeks of the transfusions, the anti-Ge3 became significantly stronger by indirect antiglobulin test, and the MMA increased from 2.2 to 79.5% reactivity. She returned 4.5 years later with an emergent need for blood and was transfused with 2 units of Ge+ RBCs after premedication with steroids and intravenous immunoglobulin. RESULTS: The first unit was transfused without incident; however, she developed clinical and laboratory signs consistent with an acute HTR with the second unit. CONCLUSION: After a comprehensive review of the literature, we believe this to be the first well-documented case of acute HTR due to anti-Gerbich alloantibodies.

Study to determine the clinical significance of HEmolysis During Orbital AtheRectomy (CLEAR study).

PURPOSE: To evaluate the incidence of clinically evident hemolysis associated with orbital atherectomy used to treat severe peripheral artery disease. METHODS: The observational CLEAR study enrolled 31 subjects (16 men; mean age 71 ± 10 years, range 44-92) with claudication (58.1%) or critical limb ischemia (38.7%) who underwent orbital atherectomy with the Diamondback 360 system at 4 US centers. The 42 lesions in 31 limbs were located in the superficial femoral (n = 19, 45.2%), popliteal (n = 8, 19.0%), and tibial arteries (n = 15, 35.8%). The majority of lesions (34, 81.0%) were de novo; moderate or severe calcification was identified in 90.5% of cases. Lesion and procedural parameters were analyzed at a core laboratory. Blood samples were collected during and post procedure and analyzed for markers of hemolysis. The primary endpoint was the occurrence of clinically significant hemolysis. The secondary endpoints included the occurrence of any clinical symptoms/signs potentially related to hemolysis. Statistical analysis was performed to identify predictors for hemolysis. RESULTS: Laboratory evidence of hemolysis was seen in 11 (35.5%) subjects. No one met the clinical event criteria, and so the primary endpoint of the study was not reached. The secondary endpoints were hypertensive crisis (1, 3.2%) and transient hemoglobinuria (3, 9.7%). Lower glomerular filtration rates, calcified plaque, long atherectomy runs, and solid crown selection were independent predictors of hemolysis. CONCLUSION: There was no clinically significant hemolysis after orbital atherectomy. The results of this study will enable users to predict conditions that predispose to high levels of red cell hemolysis following orbital atherectomy and to take appropriate measures to limit its occurrence.

Toxicity-evaluation designs for phase I/II cancer immunotherapy trials.

OBJECTIVES: To facilitate more efficient Phase I/II cancer immunotherapy trials by incorporating statistically rigorous safety analysis. SETTING: The standard Phase I oncology trial is designed to find the maximum tolerated dose (MTD) in a setting where serious drug-related toxicity is expected. However, many newer agents hope to show the efficacy without increasing the background rate of adverse events. Formal statistical designs in this setting are needed. RESULTS: The Phase I/II toxicity-evaluation design is suitable when the therapeutic dose is expected to be well below the MTD. In Phase I, the design enrolls multiple cohorts at the target dose, possibly after an initial dose titration stage, and tests a formal safety hypothesis using a standard 3 + 3 enrollment scheme. Phase I serves as an interim safety analysis before proceeding to Phase II efficacy testing. We give an exact upper confidence limit on the toxicity rate at the therapeutic dose using the combined Phase I/II toxicity data, as well as the maximum likelihood estimate of the toxicity rate. We describe an example where the design has been used for a Phase I/II trial of immunotherapy in leukemia. CONCLUSIONS: Phase I/II toxicity-evaluation designs are simple to execute and may be suitable for some cancer immunotherapy trials. We show how to compute power, expected sample size, and expected number of dose-limiting toxicities, as well as the maximum likelihood estimator and exact small sample confidence intervals for the toxicity rate at the therapeutic dose. More flexible designs are briefly discussed.

Autologous hematopoietic stem cell transplantation as an intensive consolidation therapy for adult patients in remission from acute myelogenous leukemia.

Autologous peripheral blood stem/progenitor cell transplantation (APBSCT) has been investigated as a potential therapeutic option to improve outcome in patients with acute myelogenous leukemia (AML). However, its optimal role in treatment for adults in remission has not been clearly established. We performed a retrospective analysis on 45 patients aged 21 to 73 years (median 51 years) with de novo AML who underwent APBSCT stratified by age, complete remission status, and cytogenetic risk. The 5-year disease-free survival (DFS) for all patients was 33.9% (95% confidence interval [CI], 20.1%-53.7%) and overall survival (OS) was 43.6% (CI, 29.2%-62.8%). For patients under the age of 60 years, the 5-year DFS for intermediate and high cytogenetic risk was 53.3% (CI, 23.5%-85.6%) and 50.0% (CI, 16.1%-100.0%); the 5-year OS for patients under the age of 60 years with low, intermediate, and high cytogenetic risk was 80.0% (CI, 40.0%-100.0%), 60.0% (CI, 31.2%-90.7%), and 75.0% (CI, 39.0%-100.0%), respectively. For patients over the age of 60 years, the 5-year DFS and OS for intermediate cytogenetic risk was 21.4% (CI, 7.9%-58.4%) and 21.4% (CI, 7.9%-58.4%). The DFS and OS of these patients are comparable to the historic survival of those who underwent allogeneic stem cell transplantation when adjusted by age. In addition, there was no treatment-related mortality (TRM). We conclude that APBSCT is a reasonable and safe intensive consolidation for patients with AML who do not have a suitable HLA-matched donor.

Liquid storage of marrow stromal cells.

BACKGROUND: Marrow stromal cells (MSCs) for clinical trials are inevitably stored before administration, but little is known about the effects of storage on MSCs. The effects of short-term liquid storage on the in vitro function of MSCs intended for a clinical trial were studied. STUDY DESIGN AND METHODS: Early-passage human MSCs were suspended in 0.9% saline or culture medium and stored at 4 degrees C or room temperature for up to 72 hours followed by assessment of cell loss, viability, and growth in culture. RESULTS: When stored in saline at 4 degrees C, MSC counts decreased by 5% to 20%, MSC viability decreased 17% to 37%, and MSC growth decreased 65% to 100% after 24-hour storage. Similar results were obtained by MSC storage at room temperature or in Dulbecco's modified Eagle's medium or by addition of 1% human serum albumin (HSA) from two manufacturers. Storage of MSCs in saline with HSA from a third manufacturer maintained MSC viability at prestorage levels and improved poststorage MSC growth versus saline (32 +/- 9% vs. 9 +/- 9%; p < 0.05) or saline with two other HSA preparations (4 +/- 4 and 8 +/- 11%; p < 0.05). CONCLUSION: MSCs stored at 4 degrees C or room temperature, in saline or culture medium, rapidly become nonviable. HSA preparations vary significantly in their ability to maintain poststorage MSC viability and growth. These results provide insight regarding the effects of storage on MSCs and indicate the need to screen HSA preparations before their use as additives to preserve MSCs during short-term liquid storage.

Collection and preservation of cord blood for personal use.

Unrelated-donor umbilical cord blood (CB) is a useful alternative hematopoietic stem cell source for patients without suitably matched and readily available related or unrelated stem cell donors. Expectant parents today may have the option of either donating the CB to a public CB bank or keeping and storing the CB in a private bank for potential use in the future. The alternatives are often referred to as public banking and private banking. On behalf of the American Society of Blood and Marrow Transplantation (ASBMT), we have reviewed the currently available data and opinions and offer the following recommendations: The committee acknowledges the expanding potential of indications for CB in the future, and suggests review of these recommendations at regular intervals.

Generation of T-cell lines to autologous acute myeloid leukemia cells by competitive limiting dilution culture of acute myeloid leukemia mononuclear cells.

OBJECTIVE: To develop a novel method of generating multiple autologous acute myeloid leukemia (AML) reactive T-cell lines as a step toward adoptive immunotherapy for AML. MATERIALS AND METHODS: AML peripheral blood mononuclear cells (MNC), including >90% AML blasts and 1% to 3% T cells, were seeded in limiting dilution culture in which AML blasts were induced to undergo dendritic cell (DC) differentiation. T cells were primed and activated with the addition of a cytokine combination. RESULTS: Highly reactive anti-AML T-cell lines (both CD4(+) and CD8(+)) were generated, selected, and expanded. The estimated average frequency of AML-reactive T cells or precursors was 6 +/- 3/1,000,000 AML peripheral blood mononuclear cells (n = 11). Robust intracellular interferon-gamma (IFN-gamma) release from T-cell lines was demonstrated by flow cytometry after stimulation by autologous AML cells, but not an autologous B-lymphoblastoid cell line (LCL). These T-cell lines caused specific lysis of autologous AML cells, but not autologous LCL or allogeneic AML cells, and they depleted autologous AML colony-forming cells (CFC), but not normal CFC. Most CD4(+) T-cell lines exerted strong proapoptotic effects on AML cells. AML cell apoptosis by CD4(+) T-cell lines correlated with IFN-gamma secretion. CONCLUSION: This study demonstrates a methodology for generating large numbers of AML-reactive cytotoxic T cell lines (either class I or II restricted) that may be useful clinically in adoptive immunotherapy. This study also provides estimates of AML-reactive T-cell frequency in patients with AML.

Peripheral blood progenitor cell mobilization with intermediate-dose cyclophosphamide, sequential granulocyte-macrophage-colony-stimulating factor and granulocyte-colony-stimulating factor, and scheduled commencement of leukapheresis in 225 patients undergoing autologous transplantation.

BACKGROUND: Interpatient variability in the kinetics of peripheral blood progenitor cell (PBPC) mobilization is commonly seen with conventional chemotherapy-based mobilization regimens. This necessitates the availability of leukapheresis (LP) facilities 7 days a week. STUDY DESIGN AND METHODS: The efficacy of an approach where LP was invariably commenced on Day 11 after intermediate-dose cyclophosphamide followed by sequential administration of granulocyte-macrophage-colony-stimulating factor (CSF) and granulocyte-CSF (Cy/GM/G) was retrospectively analyzed in 225 consecutive, unselected patients undergoing autologous hematopoietic stem cell transplantation for all diagnoses other than acute leukemia at our center. Cy/GM/G was scheduled to avoid weekend LP. RESULTS: After Cy/GM/G, a CD34+ cell yield of at least 2.0x10(6) per kg was achieved in 90.7 percent of patients. Optimal yield (OY; >or=5x10(6) or 10x10(6) CD34+ cells/kg depending on diagnosis) was achieved in 67.6 percent of patients. Only three patients (1.3%) required LP on Saturday or Sunday. Febrile neutropenia (FN) was encountered in 5.3 percent. PBPC yield was highest on Day 1 of LP (p<0.001). In multivariate analyses, platelet (PLT) count on Day 1 of LP (PLT-D1LP) was positively associated with achievement of OY (p<0.001). PLT-D1LP and diagnosis of myeloma were associated with a shorter time to achieve a CD34+ cell yield of at least 5x10(6) per kg (p<0.001 and p=0.002, respectively). CONCLUSION: Cy/GM/G with scheduled LP commencement on Day 11 enables optimal CD34+ cell yields in most patients undergoing autologous transplantation, despite a low risk of FN and avoidance of weekend LP.

Outcomes of 122 diverse adult and pediatric cord blood transplant recipients from a large cord blood bank.

BACKGROUND: Umbilical cord blood is a useful stem cell source for some patients. The American Red Cross Cord Blood Program was established as a national network of cord blood banks. Nine thousand cord blood units were cryopreserved for transplant use. STUDY DESIGN AND METHODS: This report summarizes the experience with the first 125 cord blood units that have been distributed for transplant for 122 patients at 36 different transplant centers worldwide. Patients were treated with a variety of conditioning regimens. RESULTS: Most patients had acute myelogeneous leukemia (21%), genetic disorders (22%), or acute lymphoblastic leukemia (18%). The median age of the patients was 11 years with a range of 2 months to 63 years. The patients ranged in size from 3 to 120 kg (median, 39 kg). The median number of days to neutrophil engraftment was 22, and the median number of days to platelet engraftment was 63. Thirty percent of patients experienced Grades III to IV acute graft-versus-host disease (GVHD). Survival at 1 year after transplant was 35 percent, with recurrent disease the major cause of death. In multivariate analysis, only age less than 18 years was a significant predictor for improved survival. Forty-two percent of patients were non-Caucasian. Engraftment, GVHD, survival, and disease-free survival were similar among Caucasian and non-Caucasian patients. CONCLUSION: Umbilical cord blood serves as a satisfactory stem cell source for a diverse group of pediatric and adult patients.

Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro.

Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, and direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and, (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes (alpha-globin, betaH-1 globin, beta-major globin, epsilon -globin, and zeta-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, epsilon-globin, gamma-globin, betaH1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies on the ES cells of secondary EB origin cultured in media containing dexamethasone showed a down-regulation of GATA-3 and an up-regulation of GATA-1, Flk-1, and Epo-R in comparison to the two other cytokines and growth factor combinations containing media. The secondary differentiation also showed an enhanced production of erythrocytic precursors in dexamethasone containing media in comparison to that in the control media. Our results indicate that dexamethasone can prove to be an effective agent which can be employed to enhance differentiation towards erythrocytic progenitors from ES cells.

Non-anthracycline based remission induction therapy for newly diagnosed patients with acute myeloid leukemia aged 60 or older.

We assessed remission rates and toxicity in 24 consecutive elderly (age>or=60) patients with untreated Acute myeloid leukemia (AML) who received the anthracycline-free combination of fludarabine, cytosine arabinoside and G-CSF (FLAG) as initial induction chemotherapy at our center. CR was achieved following one cycle of FLAG in 14 patients (58%). Another four patients cleared blasts from their bone marrow by day 30 without complete platelet recovery. Three patients died from infections prior to neutrophil recovery (12%). No other grade 3/4 toxicities and no clinically significant mucositis were seen. No significant association was found between age, WBC and cytogenetic risk group with likelihood of achieving CR. Fifteen patients proceeded to consolidation therapy and seven patients received a stem cell transplant (six autologous, one allogeneic). Primary induction with FLAG in elderly AML patients achieves a high remission rate without prohibitive mucosal or cardiac toxicity and may thus be considered as an alternative to standard anthracycline-based regimens in this setting.

Comparison of cytomegalovirus polymerase chain reaction and serology for screening umbilical cord blood components.

BACKGROUND: Recipients of umbilical cord blood (UCB) transplants are susceptible to opportunistic infections, including cytomegalovirus (CMV). To prevent CMV transmission from UCB donors, most laboratories perform serology on corresponding maternal samples and quarantine units when the mother has immunoglobulin M (IgM) anti-CMV. STUDY DESIGN AND METHODS: UCB units and associated samples (UCB plasma and red cell pellet; maternal whole blood and serum) from two cord blood banks were tested with two validated CMV polymerase chain reaction assays (UL54 and UL93 targets). Results were compared with maternal CMV serology (IgG and IgM). RESULTS: Only 4 of 48 (8.3%) quarantined CMV IgM-positive units were also CMV nucleic acid testing (NAT)-positive (651-68,600 copies/mL). In contrast, 1 of 200 "CMV-safe" UCB units (CMV IgM-equivocal or -negative) had CMV DNA (0.5%). The corresponding maternal samples were CMV NAT-negative. Positive maternal IgM serology demonstrates only modest sensitivity (80%) and specificity (82%) and poor positive predictive value (8%), when correlated with the presence of CMV DNA in UCB units. CONCLUSION: CMV NAT may be a useful adjunct to serologic screening, potentially reducing wastage of IgM-positive and NAT-negative units while also detecting potentially infectious units that would pass serologic screening. A prospective clinical trial to further evaluate the role of CMV NAT in UCB transplantation appears warranted.

Racial diversity with high nucleated cell counts and CD34 counts achieved in a national network of cord blood banks.

Banked, unrelated, partially HLA-matched, umbilical cord blood is an alternative stem cell source for patients in need of transplantation therapy who lack traditionally matched donors. A presumed advantage of cord blood is the ability to increase recruitment of donors of minority ethnic backgrounds. The American Red Cross Cord Blood Program was established in 1999 with 6 banks and 10 collection sites throughout the country. Cord blood donors self-report racial designations on questionnaires, and donor race was collected from each site. Postprocessing nucleated cell counts and CD34(+) counts were obtained on the cord blood units, and results from each racial group (white, black, Asian, Hispanic, and Native American) were compared in the natural logarithmic scale by using analysis of variance. A total of 18878 donors consented: 64% white, 16% black, 12% Hispanic, 4% Asian, 1% Native American, and 3% other. The Detroit area consented the highest percentage of black donors (87%), San Diego consented the highest percentage of Hispanic donors (59%), and Oakland consented the highest percentage of Asian donors (15%). Seven thousand eight hundred sixty-six cord blood units have been banked for transplantation. The mean preprocessing nucleated cell count was 1220 x 10(6) (range, 327-7300 x 10(6)). There was no difference among racial groups when controlled for site (P =.395). The mean CD34(+) count was 3.28 x 10(6). Blacks had a significantly lower CD34(+) count than the other racial/ethnic groups in the Midwest, Northwest, and North Carolina collection sites. A racially diverse cord blood bank can be achieved. Nucleated cell counts were similar among the different racial/ethnic groups. CD34(+) counts were lower for blacks in some collection sites.

Retention of cellular properties of PBPCs following liquid storage and cryopreservation.

BACKGROUND: G-CSF-mobilized PBPCs are routinely cryopreserved within 24 hours of collection. The ability to hold PBPCs for extended time would offer increased flexibility for patients and hospitals. Retention of PBPC properties following overnight shipping, extended liquid storage at 1 to 6 degrees C, and cryopreservation was evaluated. STUDY DESIGN AND METHODS: PBPCs were stored in liquid at 1 to 6 degrees C up to 3 days, with and without shipping, and then cryopreserved in HES (6%), DMSO (5%), and HSA (4%). Thawed samples were assayed after two procedures, on dilution and after dilution and washing. Nucleated cells, viability, CD34+ cell number, committed progenitor colonies, and long-term culture-initiating cells were measured. RESULTS: CD34+ cell number, committed colony-forming cells, and long-term culture-initiating cells were essentially maintained when samples were stored in liquid for 1, 2, or 3 days before cryopreservation or after thawing and dilution. Nevertheless, significant (p < 0.05, paired t test) losses in total nucleated cell numbers were observed if thawed PBPC samples were washed before assay. CONCLUSION: PBPCs can be maintained at 1 to 6 degrees C for up to 3 days and can be cryopreserved after extended storage with properties minimally altered. Dilution alone, without centrifugation and washing, of thawed PBPC samples is a satisfactory procedure for preparing samples for in vitro assays.

In utero or ex utero cord blood collection: which is better?

BACKGROUND: The relative nucleated cell count of umbilical cord blood (CB) correlates with improved engraftment and survival. This study compares two collection methods to assess CB content, including cell numbers. STUDY DESIGN AND METHODS: The Massachusetts CB bank used trained obstetricians and midwives to collect CB in utero before the delivery of the placenta. The banks in California, Ohio, Oregon, and Minnesota used trained American Red Cross (ARC) personnel who collected CB ex utero after the delivery of the placenta. All banks processed CB by RBC sedimentation and volume reduction. RESULTS: The volume and total nucleated cell count of collected CB before processing, as well as after processing CFU-GM and CD34+ cells, showed no advantage of either method. In utero collections resulted in more rejections of collected units (due to labeling problems, bacterial contamination, clotting, and delay between collection and processing) than ex utero collections. There were fewer medical exclusions after in utero collection. CONCLUSION: CB can be collected successfully using either the in utero or ex utero methods; both methods produce comparable nucleated cell, MNC, CD34+, and CFU-GM numbers. Bacterial contamination, low volume, clotting, and delay until processing are generally higher with in utero collection.

Mobilization of allogeneic peripheral blood progenitor cells.

It is possible to reliably obtain sufficient PBSC from most normal donors to perform allogeneic transplantation. The mobilization regimen, usually administration of a single daily dose of G-CSF at 7.5 to 10 micrograms/kg subcutaneously for 4 to 6 days, is tolerable with acceptable side effects. However, there is wide variability among individuals with respect to the extent of mobilization achieved by the regimen and the optimal timing of apheresis. Studies suggest that the likelihood of obtaining an adequate harvest of CD34+ cells, as defined locally may be enhanced by employing higher doses or different schedules of G-CSF, monitoring the mobilization and/or collection of PBPC, and using apheresis procedures processing 2 or more times blood volume. However, an optimal regimen for mobilization and harvesting for all donors has not yet been identified and a small percentage of donors may not mobilize adequately with G-CSF. Alternative regimens employing combinations of G-CSF and GM-CSF are available that may prove useful in such cases and novel cytokines that are even more effective than G-CSF in mobilizing stem cells are eagerly awaited. Based on currently available experience with normal donors, the short-term safety of G-CSF appears to be acceptable, however there exist several scenarios in which marrow harvesting may be preferable to G-CSF mobilization and apheresis collection of PBPC.