Terumo spectra optia leukapheresis of cynomolgus macaques for hematopoietic stem cell and T cell collection

Macaques are physiologically relevant animal models of human immunology and infectious disease that have provided key insights and advanced clinical treatment in transplantation, vaccinology, and HIV/AIDS. However, the small size of macaques is a stumbling block for studies requiring large numbers of cells, such as hematopoietic stem cells (HSCs) for transplantation, antigen‐specific lymphocytes for in‐depth immunological analysis, and latently‐infected CD4+ T‐cells for HIV cure studies. Here, we provide a detailed protocol for collection of large numbers of HSCs and T‐cells from cynomolgus macaques as small as 3 kg using the Terumo Spectra Optia apheresis system, yielding an average of 5.0 × 10⁹ total nucleated cells from mobilized animals and 1.2 × 10⁹ total nucleated cells from nonmobilized animals per procedure. This report provides sufficient detail to adapt this apheresis technique at other institutions, which will facilitate more efficient and detailed analysis of HSCs and their progeny blood cells.     

Engraftment after autologous hematopoietic stem cell transplantation in patients mobilized with Plerixafor: A retrospective,multicenter study of a large series of patients

Plerixafor (PLX) appears to effectively enhance hematopoietic stem-cell mobilization prior to autologous hematopoietic stem cell transplantation (auto-HCT). However, the quality of engraftment following auto-HCT has been little explored. Here, engraftment following auto-HCT was assessed in patients mobilized with PLX through a retrospective, multicenter study of 285 consecutive patients. Information on early and 100-day post-transplant engraftment was gathered from the 245 patients that underwent auto-HCT. The median number of PLX days to reach the stem cell collection goal (≥2 × 10⁶ CD34⁺ cells/kg) was 1 (range 1–4) and the median PLX administration time before apheresis was 11 h (range 1–18). The median number of apheresis sessions to achieve the collection goal was 2 (range 1–5) and the mean number of CD34⁺ cells collected was 2.95 × 10⁶/kg (range 0–30.5). PLX administration was safe, with only 2 mild and transient gastrointestinal adverse events reported. The median time to achieve an absolute neutrophil count (ANC) >500/μL was 11 days (range 3–31) and the median time to platelet recovery >20 × 10³/μL was 13 days (range 5–69). At 100 days after auto-HCT, the platelet count was 137 × 10⁹/L (range 7–340), the ANC was 2.3 × 10⁹/L (range 0.1–13.0), and the hemoglobin concentration was 123 g/L (range 79–165). PLX use allowed auto-HCT to be performed in a high percentage of poorly mobilized patients, resulting in optimal medium-term engraftment in the majority of patients in whom mobilization failed, in this case mainly due to suboptimal peripheral blood CD34⁺ cell concentration on day +4 or low CD34⁺ cell yield on apheresis.     

Analysis of factors associated with successful allogeneic peripheral blood stem cell collection in healthy donors

BackgroundThe collection of a sufficient number of stem cells is important for success of allogeneic hematopoietic stem cell transplantation (HSCT). This study aimed to investigate the factors associated with successful allogeneic peripheral stem cell (PBSC) collection in healthy donors.MethodsWe retrospectively reviewed clinical data of allogeneic PBSC collection in 175 donors from 2007 to 2017 at the National Cancer Center, Korea. This study analyzed factors associated with the CD34+ cell yield such as the characteristics of donors, including age, laboratory results before apheresis, and data of procedures on the first day. The CD34+ cell dose of ≥ 4.0 × 10⁶/kg have recently been the accepted minimum recommended dose in allogeneic HSCT settings, and this was the target dose in our study.ResultsThe factors associated with the CD34+ cell yield were age (p = 0.007), baseline platelet (PLT) (p = 0.014), and pre-collection hematopoietic progenitor cells (HPCs) (p = 0.001) by multivariate analysis. This study represented that age, baseline platelet count, and pre-collection HPC count are important predictive factors as shown in other previous studies.ConclusionOur data suggest that young age, high baseline platelet counts and high HPC counts before collection might be useful for identifying successful mobilizers.     

Poor harvest of peripheral blood stem cell in donors with microcytic red blood cells

BACKGROUND: The outcome of peripheral blood stem cell (PBSC) harvest depends on mobilization and leukapheresis. Some poor harvests might not be directly related to poor mobilizations. STUDY DESIGN AND METHODS: We retrospectively analyzed the results of 793 consecutive healthy donors who underwent PBSC donation to evaluate the impact of low mean corpuscular volume (MCV) of red blood cells on the outcomes of hematopoietic stem cell mobilization and leukapheresis. RESULTS: The circulating CD34+ cells in peripheral blood after five doses of granulocyte–colony‐stimulating factor injection were similar in donors with low MCV and those with normal MCV (68.0 × 10⁶/L vs. 69.2 × 10⁶/L, p = 0.38). The procedural settings were not different between the two groups. However, the apheresis outcome of donors with low MCV was significantly lower in total CD34+ cells, cell dose, apheresis yield, and collection efficiency than those with normal MCV (277.6 × 10⁶ vs. 455.0 × 10⁶; 4.9 × 10⁶/kg vs. 8.2 × 10⁶/kg; 16.9 × 10⁶/L vs. 27.3 × 10⁶/L; 0.285 vs. 0.388; all p < 0.0001). Similar results were noticed in subgroup analysis using the severity of microcytosis and Mentzer index for the donors with MCV of less than 80 fL. The collection efficiency was significantly correlated with the MCV (r = 0.30, p < 0.0001). CONCLUSION: Low MCV was associated with poor apheresis outcomes in PBSC donors. This effect is not related to poor mobilization of CD34+ cells into the peripheral blood. Further studies to elucidate the detailed mechanism and develop strategy to avoid poor harvest are necessary.     

Plerixafor combined with G-CSF for stem cell mobilization in children qualified for autologous transplantation- single center experience

Failure of autologous peripheral blood CD34⁺ stem cells collection can adversely affect the treatment modality for patients with hematological and nonhematological malignant diseases where high dose chemotherapy followed by hematopoietic stem cell transplantation has become part of their treatment. Plerixafor in conjunction with G-CSF is approved for clinical use as a mobilization agent. The clinical efficacy of Plerixafor in CD34⁺ cells collection was analyzed in our institution. A total of 13 patients aged 1–15,5 years received Plerixafor in combination with G-CSF: 7 with neuroblastoma, 2 with Ewing’s sarcoma and single patients with Hodgkin’s lymphoma, germ cell tumor, retinoblastoma and Wilms tumor. Twelve patients (923%) achieved CD34+ cell counts of ≥ 20 × 10⁶/L after 1–7 doses of Plerixafor. The average 9,9 - fold increase in number of CD34⁺ cells were achieved following the first dose and 429 - fold after second dose of plerixafor. Among the 13 patients, 12 yielded the minimum required cell collection of 2 × 10⁶/kg within an average of 2 doses of Plerixafor. The mean number of apheresis days was 1.75. The median total number of collected CD34⁺ cells was 982 × 10⁶/kg. Plerixafor enables rapid and effective mobilization, and collection of sufficient number of CD34⁺ cells.     

Effective collection of peripheral blood stem cells in children weighing 20 kilogram or less in a single large‐volume apheresis procedure

Introduction : Peripheral blood stem cell (PBSC) transplantation has become a routine procedure in pediatric oncology. A special group of PBSC donors are children weighing 20 kg or less. Limited vascular access and low blood volume puts them at a higher risk. Central line placement and a priming apheresis machine are recommended to avoid these complications. Patients and Methods : PBSC collections performed from July 2006 to May 2013 in children weighing less than 20 kg were included. All donors had a central venous catheter (CVC). An apheresis machine was primed with packet red blood cells. Results : Twenty‐seven PBSC collections were performed in 22 children weighing 20 kg or less, 14 for allogeneic and 8 for autologous transplantation, in order to collect at least 2 × 10⁶ CD34+ cells/kg. In the allogeneic group, median age and weight were 3 years (0.8–7) and 15.5 kg (8–20). In the autologous group, median age and weight were 3 years (2–7) and 15.35 kg (12.5–19.5). A single large‐volume apheresis was sufficient to obtain the CD34+ cells needed in 78.5% and 75% of the allogeneic and autologous groups, respectively, with a median 11.84 × 10⁶ and 5.79 × 10⁶ CD34+ cells collected per kilogram of weight of the recipient. No serious complications related to the apheresis procedure or CVC placement occurred. Conclusion : PBSC collection in a single large‐volume apheresis for allogeneic and autologous transplants in children weighing 20 kg or less is a safe and effective procedure when based on standardized protocols. J. Clin. Apheresis 30:281–287, 2015. © 2014 Wiley Periodicals, Inc.     

A prospective clinical trial evaluating the safety and efficacy of the combination of rituximab and plerixafor as a mobilization regimen for patients with lymphoma

BACKGROUND: Previous reports suggested that rituximab may impair stem cell collection and posttransplant engraftment in lymphoma patients undergoing autologous hematopoietic progenitor cell transplantation. STUDY DESIGN AND METHODS: A prospective biologic allocation study examined the effect of adding rituximab to a mobilization regimen of plerixafor and granulocyte–colony‐stimulating factor (G‐CSF) for patients with CD20+ lymphoma compared with CD20? lymphoma patients mobilized without rituximab. The primary endpoint was safety of the rituximab‐containing regimen; secondary endpoints compared the efficiency of stem cell collection, posttransplant engraftment, graft characteristics, mobilization kinetics, immune reconstitution, and engraftment durability between the cohorts of patients with CD20+ and CD20? lymphoma. RESULTS: Fifteen subjects assigned to each treatment arm were accrued. Both mobilization regimens had similar toxicities. The median number of CD34+ cells collected (7.4 × 10⁶/kg vs. 6.4 × 10⁶/kg) and the median numbers of days of apheresis needed to collect stem cells were not different between the CD20+ and CD20? cohorts. No significant differences in neutrophil engraftment (median, 13.5 days vs. 13 days) or platelet engraftment (22 vs. 21 days) or in graft durability were seen comparing patients with CD20+ versus CD20? lymphoma. There were no significant differences in the kinetics of blood T‐cell or natural killer–cell reconstitution comparing the two groups. B‐cell reconstitution was delayed in the CD20+ lymphoma group, but this did not translate into a significant increase in infectious complications. CONCLUSION: Rituximab can be safely added to the combination of plerixafor and G‐CSF as a mobilization strategy without excess toxicity or posttransplant engraftment delays for patients with chemosensitive lymphoma undergoing autologous hematopoietic progenitor cell transplantation.     

A randomized clinical trial comparing granulocyte-colony-stimulating factor administration sites for mobilization of peripheral blood stem cells for patients with hematologic malignancies undergoing autologous stem cell transplantation

BACKGROUND: A study was undertaken to investigate whether granulocyte–colony‐stimulating factor (G‐CSF) injection in lower adipose tissue–containing sites (arms and legs) would result in a lower exposure and reduced stem cell collection efficiency compared with injection into abdominal skin. STUDY DESIGN AND METHODS: We completed a prospective randomized study to determine the efficacy and tolerability of different injection sites for patients with multiple myeloma or lymphoma undergoing stem cell mobilization and apheresis. Primary endpoints were the number of CD34+ cells collected and the number of days of apheresis. Forty patients were randomly assigned to receive cytokine injections in their abdomen (Group A) or extremities (Group B). Randomization was stratified based on diagnosis (myeloma, n = 29 vs. lymphoma, n = 11), age, and mobilization strategy and balanced across demographic factors and body mass index. RESULTS: Thirty‐five subjects were evaluable for the primary endpoint: 18 in Group A and 17 in Group B. One evaluable subject in each group failed to collect a minimum dose of at least 2.0 × 10⁶ CD34+ cells/kg. The mean numbers of CD34+ cells (±SD) collected were not different between Groups A and B (9.15 × 10⁶ ± 4.7 × 10⁶/kg vs. 9.85 × 10⁶ ± 5 × 10⁶/kg, respectively; p = NS) after a median of 2 days of apheresis. Adverse events were not different between the two groups. CONCLUSION: The site of G‐CSF administration does not affect the number of CD34+ cells collected by apheresis or the duration of apheresis needed to reach the target cell dose.     

Evaluation of a new protocol for peripheral blood stem cell collection with the Fresenius AS 104 cell separator

In this report we analyzed sixty leukapheresis procedures on 35 patients with a new protocol for the Fresenius AS 104. Yields and efficiencies for MNC, CD 34+ cells, and CFU-GM indicate that the new protocol is able to collect large quantities of hemopoietic progenitors. Procedures were performed processing 8.69 ± 2.8 liters of whole blood per apheresis and modifying 3 parameters: spillover-volume 7 ml, buffy-coat volume 11.5 ml, centrifuge speed 1,500 rpm; blood flow rate was 50 ml/min and the anticoagulant ratio was 1:12. No side effects were observed during apheresis procedures except for transient paresthesia episodes promptly resolved with the administration of calcium gluconate. Yields show a high capacity of the new program to collect on average MNC 17.28 ± 10.85 × 10⁹, CD 34+ 471 ± 553.5 × 10⁶ and CFU-GM 1278.7 ± 1346.3 × 10⁴ per procedure. Separator collection efficiency on average was 49.91 ± 23.28% for MNC, 55.1 ± 35.66% for CFU-GM, and 62.97 ± 23.09% for CD 34+ cells. Particularly interesting are results for MNC yields and CD 34+ efficiency; these results make the new program advantageous or similar to the most progressive blood cell separators and capable to collect a sufficient number of progenitor cells for a graft with a mean of 1.80 ± 0.98 procedures per patient. J. Clin. Apheresis 12:82–86, 1997. © 1997 Wiley-Liss, Inc.     

Optimizing pediatric peripheral blood stem cell collection

IntroductionPediatric PBSC harvests pose specific challenges during apheresis and a knowledge of the same and variables affecting PBSC collection are very important in planning these procedures. In the present study safety profile of pediatric PBSC procedures and variables influencing the successful collection were analyzed.MethodPediatric PBSC harvest data for 3 years was reviewed for donor, procedural and product parameters and any specific challenges faced during the procedures. Successful PBSC collection was defined when CD34 dose obtained was ≥2 × 10⁶ cells/Kg of recipients’ body weight.Results85 PBSC collections performed on 46 children (age range 1.5–15 years) were included. Sixty-two procedures were on autologous donors and 23 on allogenic donors. The median CD34+ cell dose in the PBSC product per procedure was 2.12 × 10⁶ cells/Kg for autologous procedures and 4.6 × 10⁶ cells/Kg for allogenic procedures. Systemic adverse reaction was observed during only one procedure (0.01 %) and was managed conservatively. Successful dose was collected in 52 procedures (61.17 %) and was significantly associated with CD34+ count of more than 19.7/μL, monocyte count of more than 1.65 × 10⁶/μL, allogenic collection and female gender (p = 0.00001, p = 0.011, p = 0.00052, and p = 0.0001, respectively).ConclusionPBSC collection is safe in pediatric age groups and pre-procedure CD34 count of ≥20/μL on the day of collection may result in successful collection of stem cell dose. It is important to identify factors associated with failed collection for appropriate counselling and justifying pre-emptive use of stem cell mobilizing agents.     

Donor lymphocyte apheresis for adoptive immunotherapy compared with blood stem cell apheresis

Donor lymphocyte transfusion has gained considerable interest as adoptive cellular immunotherapy for prevention or treatment of relapse after allogeneic stem cell transplantation. This study was designed to compare the yield of CD3⁺, CD3⁺4⁺, CD3⁺8⁺, CD19⁺, CD3^?56⁺16⁺, and CD34⁺ cells contained in apheresis products from 61 consecutive non‐cytokine treated, human leukocyte antigen (HLA)‐matched donors for lymphocyte collection with the corresponding apheresis‐derived cell yield from 112 consecutive, HLA‐matched donors for blood stem cell collection who received recombinant human granulocyte colony stimulating factor (rhG‐CSF, filgrastim) 6 μg/kg every 12 hours until cell collection was completed. Apheresis was started on day 4 or 5 of rhG‐CSF treatment. The yield of lymphoid subsets was significantly different in the two sample groups, rhG‐CSF treated product yields exceeding untreated product yields by a median of 2.1‐fold (range: 1.3–2.6). However, the CD34⁺ cell yield in rhG‐CSF‐treated apheresis products exceeded untreated products by 26‐fold. A single untreated apheresis procedure was usually sufficient to collect a target dose of 1 × 10⁸/kg CD3⁺ cells. Untreated apheresis products contained a median of 0.2 × 10⁶/kg CD34⁺ cells. A potential engraftment dose of ≥0.5 × 10⁶ CD34⁺ cells per kg of recipient body weight was contained in 16% of 57 untreated apheresis products. One single apheresis performed in a normal, untreated donor provides a sufficient amount of CD3⁺ cells for adoptive immunotherapy. Compared with that of an rhG‐CSF stimulated apheresis product, the CD34⁺ cell count is usually, but not always, below the engraftment dose range. RhG‐CSF treatment has little effect on the yield of lymphoid subsets collected by apheresis but is highly selective of the release of CD34⁺ cells. This report provides baseline data for studies that will show whether other cytokines such as granulocyte macrophage colony stimulating factor (GM‐CSF) and/or Flt‐3 Ligand can immunomodulate allotransfusates in vivo to improve the graft‐vs.‐leukemia (GVL) effect after allogeneic stem cell transplantation, while lowering the incidence and severity of graft‐vs.‐host disease (GVHD). J. Clin. Apheresis. 16:82–87, 2001. © 2001 Wiley‐Liss, Inc.     

Blood counts in healthy donors 1 year after the collection of granulocyte-colony-stimulating factor-mobilized progenitor cells and the results of a second mobilization and collection

BACKGROUND : Granulocyte–colony-stimulating factor (G–CSF)-mobilized blood cells are being used for allogeneic transplants, but the long-term effects of G–CSF on healthy individuals are not known. Furthermore, it is not certain how many CD34+ cells can be collected in a second mobilization and collection procedure. STUDY DESIGN AND METHODS : Nineteen people were given 2, 5, 7.5, or 10 μg of G–CSF per kg per day for 5 days, and blood progenitor cells were collected by apheresis on the sixth day; this was done on two occasions separated by at least 12 months. Blood counts obtained before and after each course of G–CSF and the quantity of cells collected were compared. RESULTS : There were no differences in white cell (WBC), platelet, red cell, and WBC differential counts measured before each course of G–CSF, and all the values were in the normal range. In a subset of 12 people who received 7.5 or 10 μg of G–CSF per kg per day for both courses, the numbers of neutrophils, mononuclear cells, and CD34+ cells in the blood after each course were similar (34.1 ± 7.31 × 10⁹/L vs. 36.4 ± 12.3 × 10⁹/L, p = 0.24; 6.59 ± 2.28 × 10⁹/L vs. 5.63 ± 2.11 × 10⁹/L, p = 0.24; and 92.0 ± 55.6 × 10⁶/L vs. 119.2 ± 104.6 × 10⁸/L; p = 0.48, respectively), as were the quantities of mononuclear cells (31.0 ± 8.4 × 10⁹ vs. 31.0 ± 6.1 × 10⁹; p = 0.64) and CD34+ cells (417 ± 353 × 10⁶ vs. 449 ± 286 × 10⁶; p = 0.53) collected in the two apheresis procedures. Furthermore, there was a positive correlation between the quantity of CD34+ cells collected from each of the 12 people per liter of whole blood processed in the two procedures (r² = 0.86, p<0.001). CONCLUSION : One year after the administration of G–CSF to healthy people, their blood counts were normal and unchanged from pretreatment counts. If healthy people donate blood progenitor cells after a second G–CSF course, the quantity of CD34+ cells collected will be similar to that obtained in the first collection.     

Impact of rituximab on stem cell mobilization following ACVBP regimen in poor‐risk patients with diffuse large B‐cell lymphoma: results from a large cohort of patients

BACKGROUND: The ACVBP regimen is an efficient induction regimen for poor‐risk patients with diffuse large B‐cell lymphoma (DLBCL) before consolidative autologous stem cell transplantation. Adjunction of the monoclonal anti‐CD20 antibody rituximab (R‐ACVBP) was recently found to be superior to ACVBP alone. This study assessed the impact of rituximab on stem cell mobilization in two similar consecutive groups of patients treated with ACVBP in two prospective, controlled trials. STUDY DESIGN AND METHODS: The first trial (LNH‐98B‐3) involved 137 patients treated with ACVBP alone. In the second trial (LNH‐03‐3B), 91 patients received an R‐ACVBP regimen. Stem cell mobilization was performed after a course of (R)‐ACVBP. RESULTS: The median peak numbers of blood CD34+ cell counts recorded before the first apheresis procedure in the ACVBP and R‐ACVBP groups were 69 × 10⁶ and 63 × 10⁶/L, respectively (p = 0.55). The median numbers of CD34+ cells collected were 7.1 × 10⁶ and 6.0 × 10⁶ CD34+ cells/kg for the ACVBP and R‐ACVBP groups, respectively (p = 0.13). The median number of apheresis procedures required for gathering the minimum amount of CD34+ cells (2 × 10⁶/kg) was the same in the two groups. CONCLUSION: When compared with ACVBP alone, adjunction of rituximab does not impair stem cell mobilization.     

Peripheral Blood Stem Cell Mobilization and Collection in Pediatric Healthy Sibling Donors Weighing 20 Kilograms or Less; Algerian Experience

Peripheral blood stem cells (PBSC) are the source of allogeneic hematopoietic stem cell transplants currently used for malignant and non-malignant hematological diseases. PBSC harvest may be difficult in young children who are donors. Extracorporeal separator line priming by red blood cells or albumin alone is usually required to improve haemodynamic tolerance and efficacy of collection. We present our experience with 29 children weighing 20 kg or less mobilised between January 2005 and June 2018. The median age and weight at the time of apheresis were 5 years and 18 kg, respectively. A total of 54 PBSC were performed. The median cell yield per apheresis was 5.9 × 10⁶ CD34⁺cells/kg (2,5-13,9) recipient body weight (RBW). Despite their low weight, insertion of a femoral catheter was avoided in 58.6% of children. Nineteen donors required 2 or 3 apheresis sessions without any major complication. Twenty-nine pts with hemopathies have successfully benefited from PBSC except one case of rejection with aplastic anemia.     

Tempo of hematologic recovery correlates with peripheral blood CD34+ cell level in patients undergoing stem cell mobilization

This study was undertaken to evaluate the relationship between the time to recovery of peripheral blood counts and CD34+ cells in the peripheral blood (PB) and apheresis collections of patients undergoing intensive chemotherapy followed by rhG-CSF. Twenty-three patients with a median age of 42 years (range 17–64) with malignancies underwent peripheral blood stem cell (PBSC) collection after cyclophosphamide (CY) 4 g/m² and etoposide (600 mg/m²) followed by rhG-CSF (10 μg/kg/day). The WBC, platelet counts, CD34+ cell counts per ml of PB, and CD34+ cells in apheresis products were followed in all patients. The relationship of the time to recovery of WBC >1,000/μl, >3,000/μl, >10,000/μl and platelets >20,000/μl and 50,000/μl was compared to the average daily CD34+ cells/ml in each patient using the Spearman Correlation test. The tempo of recovery of WBC and platelets were highly correlated with the average CD34+ cell count in blood. In order to derive some useful guidelines for the timing of apheresis, the patients were divided into two groups, early recover (ER) and late recover (LR) based on the median time (day 10) to reach WBC count greater than 1,000/μl. ER patients had an average daily PB CD34+ cell count of 9.04 × 10⁴/ml (range 0.44–17.5) and a median yield of CD34+ cells of 10.43 × 10⁶/kg (range 0.60–25.95) compared to LR patients, who had 1.87 × 10⁴/ml (range 0.32–5.44) in the PB (P = .001) and a yield 3.20 × 10⁶/kg CD34+ cells (range 0.037–9.39) (P = .001). Patients recovering their WBC to 1,000/ml within 10 days of completing this regimen may undergo PBSC collection and achieve minimum-target cell doses of >2.5 × 10⁶ CD34+ cells/kg—100% of the time. J. Clin. Apheresis 13:1–6, 1998. © 1998 Wiley-Liss, Inc.     

Continuous Mononuclear Cell Collection (cMNC) protocol impact on hematopoietic stem cell collections in donors with negative collection predictors

Background

Recently, novel protocol utilizing Continuous Mononuclear Cell Collection (cMNC) have been introduced for leukapheresis. We compared the efficacy of cMNC with an older protocol – mononuclear cell collection (MNC) for CD34+ cell collection in unrelated donors with negative stem cell collection predictors.

Autologous peripheral blood progenitor cell transplantation

Harvesting of autologous peripheral blood stem cells (PBSCs) has been facilitated by using currently available, efficient apheresis technology at the time of rebound from chemotherapy while patients are receiving recombinant growth factors, i.e., granulocyte (G) or granulocyte-macrophage (GM) colony stimulating factor (CSF). Ideally pheresis should be done before patients have had extensive stem cell toxins, i.e., alkylating agents or nitrosoureas. This strategy has facilitated the use of high dose chemoradiotherapy given as a single regimen or in a divided dose for patients with solid tumors or hematologic malignancies and results in more rapid engraftment than bone marrow transplantation (BMT). Although mere are no assays which measure repopulating stem cells, enumeration of CD34⁺ cells within PBSCs is a direct and rapid assay which provides an index of both early and late long-term reconstitutive capacity, since it correlates with colony-forming unit (CFU)-GMs, as well as pre-progenitor or delta assays and long-term culture-initiating cells (LTC-IC). A threshold of ≥2 × 10⁶ CD34⁺ cells/kg recipient body weight has been reported to be required for engraftment, but may vary depending upon the clinical setting. Strategies for mobilization of normal PBSCs also increase tumor cell contamination within PB in the setting of both hematologic malignancies and solid tumors, but the significance of these tumor cells in terms of patient outcome is unclear. Recently isolation of CD34⁺ cells from PBSCs has been done using magnetic beads or immunoabsorption on columns or rigid plates in order to enrich for normal hematopoietic progenitors and potentially decrease tumor cell contamination. As for other cellular blood components, standards have been developed to assure efficient collection and processing, thawing, and reinfusion, and to maintain optimal PBPC viability. Finally, future directions of clinical research include expansion of hematopoietic progenitor cells ex vivo; use of umbilical cord or placenta as rich sources of progenitor cells; syngeneic hematopoietic stem cell transplantation; related and unrelated allogeneic hematopoietic stem cell transplantation; treatment of infections, i.e., Epstein Barr virus, or tumor relapse after allogeneic BMT using donor PBSC infusions; and gene therapy approaches.     

Counterflow centrifugation apheresis for the collection of autologous peripheral blood stem cells from patients with malignancies: a comparison with a standard centrifugation apheresis procedure

Two apheresis methods used to collect hematopoietic stem cells from peripheral blood were compared in eight patients with a variety of malignancies. The standard lymphocyte collection method was alternated with the counterflow centrifugation or lymphocyte surge protocol. The number of clonogenic cells (CFU-GM and BFU-E), the red cell volume, and the number of mononuclear cells in each collection were assessed as well as the changes in circulating leukocytes, platelets, and blood hemoglobin produced by each apheresis procedure. There was no statistically significant difference found in the number of clonogenic cells collected with either method, but the number of mononuclear cells collected with the standard procedure was significantly higher (P = 0.001). The red cell volume collected with the standard procedure was significantly higher, (P = 0.0001), but corrected for the number of mononuclear cells the difference was not significant. The counterflow centrifugation apheresis produced significantly less thrombocytopenia (P = 0.005). The counterflow centrifugation apheresis procedure used collected fewer mononuclear cells than the standard procedure, however, with less red cell contamination but a comparable number of CFU-GM and BFU-E in four hour apheresis procedures. Each collection method resulted in a comparable amount of anaemia and leukopenia but the lymphocyte surge method produced less thrombocytopenia following the collection.     

Large-bore central venous catheters for the collection of peripheral blood stem cells

In order to establish a peripheral blood stem cell graft, repeated aphereses are necessary in the majority of patients. Each apheresis requires withdrawal and reinfusion of blood with high flow rates. To guarantee these flow rates, large-bore catheters are needed for central venous access. Subcutaneously tunneled silicone catheters (Hickman) caused venous thrombosis in 10–40% of the patients. We therefore used polyurethane large-bore catheters only for the time of peripheral blood stem cells (PBSC) collection. Via a Seldinger guidewire following delineation of the right (160 patients) or left (23 patients) internal jugular vein by ultrasound, 183 apheresis catheters have been inserted when the white blood cell count was >1.0 × 10⁹/L and a measurable population of CD34⁺ cells was detected by fluorescence-activated cell sorter analysis. The median flow rate was 70 ml/min (range 50–80 ml/min). We observed the following complications: puncture of the carotid artery in 2%, pneumothorax in 0.5%, local infection in 3%, and catheter-related septicemia in only 2% of the patients. At the time of the removal of the catheters, we detected thrombosis of the internal jugular vein in 5% of the patients by ultrasound. The collection of PBSC with short-term, large-bore catheters is effective and is associated with a low incidence of infection and thrombosis.     

Mobilization of peripheral blood stem cells with paclitaxel and rhG-CSF in high-risk breast cancer patients

Preclinical studies have demonstrated the rapid and efficient mobilization of hematopoietic peripheral blood stem cells (PBSC) in a mouse model using the combination of paclitaxel with recombinant human granulocyte colony-stimulating factor (rhG-CSF). On the basis of these results, a clinical trial was initiated using rhG-CSF with paclitaxel for PBSC mobilization in high-risk breast cancer patients. The mobilized PBSC were evaluated for CD34(+) cell number, mononuclear cell content, and clonogenic potential. One-hundred and seventeen breast cancer patients received paclitaxel (300 mg/m(2)) administered as a 24-h continuous intravenous infusion. Forty-eight hours after completing paclitaxel, rhG-CSF (5 microg/kg) was initiated and continued until completion of PBSC collection. Leukapheresis was initiated once the white blood cell count reached 1.0 x 10(9)/L. Each collection was evaluated for the numbers of mononuclear cells (MNC) and CD34(+) cells. Clonogenic potential was enumerated using colony-forming units-granulocyte-macrophage (CFU-GM) and burst-forming units-erythroid (BFU-E). Patients receiving paclitaxel with rhG-CSF mobilized a large number of mononuclear cells/apheresis (mean, 3.7 x 10(8); range, 3.3-4.1) and CD34(+) cells/apheresis (mean, 7.2 x 10(6); range, 6.1-8.4). The average number of leukophereses needed was 1.8 (mean, range 1.6-2.0). Colony growth was normal with 178.9 x 10(5) and 214.8 x 10(5) colonies counted in CFU-GM and BFU-E assays, respectively. Patients engrafted platelets and neutrophils on day 10 following transplantation. In conclusion, PBSC mobilization with paclitaxel and rhG-CSF results in a large number of mononuclear cells and CD34(+) cells with normal clonogenic potential. The cells engraft normally following high-dose chemotherapy and autologous stem cell transplantation in high-risk breast cancer patients. These results demonstrate that paclitaxel with rhG-CSF is an efficient mobilizing agent in high-risk breast cancer patients.