Critical assessment of the efficiency of CD34 and CD133 antibodies for enrichment of rabbit hematopoietic stem cells.

Rabbits have many hereditary diseases common to humans and are therefore a valuable model for regenerative disease and hematopoietic stem cell (HSC) therapies. Currently, there is no substantial data on the isolation and/or enrichment of rabbit HSCs. This study was initiated to evaluate the efficiency of the commercially available anti-CD34 and anti-CD133 antibodies for the detection and potential enrichment of rabbit HSCs from peripheral blood. PBMCs from rabbit and human blood were labelled with different clones of anti-human CD34 monoclonal antibodies (AC136, 581, and 8G12) and rabbit polyclonal CD34 antibody (pCD34) and anti-human CD133 monoclonal antibodies (AC133 and 293C3). Flow cytometry showed a higher percentage of rabbit CD34+ cells labelled by AC136 in comparison to the clone 581 and pCD34 (P < 0.01). A higher percentage of rabbit CD133+ cells were also detected by 293C3 compared to the AC133 clone (P < 0.01). Therefore, AC136 clone was used for the indirect immunomagnetic enrichment of rabbit CD34+ cells using magnetic-activated cell sorting (MACS). The enrichment of the rabbit CD34+ cells after sorting was low in comparison to human samples (2.4% vs. 39.6%). PCR analyses confirmed the efficient enrichment of human CD34+ cells and the low expression of CD34 mRNA in rabbit positive fraction. In conclusion, the tested antibodies might be suitable for detection, but not for sorting the rabbit CD34+ HSCs and new specific anti-rabbit CD34 antibodies are needed for efficient enrichment of rabbit HSCs. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1278-1289, 2018.

Decision-tree algorithm for optimized hematopoietic progenitor cell-based predictions in peripheral blood stem cell mobilization.

BACKGROUND: Enumerating hematopoietic progenitor cells (HPCs) by using an automated hematology analyzer is a rapid, inexpensive, and simple method for predicting a successful harvest compared with enumerating circulating CD34+ cells. However, the optimal HPC cutoff count and the indicating factors to be considered for improved predicting have not yet been determined. STUDY DESIGN AND METHODS: Between 2007 and 2012, a total of 189 consecutive patients who proceeded to peripheral blood stem cell (PBSC) harvesting were retrospectively recruited. Baseline characteristics were analyzed to identify the risk factors for a failed harvest, which were defined as less than 2 × 10(6) CD34+ cells/kg. Variables identified by multivariate logistic regression and correlation analysis for predicting a successful harvest were subjected to classification and regression tree (CART) analysis. RESULTS: PBSCs were successfully harvested in 154 (81.5%) patients. An age of at least 60 years, a diagnosis of a solid tumor, at least five prior chemotherapy cycles, prior radiotherapy, and mobilization with granulocyte-colony-stimulating factor alone or high-dose cyclophosphamide were independent baseline predictors of poor mobilization. In CART analysis, patients with zero to two host risk factors and either higher HPC (≥28 × 10(6) /L) or mononuclear cell (MNC; ≥3.5 × 10(9) /L) counts were categorized as good mobilizers and their harvest success rate was 92.3%. By contrast, 30.3% of harvests were adequate in the patients with three to five host risk factors and lower HPC and MNC counts. CONCLUSION: A CART algorithm incorporating host predictors and HPC and MNC counts improves predictions in a successful harvest and might reduce the necessity of monitoring peripheral CD34+ cells.

Assessment of immune reconstitution status in recipients of a successful hematopoietic stem cell transplant from peripheral blood after reduced intensity conditioning.

OBJECTIVE: To document immune reconstitution status after hematopoietic stem cell transplantation (HSCT) for malignant hematologic diseases. METHODS: Hematology patients who received a reduced intensity conditioning (RIC) were followed after successful allogeneic or autologous HSCT. Patients had at least 100days post-transplant. T, B and NK cells in peripheral blood (PB), and CD34+, CD133+ progenitor cells in bone marrow (BM) and peripheral blood (PB) were determined by flow cytometry. RESULTS: Twenty-seven HSCT recipients, 19 allogeneic and 8 autologous, were studied at a median 155 (100-721) days post-transplant. In the whole group the median value of CD34+ cells was 1.03% in the bone marrow and 0.04% in PB, whereas values for CD133+ cells were 0.39% and 0.13%, respectively, without statistical differences between autologous and allogeneic recipients. Significantly more B cells (CD3-/CD56-/CD19+) were found in the autologous compared to the allogeneic group, 12.6 vs. 5.01, p=0.04. An increased number of CD8+ lymphocytes with a 0.63 CD4:CD8 relationship was documented in PB. CONCLUSION: In clinically recovered autologous and allogeneic HSCT recipients BM and PB CD34+/CD133+ hematoprogenitor homeostasis is maintained within normal ranges, with better B-cell reconstitution in the autologous group.

Innate immune system favors emergency monopoiesis at the expense of DC-differentiation to control systemic bacterial infection in mice.

DCs are professional APCs playing a crucial role in the initiation of T-cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC-depletion in humans and mice. The mechanisms of pathogen-induced DC-depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC-development, one reason for DC-depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC-depletion and the impact of different bacteria on DC-development. We show that the number of bone marrow (BM) hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram-positive and Gram-negative bacteria. This is associated with a TLR4- and IFN-γ-signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye-infection. Adoptive transfer experiments revealed that infection-induced monopoiesis occurs at the expense of DC-development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC-development leads to DC-depletion, possibly driving transient immunosuppression in bacterial sepsis.

Critical assessment of human antibody generation in humanized mouse models.

Immunodeficient mice reconstituted with human hematopoietic stem cells provide a small-animal model for the study of development and function of human hematopoietic cells in vivo. However, in the current models, the immune response, and especially the humoral response by the human immune cells is far from optimal. The B cells found in these mice exhibit an immature and abnormal phenotype correlating with a reduced capacity to produce antigen-specific affinity matured antibodies upon infection or immunization. Herein, we review the current state of knowledge of development, function and antibody production of human B cells and discuss the obstacles for the improvement of these models.

A plerixafor-based strategy allows adequate hematopoietic stem cell collection in poor mobilizers: results from the Canadian Special Access Program.

Plerixafor effectively mobilizes hematopoietic stem cells (HSCs). However, most patients' cells are successfully collected using traditional strategies and there is limited cost-effectiveness data. The objectives of this study were to: (1) summarize the published reports of mobilization using a plerixafor-based strategy during compassionate access programs and (2) describe the Canadian experience with plerixafor during its availability by Health Canada's Special Access Program. A literature search identified reports of plerixafor-based mobilization during compassionate access programs. Overall, successful collection of at least 2 × 10(6) CD34+ cells/kg was achieved in ~75% of patients, and about two-thirds of patients went on to HSCT. A greater proportion of patients had successful collections when plerixafor was used in the upfront or preemptive settings. Plerixafor was made available by Health Canada's SAP from September 2008 to December 2010. In 96 of 132 (73%) patients, there was successful collection of at least 2 × 10(6) CD34+ cells/kg. Ninety-nine (75%) patients went on to receive an autologous transplant. Plerixafor-based mobilization is effective in perceived poor mobilizers. The optimal way to incorporate plerixafor into a mobilization strategy, however, remains to be determined. Centre-specific analysis of resource utilization may help to identify the most cost-effective way to implement various plerixafor-based mobilization strategies.

Interactions of monocyte subpopulations generated from cord blood CD34(+) hematopoietic progenitors with tumor cells: assessment of antitumor potential.

Monocytes and their subsets (CD14(++)CD16(+) and CD14(+)CD16(-)) generated from cord blood CD34(+) progenitor cells were used for determination of their capacity to interact with tumor cells in vitro and in vivo. The studies in vitro included adhesion to human umbilical vein endothelial cells, cytotoxicity, production of toxic mediators: reactive oxygen and nitrogen intermediates (ROI and RNI, respectively), and finally their effect on transplantable human tumor growth in nonobese diabetic severe combined immunodeficient mice. The CD14(++)CD16(+) subset exhibited an increased adherence to human umbilical vein endothelial cells and cytotoxicity toward tumor cells in vitro. CD14(+)CD16(-) monocytes showed a higher production of reactive oxygen and nitrogen intermediates after stimulation with tumor cells, and more pronounced inhibition of tumor growth in vivo. The results revealed significant differences in the behavior of CD14(++)CD16(+) and CD14(+)CD16(-) monocyte subsets toward tumor cells, thus providing further evidence that CD34(+) cell-derived monocytes differ in this respect from blood monocytes. The protocol for generation of monocytes with antitumor reactivity described here may be useful to obtain monocytes from CD34(+) progenitor cells of cancer patients. This might offer a basis for a novel approach for various forms of cellular immunotherapy of cancer.

Interlaboratory variability of CD34+ stem cell enumeration. A pilot study to national external quality assessment within the Czech Republic.

We present the results of a pilot study concerning the interlaboratory variability of CD34+ enumeration. Three surveys, each including a set of samples, were sent to participating Czech flow cytometry laboratories. The efficacy of this exercise was determined by the reduction in interlaboratory variation and the influence of method used on assay outcome. The variability in results of CD34+ enumeration declined with time. The mean coefficient of variation (CV) of measurement among laboratories dropped, from 58% in first survey to 32% in last survey. All tested variables (gating strategy, platform methodology, sample preparation) affected the variability of the assay. Sample preparation method was associated with a significant bias of absolute CD34+ cell counts. Initially, the outcome of the measurement was also affected by the participating laboratory (identified by a unique laboratory number; ULN). However, laboratories with poorer performance modified their protocols during the study, and the ULN ceased to influence the variability. This study was successful in reducing the interinstitutional variability of CD34+ enumeration. It was shown that the implementation of a standardized protocol does not guarantee accurate measurement. Our research design represents a useful tool, which allows verification of the proper use of a standardized method, the training of operators and feedback in response to the survey results.

Evaluation of 'out-of-specification' CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products.

BACKGROUND: Immunomagnetic selection of CD34(+) hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+ cells recommended by the manufacturer for processing in a single procedure or with 1 vial of CD34 reagent is limited. METHODS: In this retrospective evaluation of 643 CliniMACS CD34-selection procedures, we validated the capacity of CliniMACS tubing sets and CD34 reagent. Endpoints of this study were the recovery and purity of CD34+ cells, T-cell depletion efficiency and recovery of colony-forming units-granulocyte-macrophage (CFU-GM). RESULTS: Overloading normal or large-scale tubing sets with excess numbers of total nucleated cells, without exceeding the maximum number of CD34+ cells, had no significant effect on the recovery and purity of CD34+ cells. In contrast, overloading normal or large-scale tubing sets with excess numbers of CD34+ cells resulted in a significantly lower recovery of CD34+ cells. Furthermore, the separation capacity of 1 vial of CD34 reagent could be increased safely from 600 x 10(6) CD34+ cells to 1000 x 10(6) CD34+ cells with similar recovery of CD34(+) cells. Finally, T-cell depletion efficiency and the fraction of CD34+ cells that formed CFU-GM colonies were not affected by out-of-specification procedures. DISCUSSION: Our validated increase of the capacity of CliniMACS tubing sets and CD34 reagent will reduce the number of selection procedures and thereby processing time for large HPC products. In addition, it results in a significant cost reduction for these procedures.

Commercial development of stem cell technology: lessons from the past, strategies for the future.

This paper presents historical and contemporary survey data on the commercial development of stem cell technology from the 1990s to the present day. We describe the first wave of industrial investment in hematopoietic stem cells during the 1990s and contrast this with the more recent expansion of the sector. In particular, we explore the cell types used, diseases targeted and business models adopted by firms. We conclude, by arguing that the commercial prospects for stem cell technologies remain highly uncertain and that innovative public policies should be adopted to prevent 'market failure'.

Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood.

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.

Hematopoiesis in the feline fetal liver: an assessment by flow cytometry.

The domestic cat is an excellent model for the development of therapeutic protocols that target hematopoietic stem cells (HSCs) because it is relatively resistant to complications related to bone marrow transplantation. To identify a plentiful source of HSC that could be used as targets for gene transduction and transplantation, the livers of 28 mid-gestation fetuses (28-52 days) and late-gestation fetuses (53 days-term) were analyzed for erythroid, myeloid, lymphoid, and uncommitted hematopoietic progenitor cells by flow cytometry. We found that the fetal liver mononuclear cells (FLMCs) contained 57% erythroid progenitors during mid-gestation, but this percentage declined to 43% as gestation progressed. Myelomonocytic cells within FLMC were more numerous in late-gestation (31%) than in mid-gestation (18%). Two monoclonal antibodies (mAb), CH 152 and CH 755, which recognize cells with the potential to reconstitute multilineage hematopoiesis in cats, were tested. Approximately, 32% of FLMC from late-gestation fetuses expressed the epitope recognized by mAb CH 152, a significant increase above the 12% positive cells in mid-gestation fetuses. Approximately, 33% of hepatic mononuclear cells expressed the epitope recognized by mAb CH 755 in both mid-term and late-term fetuses. When expressed in absolute numbers, medians of 2.7 x 10(7) CH 152-positive cells and 3.2 x 10(7) CH 755-positive cells were extracted from the late-term fetal livers of individual cats. T-lymphocytes were a minor component (<3%) of FLMC, despite their presence in the thymus and spleen. These data suggest that the late-term feline fetal liver is a suitable source of mutipotential hematopoietic cells that could be used for gene therapy protocols in the cat.

Deceptive multilineage reconstitution analysis of mice transplanted with hemopoietic stem cells, and implications for assessment of stem cell numbers and lineage potentials.

Hemopoietic stem cells (HSC) are identified through their unique ability, at the single cell level, to long-term reconstitute all blood cell lineages. Sustained myeloid reconstitution is considered the hallmark of HSC, because myeloid progenitors and their progeny have very short half-lives. Here we demonstrate that the established practice of relying on RB6-8C5 as a myeloid specific Ab can result in overestimation of HSC frequencies because the RB6-8C5 Ab also detects Ags expressed on a sizeable population of CD3(+)CD8(+) T cells, constitutively as well as following transplantation. Likewise, a high fraction of mice transplanted with limiting numbers of ex vivo expanded Lin(-)Sca(+)kit(+)CD34(-) HSC show long-term RB6-8C5(+)CD3(+) (lymphoid) but no RB6-8C5(+)CD3(-) (myeloid) reconstitution. Most noteworthy, the use of RB6-8C5 as a myeloid specific Ab can be deceptive by implicating the existence of lineage-restricted HSC capable of long-term reconstituting the myeloid and T, but not B, cell lineage. Because cross-lineage expression of "lineage-specific" markers is unlikely to be unique to the blood system, claims of unexpected cell fates should be substantiated not only by acquisition of lineage-specific markers, but also absence of markers of other lineages normally derived from the investigated stem cells.

Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): assessment of cross-reacting monoclonal antibodies.

Previous studies have shown that hematopoietic progenitor cells can be isolated from human or nonhuman primate bone marrow (BM) cells. In the present study, we studied the cross-reactivity of 13 anti-human CD34, two anti-human c-Kit, and one anti-human CD133 monoclonal antibodies (mAbs) with cynomolgus macaque (Macaca fascicularis) BM cells, using flow cytometric analysis, cell enrichment, and clonogenic assay. Among the 13 anti-human CD34 mAbs assessed, six cross-reacted as previously reported by other groups. However, only three of these six mAbs (clones 561, 563, and 12.8) recognized cynomolgus CD34+ cells that formed progenitor colonies when grown in methylcellulose culture. Similarly, of the two anti-human c-Kit mAbs (clones NU-c-kit and 95C3) that were previously reported to cross-react with cynomolgus BM cells, only one (clone NU-c-kit) resulted in a similar outcome. The anti-human CD133 mAb (clone AC133) also cross-reacted with cynomolgus BM cells, although these cells did not give rise to colonies when grown in culture. These results suggest that antibodies that cross-react with nonhuman primate cells may not identify the hematopoietic cells of interest. In addition, while the CD34 mAb (clone 561) results in the selection of hematopoietic progenitor cells of all lineages when assessed in methylcellulose culture, the c-Kit(high) fraction (NU-c-kit) exclusively identifies erythroid-specific progenitor cells after growth in culture. It is important to consider these findings when selecting cross-reacting mAbs to identify cells of hematopoietic lineages in macaque species.

Hematopoietic progenitor cells as targets for non-invasive prenatal diagnosis: detection of fetal CD34+ cells and assessment of post-delivery persistence in the maternal circulation.

Culture expansion of fetal cells from the maternal circulation will provide an increased number of cells for non-invasive prenatal diagnosis. Hematopoietic CD34+ cells are potential candidates for this application. More information is needed regarding the frequency of these cells and the phenomenon of post-delivery persistence in the maternal circulation. In this study we assessed the number of fetal CD34+ cells in the maternal circulation, the effect of culture expansion on the number of fetal cells and the persistence of fetal CD34+ cells from previous pregnancies. Fetal cells were identified by the presence of Y-chromosome sequences detected by FISH and nested PCR. Fetal CD34+ cells were detected in all samples from women carrying a male fetus. A low number of residual fetal cells from previous pregnancies was detected (1-3 XY cells in 20 ml blood) in less than 1/3 of the samples from both non-pregnant women and those pregnant with a female fetus. Culturing of CD34+ cells resulted in a significant increase in fetal cell numbers. However, the number of fetal cells persisting from previous pregnancies also increased after culture. It is proposed that information derived from CD34+ cells could potentially support data derived from other cell types for more accurate non-invasive prenatal diagnosis.

Assessment of CD24 expression on bone marrow neutrophilic granulocytes: CD24 is a marker for the myelocytic stage of development.

The understanding of normal surface maturation pattern of granulocytes plays an essential role in identifying abnormal patterns, which may be of diagnostic or pathogenetic significance in some disorders, such as myclodysplastic syndromes. CD24, which is expressed only on granulocytes and not on monocytes, has not been adequately studied. Surface marker studies on eight control marrows indicate that CD24 is expressed on bone marrow granulocytes in a pattern that is similar to CD11b while much different than the more mature markers, CD16b and CD35. Three sorting experiments show that the majority of CD24(-) cells are promyelocytes. We conclude that CD24 is expressed on the neutrophilic granulocytes at the stage of myelocytes, and hence it could potentially be used to study normal and abnormal maturation of granulocytes.

Detection of functional V(H)81X heavy chains in adult mice with an assessment of complementarity-determining region 3 diversity and capacity to form pre-B cell receptor.

Recent reports have indicated that up to 50% of all H chain proteins formed cannot associate with the surrogate L chain complex and therefore fail to form a pre-B cell receptor (pBCR), which is required for allelic exclusion and, in most cases, verifies that the H chain can assemble with the L chain to form an Ab molecule. Certain V(H) genes, such as V(H)81X, appear to be particularly prone to encoding for nonpairing (dysfunctional) H chains. It has been suggested that sequence variability at complementarity-determining region 3, especially when increased by the enzyme TdT, often precludes the ability of V(H)81X-using H chains to form pBCR. To determine whether a motif exists that accounts for the ability of H chains to pair with surrogate L chain complex/L chain, we have bred a mouse line in which H chain recombination can only occur on one allele, allowing us to compile a pool of H chains capable of forming Ab molecules in the absence of dysfunctional H chains. Somewhat unexpectedly, we have found V(H)81X H chains capable of Ab formation and cell surface expression in the presence of TdT. Scrutiny of these H chains has revealed that, although highly prone to encode for dysfunctional H chains, sequence variability is not severely limited among functional V(H)81X H chains. We also demonstrate that surface Ig expression is highly indicative of the capacity of a H chain to form pBCR.

Determination of peripheral blood stem cells by the Sysmex SE-9500.

The Sysmex SE-9500 automated haematology analyser provides an estimate of immature cells, referred to as 'haematopoietic progenitor cells' (HPC). The aim of this study was to evaluate the reliability and usefulness of the SE-9500 HPC parameter as compared with the CD34 + cell count and to determine whether the HPC count was of value in predicting the optimal harvesting time for peripheral blood stem cells (PBSC). Studies were performed on 112 samples from 21 patients with haematological malignancies and 13 healthy donors undergoing progenitor cell mobilisation. Coefficients of variation for the HPC count were 30%, 23.8%, 12.4% and 8.3% respectively for samples with low (4 x 106/l), medium (13 x 106/l), high (250 x 106/l) and very high (2413 x 106/l) counts. There was good linearity for HPC measurement in both peripheral blood (PB) and purified CD34 + cell suspensions (r > 0.995), and no detectable carryover was observed. There was an acceptable correlation between HPC and CD34 + cell counts for PB samples (r=0.669) and for CD34 + cell suspensions (r=0.859). Analysis of purified CD34 + cells using the SE-9500 HPC mode revealed that they appear both in the blast cell area and the immature granulocyte area of the analyser cell display. Quantitation of CD34 + cells and HPC during PBSC mobilisation showed good agreement between these parameters with regard to the optimal time for PBSC harvesting. These findings suggest that HPC counting with the Sysmex SE-9500 may be clinically useful for optimising the timing of PBSC collection.

Assessment of amifostine as protection from chemotherapy-induced toxicities after conventional-dose and high-dose chemotherapy in patients with germ cell tumor.

BACKGROUND: We assessed the efficacy of amifostine for protection from chemotherapy-induced toxicities in patients treated with conventional-dose paclitaxel, ifosfamide, cisplatin (TIP) and high-dose carboplatin, etoposide and thiotepa (CET) followed by peripheral blood progenitor cell (PBPC) rescue. PATIENTS AND METHODS: In a prospective single-center study 40 patients with relapsed or refractory germ-cell tumors (GCT) were treated with 3 cycles of conventional-dose TIP followed by one cycle of high-dose CET. Patients were randomized either to receive one fixed dose of 500 mg amifostine per day of conventional-dose TIP and two fixed doses of 500 mg per day amifostine during high-dose CET (group A, n = 20) or no amifostine (group B, n = 20). Prior to the first cycle of TIP, one course of 175 mg/m2 paclitaxel and 5 g/m2 ifosfamide (TI) followed by granulocyte-colony stimulating factor (G-CSF) at 10 microg/kg/day were given for PBPC mobilization. RESULTS: Toxicities and response to conventional-dose TIP and high-dose CET could be evaluated in 40 patients (100%) and 32 of 40 patients (80%), respectively. Peripheral neurotoxicity (i.e. paresthesia or sensorymotor impairment), hearing impairment, hematologic toxicity, nephrotoxicity, nausea, myalgia, skin- and liver-toxicity did not differ siginificantly between the two patient groups. Likewise, the response rates to TIP and high-dose CET were comparable in patients with or without amifostine. After a median follow-up of 18 months, 8 of 20 (40%) patients of group A and 6 of 20 (30%) patients of group B are without relapse. CONCLUSION: Repeated low doses of 500 mg amifostine additional to conventional-dose TIP or high-dose CET showed no unequivocal advantage in protection from treatment-related toxicities. Furthermore, no significant differences in response rates or survival could be observed in this small number of patients.

Flow cytometric assessment of lymphocyte subsets, lymphoid progenitors, and hematopoietic stem cells in allogeneic stem cell grafts.

Currently, bone marrow (BM), cord blood (CB), and G-CSF-mobilized peripheral blood progenitor cells (PBPCs) are the most commonly used sources for allogeneic stem cell transplantation (SCT). The aim of this study was to assess the yields and distribution of lymphocyte subsets, lymphocyte progenitors and hematopoietic stem cells (HSC) in each type of allograft by three-color flow cytometry. The yields of CD34(+)CD38(-) HSCs did not differ significantly between BM grafts (2.80 +/- 0.74 x 10(6)) and leukapheresis products (LPs) (1.82 +/- 0.64 x 10(6)), and were lowest in CB grafts (0.21 +/- 0.05 x 10(6)). For most lymphocyte subsets yields were lowest in CB grafts and significantly higher in LPs than in BM grafts. BM grafts, however, contained the highest yields of CD34(+)CD19(+)CD20(-) B cell progenitors and CD19(+)CD20(-) B cells. The relative frequencies of the naive CD45RA(+)CD45RO(-) phenotype among CD4(+) and CD8(high) T cells were highest in CB grafts (P < or = 0.001), and higher in LPs than in BM grafts (P < or = 0.02). The latter finding was in accordance with a preferential G-CSF mobilization of naive T cells relative to the total lymphocyte population (P < or = 0.014). CD3(+)CD8(low) and CD3(+)CD8(low)CD4(-) subsets, which facilitate engraftment in murine transplantation models, demonstrated a tendency towards lower frequencies among T cells in CB grafts and LPs compared to BM grafts. This observation coincided with a significantly reduced mobilization of subsets potentially enriched for facilitating cells as compared to the total lymphocyte population (P < or = 0.036). The CD34(+) compartment of CB grafts contained a significantly higher percentage (12.1%) of CD34(+)CD7(+)CD3(-) T cell progenitors than those of BM grafts (5.1%) and LPs (3.6%). In addition, CB lymphocytes contained the highest fraction of CD3(-)CD16/56(+) NK cells (P < or = 0.013) and almost no CD3(+)CD16/56(+) NKT cells (P < 0.001) compared to adult cell sources. In summary, LPs, CB allografts and BM allografts differ widely with respect to the cellular composition of their lymphocyte compartments, which is partially affected by a varying mobilization efficiency of G-CSF for distinct lymphocyte subsets.