Hematopoietic Progenitor Cell Products Research Articles

Microbial contamination of hematopoietic progenitor cell products

IntroductionMicrobial screening for contamination is a part of hematopoietic progenitor cell (HPC) collection and infusion procedure. We aimed to find out our microbial contamination rates during collection, processing and infusion steps of HPC products. We also evaluated the clinical course of patients who received contaminated HPC products. Patients-methodsWe retrospectively analyzed microbial contamination records of HPC grafts between 2010 and 2012. HPC products of autologous donors were evaluated for contamination at three steps: at the end of mobilization, following processing with DMSO and just before stem cell infusion. Grafts of allogeneic donors were assessed only before HPC transplantation (HCT). Microbiological analysis of HPC samples were performed with an automated system (BacT/Alert®). ResultDuring the study period a total of 492 mobilization procedures were performed on 329 (214 autologous and 115 allogeneic) donors. Bacterial contamination has been detected in 103 of 1630 samples (6%). Ninety-seven out of 1162 blood samples (8%) from 265 patients who were treated with HCT were contaminated. Forty-six patients (41 autologous and 5 allogeneic) were transplanted with contaminated HPC products. During HCT 42 patients experienced febrile neutropenic attack and 34 of them had positive blood culture results. In none of these 34 patients the isolated pathogens were the same organisms with those found in the final contaminated stem cell product before stem cell infusion. None of the patients who received contaminated products died because of sepsis within the posttransplant 30days. There was no significant difference between patients who received contaminated and non-contaminated products in terms of the first day of fever, duration of fever, engraftment kinetics and duration of hospitalization. ConclusionOur results suggest that microbial contamination of HPC products is an issue to be prevented, although it may not have a major impact on the general success of HCT.

Microbial contamination of hematopoietic progenitor and other regenerative cells used in transplantation and regenerative medicine

Microbial contamination of hematopoietic progenitor cells (HPCs) and other regenerative cells used in transplantation and regenerative medicine can occur during collection and after in vitro manipulation, including purging, cryopreservation, thawing, and infusion. Microbiologic culture findings on consecutive HPCs and other cell preparations at a single institution derived from peripheral blood, marrow, cord blood, and mesenchymal stromal cells during all phases of manipulation were retrospectively examined from 2005 through 2011. Results were classified as confirmed positive, false positive, and indeterminate. During the 6-year surveillance period, 365 patients underwent 912 procedures involving HPC or other cell-based transfusion. True positive microbial contamination was found in five of 663 (0.8%) peripheral blood and two of 34 (5.9%) marrow preparations (p = 0.04), while no contamination was found in 118 preparations from other sources. True-positive microbial contaminants included coagulase-negative staphylococci in autologous HPC products derived from peripheral blood from two patients with asymptomatic central venous catheter infections at time of apheresis and Propionibacterium acnes in one apheresis and two marrow products. Organism loads were low in all cases (≤500 colony-forming units/mL), and no adverse sequelae occurred in four patients that received contaminated products. The incidence of microbial contamination of progenitor cell products in our institution over a 6-year period was low (0.8% overall), with contaminants originating from infected central venous catheters or from skin flora. All contaminants were bacterial species of low virulence, present in low titers and, if transfused, did not result in adverse reactions.

Sterility testing of apheresis hematopoietic progenitor cell products using an automated blood culture system

AABB Standards require monitoring of hematopoietic progenitor cell (HPC) products for microbial contamination. To date, there is no automated blood culture system cleared by the Food and Drug Administration for this application. Our objective was to validate the VersaTREK system (TREK Diagnostic Systems) for sterility testing of apheresis HPC products. Four aerobic bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mitis, and Bacillus cereus), five anaerobic bacteria (Fusobacterium necrophorum, Clostridium perfringens, Bacteroides fragilis, Prevotella loescheii, and Propionibacterium acnes), and one fungus (Candida albicans) were spiked into apheresis HPC products at concentrations of 10, 10(2) , 10(3) , and 10(4) colony-forming units (CFUs)/mL. Aerobic and anaerobic bottles were incubated until positive or for up to 5 days. DNA was simultaneously extracted for polymerase chain reaction amplification of 16S ribosomal RNA (rRNA) gene. All aerobic bacteria grew in both bottles at all concentrations tested within 24 hours, and the time to positivity (TTP) was significantly shorter with aerobic bottles. C. albicans grew in the aerobic media at all concentrations within 30 hours. Anaerobes grew in the anaerobic bottle at all concentrations within 5 days. No bacteria were detected by using 16S rRNA gene amplification at 10(4) CFUs/mL. Compared to culture, 16S rRNA gene amplification of HPCs does not improve sensitivity or turnaround time for HPC sterility testing. The VersaTREK system is a reliable tool for detecting microbial contamination of apheresis HPC products with a limit of detection of less than or equal to 10 CFUs/mL. Inclusion of both the aerobic and the anaerobic culture bottles achieves the shortest TTP for all species tested.

Avoiding room temperature storage and delayed cryopreservation provide better postthaw potency in hematopoietic progenitor cell grafts

Conditions for maintaining hematopoietic progenitor cells (HPCs) before cryopreservation remain controversial. An understanding of the impact of time and temperature during nonfrozen storage can contribute to the maintenance of the quality of products, improving transplantation outcomes. The objective of this study was to determine the influence on cell potency of thawed products from three sources of HPCs after prolonged storage at different temperatures before cryopreservation. Viable cell counts by flow cytometry and colony-forming unit (CFU) recoveries were assessed on cord blood (CB), mobilized peripheral blood stem cell (PBSC), and bone marrow (BM) samples over 72 hours using two different storage conditions, refrigerated (4-8°C) or room temperature (19-22°C). To determine the effects of delayed freezing on progenitor recoveries, paired samples were evaluated before and after cryopreservation. All samples maintained at refrigerated temperatures resulted in higher recoveries than those at room temperature in all variables assessed. Specifically, when assessing for CFU yields after thawing, the impact of time on BM resulted in a significant loss as soon as 24 hours (n = 10, 36.4 ± 28.0%, p = 0.003). This decrease was also observed for PBSCs and CB but at 48 hours of fresh storage (PBSCs n = 11, 32.7 ± 26.2%, p = 0.006; CB n = 10, 39.6 ± 26.4%, p = 0.001). Our data suggest that HPC products are better maintained at refrigerated temperatures before cryopreservation. Delaying cryopreservation should be minimized to avoid significant losses in cell potency.

Quality and Safety of Hematopoietic Progenitor Cell (HPC) Products Cryopreserved by Simple Passive Gradual Freezing Method

Abstract 4410To provide quality HPC products for transplantation, harvests of bone marrow (BM), Peripheral Blood Stem Cells (PBSCs) and Cord Blood (CB) are routinely cryopreserved by gradual cooling from ambient temperature to −90°C using Controlled Rate Freezing (CRF) systems before placement in liquid/vapor nitrogen storage. CRF systems generally utilize a freezing chamber and computer-controlled freezing cycle with an established sequence of steps where timing, target temperatures and cooling rates are defined. System failures that endanger product quality may occur due to impurities in liquid nitrogen, mechanical/software problems and probe malfunction. A more convenient and economical freezing may be performed by simple “passive freezing” (PF) where products are placed directly into an ultra-low freezer (−80°C). Continuous temperature monitoring during the freeze process is achieved using individual data loggers. In the PF process, products remain undisturbed during the freeze process, and are removed for placement in liquid/vapor nitrogen storage when the target product temperature is reached (−80°C). Probe specific freezing curves are generated from downloaded data logger information.In an internal validation study, the St. Louis Cord Blood Bank (SLCBB) investigated quality and safety of HPC products cryopreserved using this PF method. Evaluation was based on the analysis of freezing kinetics, post-thaw cell recoveries, and available transplant outcome data in reference to those obtained by the conventional CRF method. A total of twelve (12) red blood cell and plasma reduced CB products cryopreserved in 10% DMSO/Dextran by PF were thawed according to a validated albumin/dextran reconstitution thaw method. Compared to the programmed CRF system, PF product cooling rate to a temperature of −60°C was faster (1.2– 2.0°C/min compared to 1°C/min), ultimately followed by slower cooling rate to a product target temperature of −80°C (approximately 0.4°C/min compared to 10°C/min). The kinetics of PF curves resulted in longer freezing cycles with an average freeze time of 180 minutes compared to 80 minutes by CRF method. Post-thaw cell recoveries of the PF data set were comparable to those obtained from an established thaw control group of RBC and plasma reduced CB cryopreserved by CRF system (n = 25). Average percent recoveries for CB products cryopreserved by PF methodology compared to CRF system were reported as follows, respectively: viable nucleated cells (NC) by trypan blue = 84% +/− 5% compared to 71% +/− 6%, viable (7AAD) CD34+ cells = 67% +/− 7% compared to 63% +/− 15%, and colony forming unit (CFU) = 73% +/− 7% compared to 72% +/− 14%.Subsequently, the SLCBB reviewed post-thaw cell recoveries of ten (10) PBSC harvests cryopreserved by PF and thawed at the SLCBB for clinical transplantation. Similar to the CB evaluation, results indicated a high quality HPC product post-thaw with average percent recoveries reported as follows: viable nucleated cells (NC) by trypan blue = 79% +/− 10%, viable (7AAD) CD34+ cells = 81.2% +/− 20.2, and CFU = 73.6% +/− 21.9%. All PBSC products were thawed and infused with no reports of infusion-related adverse events. These PBSC products engrafted shortly within predicted time; absolute neutrophil count (ANC) > 500/uL was reported within 12 days, while platelets > 20,000/uL was achieved within 22 days.Retrospective review of more than 1,800 CB products distributed by the SLCBB for transplant showed that five (5) PF products were transplanted as singlet products to treat patients with myeloid malignancies. Transplant outcome data received by CIBMTR for those cases indicated 100% success of engraftment and 100% survival at 100 days post-infusion. Engraftment data reports indicated ANC > 500 was achieved within 6–30 days, and platelet count > 20,000 was achieved within 25–163 days. The transplant outcome of PF product is comparable to that reported historically for CRF CB products. Conclusions:In vitro results and clinical findings support the quality and safety of HPC products cryopreserved using PF methodology and would recommend PF as valid alternative to the use of CRF systems. Disclosures:No relevant conflicts of interest to declare.

Endothelialization and altered hematopoiesis by persistent Etv2 expression in mice

Etv2 is a master gene for the commitment of hematopoietic/endothelial cells and is a potent inducer of endothelial/hematopoietic cells from embryonic stem cells. Etv2 is highly expressed in endothelial/hematopoietic precursors but is downregulated when they are differentiated, indicating that Etv2 should have transient but not constitutive function. However, relatively little attention has been paid to the importance of transient Etv2 expression. To determine whether transient Etv2 expression is essential to normal development and cell differentiation, we generated mice that constitutively express Etv2 from a Cre-activatable ROSA26 locus in endothelial/hematopoietic, somite, or neuronal lineages. Constitutive Etv2 expression caused profound phenotypes in hematopoietic/endothelial cells, with little effect on somite or neuronal lineages. In hematopoietic/endothelial lineages, constitutive Etv2 expression induced by Tie-2 Cre transgene caused abnormal yolk sac vasculature. Prolonged vascular endothelial cadherin expression and decreased B lymphopoiesis were observed in Etv2 expressing vascular endothelial cadherin(+)/CD45(+) cells, indicating that Etv2 forces endothelial program on hematopoietic cells. Etv2 expression in adult hematopoietic cells by Vav-iCre transgene also conferred an endothelial phenotype on hematopoietic stem cells and suppressed hematopoiesis, with erythropoiesis severely affected. We conclude that constitutive Etv2 expression perturbs vascular development and hematopoiesis. While promoting hematopoiesis/vasculogenesis, Etv2 expression should be tightly regulated to achieve normal vascular development and hematopoiesis.

Aldehyde dehydrogenase as an alternative to enumeration of total and viable CD34+ cells in autologous hematopoietic progenitor cell transplantation

We validated the correlation of aldehyde dehydrogenase ALDH(br) cells with total and viable CD34(+) cells in fresh and thawed hematopoietic progenitor cell (HPC) products, and looked for a correlation with time to white blood cell (WBC) and platelet engraftment after autologous transplantation, using simple linear regression analyzes. We found a significant correlation between pre-freeze ALDH(br) cell numbers and pre-freeze total CD34(+) (P < 0.001), viable CD34(+) (P < 0.001) and post-thaw viable CD34(+) (P < 0.001) cell numbers. We suggest that ALDH(br) may be substituted for CD34(+) cell numbers when evaluating HPC. As post-thaw viability testing apparently adds no significant information, we suggest that it may not be necessary. Finally, neither marker correlated with time to engraftment in our patients, supporting previous data suggesting the existence of a threshold dose for timely engraftment around 2.5 × 10(6) cells/kg.

How do I approach ABO‐incompatible hematopoietic progenitor cell transplantation? (CME)

H uman leukocyte antigen (HLA) matching is critically important for successful hematopoietic progenitor cell (HPC) transplantation because the rates of engraftment and transplant outcomes are strongly influenced by the degree of match. This is largely because HLA antigens are expressed on immature pluripotent stem cells. In contrast, ABO incompatibility is not a barrier to successful HPC transplantation because ABO blood group antigens are not expressed on pluripotent or early committed HPCs. HLA and ABO antigens are encoded by different genes; thus it is common that potential donors who are fully HLA matched have an ABO incompatibility. Approximately 40 to 50% of HPC transplants are ABO incompatible and are now performed using bone marrow (HPC-M), apheresis-derived peripheral blood progenitor cells (HPCA), and umbilical cord blood (HPC-C) as HPC sources. Three types of ABO incompatibility exist: major, minor, and bidirectional. A total of 20 to 25% of HPC transplants have a major ABO incompatibility, 20 to 25% a minor ABO incompatibility, and up to 5% a bidirectional incompatibility. Each presents a unique set of potential adverse consequences, including early acute hemolysis that can be ameliorated through HPC product processing to remove incompatible red blood cells (RBCs) or plasma (Table 1). In these cases, the recipient must be closely monitored both at the time of the infusion and in the posttransplant period because serious events can occur many days later. Accreditation agencies such as AABB and the Foundation for the Accreditation of Cellular Therapy (FACT) require that all donors be tested for ABO group and D type before the collection of HPC-A or HPC-M and that HPC-C be typed after product processing before cryopreservation. The testing and its required documentation in the medical record need to be done in accordance with institutional standard operating procedures. Current FACT standards also require that “for allogeneic cellular therapy products containing RBCs at the time of administration, a test for ABO group and Rh type shall be performed on the first product collected, or on blood obtained from the donor at the time of the first collection.” At our institution, the donor ABO group and D type is tested as part the donor evaluation process. Additional donor ABO group and D testing is performed on each collection day from blood samples obtained at time of collection. If donor blood sample is not available for a particular collection, confirmatory typing is performed on a product sample on the day of product processing. For all fresh HPC products received from outside facilities, confirmatory ABO group and D testing is performed before further product processing. HPC-C units are received in the frozen state and are not subject to repeat testing.

Storage characteristics of cord blood progenitor cells: report of a multicenter study by the cellular therapies team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative

Most hematopoietic progenitor cell (HPC) products are infused or processed shortly after collection, but in some cases this may be delayed for up to 48 hours. A number of variables such as temperature and cell concentration are of critical importance for the integrity of HPCs during this time. We evaluated critical variables using cord blood HPC units that were divided equally and stored at 4 °C versus room temperature (RT) for up to 96 hours. Total nucleated cell (TNC) and mononuclear cell (MNC) counts, viable CD34+ cell counts, and CD45+ cell viability as well as colony-forming unit-granulocyte-macrophage (CFU-GM) present over time at each temperature were determined. Overall, the data indicate that with the exception of viable CD34+ cells, there was a significant decrease in each variable measured for 72 to 96 hours and, with the exception of viable CD34+ cells and CFU-GM, the reductions were significantly greater in RT units than 4 °C units. There was an increase in viable CD34+ count for units where TNC count was greater than 8.5 × 10(9) /L, compared with units where TNC count was less than 8.5 × 10(9) /L, that was different for each storage temperature. Cord blood HPC collections maintained at 4 °C retained higher TNC counts, MNC counts, and CD45+ cell viability over a 72- to 96-hour storage period.

Dual Roles of CD8+ T Cell In Hematopoietic Progenitor Cell Mobilization and Engraftment

AbstractAbstract 349Failure to collect enough CD34+ cells following poor hematopoietic progenitor cell (HPC) mobilization is generally thought to be due to marrow damage following previous therapies. However, marrow damage alone cannot account for all poor mobilizations since there are donors who have good marrow cellularity but fail to adequately mobilize. In addition, the relationship between CD34+ cell dose and engraftment rate is not linear, suggesting other factors play a role during engraftment. Previous animal studies suggest CD8+ T cells are important for CD34+ cell homing to the marrow. We hypothesized that CD8+ T cells have dual roles by inhibiting CD34+ cell mobilization and facilitating engraftment.To test our hypothesis, we followed 192 autologous HPC donors to assess the role of CD8+ T cells in HPC mobilization and engraftment. The median donor age was 58.5 years (range 18 – 76), and 110 of the 192 patients were males. Donors were diagnosed with plasma cell disease, primarily multiple myeloma, or non-Hodgkin's lymphoma. CD34+ and CD8+ T cell content in the patients' apheresis products were enumerated using flow cytometry, quantitative RT-PCR (qPCR) and immunohistochemistry. The median CD34+ cell/kg was 4.7 × 106.Patients were categorized as low, moderate or high mobilizers according to their total CD34+ cell collection quartile distribution: <3.7, 3.8 – 5.5 and ≥5.6 × 106/Kg respectively. We found that HPC products from low mobilizers contained significantly more CD8+ T cells (median 1.25 × 108/Kg) than HPC products from moderate and high mobilizers (Table 1 p = 0.04). Therefore, the ratio of CD8+ T cells to CD34+ cells was 4 – 8 folds higher in low mobilizers than in moderate and high mobilizers. Moreover, CD8 antigen expression measured using qPCR, normalized with 18s ribosomal RNA, was significantly higher in low mobilizers than in moderate or high mobilizers. Similar analysis with CD4+ T cells did not demonstrate significant correlation with mobilization.To assess the role of CD8+ T cells in HPC mobilization, we purified CD8+ T and CD34+ cells from both low and moderate or high mobilizers. Using chemotaxis assay, we assessed the effect of CD8+ T cells on CD34+ cell migration away from high stromal cell derived factor (SDF1-a) concentrations. Purified CD34+ cells were plated in bone marrow epithelial cell coated trans-wells and co-cultured with purified CD8+ T cells at 1:2 and 1:5 ratios in the presence of SDF-1a at 0, 200 and 400ng/ml concentrations. Cells that migrated through the micro-pores of the trans-well into the lower wells were enumerated and analyzed. Our studies showed CD8+ T cells inhibit migration of CD34+ cells in-vitro independent of SDF1-a concentration. (Figure 1) The inhibitory properties of CD8+ T cells were more pronounced on CD34+ cells from low mobilizers than on CD34+ cells from moderate or high mobilizers.We also assessed if CD8+ cell content was associated with days to neutrophil engraftment. Our analysis showed the higher the CD8+ T cell content per unit CD34+ cell, the faster the rate of neutrophil engraftment.In summary, HPC products from low mobilizers contained significantly higher numbers of CD8+ T cells. CD8+ T cells, independent of SDF1-a concentration, significantly inhibited migration of purified CD34+ cells. In addition, CD8+ T cell content per unit CD34+ cell was inversely proportional to the time to neutrophil engraftment. These findings suggest CD8+ T cells may play dual roles by inhibiting CD34+ cell mobilization and facilitating its homing and engraftment.Table 1Association of CD8+ T cell number with CD34+ cell mobilization capacityMobilization QuartilesLowModerateHighp-valueMedian CD34+ Cell × 106/Kg3.034.556.73<0.001Median CD8+ T cell × 108/Kg1.250.500.330.04CD8+ T: CD34+ cell ratio41115 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Survivin Modulates Gene Expression That Connects through An Extensive Functional Signaling Network and Regulates Proliferation of Hematopoietic Stem Cells through Evi-1

AbstractAbstract 87Survivin is a member of the inhibitor of apoptosis protein (IAP) family that antagonizes caspases and has been implicated in regulation of apoptosis, cell division and cell cycle both in cancer cells and normal tissues. Survivin is essential for activation of Aurora kinase that phosphorylates Histone H3, an event required for transcriptional regulation and cytokinesis. Although there is no evidence that Survivin directly regulates gene transcription, Bir1, a C. elegans homologue of Survivin, regulates transcription, most likely through Histone phosphorylation by Aurora kinase. We previously showed that Survivin is expressed and growth factor regulated in human CD34+ cells (Fukuda & Pelus. Blood 2001 & 2002). Antagonizing Survivin impairs production of mouse bone marrow hematopoietic progenitor cells in vitro (Fukuda et al. Blood 2004) and conditional Survivin gene deletion in vivo in mice leads to bone marrow ablation as a result from loss of hematopoietic stem and progenitor cells (Leung et al. JEM 2007). However, little is known about signaling pathways downstream of Survivin in hematopoietic stem cells (HSC). Herein, we investigated the mechanism of action of Survivin by creating Survivin gene signature network following gene deletion in HSC and by functional rescue on impaired HSC activity induced by Survivin deletion.Survivin deletion in Tamoxifen-Cre Survivinflox/flox mice led to significant changes in 2,228 mRNA in marrow CD34neg, c-kit+, Sca-1+, lineage− cells. Molecular network of the genes affected by Survivin deletion in HSC were created using DAVID (Huang et al. Nature Protocol 2009) and Cytoscape (Cline et al. Nature Protocol 2007). Among the signaling pathways defined by KEGG (www.kegg.org), phosphatidylinositol signaling pathway was most significantly affected in HSC by Survivin gene deletion (P<0.05). In addition, at least 56 genes regulated by Survivin were connected through an extensive network structured by 183 genes surrounding the phosphatidylinositol signaling pathway. These include Stathmin1, p38, NFkB, Jun D and Evi-1, a transcriptional factor that regulates HSC proliferation and prevents stress-induced cell death. Furthermore, knocking down Survivin induced coincident repression of 43 HSC related genes such as Gata2, Pbx1 and Sal2 in addition to Evi-1. This is consistent with down-regulation of Gata2, Pbx1 and Sal2 by Evi-1 ablation in HSC in vivo (Goyama et al. 2008) and suggests that Evi-1 may function downstream of Survivin to maintain HSC function. To test this hypothesis, marrow cells from conditional Survivin−/− mice were transduced with vector or Evi-1 and transplanted into lethally irradiated normal recipients. Subsequent Survivin deletion in vivo significantly impaired hematopoietic recovery (53.0±4.4% vs 1.6±0.3% donor chimerism in Survivin+/+ and Survivin−/− cells, respectively, P<0.01), whereas Survivin−/− marrow cells over-expressing Evi-1 showed a marginal but significant increase in hematopoietic recovery at 6 months post transplantation (7.9±0.7% donor chimerism, P<0.01 compared to control vector in Survivin−/−). Gata2 over-expression did not affect hematopoietic recovery (0.6±0.1% donor chimerism). None of the mice transplanted with cells harboring ectopic Evi-1 developed leukemia after 12 months, making it unlikely that recovery of donor chimerism is a consequence of clonal expansion of transduced cells by Evi-1.These results suggest that Evi-1 lies down stream of Survivin in HSC and that repression of Evi-1 expression is a functional consequence of Survivin ablation rather than an indirect effect resulting from apoptosis and/or cell cycle alteration of HSC induced by Survivin deletion. Our data indicate that Survivin regulates HSC proliferation at least through Evi-1. Alteration of genes with multiple cellular functions as a consequence of Survivin deletion and the presence of a functional network downstream of Survivin suggest that Survivin regulates multiple effector pathways in HSC, independent of its activity as a caspase inhibitor. Disclosures:No relevant conflicts of interest to declare.

Viability And Potency Of Hematopoietic Progenitor Cells After Prolonged Cryopreservation At -80 °C

Hematopoietic progenitor cell (HPC) products cryopreserved in the liquid or vapor phase of liquid nitrogen (LN) remain viable and potent for years. Cryopreserved HPC products when stored at -80°C in mechanical freezers are known to remain viable and potent for up to 180 days. We demonstrated viability and potency of HPC products stored at -80°C for up to 12 years. Products were cryopreserved in 6% hydroxyethyl starch, 5% DMSO and 4% human serum albumin using non-controlled rate freezing.

Controle de esterilidade de produtos de células progenitoras hematopoéticas do sangue periférico

A taxa de contaminação microbiana dos produtos de células progenitoras hematopoéticas do sangue periférico é baixa. Neste estudo pesquisou-se a prevalência de hemoculturas positivas em células progenitoras hematopoéticas do sangue periférico (CPHSP) no Serviço de Hemoterapia do Hospital de Clínicas de Porto Alegre. Do total de 618 coletas realizadas no período de 2000 a 2007, 26 (4,2%) apresentaram contaminação por bactérias. O Staphylococcus coagulase-negativo foi predominantemente isolado nas hemoculturas. A antibioticoterapia pré e pós-infusão foi estabelecida de acordo com o microorganismo e seu antibiograma, sendo que, em cinco das doze infusões contaminadas realizadas, não foram administrados antimicrobianos profilaticamente. Episódios febris foram observados em sete pacientes (58%), enquanto cinco (42%) não apresentaram febre. Das doze infusões contaminadas realizadas, seis (50%) apresentaram hemocultura pós-descongelamento positivas, enquanto as restantes (50%) foram negativas. Isto se deve às propriedades bactericidas do DMSO, de células fagocitose-ativas e de temperaturas muito baixas atingidas na criopreservação. Autores têm relatado sucesso neste procedimento após a infusão desses produtos contaminados com o mínimo de consequências clínicas.

Solving an unusual clotting event during thawing of peripheral blood hematopoietic progenitor cell product

Although rare, hematopoietic progenitor cell (HPC) products clotting upon thawing presents enormous challenge. Peripheral blood HPC product was collected from a 10-year-old sibling donor, using acid-citrate-dextrose Formula A (ACD-A) ratio of 1:15 and additional heparin. Most of the product was freshly infused with no apparent clotting, and two additional aliquots were cryopreserved in dimethyl sulfoxide and human serum albumin for future infusion. Two months later one aliquot was thawed and issued in a syringe. At the time of issue no clotting was observed, but the infusion was stopped due to massive product clotting (see figure, A). Cell clumping is caused by multiple factors such as plasma cryofibrinogens and can be overcome by adding anticoagulants and altering cryopreservation practices. 1,2 We experienced only one similar event of 500 products collected over the past 5 years. Since our investigation found no unusual practices in the collection and processing of the recent product, we took extra measures to prevent clotting when thawing the last aliquot. These included adding ACD-A in 1:8 ratio, further dilution with Plasmalyte A, and filtration. The product was issued and administered successfully. To test our practice, a small postthaw sample was transferred into a tube before addition of the anticoagulant. This original unmanipulated sample clotted within 1 hour while the manipulated one did not (see figure, B), proving that ACD-A prevented clotting. Most collection facilities try to avoid using higher volumes of ACD-A due to its possible toxicity. 3 Adding anticoagulant to the final apheresis product might be an optional way to reduce the probability of similar events.

Association Of Hematopoietic Stem Cell Product Culture And Clinical Infection In Patients Receiving Myeloablative Chemotherapy With Autologous Hematopoietic Stem Cell Reinfusion

Purpose: The purpose of this descriptive retrospective QA project is to examine the association between hematopoietic progenitor cell (HPC) product cultures at the time of reinfusion and the subsequent development of infection. Methods: Using a descriptive, retrospective design, data were collected from the records of 68 patients who received autologous peripheral blood HPC reinfusions after undergoing myeloablative chemotherapy at a single institution. Consistent with the oncology service QA program, all of the products were tested for microbial contamination at the time of stem cell processing. At the time of reinfusion, a sample from each thawed product was sent to the microbiology lab. Following the HPC reinfusion, blood cultures were sent on patients at the time of their first fever and at least once daily until they were afebrile and cultures were negative. Blood cultures were sent from each lumen of the central venous catheter. Peripheral blood cultures were not routinely drawn. Results: Between March 2002 and September 2008, 68 patients received 148 HPC reinfusions. All of the reinfused HPC products were negative for bacterial contamination at the time of processing. Nineteen of the reinfused HPC cultures, which were reinfused into 13 patients, were positive. Two of these patients developed positive blood cultures. Patient #1 had HPC product culture positive for Bacillus species and Staphhylococcus epidermis. Blood cultures were positive only for S. epidermis on day +15. Patient #2 had HPC product cultures positive for Streptococcus oralis. Blood cultures were positive for Streptococcus mitis on day +4 and S. epidermis on day +6. Conclusions: There does not appear to be a strong correlation between positive product cultures and the subsequent development of positve blood cultures. Microbial testing at the time of HPC reinfusion may not be necessary.

Application of pulsed‐field gel electrophoresis to identify the source of bacterial contamination of peripheral blood progenitor cell products

Positive microbial cultures of peripheral blood progenitor cell (PBPC) products, although estimated to be low, are serious events in the manufacture of hematopoietic progenitor cell (HPC) products that warrant a thorough investigation to determine the contamination source. Two patients underwent autologous PBPC collection. The first patient was admitted before the collection and was febrile intermittently throughout hospitalization. The second patient spiked a low-grade fever by the end of the procedure. The HPC products from each patient were cultured during processing and before infusion. Blood cultures were drawn during febrile episodes and before transplant. Bacterial identification and antimicrobial susceptibilities were performed on all positive cultures. All strains were typed using pulsed-field gel electrophoresis (PFGE) to determine their relatedness. The blood cultures from both patients and their corresponding HPC products grew Staphylococcus epidermidis. The PFGE pattern of the S. epidermidis recovered from each patient blood was indistinguishable from the one recovered from the corresponding HPC product. The gel pattern of the strains recovered from the first patient differed by four bands from the one recovered from the second. For each patient, the antibiotic susceptibility patterns of the blood cultures and the HPC products were identical. Infusion of the contaminated HPC had no adverse event, and the patients engrafted successfully. By use of PFGE technology, the contamination source of PBPC products was identified. It is concluded that the contamination resulted from intermittent bacteremia in the donors and was not introduced during laboratory manufacturing.

Evaluation of ‘out-of-specification’ CliniMACS CD34-selection procedures of hematopoietic progenitor cell-apheresis products

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. 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). 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. 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.

Microbial contamination of hematopoietic progenitor cell products: clinical outcome

We reviewed the results of routine microbiological assays of 3078 infused hematopoietic progenitor cell (HPC) products for autologous and allogeneic transplantation between January 2001 and December 2005. Thirty-seven (1.2%) contaminated products were found. All patients receiving contaminated infusions received empirical antibiotic prophylaxis according to the assay result. None of these patients developed a positive blood culture with the same agent, developed infections that could be attributable to the contaminated product or experienced any clinical sequelae. Coagulase-negative Staphylococcus was found in 32 (86.5%) products. Admission lengths and time to engraftment were within the expected time frame for autologous and allogeneic transplants. Microbial contamination of HPC products occurs at a low frequency; prophylactic use of antibiotics based on the microbiological assay appears to be effective in preventing clinical complications.

Sterility testing of hematopoietic progenitor cell products: a single‐institution series of culture‐positive rates and successful infusion of culture‐positive products

Administration of culture-positive hematopoietic progenitor cells (HPCs) causing adverse events has been a hypothesized yet largely unmeasured risk of the clinical practice of HPC transplantation. To enhance patient safety, the FDA has issued regulations prohibiting the use of culture-positive HPCs. Numerous studies have reported the infusion of culture-positive HPCs; however, the low frequency of adverse events prevents accurate determination of this risk. Product culture results and clinical outcomes from January 1998 through March 2006 representing 7233 HPC collections for 2118 transplants at a single institution were reviewed. A total of 119 units of HPCs (1.6%) intended for 95 patients were culture-positive. Of the 69 patients transplanted with culture-positive HPCs, 5 received products with cultures pending, and 64 received products with the positive culture results known. One of 69 patients had a new positive blood culture 5 days after infusion with the same species as the product. There was not a clinically relevant difference in the rate of infusion-related symptoms reported for patients who received culture-positive products compared to all infusions. The survival of patients who received culture-positive products (n = 69) was not different from all HPC recipients (n = 2046; p = 0.419). No infusion-related risks of culture-positive HPCs to patient safety were identified. Our data suggest that the decision to use culture-positive HPCs must be made in the context of the global risks associated with transplants such as remobilization, replacement product availability, and the nature of the organism.

β-Catenin Expression in Cultured Bone Marrow Stromal Cells Is Required To Maintain Production of Osteoblasts and Hematopoietic Progenitor Cells.

The canonical Wnt signaling pathway is defined by Wnt ligand-mediated stabilization and nuclear translocation of β-catenin to induce target gene expression. This pathway has been demonstrated to regulate differentiation of mesenchymal tissue, which includes the cell types (e.g. osteoblasts, myofibroblasts, adipocytes) that comprise the stromal cells of the hematopoietic microenvironment. We hypothesized that loss of canonical Wnt signaling would result in disruption of the ability of stromal cells to support hematopoiesis. To test this hypothesis, we generated transgenic mice that expressed conditonal loss of function β-catenin alleles along with Cre-recombinase under the control of the inducible Mx1 promoter, which is active in bone marrow. We induced excision of β-catenin by injecting β-cateninlox/lox Mx-cre+/cre mice with 10 doses of 300 mg/ml pIpC. Whole bone marrow from treated (KO) and untreated (WT) animals was used to establish Dexter stromal cultures with an input of 1 × 106 cells/cm2 culture surface area. PCR performed on DNA isolated from KO stromal cells showed that nearly 100% deletion of β-catenin occurred with this regimen. To determine the ability of KO stroma to support hematopoiesis, irradiated KO and WT stromal cultures were seeded with 4 × 104 normal lin− cells/cm2. There were no differences in cell expansion, cell cycle activity, or apoptosis between hematopoietic cells cultured on WT vs. KO stroma. We determined the capacity of β-catenin deficient stroma to maintain hematopoietic progenitors by measuring myeloid CFU formation after 1, 2, and 3 weeks in culture. After 1 week, hematopoietic cells cultured on WT stroma contained 5-fold more CFU-GM (151.7 ± 21.4 CFU-GM/1×104 cells) than cells cultured on KO stroma (28.7 ± 4.9; n = 6, p < .001). Similar differences in CFU-GM formation were observed after 2 weeks (WT 46.5 ± 8.0 vs. KO 10.3 ± 1.7; n = 6, p< .001) and 3 weeks (WT 16.5 ± 2.8 vs. KO 2.6 ± 1.5; n = 6, p < .001) in culture. This decrease in the production of hematopoietic progenitor cells was not due to decreased numbers of stromal cells as the average number of KO stromal cells (4.8 ± 0.07 × 104/cm2) was greater than WT (3.7 ± 0.7 × 104/cm2; n = 3, p = .05). We also determined the ability of WT and KO mesenchymal progenitors to generate fibroblast colonies (CFU-F) and found no difference between WT (17 ± 1.8 CFU-F/1 × 106 bone marrow cells) and KO (15.8 ± 3.5; n = 4, p = .54). Canonical Wnt signaling has been proposed to regulate the differentiation of mesenchymal stem cells into osteoblasts. Since osteoblasts contribute to the proper regulation of hematopoiesis, we hypothesized that the depletion of hematopoietic progentiors in KO stromal cultures is due to a reduction in the number of osteoblasts. To detect osteoblasts in vitro, we performed histochemical staining to detect alkaline phosphatase (ALP) activity in WT and KO stromal cultures and scored the positive cells. We observed a significant 50% reduction in the percentage of ALP+ cells in KO stroma (13.2 ± 4.8%) compared to WT (28.0 ± 7.9%) (n = 3, p = .05). In summary, these data indicate that loss of canonical Wnt signaling results in decreased support of hematopoietic progenitors and osteoblasts. From these data, we propose a model in which canonical Wnt signaling is necessary to maintain normal numbers of osteoblasts within the bone marrow stroma and that loss of β-catenin leads to a decrease in the number of osteoblasts and a subsequent reduction in the ability of the stroma to support hematopoiesis.