Peripheral Blood Hematopoietic Progenitor Research Articles

Discovery and evaluation of ZT55, a novel highly-selective tyrosine kinase inhibitor of JAK2V617F against myeloproliferative neoplasms

BackgroundThe JAK2-STAT signaling pathway plays a critical role in myeloproliferative neoplasms (MPN). An activating mutation in JAK2 (V617F) is present in ~ 95% of polycythemia vera, essential thrombocythemia, and primary myelofibrosis cases. This study aims to explore the selective JAK2V617F inhibitor, evaluate the efficacy and possible mechanism of ZT55 on MPN.MethodsHTRF assays were conducted to evaluate the selective inhibition of ZT55 for JAKs. Cell apoptosis, proliferation, and cycle arrest assays were performed to examine the effect of ZT55 on HEL cell line with JAK2V617F mutation in vitro. Western analysis was used to monitor the expression and activity of proteins on JAK2/STAT pathway. A mice xenograft model was established to evaluate the antitumor efficacy of ZT55 in vivo. Peripheral blood samples from patients with the JAK2V617F mutation were collected to estimate the effect of ZT55 on erythroid colony formation by colony-forming assay.ResultsWe found that ZT55 showed a selective inhibition of a 0.031 μM IC50 value against JAK2. It exhibited potent effects on the cellular JAK-STAT pathway, inhibiting tyrosine phosphorylation in JAK2V617F and downstream STAT3/5 transcription factors. ZT55 inhibited the proliferation of the JAK2V617F-expressing HEL cell line, leading to cell cycle arrest at the G2/M phase and induction of caspase-dependent apoptosis. Notably, ZT55 also significantly suppressed the growth of HEL xenograft tumors in vivo. Further evaluation indicated that ZT55 blocked erythroid colony formation of peripheral blood hematopoietic progenitors from patients carrying the JAK2V617F mutation.ConclusionThese results suggest that ZT55 is a highly-selective JAK2 inhibitor that can induce apoptosis of human erythroleukemia cells by inhibiting the JAK2-STAT signaling.

Processing of G-CSF Mobilized Peripheral Blood Hematopoietic Progenitor Cells (PB-HPC) from Matched- Related or Unrelated Donors Undergoing CD34 Enrichment with T Cell Addback in Children, Adolescents and Adults with Malignant and Nonmalignant Diseases: Rapid Engraftment, Immune Cell Reconstitution and Sustained Donor Chimerism

Sixty to seventy percent of patients undergoing HLA identical transplants will develop grade II-IV acute GVHD, despite using prophylactic immunosuppressants. Several studies have demonstrated that manipulation utilizing ex-vivo CD34+ positively selected grafts result in 4-5 log T-cell depletion with limited T-cell add back follow with early engraftment and decreased aGVHD (Silva et al, Blood Journal, 2015 & Chakrabarti et al, BMT, 2003). We previously demonstrated the safety and efficiency of CD34+ enriched cells with 2 × 105 CD3 (MNC)/kg add back in pediatric SCD patients resulting in rapid engraftment, donor chimerism, and low incidence of aGVHD (Geyer/Cairo et al, BJH, 2012). Utilizing CD34+ cell selection, children, adolescents, and adults with a matched or partially matched family donor or matched unrelated donors allogeneic PB-HPC transplant can safely receive and tolerate with a low incidence of serious (Grade III/IV) acute and chronic GVHD. Patients received CD34+ enriched products processed by using the CliniMACS CD34+ Reagent System (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD34+ enriched HPC product was either a fresh infusion or cryopreserved and stored until time of transplantation. A target of 5 × 106 CD34+enriched/kg recipient weight with a T cell add back of 2 × 105 CD3+/kg (MNC) was infused. Sixteen patients underwent BMT with median age of 55 years (23 months to 71 years). Patients' disease status was as follows: complete remission (CR) 2 in four AML patients, CR1 in 9 AML patients, Lymphohistiocytosis (n=1), Macrophage Activating Syndrome (n=1), CR2 in one Non-Hodgkins Lymphoma patient. Nine patients received 10/10 HLA-matched unrelated donor BMT, two 9/10 HLA-matched unrelated donors, five had 6/6 HLA-matched sibling transplant. PB-HPC products contained 0.59 × 1010 CD3/kg (±0.23), and 6.77 × 106 CD34/kg (±1.27). After CD34 enrichment, the PB-HPC CD34% recovery was 73.1±3.08% with a log T cell depletion of 4.9 (±0.11). The target transplant dose per patient was 5 × 106 CD34 enriched/kg. Sixteen and patients had myeloid and 13 patients had platelet engraftment with a median of 11.5 days and 16 days, respectively (Fig. 1a and Fig. 1b). Two patients died prior to platelet engraftment one due to multi-organ system failure following septic shock and the other patient due to refractory AML. One patient died after myeloid and platelet engraftment secondary to disease progression. Early and late peripheral blood chimerism were ≥ 98.4% at 19.9 days (±12) and 92.5% at 180 days (n=4). The probability of grade II-IV aGVHD was 20% (CI95: 0.1-66.9) (Fig 2). One patient had grade II skin aGVHD and one patient had grade II gut aGVHD. No patient had cGVHD. This study demonstrates the safety and efficiency of CD34+ enriched PB-HPC products with T-cell addback in AlloSCT recipients with a low probability of acute GVHD.

Dissecting Ex Vivo Expansion of Mobilized Peripheral Blood Hematopoietic Stem and Progenitor Cells By Single Cell RNA Sequencing

Hematopoietic stem and progenitor cell (HSPC) expansion remains an important unmet goal for ex vivo gene therapy based on gene addition and editing to compensate for the negative impact of the gene transfer procedure enabling faster engraftment and less complications. Additionally, ex vivo expansion of corrected cells may improve efficacy at more sustainable manufacturing costs by downscaling transduction. To date, our knowledge of precise mechanisms of action of expansion compounds is limited, and it remains unclear whether cord blood expansion protocols also maintain stemness of mobilized peripheral blood CD34+ cells (mPB), the preferred HSPC source for gene therapy.We performed serial (day 0,4,8) droplet-based single cell RNA sequencing (scRNAseq) on lentivirally transduced mPB expanded with UM171 to dissect cellular heterogeneity, monitor population dynamics over time and identify a transcriptional profile of primitive cells in culture.By associating published HSPC gene expression profiles to our scRNAseq dataset from uncultured mPB, we found that 45% of cells harbored a myelo-lymphoid signature. Smaller cell clusters expressed a shared erythroid (ERY) and megakaryocytic (MK) signature (20%), or a more primitive multipotent HSC-like signature (15%) characterized by enhanced JAK/STAT signaling and expression of HSC associated genes (AVP, HOPX, ID3). Unsupervised ordering of cells within pseudotime separated emerging MK/ERYpoiesis (FCER1A, HBD) from lympho-myelopoiesis (CD52, JUN), with intermediate states of more primitive progenitors located in between.After 4 days in culture, we noted a general increase in nuclear and mitochondrial gene transcription with activation of oxidative metabolism, paralleled by cell cycle activation, as expected from cytokine stimulation. By d8 of culture these changes leveled off but remained higher than uncultured cells. Of note, cells at d8 revealed an activation of cellular stress response pathways (e.g. TNFa, IFNg) hinting towards a compromised culture that may eventually exhaust HSC.Unsupervised clustering of cultured mPB highlighted a dramatic expansion (70-80%) of MK/ERY progenitor cells with high cycling activity with only 20-30% cells showing myelo-lymphoid transcriptional features. In line, pseudotime analysis highlighted a main ERY and MK trajectory separated from that of cells characterized by the expression of HSPC genes (HOPX, SPINK2) and of an emerging myeloid trajectory (MPO).To profile HSC in culture, we sorted and sequenced CD34+90+201+ cells from d4 expansion culture (3% of total cells), which we show to contain >70% of SCID repopulating potential. ScRNAseq revealed transcriptional similarity with the myelo-lymphoid progenitor cluster identified in the unsorted d4 culture. Unsupervised clustering of the CD34+90+201+ population revealed cell cycle dependent heterogeneity, identifying a highly quiescent cluster with expression of HSC-like signatures. This cluster was also characterized by relatively low gene expression, possibly reflecting a non-activated cell state consistent with primitive HSPC. Pseudotime analysis produced a four-branched minimum spanning tree, which retained a clear cell cycle and metabolic effect. Top variable genes included cell cycle, glycolytic, mitochondrial and ribosomal genes, identifying different metabolic modules along the branched trajectory. These results highlight that cell heterogeneity within a purified, HSC-enriched population is driven mainly by metabolic activation and cell cycle status.As a complementary approach, we purified LT-HSC from uncultured mPB (CD34+38-90+45RA-49f+), marked them with CFSE and expanded them in UM171 culture. LT-HSCs expanded on average 3.5 fold in 7 days, with the following distribution: 0 divisions: 3%; 1: 26%; 2: 47%; 3: 21%; 4: 3%. We performed scRNAseq on LT-HSC pre culture and after 7d separating a highly proliferative (≥2 divisions) and quiescent (0 - 1 division) fraction, allowing us to obtain unprecedented insight into the response of engrafting cells to ex vivo culture and set a framework to dissect self-renewal (HSC expansion), HSC maintenance and loss through differentiation as potential culture outcomes. Our combined functional/transcriptomic approach will define new HSC markers in culture and greatly facilitate side-by-side comparison of different expansion protocols towards rapid clinical translation. DisclosuresNo relevant conflicts of interest to declare.

Myocardial infarction does not affect circulating haematopoietic stem and progenitor cell self-renewal ability in a rat model.

What is the central question of this study? Although peripheral blood haematopoietic stem and progenitor cells are potentially important in regeneration after acute myocardial infarction, their self-renewal ability in the post-acute phase has not yet been addressed. What is the main finding and its importance? In rat peripheral blood, we show that myocardial infarction does not negatively affect circulating haematopoietic stem and progenitor cell self-renewal ability 2 weeks after acute infarction, which suggests a constant regenerative potential in the myocardial infarction post-acute phase. Given the importance of peripheral blood haematopoietic stem and progenitor cells (HPCs) in post-acute regeneration after acute myocardial infarction (MI), the aim of the present study was to investigate the number and secondary replating capacity/self-renewal ability of HPCs in peripheral blood before and 2weeks after MI. In female Lewis inbred rats (n=9), MI was induced by ligation of the left coronary artery, and another nine underwent sham surgery, without ligation, for control purposes. Myocardial infarction was confirmed by troponinI concentrations 24h after surgery. Peripheral blood was withdrawn and fractional shortening and ejection fraction of the left ventricle were assessed before (day0) and 14days after MI or sham surgery (day14). After mononuclear cell isolation, primary and secondary functional colony-forming unit granulocyte-macrophage (CFU-GM) assays were performed in order to detect the kinetics of functional HPC colony counts and cell self-renewal ability in vitro. The CFU-GM counts and cell self-renewal ability remained unchanged (P>0.05) in both groups at day14, without interaction between groups. In the intervention group, higher day0 CFU-GM counts showed a relationship to lower fractional shortening on day14 (ρ=-0.82; P<0.01). Myocardial infarction did not negatively affect circulating HPC self-renewal ability, which suggests a constant regenerative potential in the post-acute phase. A relationship of cardiac contractile function 14days after MI with circulating CFU-GM counts on day0 might imply functional colony count as a predictive factor for outcome after infarction.

Performance and safety of femoral central venous catheters in pediatric autologous peripheral blood stem cell collection.

Autologous peripheral blood hematopoietic progenitor cell collection (A-HPCC) in children typically requires placement of a central venous catheter (CVC) for venous access. There is scant published data regarding the performance and safety of femoral CVCs in pediatric A-HPCC. Seven-year, retrospective study of A-HPCC in pediatric patients collected between 2009 and January 2017. Inclusion criteria were an age ≤ 21 years and A-HPCC using a femoral CVC for venous access. Femoral CVC performance was examined by CD34 collection rate, inlet rate, collection efficiency (MNC-FE, CD34-FE), bleeding, flow-related adverse events (AE), CVC removal, and product sterility testing. Statistical analysis and graphing were performed with commercial software. A total of 75/119 (63%) pediatric patients (median age 3 years) met study criteria. Only 16% of children required a CVC for ≥ 3 days. The CD34 collect rate and CD34-FE was stable over time whereas MNC-FE decreased after day 4 in 80% of patients. CD34-FE and MNC-FE showed inter- and intra-patient variability over time and appeared sensitive to plerixafor administration. Femoral CVC showed fewer flow-related AE compared to thoracic CVC, especially in pediatric patients (6.7% vs. 37%, P = 0.0005; OR = 0.12 (95%CI: 0.03-0.45). CVC removal was uneventful in 73/75 (97%) patients with hemostasis achieved after 20-30 min of pressure. In a 10-year period, there were no instances of product contamination associated with femoral CVC colonization. Femoral CVC are safe and effective for A-HPCC in young pediatric patients. Femoral CVC performance was maintained over several days with few flow-related alarms when compared to thoracic CVCs.

Comparison of Amicus and COBE Spectra for allogenic peripheral blood stem cell harvest: Study from tertiary care centre in India

IntroductionMost common source of stem cell graft for both autologous and allogenic haematopoietic transplants are peripheral blood haematopoietic progenitor stem cells. Adequate collection of the CD34+ cells and safety of the allogenic donor during the leukapheresis are of prime importance to an apheresis physician. Our retrospective analysis is a comparison between of two platforms namely, COBE Spectra and Amicus, for CD34+ mononuclear cell collection. Material and methodThe study included the data of GSCF (Granulocyte-Colony-Stimulating Factor) mobilized allogenic PBSC collections at our centre from January 2015 to June 2016. The apheresis platforms used were COBE Spectra and Amicus. Blood cell counts were done using LH750 Beckman Coulter (Florida, Miami, USA). CD45+ & CD34+ cell counts were done using BD FACS Canto-II Flow-Cytometer by ISHAGE guidelines. ResultsA total of 170 PBSC (100 COBE Spectra & 70 Amicus) harvests were done on 143 donors, of which 116 completed the collection in a single session and 27 required a second session. Demographic details and pre harvest peripheral blood counts for both the groups did not show any statistical differences. Amicus processed higher blood volume with higher ACD exposure and procedure time compared to COBE Spectra. Higher platelets loss was with COBE Spectra harvests with higher product volumes collection. Collection efficiency (CE2), collection ratio, CD34+ cells dose was similar on both the platforms. RBC contamination, absolute lymphocyte and monocytes counts were significantly higher with Amicus harvest product compared with COBE Spectra. A total of 14 (8.2%; citrate toxicity) adverse reactions were reported out of 170 allogenic PBSC collections. Discussion/conclusionOur study suggests that both Amicus and COBE Spectra platforms offer comparable results for allogenic PBSC collections. Amicus offers a concentrated PBSC product with lesser volume and platelets loss but higher RBC contamination.

Zoledronic acid alters hematopoiesis and generates breast tumor-suppressive bone marrow cells

BackgroundThe bone-targeting agent zoledronic acid (ZOL) increases breast cancer survival in subsets of patients, but the underlying reasons for this protective effect are unknown. ZOL modulates the activity of osteoclasts and osteoblasts, which form hematopoietic stem cell niches, and therefore may affect hematopoietic cells that play a role in breast cancer progression.MethodImmunocompetent and immunocompromised strains of mice commonly used for breast cancer research were injected with a single, clinically relevant dose of ZOL (100 μg/kg) or vehicle control. The effects of ZOL on the bone marrow microenvironment (bone volume, bone cell number/activity, extracellular matrix composition) were established at various time points following treatment, using micro-computed tomography (μCT) analysis, histomorphometry, ELISA and immunofluorescence. The effects on peripheral blood and bone marrow hematopoietic progenitor populations were assessed using a HEMAVET® hematology analyzer and multicolor flow cytometry, respectively. Tumor support function of bone marrow cells was determined using an in vivo functional assay developed in our laboratory.ResultsUsing multiple mouse strains, we observed transient changes in numbers of hematopoietic stem cells, myeloid-biased progenitor cells, and lymphoid-biased cells concurrent with changes to hematopoietic stem cell niches following ZOL administration. Importantly, bone marrow cells from mice treated with a single, clinically relevant dose of ZOL inhibited breast tumor outgrowth in vivo. The ZOL-induced tumor suppressive function of the bone marrow persisted beyond the time point at which numbers of hematopoietic progenitor cells had returned to baseline.ConclusionsThese findings provide novel evidence that alterations to the bone marrow play a role in the anti-tumor activity of ZOL and suggest possibilities for capitalizing on the beneficial effects of ZOL in reducing breast cancer development and progression.

332 - Familial Haploidentical (FHI) T-Cell Depleted (TCD) with T-Cell Addback Stem Cell Transplantation for Patients with High-Risk Sickle Cell Disease (SCD) (IND 14359)

Allogeneic stem cell transplantation (AlloSCT) from HLA-matched unaffected sibling donors (MSD) has been successful for high-risk SCD, and is the only known curative therapy (Freed/Cairo et al, BMT, 2012). We have recently demonstrated 100% event free survival and absence of sickle cell symptoms following reduced toxicity conditioning and HLA matched sibling bone marrow or cord blood AlloSCT (Bhatia/Cairo et al, BMT, 2014). However, 5 out of 6 children who might benefit from this therapy lack an HLA matched family donor. Identifiable matched unrelated adult donors (URD) in this ethnic group are extremely limited and results from unrelated cord blood transplants are poor (Radhakrishnan K/Cairo et al., BBMT 2013, Kamani et al., BBMT, 2012). We previously demonstrated the use of positive CD34 selection followed by T cell add back (2 x 105 CD3/kg) from unrelated donors in pediatric recipients with both malignant and nonmalignant disease lead to 100% engraftment with minimal acute GVHD (aGVHD). In a high-risk FHI TCD thalassemia study, 16/22 cases engrafted without aGVHD and with 90% overall survival (Sodani et al., Blood, 2010). FHI TCD AlloSCT could expand the donor pool and improve outcomes for patients with high risk SCD. This SCD consortium trial is investigating the safety, feasibility, EFS, donor chimerism, graft failure, aGVHD and chronic GVHD (cGVHD), and infectious mortality after FHI TCD AlloSCT in high-risk SCD patients ([Figure 1][1]). High risk features included one or more of the following: ≥1 CVA, ≥2 ACS, ≥3 VOC in past 2 years, or 2 abnormal TCDs. Patients (2-20.99 yrs) without an 8/8 HLA MSD or URD and who have ≥1 high-risk SCD features were eligible. Patients received hydroxyurea 60 mg/kg/d and azathioprine 3mg/kg/d, day -59 – day -11, fludarabine (30mg/m2/d x5d), busulfan (3.2 mg/kg/d x4d [<4yrs of age: 4 mg/kg/d x4d]), thiotepa (10 mg/kg/d x1d), cyclophosphamide (50mg/kg/d x4d), R-ATG (2mg/kg/d x4d), and TLI (500cGy) followed by FHI T-cell depleted AlloSCT. AGVHD prophylaxis included tacrolimus single agent. We utilized the CliniMACS (IND 14359) to enrich for peripheral blood hematopoietic progenitor cells (HPC's); target dose of 10 x 106 CD34+ cells/kg with 2 x 105 CD3+ T cells/kg added back as a final CD3/kg concentration. Six patients have received AlloSCT to date ([Figure 2][2]). All patients utilized maternal donors who encountered no complications during collection. All had early neutrophil engraftment (median day +9), ≥98% whole blood chimerism and ≥85% RBC donor chimerism, no aGVHD or cGVHD ([Figure 2][2]). One patient developed late hepatic SOS and died at day +59; the remainder are alive and free of disease (day +12 to +642). One more patient has been enrolled and has begun conditioning and others are in the early part of their transplant process. Early results indicate FHI TCD AlloSCT is feasible in high-risk SCD patients who lack a MSD or URD. A larger cohort with longer term follow-up is needed to assess long-term safety and outcomes (Supported by FDA 5R01FD004090 and a grant from Otsuka) (IND #14359 and NCT 01461837). http://www.sicklecelltransplantconsortium.org ![Figure 1][3] Figure 1 ![Figure 2][3] Figure 2 Disclosures No relevant conflicts of interest to declare. [1]: #F1 [2]: #F2 [3]: pending:yes

Basal CD34+ Cell Count Predicts Peripheral Blood Hematopoietic Progenitor Cells Mobilization in Healthy Donors after Administration of G-CSF

BACKGROUND: Peripheral blood (PB) hematopoietic progenitor cells (HPC) mobilized with G-CSF are the first-choice source for allogeneic stem cell transplantation. We carried out a prospective study on healthy donors (HDs), to identify donor characteristics that could influence the effectiveness of mobilization.STUDY DESIGN AND METHODS: PB-HPC allogeneic donations from sibling HDs were analyzed. We tested somatic variables (sex, age, weight, height, volemia) and blood counts (WBC, platelets, hemoglobin, CD34+ cell count). Two different determinations of CD34+ cells were done in each HD: baseline (before G-CSF administration) and in PB on the morning of the fifth day (after G-CSF administration). HDs received G-CSF subcutaneously at a dose of 10 µg/kg per day.RESULTS: 128 consecutive HDs (66 males) with a median age of 43 years were enrolled. The mean value of CD34+ on day 5 was 90.8 cells/µL, 84.2 cells/µL in females and 97.2 cells/µL in males. The median values of CD34+ on day 5 were 75.5 cells/µL for the overall sample, 74 cells/µL in females and 80 cells/µL in males. On univariate correlation analysis, donor weight r=0.19, P<0.05, height (r=0.20, P<0.05), volemia (r=0.18, P<0.05) and baseline CD34+ (r=0.38, P<0.001) were significant predictors of CD34+ on day 5. Data analysis by gender showed that CD34+ on day 5 were predicted by age (r=-0.30, P<0.05), baseline WBC (r=0.25, P<0.05), baseline CD34+ (r=0.31, P<0.05) in males and by baseline CD34+ (r=0.45, P<0.001) in females. We performed two multivariate analyses either by using ordinary regression (to predict the mean value of CD34+ on day 5) or median regression (to predict the median value of CD34+ on day 5) according to the values of CD34+ at baseline (see table) in the whole study population and separately in females. These results indicate that in the whole study population and by using ordinary regression, a baseline value of CD34+ ranging from 0.5 to 2 cells/µL predicts a mean CD34+ value of 75.7 cells/µL at 5 days whereas a baseline value of CD34+ ≥3 cells/µL predicts a mean CD34+ value of 107.5 cells/µL at 5 days. Furthermore, in the whole study population but by using the median regression, a baseline value of CD34+ ranging from 0.5 to 2 cells/µL predicts a median CD34 + value of 64.6 cells/µL on day 5 whereas a baseline value of CD34+≥3 predicts a median CD34 value of 101.1 cells/µL on day 5.CONCLUSIONS: Baseline, HDs PB CD34+ cell counts correlated with the effectiveness of allogeneic HPC mobilization and could be useful to plan the collection. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Elevation of Serum Pro-Brain Natriuretic Peptide in G-CSF Stimulated Hematopoietic Progenitor Cell Donors

Introduction:Healthy donors who are treated with GCSF before peripheral blood hematopoietic progenitor cell (HPC) collection may develop side effects and aberrant laboratory values that have been described with the mobilization regimen. Alternatively, unexpected abnormal laboratory results could reflect occult complications or underlying disease. While many serum chemistry test results are affected by G-CSF, there have been no published reports describing an effect on BNP (pro-brain natriuretic peptide), a serum marker of heart failure. We encountered a donor who developed an elevated BNP after GCSF-stimulated HPC collection, and investigated its possible association with GCSF administration in healthy donors.Case Report:A 41-year old woman was evaluated for matched allogeneic peripheral HPC donation. History and physical exam were unremarkable, and standard laboratory values, EKG and chest X-ray revealed no abnormalities. The donor was mobilized with 10mcg/kg GCSF daily for 5 days. Her only complaint was of headache starting on day 4 that was relieved partially with acetaminophen. No other side effects were recorded.Her pre-apheresis peripheral CD34+ count was 87/mcL. At the start of apheresis HPC collection, the donor reported headache 7/10 in intensity and intolerance to light, unrelieved by acetaminophen but partially relieved with oxycodone. She complained of nausea during collection, but the procedure was otherwise successful and uneventful (15L of whole blood processed). Her headache persisted at the time of discharge, and the donor was counseled and prescribed one additional dose of oxycodone.Five hours after discharge the donor reported “the worst headache of her life”, nausea and difficulty breathing. She was evaluated at the ER, where she had stable vital signs and normal oxygen saturation. Her physical exam was unremarkable. Head CT was negative for intracranial hemorrhage or mass effect. A panel of laboratory analyses revealed leukocytosis, decreased platelets, and increased alkaline phosphatase and liver enzymes, which are documented findings associated with GCSF stimulation and large-volume apheresis. Given the donor’s complaint of dyspnea, serum BNP was measured and found increased to 483pg/mL (normal <124pg/mL). The donor had a normal pulmonary examination and no clinical evidence of cardiac insufficiency; a chest X-ray and an echocardiogram were also within normal limits. Her symptoms resolved with IV hydromorphone and she was discharged in stable condition. A follow-up serum BNP 4 weeks after donation was within normal range (105pg/mL).Investigation:A preliminary study evaluated 11 additional HPC donors who were stimulated with 10mcg/kg GSCF for 5 days (Table 1). Nine of twelve (75%) donors were male, and seven were Caucasian. Ten of the total 12 donors demonstrated an increase in BNP levels compared to pre-stimulation results. Follow-up of four total donors 9-17 days after donation demonstrated return of BNP levels to baseline. None of the donors had clinical evidence of heart failure during stimulation, donation, or follow-up. The average increase in BNP was 4.2 fold, and in a total of 3 donors it exceeded the normal range for the laboratory. The presence or degree of BNP elevation did not correlate with age, gender, severity of GCSF side effects, liver panel results, or pre-apheresis peripheral CD34+ count.Conclusion:This case and preliminary study indicate that an increase in BNP levels can be observed in approximately 80% of HPC donors stimulated with 5 days of 10mcg/kg GCSF. The increase in BNP in these donors is not associated with ventricular dysfunction or symptoms of cardiac congestion, and it returns to baseline levels in 2-4 weeks. Two possible molecular mechanisms for this BNP increase include 1) activation of gp130 by increased circulating cytokines associated with GCSF; or 2) direct transcriptional activation of the BNP gene by cardiomyocyte GCSF receptor activation of the JAK-STAT pathway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Factors Influencing Long-Term Hematopoietic Function Following Autologous Stem Cell Transplantation

Background/Objective:Mobilized peripheral blood hematopoietic progenitor cells are the most common stem cell source for autologous hematopoietic stem cell transplantation (auto-HSCT). Successful short-term stem cell engraftment requires collection of at least 2x106 CD34+ cells/kg. The American Society of Bone Marrow Transplantation (ASBMT) recommends a stem cell infusion target of 3-5 x106 cells/kg (Giralt et al. 2014). However, the number of CD34+ cells to reinfuse to ensure long-term engraftment has not been established. Plerixafor, a reversible CXCR4 antagonist, increases CD34+ cell yield at collection even in patients who are predicted poor mobilizers (PPM). Although plerixafor could be used universally for all collections, this may not be the most cost-effective strategy (Veltri et al. 2012). This study sought to determine the minimum number of CD34+ cells/kg required for adequate long-term hematopoiesis, identify factors associated with poor long-term hematopoiesis, and determine if plerixafor mobilization improved long-term peripheral blood counts.Methods:A retrospective chart review was conducted on patients who underwent auto-HSCT between January 2004 and September 2013 at The Ottawa Hospital, for management of hematological malignancies. Peripheral blood cell counts were collected from 1 to 5 years after auto-HSCT, or until disease relapse. Poor long-term hematopoiesis was defined as an ANC <1 x109/L, hemoglobin <100 g/L, or platelets <100 x109/L. Patients were stratified into groups based on the infused CD34+ concentration (in cells/kg), and the proportion of patients with poor long-term hematopoiesis at 1, 2, 3, 4, and 5 years post auto-HSCT was compared with chi square tests. Long-term clinical outcomes (platelet and packed red blood cell transfusions, and post auto-HSCT infection rates) were compared between plerixafor-mobilized patients and PPM (defined as patients with pre-collection CD34+ <2 x 106 cells/kg) with standard mobilization regimens.Results:This study included 560 patients who underwent auto-HSCT, 210 with multiple myeloma and 350 with lymphoma. At 1 and 5 years post auto-HSCT 377 and 104 patients were included, respectively. A dose dependent improvement 1 year after auto-HSCT was seen in patients who received 0-2.99 x 106 CD34+ cells/kg (24.4%, n= 41) compared to patients who received 5-9.99 x 106 CD34+ cells/kg (11%, n=154, p=0.051) and ³10 x 106 CD34+ cells/kg (4.5%, n=66, p=0.006). Though there was a trend towards lower CD34+ infusions and poorer hematopoietic function (see table 1), there was no statistically significant difference in hematopoietic function based on CD34+ infusion concentrations after 1 year post auto-HSCT. 10 patients received <2 x106 CD34+ cells/kg, of whom the rate of inadequate hematopoiesis was 33% at 1 year (n=6) and 0% (n=1) at 5 years post auto-HSCT. Factors that increased the risk of poor hematopoiesis over the course of study follow up, based on a univariate analysis, included advanced age (OR 1.189, p=0.05), multiple prior collections (OR 2.978, p=0.035), and prior treatment with more than two chemotherapy lines (OR 2.571, p=0.02). Plerixafor-mobilized patients (n=25), compared to PPM (n=197), had a significantly higher median CD34+ cell collection (4.048 x109/L and 2.996 x109/L cells/kg, respectively, p=0.005). There was no significant difference in overall cytopenias, transfusion requirements, or infection rates between plerixafor-mobilized and PPM patients over the course of the study follow up.Conclusion:Low pre-collection CD34+ counts, advanced age, multiple prior collections, and more than two prior chemotherapy treatments adversely affected long-term hematopoiesis post auto-HSCT. We support the transfusion target of 3-5 x 106 cells/kg, as proposed by the ASBMT, given that at 5 years post auto-HSCT there was no statistical or clinically significant difference in hematopoietic function with higher CD34+ infusion targets. While mobilization with plerixafor significantly increased overall CD34+ cell collection when compared with PPM, long-term hematopoietic function and clinical outcomes were not different. This finding supports the practise of limiting plerixafor use only to patients who are PPM, thereby facilitating adequate stem cell collection and early engraftment, as opposed to universal plerixafor mobilization. [Display omitted] DisclosuresSabloff:Lundbeck: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Canada: Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Alexion: Honoraria.

Algorithms utilizing peripheral blood hematopoietic progenitor cell counts in lieu of some CD34+ cell counts predict successful peripheral blood stem cell collections with substantial time and cost savings.

Hematopoietic progenitor cell (HPC) counts from Sysmex hematology analyzers have been shown to correlate with peripheral blood (PB) CD34+ cell counts by flow cytometry. Algorithms utilizing HPC counts to guide stem cell collections have been proposed but rarely tested. This study describes the development and validation of algorithms utilizing HPC and PB CD34+cell counts to predict adequate peripheral blood stem cell (PBSC) collections for chemomobilized and cytokine-mobilized individuals. Utilizing a test set of 83 PB samples from chemomobilized or cytokine-mobilized PBSC collection patients, PB CD34+ counts were correlated with HPC counts and a receiver operating characteristic curve was constructed. Cut-offs of ≤0.5 HPC/μl and ≥7 HPC/μl were established to maximize sensitivity and specificity for using HPC to predict PB CD34+ ≥ 10 cells/μl. These cut-offs were subsequently validated using a separate prospective validation set of 88 HPC/CD34+ cell sample pairs. Using the algorithms, all patients in the prospective validation data set achieved adequate collections of ≥1 × 106 CD34+ cells/kg, and a 67% reduction in the number of CD34+ cell counts performed was achieved. This lead to a direct cost savings of at least $18,700 USD over a 21-month period (88% reduction in direct costs). Use of the algorithms provides significant time and cost savings for the laboratory while accurately predicting (i) timing of PBSC collections to obtain adequate CD34+ product yields for chemomobilized patients and (ii) when to administer plerixafor to cytokine-mobilized patients to improve the likelihood of achieving adequate collections.

Procedure-related complications and adverse events associated with pediatric autologous peripheral blood stem cell collection.

Autologous peripheral blood hematopoietic progenitor cell collection (A-HPCC) in pediatric patients is considered relatively safe although technically challenging. Very little is known regarding the incidence, risk factors and impact of procedure-related adverse events (AE) on pediatric A-HPCC outcomes. Prospective 4.5-year review of AE associated with pediatric A-HPCC. AE were graded by severity and type. Potential demographic and procedural risk factors, and the impact on product quality, were compared by t-test, chi-square, and linear regression. Sixty-two children underwent 110 A-HPCC, including 36 (58%) under 20 kg. Fifty-five AE were documented in 25.4% A-HPCCs and 39% of children (citrate 25%, access 19%, technical 11%, cardiovascular 0%, allergic 1.8%). No AE were noted in children < 10 kg anticoagulated with heparin. Access and technical AE accounted for 73% of severe AE, with line-related problems underlying most technical AE (87.5%, P = 0.006). AE were more likely in older (P = 0.012), heavier patients (P = 0.02), who frequently required more than one A-HPCC (P = 0.012). In contrast, young children were more likely to experience citrate AE with gastrointestinal symptoms (median age, 6 years; P = 0.076). AE had no impact on CD34 collection rates; however, mean CD34 yields (4.2 vs. 20.4 million/kg; P = 0.0035) were decreased in patients with technical AE due to lower peripheral CD34 counts and a high number of aborted procedures (37%). Venous access and flow-related issues are a major factor associated with moderate and severe AE, effecting ∼10% of patients. AE are more frequent with increasing patient age, weight, and number of procedures. J. Clin. Apheresis 32:35-48, 2017. © 2016 Wiley Periodicals, Inc.

Abnormal proplatelet formation and emperipolesis in cultured human megakaryocytes from gray platelet syndrome patients.

The Gray Platelet Syndrome (GPS) is a rare inherited bleeding disorder characterized by deficiency of platelet α-granules, macrothrombocytopenia and marrow fibrosis. The autosomal recessive form of GPS is linked to loss of function mutations in NBEAL2, which is predicted to regulate granule trafficking in megakaryocytes, the platelet progenitors. We report the first analysis of cultured megakaryocytes from GPS patients with NBEAL2 mutations. Megakaryocytes cultured from peripheral blood or bone marrow hematopoietic progenitor cells from four patients were used to investigate megakaryopoiesis, megakaryocyte morphology and platelet formation. In vitro differentiation of megakaryocytes was normal, whereas we observed deficiency of megakaryocyte α-granule proteins and emperipolesis. Importantly, we first demonstrated that platelet formation by GPS megakaryocytes was severely affected, a defect which might be the major cause of thrombocytopenia in patients. These results demonstrate that cultured megakaryocytes from GPS patients provide a valuable model to understand the pathogenesis of GPS in humans.

Evaluation of circulating haematopoietic progenitor cells in patients with Trauma Haemorrhagic shock and its correlation with outcomes.

Background:Haemorrhagic shock accounts up to 50% of early trauma deaths. Hematopoietic failure has been observed in experimental animals and human following shock and injury. One of the facets of bone marrow failure is multiple organ dysfunction syndrome and is commonly seen in patients recovering from severe trauma and hemorrhagic shock. Bone Marrow (BM) dysfunction is associated with mobilization of hematopoietic progenitor cells (HPCs) into peripheral blood. Present study explored the association of peripheral blood hematopoietic progenitor cells (HPCs) with mortality in trauma haemorrhagic shock patients (T/HS).Materials and Methods:Prospective cohort studies of patients presenting within 8 hrs of injury with T/HS to the Department of Emergency Medicine, Jai Prakash Narayan Apex Trauma Center, All India Institute of Medical Sciences were recruited. Peripheral blood samples were collected in each patient for measurement of peripheral blood HPCs. Peripheral blood progenitor cell (PBPC) quantification was performed by measuring HPCs counts using the haematology analyzer (Sysmex XE-2100). Clinical and laboratory data were prospectively collected after consent. Ethical approval was taken and data was analysed by Stata 11.2.Results:39 patients with trauma hemorrhagic shock and 30 normal healthy controls were recruited. HPCs were significantly higher (P < 0.001) in the T/HS as compared to control. Among study group, 14 patients died within 24 h. at the hospital admission, and found HPCs concentrations were highly significant (<0.001) in non-survivors (n = 14) when compared with survivors (n = 25) among T/HS patients.Conclusions:Our studies suggest the peripheral blood HPCs may be early prognostic marker for mortality among patients who presented with trauma hemorrhagic shock on admission. But the exact molecular mechanism and signalling pathway involved in the change of the behaviour of bone marrow microenvironment is still unclear.

Improved Prediction of CD34+ Cell Yield before Peripheral Blood Hematopoietic Progenitor Cell Collection Using a Modified Target Value–Tailored Approach

The most commonly used stem cell source for both autologous and allogeneic transplantation is mobilized peripheral blood hematopoietic progenitor cells collected by apheresis. In the 1990s, an Italian group used the correlation between the preapheresis peripheral blood CD34+ cell count and the final number of CD34+ cells collected to devise a formula for “target value–tailored” (TVT) apheresis. Using local patient data, the Canadian Blood Services Stem Cell Laboratory created a similar model to determine the blood volume to process during apheresis collection. The objectives of this study were to determine the correlation between the number of CD34+ cells predicted by the TVT formula and the actual number of CD34+ cells collected and to determine whether the TVT formula remains predictive when applied to an external data set. All apheresis collections performed at the Ottawa Hospital between January 1, 2003 and October 2, 2013 were reviewed. The primary outcome was the correlation between the number of CD34+ cells predicted by the TVT formula and the actual number of CD34+ cells collected on day 1 of apheresis. For the external data set, all autologous collections performed at the London Health Sciences Centre between December 1, 2008 and December 1, 2013 were reviewed. The external data set was divided into test and validation sets to determine whether a model could be created to predict the final number of CD34+ cells collected on day 1 based on the preapheresis CD34+ count. A total of 1252 collections were included in the analysis. The Ottawa data set included 1012 collections, 836 of which were autologous and 176 of which were from donors. Of the autologous collections in Ottawa, 764 (92.5%) were first collections. In 759 (91%) collections, chemotherapy plus granulocyte colony–stimulating factor (G-CSF) was used as the mobilization regimen. In 747 collections (89%), only 1 collection day was required to achieve the desired number of CD34+ cells. The TVT estimate was highly predictive of the number of CD34+ cells × 106/kg actually collected on apheresis day 1 (r = .90, P < .0001). The London data set included 240 autologous collections. All mobilizations were with G-CSF alone. For the test set, the precollection CD34+ count was highly predictive of the number of CD34+ cells × 106/kg collected on day 1 of apheresis. Applying this model to the validation set, the correlation between the predicted and final and day 1 CD34+ cells × 106/kg count was .9186 (P < .0001). Using a modified TVT approach, the preapheresis CD34+ count can be used to accurately predict the number of CD34+ cells × 106/kg collected on day 1. This approach can be applied at other centers and for different diseases and mobilization regimens. This method can be used to individualize the blood volume processed and, thus, optimize resource utilization.

An international survey of paediatric apheresis practice

IntroductionPaediatric apheresis (PA) has distinct characteristics compared to adult apheresis and requires specialized knowledge and experience to perform safely, particularly in low‐weight patients. As evidence‐based medicine advances the field of therapeutic apheresis, increased attention must be paid to paediatric patients with conditions for which apheresis is indicated.MethodsAn electronic survey of &gt;5000 potential participants throughout the world was conducted to ascertain the scope and the current state of practice.ResultsThe survey elicited 159 responses from 12 countries; 107 of the responses provided sufficient information for analysis. Participants performed an average of 176 PA procedures/year (range 1–2000). The types of PA procedures were therapeutic plasma exchange (TPE, 92% of centres), red cell exchange (RCE, 86%), leucocyte depletion (LD, 87%) and peripheral blood hematopoietic progenitor cell collection (HPC, 72%). More than 65% of the centres had treated children older than 5 years with PA. Many centres had also performed PA on younger children; 40% have treated patients &lt;12 months of age; 61% had treated patients 1–5 years old. About 36% of centres reported that they would perform apheresis regardless of patient weight; 18% used a 5 kg threshold, 11% used 5–10 kg and 17% used 10 kg as their weight threshold.ConclusionThis report is the largest single survey of centres performing PA. The results provide information about the scope and diversity of PA and identify areas where considerable variability in practice exists. Further exploration of these differences could establish best practices in PA through international research and collaboration.

Mobilization and collection of CD34(+) cells for autologous transplantation of peripheral blood hematopoietic progenitor cells in children: analysis of two different granulocyte-colony stimulating factor doses.

IntroductionThe use of peripheral hematopoietic progenitor cells (HPCs) is the cell choice in autologous transplantation. The classic dose of granulocyte-colony stimulating factor (G-CSF) for mobilization is a single daily dose of 10 μg/kg of patient body weight. There is a theory that higher doses of granulocyte-colony stimulating factor applied twice daily could increase the number of CD34+ cells collected in fewer leukapheresis procedures.ObjectiveThe aim of this study was to compare a fractionated dose of 15 μg G-CSF/kg of body weight and the conventional dose of granulocyte-colony stimulating factor in respect to the number of leukapheresis procedures required to achieve a minimum collection of 3 × 106 CD34+ cells/kg body weight.MethodsPatients were divided into two groups: Group 10 – patients who received a single daily dose of 10 μg G-CSF/kg body weight and Group 15 – patients who received a fractioned dose of 15 μg G-CSF/kg body weight daily. The leukapheresis procedure was carried out in an automated cell separator. The autologous transplantation was carried out when a minimum number of 3 × 106 CD34+ cells/kg body weight was achieved.ResultsGroup 10 comprised 39 patients and Group 15 comprised 26 patients. A total of 146 apheresis procedures were performed: 110 (75.3%) for Group 10 and 36 (24.7%) for Group 15. For Group 10, a median of three (range: 1–7) leukapheresis procedures and a mean of 8.89 × 106 CD34+ cells/kg body weight (±9.59) were collected whereas for Group 15 the corresponding values were one (range: 1–3) and 5.29 × 106 cells/kg body weight (±4.95). A statistically significant difference was found in relation to the number of apheresis procedures (p-value <0.0001).ConclusionsTo collect a minimum target of 3 × 106 CD34+ cells/kg body weight, the administration of a fractionated dose of 15 μg G-CSF/kg body weight significantly decreased the number of leukapheresis procedures performed.

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.