Hematopoietic Progenitor Cell Donor Research Articles

Ethylene oxide-type hypersensitivity reactions in G-CSF mobilized, peripheral blood hematopoietic progenitor cell donors and review.

Ethylene oxide (EtO) is a volatile, ringed toxic ether used to sterilize heat-labile plastics including apheresis sets. In the 1980s, EtO-associated severe hypersensitivity reactions during hemodialysis led to widespread adoption of alternative sterilization for dialysis kits but not apheresis tubing sets. We now report several cases of EtO-type hypersensitivity reactions in autologous donors undergoing hematopoietic progenitor cell collection (HPCC). A 10-year retrospective review of allergic EtO-type reactions in adults undergoing HPCC on the COBE Spectra and SPECTRA Optia was performed. Donor medical history and absolute eosinophil counts were compared between cases and 34 HPCC controls. Published EtO reactions during extracorporeal procedures were reviewed with statistical analysis. Graphics and statistics were performed using commercial software. Three autologous HPCC donors experienced EtO-type reactions within 15 min of initiating HPCC, for a 10-year incident rate of 0.08% per procedure and 0.18% per donor. All three reactions occurred using the Spectra Optia and IDL tubing set, for an Optia/IDL specific rate of 0.2% per procedure and 0.5% per donor. There was no correlation between EtO reactions, eosinophil counts, or saline prime dwell times. No patient had classic predisposing risk factors for EtO hypersensitivity. Two patients required medical intervention whereas the third responded by pausing the procedure and slowing the inlet rate. EtO-type hypersensitivity reactions can be observed during HPCC, especially with the Optia IDL tubing set. EtO reactions may be missed due to their rarity and staff unfamiliarity with this clinical entity.

Increased serum pro-B-type natriuretic peptide in hematopoietic progenitor cell donors stimulated with G-CSF.

Hematopoietic stem cells (HPCs) donors mobilized by granulocyte-colony-stimulating factor (G-CSF) can develop various signs and symptoms. proBNP (pro-B-type natriuretic peptide) is a serum marker of heart failure. A donor who developed severe adverse reactions after G-CSF mobilization was found to have high serum proBNP levels. We followed additional donors who received identical mobilization regimen to investigate the prevalence of this phenomenon. Eighteen healthy donors underwent a mobilization regimen of 10μg/Kg G-CSF daily for 5 days prior to allogeneic HPC collection using Spectra Optia between January 2016 and February 2017 were included in this study. Serum proBNP levels were measured before and after G-CSF stimulation and immediately after apheresis. Apheresis collection parameters and other laboratory results were also reviewed. The majority of donors (86.7%) had post-G-CSF elevation of serum proBNP. Seven of those had elevated proBNP above upper normal range (124 pg/ml). The subgroup of donors with normal proBNP is younger (median age of 37 vs 42 years), with majority being male (90.9% vs 28.6%) and with smaller processed blood volume (2.2 vs 3 × total blood volume). This case series demonstrates an increase of serum proBNP can be common in HPC donors stimulated with 5 days of 10 mcg/kg G-CSF. This is an adverse reaction that has not been described before. The temporary elevation of proBNP in these donors is not associated with ventricular dysfunction of the heart. The risk factors for marked elevation of proBNP post-G-CSF should be further investigated.

Combining one-step Sanger sequencing with phasing probe hybridization for HLA class I typing yields rapid, G-group resolution predicting 99% of unique full length protein sequences.

Sanger-based DNA sequencing of exons 2+3 of HLA class I alleles from a heterozygote frequently results in two or more alternative genotypes. This study was undertaken to reduce the time and effort required to produce a single high resolution HLA genotype. Samples were typed in parallel by Sanger sequencing and oligonucleotide probe hybridization. This workflow, together with optimization of analysis software, was tested and refined during the typing of over 42,000 volunteers for an unrelated hematopoietic progenitor cell donor registry. Next generation DNA sequencing (NGS) was applied to over 1000 of these samples to identify the alleles present within the G group designations. Single genotypes at G level resolution were obtained for over 95% of the loci without additional assays. The vast majority of alleles identified (>99%) were the primary allele giving the G groups their name. Only 0.7% of the alleles identified encoded protein variants that were not detected by a focus on the antigen recognition domain (ARD)-encoding exons. Our combined method routinely provides biologically relevant typing resolution at the level of the ARD. It can be applied to both single samples or to large volume typing supporting either bone marrow or solid organ transplantation using technologies currently available in many HLA laboratories.

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.

Venous access for hematopoietic progenitor cell collection: An international survey by the ASFA HPC donor subcommittee

Hematopoietic Progenitor Cell (HPC) collection by apheresis is performed in patients and donors to obtain HPCs for transplantation. Although studies have shown these procedures to be safe, successful collection cannot be performed without establishment of venous access. This project's objective was to ascertain the current practices of donor vein assessment and central venous catheter (CVC) usage. The American Society for Apheresis (ASFA) HPC subcommittee created an electronic survey about precollection vein assessment and line placement, care, and removal in autologous and allogeneic donors. It was distributed to >5,000 possible participants, with one response analyzed per institution. One hundred centers performing autologous and/or allogeneic procedures provided adequate responses for analysis. Donor vein assessment is most often performed by apheresis staff more than 1 week prior to collection. For patients with questionable access, the next step performed most often is secondary assessment for autologous procedures and CVC placement for allogeneic procedures. Most centers use interventional radiology to place CVCs in jugular veins on collection day with placement verification through electronic medical records. Verbal and written postinsertion CVC care instructions are routinely provided. The apheresis team frequently provides postinsertion CVC care for autologous patients. Heparin is used most often for CVC lock. When used, tissue plasminogen activator is usually instilled for up to 60 min. These data summarize the largest single survey of donor vein assessment at institutions performing HPC collections by apheresis. The variations identified in donor venous access practice warrant further investigation and consensus to establish best practices. J. Clin. Apheresis 31:529-534, 2016. © 2015 Wiley Periodicals, Inc.

Dual roles of autologous CD8+ T cells in hematopoietic progenitor cell mobilization and engraftment.

Poor marrow cellularity alone cannot explain poor hematopoietic progenitor cell (HPC) mobilization. This study assessed the role of CD8+ T cells in HPC cell mobilization and engraftment. Mobilization and engraftment were assessed in 192 autologous HPC donors. CD34+, CD4+, and CD8+ T-cell contents in apheresis products were evaluated. Using a chemotaxis assay, we assessed the effect of purified autologous CD8+ T cells from low and high mobilizers on HPC migration from high to low stromal cell-derived factor (SDF-1α) concentration gradients. We also assessed CD8+ T-cell content association with days to neutrophil engraftment. The median number of CD34+ cells/kg was 4.7 × 10(6) . Patients were categorized according to their total CD34+ cell collection quartile distribution into low, moderate, and high mobilizers. We found that HPC products from low mobilizers contained more CD8+ T cells than HPC products from moderate and high mobilizers. Chemotaxis assays showed depletion of CD8+ T cells enhances HPC mobilization independent of SDF-1α concentration. Neutrophil engraftment analysis showed that the higher the CD8+ T-cell content per unit CD34+ cell, the faster the rate of engraftment. Our findings suggest CD8+ T cells inhibit HPC mobilization and facilitate homing and engraftment.

Identification of hematopoietic progenitor cell donor characteristics predicting successful mobilization: results of an Italian multicenter study

Peripheral blood (PB) hematopoietic progenitor cells (HPC) collected by apheresis are the first-choice source for allogeneic stem cell transplantation. The target HPC dose is usually considered to be 4 × 10(6) CD34+ cells/kg of the recipient, but higher doses are required in reduced-intensity conditioning and haploidentical transplants. Thus, prolonged stimulation and repeated collections or failure to reach HPC target may occur, increasing risks for donors and recipients. We carried out a retrospective multicenter study on healthy donors, to identify donor variables which may correlate with HPC mobilization. HPC allogeneic donations from sibling and unrelated donors performed in two centers from 1995 to 2012 were analyzed. We defined a mobilization cutoff of 50 × 10(6) CD34+ cells/L and tested somatic variables, blood counts, and granulocyte-colony-stimulating factor (G-CSF) dose and molecular form. A total of 360 donors were analyzed (male, 201; female, 159; sibling, 348; unrelated, 12; median [range] age, 44.8 [13-80] years). Median peak CD34+ in PB was 54.4 × 10(6) /L (range, 5 × 10(6) -299 × 10(6) ). By multivariate analysis, we identified the following variables to correlate with good mobilization: 1) male sex (p<0.0005); 2) younger age (p=0.007); 3) higher baseline (premobilization) white blood cell (WBC) count (p<0.0005); 4) higher G-CSF dosage (p<0.0005); and 5) use of lenograstim rather than filgrastim (p<0.002). In healthy donors it is possible to predict successful HPC mobilization by donor sex, age, WBC count, and G-CSF form and dose. Furthermore, based on these data, it may be possible, at least in parental setting, to modulate G-CSF dosage on the basis of donor characteristics.

Subsequent donation requests among 2472 unrelated hematopoietic progenitor cell donors are associated with bone marrow harvest

Approximately 1 in 20 unrelated donors are asked to make a second donation of hematopoietic progenitor cells, the majority for the same patient. Anthony Nolan undertook a study of subsequent hematopoietic progenitor cell donations made by its donors from 2005 to 2011, with the aims of predicting those donors more likely to be called for a second donation, assessing rates of serious adverse reactions and examining harvest yields. This was not a study of factors predictive of second allografts. During the study period 2591 donations were made, of which 120 (4.6%) were subsequent donations. The median time between donations was 179 days (range, 21-4016). Indications for a second allogeneic transplant included primary graft failure (11.7%), secondary graft failure (53.2%), relapse (30.6%) and others (1.8%). On multivariate analysis, bone marrow harvest at first donation was associated with subsequent donation requests (odds ratio 2.00, P=0.001). The rate of serious adverse reactions in donors making a subsequent donation appeared greater than the rate in those making a first donation (relative risk=3.29, P=0.005). Harvest yields per kilogram recipient body weight were equivalent between donations, although females appeared to have a lower yield at the subsequent donation. Knowledge of these factors will help unrelated donor registries to counsel their donors.

Clinically significant anti‐A1 in a presumed ABO‐identical hematopoietic stem cell transplant recipient: a case report

BACKGROUND: Subgroups of the blood group A (ABO) are generally not considered ABO incompatible for hematopoietic progenitor cell (HPC) transplant.CASE REPORT: A 54‐year‐old female presented for HPC transplantation for acute leukemia. No HLA‐matched donor was identified, so she received a peripheral blood stem cell graft from an HLA‐mismatched unrelated donor. On pretransplant testing, both the donor and the recipient typed as blood group A. On Day +67 after transplant, the recipient had a transfusion reaction consisting of an increase in temperature, rigors, and shaking chills during infusion of a unit of group A red blood cells (RBCs). A transfusion reaction workup revealed an ABO discrepancy with both anti‐A (1+) and anti‐B (3+) identified in the patient's serum as well as a positive direct antiglobulin test with monoclonal anti‐IgG antisera. Anti‐A1 were identified serologically and in an eluate. Hemolysis was clinically significant, requiring blood transfusion. No ABO typing discrepancies were found on pretransplant testing in either the recipient or the donor. DNA sequencing for blood group A subgroups performed after the transfusion reaction on blood collected before the transplant showed the donor to be type A1 and the recipient as A2. Unfortunately, the patient experienced graft failure requiring reconditioning and reinfusion of additional cells from the original HPC donor. On Day +94 after the second transplant, the patient died with severe acute gastrointestinal graft‐versus‐host disease.CONCLUSION: This report describes a blood group A2 patient who developed an anti‐A1 causing clinically significant hemolysis after HPC transplant from an A1 donor.

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.

20: Delayed Immune Recovery after Umbilical Cord Blood Transplantation (UCBT) Is Characterized By Thymic Regeneration Failure

UCBT is a potentially curative modality for many patients with relapsed or high-risk hematological malignancies who lack suitably matched hematopoietic progenitor cell donors. The advent of transplant approaches that combine multiple CB units to facilitate transplantation into adult recipients, as well as advances in conditioning regimens and supportive care, have significantly improved outcomes following UCBT. Despite these advances, infections and/or relapse associated with impaired T cell reconstitution remains an important source of morbidity and mortality.

Delayed Immune Recovery after Umbilical Cord Blood Transplantation (UCBT) Is Characterized by Thymic Regeneration Failure.

UCBT is a potentially curative modality for many patients with relapsed or high-risk hematological malignancies who lack suitably matched hematopoietic progenitor cell donors. The advent of transplant approaches that combine multiple CB units to facilitate transplantation into adult recipients, as well as advances in conditioning regimens and supportive care, have significantly improved outcomes following UCBT. Despite these advances, infections and/or relapse associated with impaired T cell reconstitution remains an important source of morbidity and mortality. We prospectively assessed the quantitative and functional recovery of CD4+ and CD8+ T cells in 34 consecutively transplanted UCBT recipients. Functional studies of CD4+ and CD8+ T cells stimulated with a superantigen (staphylococcal enterotoxin B, SEB) and cytomegalovirus (CMV) antigens revealed that SEB-stimulated IFNγ responses by cytokine flow cytometry (CFC) were significantly lower in the UCBT recipients than in control bone marrow or peripheral blood stem cell transplant (SCT) recipients (median values of 0.99% (n=12) vs. 24.8% (n=24) in the CD4 compartment and 0.62% (n=14) vs. 6.37% (n=9) in the CD8 compartment, respectively). Despite this, many UCBT recipients had measurable donor-derived CMV-specific CD4+ and CD8+ T cells by 3 months (4 of 11, 44% in CD4+ T cells and 6 of 12, 50% amongst CD8+ T cells), indicating that primary immune responses were derived from the naïve CB graft precursors. Immunophenotypic studies demonstrated that a higher proportion of effector and late-effector memory cells (i.e., CCR7-CD45RA− and CCR7-CD45RA+ T cells) were present in UCBT recipients than SCT recipients. The median fraction of CCR7-negative T cells was 65% (n=12, UCBT recipients) vs. 30% (n=21, other SCT recipients) in the CD4 compartment, and 76% vs. 31%, respectively, in the CD8 compartment. Furthermore, the subgroup of UCBT recipients with a ratio of CCR7+ to CCR7-negative CD4+ T cells above the median at day 30 after SCT survived significantly longer (666 days vs. 224 days, p=0.03). Finally, we compared thymic regeneration (using a δ-chain T cell receptor excision, δTREC assay) in UCBT recipients with three other groups of SCT recipients, including longitudinally assessed autologous and allogeneic SCT recipients, as well another group of allogeneic SCT recipients assessed cross-sectionally. Thymic recovery was reduced at one and three months after UCBT, relative to all other SCT recipient groups. Median δTREC values (reported as δTRECs/million PBMC) at three months were 0.0 in UCBT recipients (n=12), 12 in autologous SCT recipients (n=11), 124 in allogeneic SCT recipients (n=27), and 2150 in a second group of allogeneic SCT recipients (n=31) (p<0.04 for all observations). These results demonstrate that T cell immune recovery in UCBT recipients is characterized by delayed functional recovery and a relative predominance of late memory (CCR7−) T cells, relative to other SCT recipients. However, naïve donor-derived CB cells are capable of primary pathogen-specific expansion in vivo. Finally, these data suggest that efforts to improve T cell immune reconstitution after UCBT should be directed at improving thymic regeneration, which is likely to be important in facilitating the recovery of naïve and early memory T cells.