Immunological recovery following HLA-matched CD3+ TCR αß+/CD19+ depleted hematopoietic stem cell transplantation in children.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative option for children with both malignant and nonmalignant diseases. T-cell depletion techniques may result in reduced transplant-related mortality compared with unmanipulated grafts due to a lower incidence of GvHD. METHODS: Immune recovery and outcome were analyzed in a cohort of 23 patients with malignant and nonmalignant diseases who received CD3+TCRαß+ T- and B-cell-depleted allografts from matched donors after reduced-intensity or myeloablative conditioning. The median number of CD34+, CD3+TCRαß+, and CD19+B-cells infused was 12.7 × 106 /kg, 16.8 × 103 /kg, and 96 × 103 /kg bodyweight. RESULTS: With a median follow-up of 36 (range 1-73) months, overall survival and disease-free survival at 3 years were 65.2% and 60.8%. Eight patients died, six due to the underlying disease and two of extended visceral cGvHD. Immune reconstitution, disease-free, and overall survivals were similar compared with a historical cohort of 23 patients transplanted with matched unmanipulated bone marrow. A significant lower rate of higher grade (III-IV) aGvHD was observed in the manipulated HSCT group (8.7% vs. 26%; p = 0.001), whereas the incidence of cGvHD was equal. CONCLUSIONS: Our data suggest that this graft manipulation strategy could be a safe and effective alternative to conventional HSCT techniques in matched donors.

Does Offline Beat Inline Treatment: Investigation into Extracorporeal Photopheresis.

BACKGROUND: Extracorporeal photopheresis is a therapy based on the induction of apoptosis to cells harvested from peripheral blood, followed by direct retransfusion. Currently, there are two approaches: inline procedures, where cell harvesting, 8-methoxypsoralen (8-MOP) incubation, and UV irradiation is performed with a single device, and offline procedures, with collection in one device, followed by 8-MOP incubation/UV irradiation using a second device. STUDY DESIGN AND METHODS: In a prospective crossover study, we compared an inline (Cellex, Therakos) with an established offline procedure (Optia, Terumo, and MacoGenic G2, Macopharma) in 6 patients, focusing on cell composition and apoptosis induction after 24 h. In total, 32 photopheresis treatments per device were performed. RESULTS: We observed an overall 2-fold higher number of apoptotic "target" cells for each patient with offline treatment. All yields were stratified per patient. Yields were compared as ratio offline/inline for CD3+ (2.5-fold), CD4+ (2-fold), CD8+ (2.8-fold), CD56+ (2.8-fold), CD19+ (1.8-fold), CD15+ (0.5-fold), and CD14+ (2.2-fold) cells. Apoptosis induction was measured after 24 h with Annexin V/7-AAD for early and late apoptosis rates of CD3+ (CD4+, CD8+) and CD56+ cells. CD3+ cells of the inline treatment had an average of 88% (26% early, 62% late) of apoptotic cells compared to 75% (34% early, 41% late) in the offline treatment. Procedure duration ranged from 80 to 100 min inline, with a maximum of 1,500 mL processed blood, and 125-140 min offline, with at least 3,000 mL processed blood, depending on blood flow. Average hematocrit levels of the products were 2.7% inline versus 1.7% offline. CONCLUSIONS: The offline procedure, as established in our department, provides more apoptotic cells for treatment. The increased number of mononuclear cells collected outweighs a slightly reduced apoptosis rate after 24 h in comparison to the inline procedure. Besides this, the final decision for one or the other procedure has to take into account additional aspects, such as peripheral white blood cell count, hematocrit, and weight of the patient, required before apheresis, extracorporeal volume, and, last but not least, overall costs. The final criterion, however, has to be the reported clinical efficacy of the system applied.

Successful Unrelated Stem Cell Transplantation in an Infant With Congenital Acute Myelogenous Leukemia FAB M5 Showing Massive Cutaneous Infiltrations--A Challenging Multidisciplinary Approach.

The multidisciplinary management of a male neonate presenting with congenital acute myelogenous leukemia of monoblastic phenotype is reported using conventional chemotherapy, high dose conditioning, and matched unrelated donor stem cell transplantation. These therapies were combined to add a graft versus leukemia effect to the treatment. Although chimerism studies showed a decrease of donor white blood cells, T-cells remained stable of allogeneic origin. We hypothesize that a continuous graft versus leukemia effect results in minimal residual disease negativity for now more than 18 months since stem cell transplantation.

Alternative donor HSCT in refractory acquired aplastic anemia - prevention of graft rejection and graft versus host disease by immunoablative conditioning and graft manipulation.

Early alternative donor HSCT is a potentially curative therapeutic option for patients with AAA not responding to IST. Seven patients (median age at diagnosis, 11 yr) with refractory AAA without a MSD underwent HSCT from matched unrelated (n = 6) or haploidentical (n = 1) donors. Conditioning regimens included CY (n = 7), muromonab-CD3/ATG (n = 7), TT (n = 6), FLU (n = 5), and TLI (n = 2). Grafts were either CD34 purified and/or CD3/19 depleted and contained a median of 10.17 × 10(6) /kg CD34 and 5.5 × 10(4) /kg CD3 cells. All patients engrafted rapidly. Median time to leukocyte engraftment was 10 days. With a median follow-up of 26 (range, 11-153) months, six patients are alive and well with complete donor hematopoiesis. One heavily pretreated patient developed GVHD grade III and died from progressive renal failure (resulting from microangiopathic hemolytic anemia) and disseminated aspergillosis. Early alternative donor HSCT can help to avoid complications from prolonged IST and presumably improve survival of patients with refractory AAA. Administration of high doses of CD34 purified and/or CD3/19 depleted stem cells following novel immunoablative conditioning may prevent graft rejection and GVHD. However, a long interval from diagnosis to HSCT seems to be associated with poor outcome.

Apheresis affects bone and mineral metabolism.

BACKGROUND: Apheresis is a procedure to selectively obtain blood components. For the collection process citrate is routinely used. It inhibits coagulation by binding to ionized calcium and leads to metabolic alkalosis. OBJECTIVE: Whether regular apheresis affects bone and mineral metabolism is unknown. The intention of this study was to investigate 1) the acute effects of apheresis on acid-base balance, bone and mineral metabolism and 2) to compare bone mineral density (BMD) at the lumbar spine and hip of donors to matched control subjects. DESIGN: In this open, observational, single-center, cross-sectional study we enrolled 102 regular plasma and thrombocyte donors to pursue objective 1) and compared those to 102 matched controls (CTR) for objective 2). RESULTS: Platelet donation led to significant decreases in serum ionized calcium (-17%) and phosphate (-18%), to marked increases in base excess (57%) and PTH levels (192%) during apheresis. Baseline biochemical comparisons between donors and CTR revealed significantly lower values for donors for serum calcium, albumin, and 25-hydroxyvitamin D levels. Mean Z-score at the lumbar spine adjusted for BMI, average physical activity and daily calcium intake was lower for donors (-0.28+/-0.11) when compared to CTR subjects (0.06+/-0.11, P<0.05). Total and neck femoral BMD was also lower in the donor group, however, this difference was not significant. CONCLUSIONS: Exposure to citrate during the apheresis procedure acutely affects mineral and bone metabolism. Regular donations of blood components compromised BMD at the lumbar spine. If confirmed, strategies to prevent long-term effects on bone need to be formulated.

Peripheral blood stem cell mobilization with pegfilgrastim compared to filgrastim in children and young adults with malignancies.

BACKGROUND: Pegfilgrastim, the long acting agent of rh-GCSF, has been shown to be as effective as Filgrastim in children undergoing cytotoxic chemotherapy by reducing the duration of neutropenia. Recent studies in adults have also shown that Pegfilgrastim is effective to mobilize CD34+ stem cells, resulting in earlier peripheral stem cell collections (PSCC). The aim of the study was to compare the efficacy of Pegfilgrastim with Filgrastim for CD34+ stem cell mobilization in children. PROCEDURE: Three groups of patients were compared: Group 1: six patients with Ewing Sarcoma stimulated with Filgrastim; Group 2: five patients with Ewing Sarcoma, Ependymoma, and Neuroblastoma; Group 3: four patients with relapsed neoplasm. Patients of Group 2 and 3 were stimulated with Pegfilgrastim followed by peripheral stem cell collection. Two patients in Group 3 needed further cytokine stimulation with Filgrastim combined with stem cell factor, Ancestim. RESULTS: In Groups 1-3, a median of 4, 3, and 3 PSCC between day 12-24, 6-13, and 8-30 were performed, yielding a median of 14.2, 24.0, and 10.3 x 10(6) CD34+ stem cells/kg BW, respectively. CONCLUSIONS: Group 2 data show that stem cell mobilization with Pegfilgrastim in children when performed during primary or without previous long lasting chemotherapy seems to produce earlier CD34+ peaks and better CD34+ yields than in Group 1. CD34+ cell mobilization with Pegfilgrastim in Group 3-patients with previous long lasting chemotherapy was possible.

Thrombin generation before and after multicomponent blood collection.

BACKGROUND: Apheresis technology has made tremendous progress up to the development of automated blood component collection, which offers increased efficiency in donor blood use, but the concern about the contact of donor blood with artificial surfaces remains. Activation of the hemostatic system is a major issue in this context and is controversial. The aim of this study was to estimate the effect of apheresis on continuous thrombin generation (TG), representing a new tool to examine the overall function of the plasmatic clotting system. STUDY DESIGN AND METHODS: Twenty-six voluntary blood donors, fulfilling the law requirement for apheresis donation, participated in the study. Two units of platelets (6 x 10(11)) and 1 unit of red cells (250 mL; hematocrit level, 80%) were collected using two types of cell separators (Amicus, Fenwal, Inc.; and Trima Accel, Gambro BCT). Each donor underwent collection on both apheresis systems with at least 8 weeks in between. Samples of blood were collected before, immediately after, and 48 hours after apheresis. TG was measured using a slow fluorogenic substrate by means of calibrated automated thrombography (CAT). RESULTS: CAT data changed only slightly, and no significant changes were seen before, immediately after, and 48 hours after apheresis (p > 0.05). The variables did not differ significantly between the two different apheresis systems (p > 0.05). CONCLUSION: Using a CAT-based technique, no change in variables of continuous TG were observed, suggesting that multicomponent blood collection did not lead to severe alterations in the hemostatic system of the donors.