Loss Of Nucleated Cells Research Articles

Investigation of potential adverse central nervous system effects after long term oral administration of gadolinium in mice.

ObjectivesTo examine potential gadolinium (Gd) accumulation in the brain of healthy mice after long-term oral administration of Gd-containing food pellets and to investigate whether Gd leads to adverse central nervous system (CNS) effects, specifically focussing on locomotor impairment in Gd exposed compared to control animals.Materials and methodsThe local Animal Experimental Ethics Committee approved all procedures and applications. Fifteen female C57Bl/6 mice were orally exposed to a daily intake of 0.57 mmol Gd chloride/ kg body weight over a period of 90 weeks from the age of 4 weeks on. Gd-free, but otherwise equivalent experimental diets were given to the control group (N = 13). The animals were monitored daily by animal caretakers regarding any visible signs of distress and evaluated clinically every four weeks for the first 60 weeks and afterwards every two weeks for a better temporal resolution of potential long-term effects regarding impairment of motor performance and loss of body weight. The individual Gd content was measured using mass spectrometry in a sub-cohort of N = 6 mice.ResultsThe absolute brain Gd levels of the Gd-exposed mice were significantly increased compared to control mice (0.033± 0.009 vs. 0.006± 0.002 nmol Gd/ g brain tissue). Long-term oral Gd exposure over almost the entire life-span did not lead to adverse CNS effects including locomotor changes (rotarod performance, p = 0.1467) in healthy mice throughout the study period. Gd-exposed mice showed less increased body weight compared to control mice during the study period (p = 0.0423). Histopathological alterations, such as hepatocellular vacuolization due to fatty change in the liver and a loss of nucleated cells in the red pulp of the spleen, were found in peripheral organs of both groups.ConclusionsLow levels of intracerebral Gd caused by chronic oral exposure over almost the entire life span of mice did not lead to alterations in locomotor abilities in healthy mice throughout the normal aging process.

Analysis of 120 pediatric patients with nonmalignant disorders transplanted using unrelated plasma‐depleted or ‐reduced cord blood

Unrelated cord blood (CB) is an important stem cell source for unrelated hematopoietic cell transplantation (HCT) of patients with nonmalignant disorders. Processing methods to prepare red blood cell-reduced CB units incur significant nucleated cell loss. In contrast, plasma depletion or reduction (PDR) processing of CB units entails the removal of only a portion of the plasma with minimal nucleated cell loss. However, there are relatively limited data regarding outcomes of CB transplants using units processed by PDR. A Center for International Blood and Marrow Transplant Research (CIBMTR)-audited analysis was performed on 120 pediatric patients with nonmalignant disorders transplanted between November 2001 and January 2008 at 29 US and 17 international centers using PDR CB units from two CB banks. Transplant characteristics were as follows: median age, 3.5 years (range, 0.1-14 years); median patient weight, 15 kg (range, 4-61 kg); 58% male; HLA matches (intermediate-resolution HLA-A and HLA-B and high-resolution HLA-DRB1) of the units used in these patients six of six in 26, five of six in 48, four of six in 47, and three of six or two of six in 6; median prefreeze total nucleated cell dose, 10.5×10(7)/kg; median prefreeze CD34+ dose, 3.7×10(5)/kg; and nonmyeloablative regimen in 24%. The median times to myeloid and platelet engraftment were 21 and 49 days, respectively. The cumulative incidence of reported Grade II to IV acute graft-versus-host disease (aGVHD) was 38±5%, and 19±4% had Grade III to IV aGVHD. The Kaplan-Meier estimates of 3-year transplant-related mortality, overall survival, and disease-free survival were 20±4, 79±4, and 70±6%, respectively. These data demonstrate the effectiveness of PDR CB units for HCT.

Cell recovery comparison between plasma depletion/reduction- and red cell reduction-processing of umbilical cord blood

Limited cell dose has hampered the use of cord blood transplantation (CBT) in adults. One method of minimizing nucleated cell loss in cord blood (CB) processing is to deplete or reduce plasma but not red blood cells - plasma depletion/reduction (PDR). The nucleated cell loss of PDR was studied, and determined to be less than 0.1% in the discarded supernatant plasma fraction in validation experiments. After testing and archival sampling, the median nucleated cell recovery for PDR processing was 90%, and median CD34(+) cell recovery 88%. In a CB bank inventory of 12 339 products with both pre- and post-processing total nucleated cells (TNC), PDR processing resulted in median post-processing TNC recoveries of 90.0% after testing and archival samples removal. Using the same 10 CB units divided into two halves, we compared directly the recovery of PDR against hydroxyethyl starch red cell reduction (RCR) for TNC, CD34(+) cells and colony-forming units (CFU-GM, CFU-E, CFU-GEMM and total CFU) after parallel processing. We also compared the loss of very small embryonic-like stem cells (VSEL). We demonstrated significantly higher recoveries using PDR for TNC (124%), CD34(+) cells (121%), CFU-GM (225%), CFU-GEMM (201%), total CFU (186%) and VSEL (187%). The proportion of high TNC products was compared between 10 912 PDR and 38 819 RCR CB products and found to be 200% higher for products that had TNC ≥150 × 10(7) (P = 0.0001) for the PDR inventory. Our data indicate that PDR processing of CB provides a significantly more efficient usage of this valuable and scarce resource.

Essential Role of p400/mDomino Chromatin-remodeling ATPase in Bone Marrow Hematopoiesis and Cell-cycle Progression

p400/mDomino is an ATP-dependent chromatin-remodeling protein that catalyzes the deposition of histone variant H2A.Z into nucleosomes to regulate gene expression. We previously showed that p400/mDomino is essential for embryonic development and primitive hematopoiesis. Here we generated a conditional knock-out mouse for the p400/mDomino gene and investigated the role of p400/mDomino in adult bone marrow hematopoiesis and in the cell-cycle progression of embryonic fibroblasts. The Mx1-Cre- mediated deletion of p400/mDomino resulted in an acute loss of nucleated cells in the bone marrow, including committed myeloid and erythroid cells as well as hematopoietic progenitor and stem cells. A hematopoietic colony assay revealed a drastic reduction in colony-forming activity after the deletion of p400/mDomino. Moreover, the loss of p400/mDomino in mouse embryonic fibroblasts (MEFs) resulted in strong growth inhibition. Cell-cycle analysis revealed that the mDomino-deficient MEFs exhibited a pleiotropic cell-cycle defect at the S and G(2)/M phases, and polyploid and multi-nucleated cells with micronuclei emerged. DNA microarray analysis revealed that the p400/mDomino deletion from MEFs caused the impaired expression of many cell-cycle-regulatory genes, including G(2)/M-specific genes targeted by the transcription factors FoxM1 and c-Myc. These results indicate that p400/mDomino plays a key role in cellular proliferation by controlling the expression of cell-cycle-regulatory genes.

Autologous bone marrow transplantation in a dog with lymphoma: a clinical study

The objective of this study is to provide the first report of bone marrow transplantation (BMT) in dogs in Brazil. A Rottweiler with cutaneous lymphoma was submitted to a twelve-week Madison-Wisconsin chemotherapy protocol followed by autologous bone marrow transplantation. For this, 10mL kg-1 of bone marrow was collected simultaneously from both iliac crests and cryopreserved in a freezer at -80°C. The conditioning step was performed by administering cyclophosphamide by intravenous route at 400mg m-2. Bone marrow was reinfused after defrosting in a water bath at 37°C. Bone marrow nucleated cell counts before and after freezing, showed a small relative loss of nucleated cells (35.10 and 31.80x10³µL-1 , respectively). Cyclophosphamide induced neutropenia which was reverted by a granulocyte colony-stimulating factor (G-CSF) capable of stimulating hematopoetic reconstitution. On the day 360 after transplant the patient was found to be in complete remission. This study indicates that autologous BMT in a dog with lymphoma submitted to myelosuppressive chemotherapy was potentially safe and effective.

Analysis of 120 Pediatric Patients with Non-Malignant Disorders Transplanted Using Unrelated Plasma Depleted Cord Blood and the Role of Post-Thaw Wash

Unrelated cord blood (UCB) is an important stem cell source for unrelated hematopoietic cell transplantation of patients with non-malignant disorders. Cell dosage is a critical factor for successful UCB hematopoietic stem cell transplantation (HSCT). The red cell reduced (RCR) and post-thaw wash techniques that are widely used incur significant nucleated cell loss. Three strategies were employed to maximize cell dose and improve outcome–use of cord blood processed with plasma depletion without red blood cell reduction (PD CB), avoidance of post-thaw wash, and the use of double cords (2X) when necessary. A CIBMTR-audited analysis was performed on all 120 pediatric patients with non-malignant disorders transplanted with PD CB at 29 U.S. and 17 international centers. Transplant characteristics: median age 3.5 years (range 0.1–14); median patient weight 15 kg (range 4–61); male 58%. The majority of patients (n=58; 55%) were Asian. Twenty-two (21%) patients were Hispanic, 15 (14%) were Caucasian, 6 (6%) were African-American, and three (3%) were of Middle Eastern background. HLA ABDR matches: 6/6–26; 5/6–48; 4/6–47; 3/6 or 2/6–6; median pre-freeze nucleated cell dose 10.5×107/kg; median pre-freeze CD34+ dose 3.7×105/kg; non-myeloablative regimen 24%; 58% infused without post-thaw wash (NW). Myeloid engraftment defined as ANC≥500 and 6-month platelet engraftment defined as ≥ 20K and ≥ 50K are 89±8%, 88±8%, and 84±6% respectively. The median time to myeloid and platelet engraftment are 21 days (range 11–64), 49 days (range 13–155), and 61.5 days (range 21–205) respectively. No major adverse event was observed in either the W or the NW group. The cumulative incidence of reported grade II–IV acute GVHD was 38±5%, and 19±4% had grade III–IV acute GVHD. 36±6% developed limited chronic GVHD, and 12±4% developed extensive chronic GVHD. With a median follow-up of 329 days (range 3–1928 days), the Kaplan-Meier estimates of 1-year TRM, OS and diseasefree survival were 20±6%, 88±6% and 72±6% respectively. Foregoing post-thaw wash for PD CB transplantation improved neutrophil (RR=1.75; p=0.01) and platelet engraftment (RR=1.72; p=0.02) and reduced TRM (RR=0.38; p=0.04). This series demonstrated that unrelated PD CB transplantation can be performed safely and effectively in children with life-threatening, non-malignant disorders. Additionally, the results demonstrate possible improvement in myeloid and platelet engraftment, overall and disease-free survival when post-thaw wash is not employed.Table 1. Summary of overall resultsOutcomeAll Patients N = 120Washed CB N = 48Unwashed CB N = 71RR (Wash=Ref)P-valueANC500 Engraftment Cumulative Incidence Median # Days to Engraftment87±6% d+2186±9% d+2589±8% d+191.750.01Platelet 20K Engraftment Cumulative Incidence Median # Days to Engraftment81±6% d+4975±9% d+5288±9% d+431.720.02Autologous Recovery3±2%2±2%4±3%1.060.95Acute GvHD II–IV Acute GvHD III–IV38±5% 19±4%31±7% 17±6%45±7% 21±6%1.74 1.380.11 0.50Chronic GvHD Limited Chronic GvHD Extensive36±6% 12±4%14±6% 19±6%60±10% 6±4%5.69 0.24<0.001 0.08Transplant-Related Mortality–100 Day Transplant-Related Mortality–3 Yr10±3% 20±4%11±5% 34±8%9±4% 11±4%0.380.04Overall Survival–1 Yr Overall Survival–3 Yr79±4% 79±4%66±8% 66±8%88±4% 88±4%0.430.06Disease-Free Survival–1 Yr Disease-Free Survival–3 Yr72±5% 70±6%58±9% 51±10%84±5% 84±5%0.480.07

104: An audited retrospective analysis of cord blood transplantation (CBT) of 230 patients using plasma depleted cord blood units (PD CB)

Limited cell dose has hampered widespread use of CBT. The red cell depletion (RD) techniques that are widely used incur significant nucleated cell loss after processing; therefore, to minimize cell loss and still reduce volume during processing, a technique was devised to deplete plasma (PD) but not red blood cells. An updated retrospective audited analysis of 230 consecutive patients with engraftment and/or survival data from 81 transplant centers over 5 continents up to September 2006 was performed.

Optimising methods of red cell sedimentation from cord blood to maximise nucleated cell recovery prior to cryopreservation

Human cord blood is now an established source of stem cells for haematopoietic reconstitution. Red blood cell (RBC) depletion is required to reduce the cord blood unit volume for commercial banking. Red cell sedimentation using hydroxy ethyl starch (HES) is a standard procedure in most cord blood banks. However, while standardising the procedure for cord blood banking, a significant loss of nucleated cells (NC) may be encountered during standard HES sedimentation protocols. This study compares four procedures for cord blood processing to obtain optimal yield of nucleated cells. Gelatin, dextran, 6% HES and 6% HES with an equal volume of phosphate-buffered saline (PBS) were compared for RBC depletion and NC recovery. Dilution of the cord blood unit with an equal volume of PBS prior to sedimentation with HES resulted in maximum NC recovery (99% [99.5±1.3%). Although standard procedures using 6% HES are well established in Western countries, they may not be applicable in India, as a variety of factors that can affect RBC sedimentation (e.g., iron deficiency, hypoalbuminaemia, thalassaemia trait, etc.) may reduce RBC sedimentation and thus reduce NC recovery. While diluting cord blood with an equal volume of PBS is a simple method to improve the NC recovery, it does involve an additional processing step.

Hematopoietic Stem Cell Transplantation (HSCT) for Benign Indications Using Plasma Depleted Umbilical Cord Blood Units (UCB) That Were Not Depleted of Red Blood Cells.

UCB is an attractive source for unrelated HSCT of benign indications; however, cell dosage is a critical factor for UCB HSCT. The red cell depletion (RD) and post-thaw wash techniques that are widely used incur significant nucleated cell loss; therefore, two strategies to minimize cell loss are to deplete plasma, but not the red blood cells (PD) during processing and forego post-thaw wash. A retrospective audited analysis was performed on 61 patients with benign disorders who were transplanted with 68 PD UCB units (8 double cords) with 29 thalassemias, 8 AA, 5 WAS, 5 SCID, 2 osteoporosis, 2 sickle cell disease, 2 Hemophagocytic Lymphohistiocytosis, 2 Hurler Syndrome, 1 CGD, 1 Fanconi's Anemia, 1 Leroy I-Cell Disease, 1 Lymphohistiocytosis, 1 OIMD, and 1 Alpha Mannosidosis. Transplant characteristics: patient median age 4.25 years old (range 0.3–39); median weight 17 kg (range 5–76); male 56%; median # HLA ABDR matches of 5.0 (12–6/6; 19–5/6; 23–4/6; 5–3/6, 1–2/6); median pre-freeze TNC dose 8.1 x 107/kg; median post-thaw TNC dose as reported by TC 7.7 x 107/kg; median pre-freeze CD34 dose 3.1 x 105/kg; transplants outside of U.S.− 32 (52%); non-myeloablative − 9; 44% post-thaw washed (W), 56% infused without post-thaw wash (NW). The Kaplan-Meier estimates of 3-month ANC500 and 6-month platelet 20K and 50K engraftment are 87±5%, 83±6%, and 84±6% respectively. The median time to engraftment for ANC 500, platelet 20K, and 50K are 20 days (range 11–64), 46 days (range 13–153), and 61 days (range 21–171) respectively. No major adverse event was observed in either the W or the NW group, and the median time to engraftment for ANC 500, platelet 20K and 50K for W vs. NW were 21 vs. 19 days, 55 vs. 44.5 days, and 76 vs. 59 days respectively. The incidence of reported grade II–IV acute GVHD was 29%, and 10% had grade III–IV acute GVHD. 33% developed limited chronic GVHD, and 15% developed extensive chronic GVHD. With a median follow-up of 219 days (range 4–1402 days), the Kaplan-Meier estimates of 1-year TRM, OS and disease-free survival were 20±6%, 78±6% and 72±6% respectively. These results demonstrate that HSCT using unrelated PD UCB can be performed safely with outstanding results in patients with benign disorders, and post-thaw washing may delay engraftment of HSCT using PD UCB.

Hematopoietic Stem Cell Transplantation (HSCT) Using Plasma Depleted Umbilical Cord Blood (UCB) That Were Not Red Cell Depleted.

UCB is an attractive source for HSCT; however, limiting cell dose has hampered its widespread use. The red cell depletion (RCD) techniques that are widely used incur significant nucleated cell loss after processing; therefore, to minimize cell loss and still reduce volume during processing, a technique was devised to deplete plasma (PD) but not red blood cells. A large racially diverse inventory of over 25,000 PD UCB is available on stem cell registries. Of the 265 PD UCB transplanted into 240 patients up to May 2006, an updated retrospective audited analysis of all 205 patients with engraftment and/or survival data was performed. The characteristics for the patients were: median age 8.8 yo, range 0.3–59, 76 ≥16 yo (32%); median weight 29 kg, range 5–137, 84 ≥50kg (35%); male 59%; median # HLA ABDR matches 4.0; median pre-freeze TNC dose 5.31 × 107/kg (n=229); transplant center reported median post-thaw TNC dose 4.92 × 107/kg (n=112); median pre-freeze CD34 dose 1.7 × 105/kg; malignant indications 71%; transplants outside U.S. 39%; double transplant 23%; and non-myeloablative 15%. For all engrafted patients, the median time to engraftment for ANC 500 (n=192), platelet 20K (n=174) and 50K (n=169) were 22.0 days (range 7–64), 49.5 days (range 12–181), and 63.5 days (range 21–374) respectively. Kaplan-Meier estimates of engraftment of ANC500, platelet 20K and 50K engraftment were 88±3%, 82±4% and 76±4% respectively. The incidence of grade III–IV acute GVHD and extensive chronic GVHD were 13% and 14% respectively. One-year relapse rate was 23±4% for all patients (n=190), and TRM was 29±3% (n=203) at 1 year. With a median follow-up of 178 days (range 4–1402 days) for the surviving patients, the Kaplan-Meier estimates of 1-year OS (n=203) and DFS (n=190) are 59±4% and 54±4% respectively for all patients. These results demonstrate that HSCT using PD UCB can be performed safely and effectively.

Hematopoietic Stem Cell Transplantation (HSCT) for Malignancies Using Plasma Depleted Umbilical Cord Blood (UCB) That Were Not Red Blood Cell Depleted.

Cell dosage is a limiting factor for UCB HSCT, especially for adult patients. Most UCB banks practice red cell depletion (RCD) techniques to save storage space, which incur significant nucleated cell loss after processing. One method to minimize cell loss and still reduce volume after processing is to deplete plasma (PD), but not the red blood cells. Not washing UCB (NW) after thawing also minimizes cell loss. A large racially diverse PD UCB inventory of over 25,000 units is now available on stem cell registries. There were 70 ALL, 54 AML, 19 CML, and 45 others transplanted for malignant indications. A retrospective audited analysis was performed on 170 patients with engraftment and/or survival information. Of the ALL/AML/CML cases with available information, there were 32 1CR/CP, 33 2CR, and 13 3CR/CP. The median age of patients was 13 years old (range 05–59); median weight 50 kg (range 5–137); male 45%. Transplant characteristics indicated a median # HLA ABDR matches of 4.0; median pre-freeze TNC dose 4.1 × 107/kg; median post-thaw TNC dose as reported by TC 3.7 × 107/kg; median pre-freeze CD34 dose 1.4 × 105/kg; transplants outside of U.S. 34%; double unit transplant 30%; non-myeloablative 16%. Sixty-one percent of the transplanted UCB were washed post-thaw (W), 39% were infused without post-thaw wash (NW), with 20% of the units without available post-thaw data. Kaplan-Meier estimates of 3-month ANC500 and 6-month platelet 20K and 50K engraftment are 89±3%, 81±5%, and 73±5% respectively. Median time to engraftment for ANC 500, platelet 20K, and 50K were 22 days (range 7–69 days), 52.5 days (range 12–181 days), and 64 days (range 25–374 days) respectively. Median time to engraftment for post-thaw washed units (W) versus PD CBU not washed and infused directly after thawing (NW) were 24 vs. 22 days for ANC500, and 57 vs. 48 days for platelet 20K respectively. The incidence of reported grade II-IV and III-IV acute GVHD were 32% and 14% respectively. Twelve percent developed limited chronic GVHD and 14% developed extensive chronic GVHD. With a median follow-up of 170 days (range 4–1402 days), the Kaplan-Meier estimates of 1-year TRM, OS and relapse-free survival were 31±4%, 51±4% and 47±4% respectively. These results demonstrate that HSCT using unrelated PD UCB can be performed safely and effectively in patients with malignancies, and post-thaw wash may not be necessary.

Clinical Outcome of Hematopoietic Stem Cell Transplantation (HSCT) Using Plasma Depleted Umbilical Cord Blood Units (UCB) That Were Not Depleted of Red Blood Cells Prior to Cryopreservation.

Background: UCB is an attractive source for HSCT because of less stringent HLA matching requirements, lower incidence and severity of GvHD, and ready availability of a physical inventory. However, unlike BMT or PSCT, the limiting cell dose associated with UCB units has hampered its widespread use. The red cell depletion (RCD) techniques that are widely used by UCB banks incur significant nucleated cell loss after processing. One method of minimizing cell loss during processing is to deplete plasma (PD) but not red blood cells. A large racially diverse inventory of 18,000 PD UCB is now available on stem cell registries; however, clinical outcome for HSCT using PD UCB products is unavailable.Hypothesis: Usage of PD UCB in unrelated HSCT will result in acceptable clinical outcome for myeloid (ANC 500) and platelet engraftment, overall survival (OS), event-free-survival (EFS), and transplant related mortality (TRM).Methods: A retrospective analysis limited to patients without prior transplants and transplanted during remission (“eligible”) was performed on 98 HSCT using PD UCB. An additional 20 “high risk” patients with prior transplants or transplanted during relapse were included in the engraftment and survival analysis.Results: Average loss of less than 0.1% nucleated cells was found in the discarded plasma fraction after PD UCB processing (n=27), though cell clumping limited the cellular fraction recovery to 90%. Outcome on engraftment and/or survival was available for 98 patients (median age 6.7 yo, range 0.3–54 yo, 24 >16 yo; median weight 23.5 kg, range 4.5–88 kg, 27 >50kg; male 60%; median # HLA ABDR matches 4.0; median pre-freeze TNC dose 5.7 x 107/kg; transplant center reported median post-thaw TNC dose 5.5 x 107/kg; median pre-freeze CD34 dose 2.0 x 105/kg; malignant indications 71%; transplants outside U.S. 37%; double unit transplant 14%; and non-myeloablative 7%). The median time to engraftment for ANC 500 (n=87), platelet 20K (n=72) and 50K (n=68) were 22.0 days (range 7–49 days), 49.5 days (range 13–94 days), and 58.0 days (range 21–132 days) respectively. The unadjusted cumulative incidence of ANC500 and platelet 20K and 50K engraftment were 94±3%, 81±5% and 80±5% respectively for 98 eligible patients, and 90±3%, 77±5% and 75±5% respectively for the eligible plus high risk patients. The incidence of grade III–IV acute GVHD and extensive chronic GVHD were 15% and 14% respectively. Relapse rate for malignancies (n=67) and TRM (n=98) were 20±6% and 20±4% respectively at 1 year. With a median follow-up of 297 days (range 50–1,263 days), the Kaplan-Meier estimates of 1-year survival (n=98) and disease-free survival (n=67) are 73±5% and 59±7% respectively for the eligible patients, and 65±5% (n=118) and 50±6% (n=85) respectively for the eligible plus high risk patients.Conclusion: These results demonstrate that HSCT using PD UCB can be performed safely and effectively.

Hematopoietic Stem Cell Transplantation (HSCT) for Benign Indications Using Umbilical Cord Blood Units (UCB) That Were Depleted of Plasma, but Not of Red Blood Cells.

Background: UCB is an attractive unrelated source for HSCT of benign indications such as hemoglobinapathies, genetic diseases and metabolic disorders, because of less stringent HLA matching requirements, lower incidence and severity of GvHD, and ready availability of a physical inventory; however, unlike BMT or PSCT, cell dosage is a critical factor in success of UCB HSCT. The red cell depletion (RCD) techniques that are widely used by UCB banks incur significant nucleated cell loss after processing. One method of minimizing cell loss during processing is to deplete plasma, but not the red blood cells (PD). A large racially diverse PD UCB inventory of 18,000 units is now available on stem cell registries. We now report our observations of using unrelated PD UCB HSCT for benign indications.Hypothesis: Usage of unrelated PD UCB for HSCT of benign indications will result in acceptable clinical outcome for myeloid (ANC 500) and platelet engraftment, overall survival (OS), and transplant related mortality (TRM).Methods: A retrospective analysis was performed on 28 patients with benign disorders who were transplanted with PD UCB in 15 centers and 7 countries, with 13 thalassemias, 5 SAA, 5 WAS, 2 SCID, 1 sickle cell disease, 1 osteopetrosis and 1 metabolic disorder.Results: The median age of patients was 3.7 years old (range 03–27 years old); median weight 16.0 kg (range 4.5–43 kg); male 57%. Transplant characteristics indicated a median # HLA ABDR matches of 4.0 (5 6/6; 7 5/6; 12 4/6; 3 3/6; 1NA); median pre-freeze TNC dose 7.7 x 107/kg; median post-thaw TNC dose as reported by TC 7.7 x 107/kg; median pre-freeze CD34 dose 3.1 x 105/kg; transplants performed outside of U.S. 68%; double unit transplant 13%; non-myeloablative 6%. The median time to engraftment for ANC 500 (n=21), platelet 20K (n=20), and 50K (n=18) are 14.5 days (range 11–41 days), 47.0 days (range 13–82 days), and 55.0 days (range 21–96 days) respectively. The unadjusted cumulative incidence (C.I.) of ANC500 and platelet 20K and 50K engraftment are 89±7%, 89±7%, and 87±8% respectively. The incidence of reported grade II–IV acute GVHD was 33%, and none had grade III–IV acute GVHD. 50% developed limited chronic GVHD (7/14), and so far only one patient was reported to have extensive chronic GVHD. With a median follow-up of 356 days (range 93–1,100 days), the Kaplan-Meier estimates of 1-year TRM, OS and disease-free survival were 11±6%, 89±6% and 89±6% respectively.Conclusion: These results demonstrate that HSCT using unrelated PD UCB can be performed safely and effectively in patients with benign disorders. Further study of HSCT using unrelated PD UCB in patients with non-malignant hematological disorders is warranted.

Recombinant human deoxyribonuclease for hematopoietic stem cell processing.

Numerous reagents are used in the collection processing and storage of hematopoietic progenitor cells for transplantation. To decrease potential variations in the final component for transplantation, these reagents should be uniform in safety, potency, and efficacy. Pharmaceutical-grade reagents are ideal but often are not available. Recently, recombinant human deoxyribonuclease (DNase) was approved for the treatment of patients suffering from the pulmonary complications of cystic fibrosis. We tested this pharmaceutical for toxicity to hematopoietic progenitor cells. These cells were exposed to a range of incubation concentrations for both the recombinant enzyme and bovine DNase previously used in this laboratory. No loss of nucleated cells or hematopoietic progenitors was observed after short-term incubation (1 h) or with direct addition to the culture medium. No incremental toxicity was observed in using recombinant enzyme with murine anti-B cell antibodies and rabbit complement in an immunologic purge technique. A variable effect on cell recovery after thawing of cryopreserved bone marrow cells was observed for both enzyme sources. These data suggest that the pharmaceutical-grade, recombinant human DNase may substitute for previously used reagent-grade protein from animal sources.

Cryopreservation and subsequent viability of human bone marrow in hematologic malignancies: Comparison of two different methods of cell reconstitution

Bone marrow cells collected from patients with hematologic malignancies were cryopreserved using DMSO as a cryoprotective agent. The growth kinetics of hemopoietic stem cells frozen to −196 °C was monitored immediately after thawing by the semisolid agar CFU-C assay and two different methods of cell reconstitution were compared. In the first procedure, thawed cells were plated after the removal of DMSO by washing the cell suspension; in the second, cell suspensions were cultured after a simple 1:1 dilution of DMSO with medium. The numbers of CFU-C per 2 × 10 5 cells plated was higher by washing out the DMSO in all the groups studied. However, the absolute numbers of CFU-C contained in the whole ampoules after the freezing procedures was approximately the same using both methods. It is concluded that washing the cells only apparently yielded a better cloning efficiency, suggesting that such a procedure led to a higher mature nucleated cell loss with the consequence of a CFU-C concentration. This trend seems particularly evident in cells from the AML and CML patients.

Splenic Necrosis in Mice Infected with Cytomegalovirus

Necrosis of the spleen is seen in mice of certain strains after injection of large doses of murine cytomegalovirus. Necrosis begins in perifollicular areas containing infected cells which do not appear to be macrophages, and in severe cases the entire spleen is involved. Necrosis is first visible three days after infection and is not prevented by treatment of mice with corticosteroids, cyclophosphamide, or theta antiserum. There is a striking loss of nucleated cells from the spleen at two to four days after infection, in the absence of splenic necrosis. The pathogenesis of splenic necrosis, which is seen also in certain other viral infections, remains unexplained.

Thymus Cell Death Following Irradiation

JACKSON, K. L., CHRISTENSEN, G. M., AND FORKEY, D. J. Thymus Cell Death following Irradiation. Radiation Res. 34, 366-377, (1968). Rat thymocytes were studied in vivo and in vitro following 1000 R of Xirradiation to determine whether in vivo loss of nucleated cells is due primarily to mitotic inhibition or to interphase death. In vivo the rate of loss of live nucleated cells exceeded the rate of loss of total nucleated cells, and both rates exceeded that predicted on the basis of inhibition of cell division, implicating interphase death as a mechanism. Total thymus DNA decreased, but DNA associated with cell debris increased after irradiation, suggesting that cells killed by irradiation undergo lysis and that the resulting cell debris is removed more slowly than the cells break down. After in vitro irradiation thymocytes died in interphase at the same rate that live cells disappeared from the gland after in vivo exposure. It is concluded that the major mechanism for cell loss from rat thymus following whole-body exposure is radiation-induced interphase death followed by cell lysis.

Reduction of Radiation Mortality through Magnetic Pre-treatment

THE effect of whole body irradiation on rodents with lethal doses of X-rays or γ-rays is one of the most widely investigated phenomena. Mortality depends on the total dose, intensity at which the dose is delivered, strain, sex and age of the animal. Both serial histological examination and direct counts demonstrate the rapid loss of nucleated cells from the bone marrow. The animals' failure to replenish the granulocytes, platelets and erythrocytes from the circulating blood is primarily responsible for the death, as a consequence of septicaemia, haemorrhage and anaemia.