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Journal ArticleDOI

Difference in Viability of CD34+ Cells in Cryopreserved Cord Blood According to Evaluation Methods

01 Jun 2009-The Korean Journal of Hematology (The Korean Society of Hematology)-Vol. 44, Iss: 2, pp 92-99

TL;DR: After thawing the cryopreserved UCB, 89% of the total MNCs and 84% of CD34+ cells were viable as identified by trypan blue exclusion assay, and the cell death rate was found to be 47% by Annexin-V/PI staining and less than 5% by DNA contents analysis.

AbstractBackground: On performing umbilical cord blood (UCB) transplantation, faster engraftment may lead better clinical outcome. Because transplanted viable cell count in UCB is related to the engraftment, accu- rate evaluation of viability of CD34+ cells in cryopreserved UCB has clinical implication. We examined the difference in viability of cells in cryopreserved UCB according to the duration of cryopreservation and different methods. Methods: A total of 60 UCB samples which were cryopreserved for 1 to 4 years were used in this study. Viability of cryopreserved cells were examined with trypan blue exclusion assay, DNA contents analysis, caspase-3 activation test, intracellular esterase activity and Annexin-V/PI staining. Results: After thawing the cryopreserved UCB, 89% of the total MNCs and 84% of CD34+ cells were viable as identified by trypan blue exclusion assay. In the CD34+ cell population, the cell death rate was found to be 47% by Annexin-V/PI staining and less than 5% by DNA contents analysis. However, cspase-3 activity failed to document apoptosis. The intracellular esterase activity test also showed a cell death rate of about 10 ∼20% at 2, 4, and 6 hours after thawing.

Topics: Cord blood (51%), Trypan blue (51%), Transplantation (51%), Population (51%)

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Book ChapterDOI
TL;DR: It has proved feasible to categorize most if not all dying cells into one or the other of two discrete and distinctive patterns of morphological change, which have, generally, been found to occur under disparate but individually characteristic circumstances.
Abstract: Publisher Summary The classification of cell death can be based on morphological or biochemical criteria or on the circumstances of its occurrence. Currently, irreversible structural alteration provides the only unequivocal evidence of death; biochemical indicators of cell death that are universally applicable have to be precisely defined and studies of cell function or of reproductive capacity do not necessarily differentiate between death and dormant states from which recovery may be possible. It has also proved feasible to categorize most if not all dying cells into one or the other of two discrete and distinctive patterns of morphological change, which have, generally, been found to occur under disparate but individually characteristic circumstances. One of these patterns is the swelling proceeding to rupture of plasma and organelle membranes and dissolution of organized structure—termed “coagulative necrosis.” It results from injury by agents, such as toxins and ischemia, affects cells in groups rather than singly, and evokes exudative inflammation when it develops in vivo. The other morphological pattern is characterized by condensation of the cell with maintenance of organelle integrity and the formation of surface protuberances that separate as membrane-bounded globules; in tissues, these are phagocytosed and digested by resident cells, there being no associated inflammation.

7,342 citations


Journal ArticleDOI
TL;DR: It is necessary to select patients suitable for vaginal or laparoscopic mesh placement for Fanconi's anemia preoperatively on the basis of prior history and once they provide informed consent for surgery.
Abstract: The clinical manifestations of Fanconi’s anemia, an autosomal recessive disorder, include progressive pancytopenia, a predisposition to neoplasia, and nonhematopoietic developmental anomalies [1-3]. Hypersensitivity to the clastogenic effect of DNA-cross-linking agents such as diepoxybutane acts as a diagnostic indicator of the genotype of Fanconi’s anemia, both prenatally and postnatally [3-6]. Prenatal HLA typing has made it possible to ascertain whether a fetus is HLA-identical to an affected sibling [7]. We report here on hematopoietic reconstitution in a boy with severe Fanconi’s anemia who received cryo-preserved umbilical-cord blood from a sister shown by prenatal testing to be unaffected by the disorder, to have a normal karyotype, and to be HLA-identical to the patient. We used a pretransplantation conditioning procedure developed specifically for the treatment of such patients [8]; this technique makes use of the hypersensitivity of the abnormal cells to alkylating agents that cross-link DNA [9,10] and to irradiation [11] In this case, the availability of cord blood obviated the need for obtaining bone marrow from the infant sibling. This use of cord blood followed the suggestion of one of us that blood retrieved from umbilical cord at delivery, usually discarded, might restore hematopoiesis – a proposal supported by preparatory studies by some of us [12] and consistent with reports on the presence of hematopoietic stem and multipotential (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells in human umbilical-cord blood (see the references cited by Broxmeyer et al. [12]).

1,997 citations


Journal ArticleDOI
TL;DR: Placental blood is a useful source of allogeneic hematopoietic stem cells for bone marrow reconstitution and is associated with the severity of GVHD, type of leukemia, and stage of the disease.
Abstract: Background A program for banking, characterizing, and distributing placental blood, also called umbilical-cord blood, for transplantation provided grafts for 562 patients between August 24, 1992, and January 30, 1998. We evaluated this experience. Methods Placental blood was stored under liquid nitrogen and selected for specific patients on the basis of HLA type and leukocyte content. Patients were prepared for the transplantation of allogeneic hematopoietic cells in the placental blood and received prophylaxis against graft-versus-host disease (GVHD) according to routine procedures at each center. Results Outcomes at 100 days after transplantation were known for all 562 patients, and outcomes at 1 year for 94 percent of eligible recipients. The cumulative rates of engraftment among the recipients, according to actuarial analysis, were 81 percent by day 42 for neutrophils (median time to engraftment, 28 days) and 85 percent by day 180 for platelets (median, day 90). The speed of myeloid engraftment was as...

1,339 citations


Journal ArticleDOI
TL;DR: It was determined that granulocyte-macrophage, erythroid, and multipotential progenitor cells remained functionally viable in cord blood untreated except for addition of anticoagulant for at least 3 days at 4 degrees C or 25 degrees C (room temperature), though not at 37 degrees C, implying that these cells could be satisfactorily studied and used or cryopreserved for therapy.
Abstract: The purpose of this study was to evaluate human umbilical cord blood as an alternative to bone marrow in the provision of transplantable stem/progenitor cells for hematopoietic reconstitution Although no direct quantitative assay for human hematopoietic repopulating cells is at present available, the granulocyte-macrophage progenitor cell (CFU-GM) assay has been used with success as a valid indicator of engrafting capability We examined greater than 100 collections of human umbilical cord blood for their content of nucleated cells and granulocyte-macrophage, erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells, in many cases both before and after cryopreservation First it was determined that granulocyte-macrophage, erythroid, and multipotential progenitor cells remained functionally viable in cord blood untreated except for addition of anticoagulant for at least 3 days at 4 degrees C or 25 degrees C (room temperature), though not at 37 degrees C, implying that these cells could be satisfactorily studied and used or cryopreserved for therapy after transport of cord blood by overnight air freight carriage from a remote obstetrical service Granulocyte-macrophage progenitor cells from cord blood so received responded normally to stimulation by purified recombinant preparations of granulocyte-macrophage, granulocyte, and macrophage colony-stimulating factors and interleukin 3 The salient finding, based on analysis of 101 cord blood collections, is that the numbers of progenitor cells present in the low-density (less than 1077 gm/ml) fraction after Ficoll/Hypaque separation typically fell within the range that has been reported for successful engraftment by bone marrow cells Another observation of practical importance is that procedures to remove erythrocytes or granulocytes prior to freezing, and washing of thawed cells before plating, entailed large losses of progenitor cells, the yield of unwashed progenitor cells from unfractionated cord blood being many times greater The provisional inference is that human umbilical cord blood from a single individual is typically a sufficient source of cells for autologous (syngeneic) and for major histocompatibility complex-matched allogeneic hematopoietic reconstitution

1,170 citations


Journal ArticleDOI
01 Sep 2002-Blood
TL;DR: There is a high probability of survival in recipients of UCB grafts that are disparate in no more than 2 human leukocyte antigens when the grafts contain at least 1.7 x 10(5) CD34(+) cells per kilogram of recipient's body weight, and graft selection should be based principally on CD34 cell dose when multiple UCB units exist with an HLA disparity of 2 or less.
Abstract: The potential benefits of unrelated donor marrow transplantation are offset by the immunologic complications of graft-versus-host disease (GVHD) and infection. Therefore, we used cryopreserved umbilical cord blood (UCB) as a strategy to reduce the risks of GVHD and treatment-related mortality (TRM) and improve survival. Data on 102 patients (median age 7.4 years) who received transplants between 1994 and 2001 for the treatment of malignant (n = 65; 68% were high-risk patients) and nonmalignant (n = 37) diseases were evaluated. Log-rank tests and Cox regression analyses were used to determine the effects of various demographic, graft-related, and treatment factors on engraftment, GVHD, TRM, relapse, and survival. As of October 15, 2001, the median follow-up was 2.7 years (range, 0.3-7.2). Incidences of neutrophil and platelet engraftment were 0.88 (CI, 0.81-0.95) and 0.65 (CI, 0.53-0.77), respectively. Notably, incidences of severe acute and chronic GVHD were 0.11 (CI, 0.05-0.17) and 0.10 (CI, 0.04-0.16), respectively. At 1 year after transplantation, proportions of TRM and survival were 0.30 (CI, 0.21-0.39) and 0.58 (CI, 0.48-0.68), respectively. In Cox regression analyses, CD34 cell dose was the one factor consistently identified as significantly associated with rate of engraftment, TRM, and survival. Despite the low incidence of GVHD, the proportion of patients with leukemia relapse at 2 years was 0.17 (CI, 0.00-0.38) and 0.45 (CI, 0.28-0.61) for patients with standard and high-risk disease, respectively. There is a high probability of survival in recipients of UCB grafts that are disparate in no more than 2 human leukocyte antigens (HLAs) when the grafts contain at least 1.7 x 10(5) CD34(+) cells per kilogram of recipient's body weight. Therefore, graft selection should be based principally on CD34 cell dose when multiple UCB units exist with an HLA disparity of 2 or less.

982 citations


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