RESUMEN
Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts. In the present study, we review and compare the major counting techniques of stem and progenitor cells. The methods are: the Milan/Mullhouse protocol, two-platform ISHAGE (International Society of Hematotherapy and Graft Engineering) and single-platform ISHAGE analysis system. According to the Milan/Mulhouse protocol, HSCs are identified by CD34 antibody staining and easy gating strategy. The ISHAGE guidelines for detection of CD34+ cells are based on a four-parameter flow cytometry method (CD34PE/CD45PerCP staining, side and forward angle light scatter) thus employing multiparameter gating strategy. With two-platform ISHAGE protocol, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser. The single-platform ISHAGE method to determine the absolute CD34+ count directly from a flow cytometer includes the use of Trucount tubes (Becton Dickinson) with a known number of fluorescent beads. CD34+ cells were quantified in mobilized peripheral blood, collected by leukapheresis, and bone marrow from 42 samples from patients with hematological malignancies. The differences against the means display low disagreement between the Milan/Mulhouse and ISHAGE protocols, with discrepancies of up to 2.5% (two-platform ISHAGE)--2.6% (single-platform ISHAGE) in enumeration of CD34+ cells in leukapheresis product and 4.8% (two-platform ISHAGE)--4.9% (single-platform ISHAGE) in bone marrow. Our results show high correlation among all three methods. Since the three protocols are compatible, choosing the most convenient in terms of costs, simplicity and compliance with clinical results appears to be a logical consequence.
Asunto(s)
Antígenos CD34/metabolismo , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Células Madre Hematopoyéticas/metabolismo , Leucaféresis , Trasplante de Células Madre , Células Madre/metabolismo , Anticuerpos Monoclonales , Recuento de Células , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Reproducibilidad de los ResultadosRESUMEN
Dendritic cell (DC) counts and function were assayed in peripheral blood of lymphoma and solid tumor patients before and after chemotherapy. The DC counts declined significantly within the first week from the start of chemotherapy, recovered in the second week, and exceeded the baseline values in the third week. DC recovery was usually similar after the first and after the last cycle of chemotherapy. DC1 and DC2 subsets followed the pattern of reconstitution found for the DC population as a whole. Monocytes and granulocytes recovered 1-2 weeks later than DC. The primary proliferative response to keyhole lympet hemocyanin (KLH), totally DC-dependent, declined within the first week from the start of chemotherapy, and in the majority of patients (including those initially unresponsive) recovered along with DC counts. The recovered responsiveness to KLH, but not to anti-CD3 antibody, disappeared at the end of chemotherapy in lymphoma and some solid tumor patients. Prolonged depletion of CD4+ T cells could contribute to the loss of responsiveness in lymphoma patients receiving multiple cycles of chemotherapy. However, in some solid tumor patients, the reactivity to KLH was absent, despite the reconstitution of both DC and CD4+ T-cell counts. Our data show that numerical reconstitution of DC is not necessarily accompanied by functional recovery. The early recovery of DC should be considered while designing protocols for DC collection for immunotherapy.
Asunto(s)
Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Adulto , Anticuerpos Monoclonales/metabolismo , Complejo CD3/biosíntesis , Linfocitos T CD4-Positivos/metabolismo , División Celular , Células Dendríticas/citología , Femenino , Hemocianinas/metabolismo , Humanos , Inmunofenotipificación , Inmunoterapia/métodos , Interleucina-2/metabolismo , Linfoma/sangre , Linfoma/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Factores de TiempoRESUMEN
Following the first successful cord blood transplantation in 1988, several hundreds of patients were treated using same protocol. The main limitation of the wide use of cord blood as a source of haematopoietic cells is the number of available units of this tissue. To make possible selection of HLA-matched cells for individual patient, several thousands of cord blood samples must be collected and stored in liquid nitrogen. The network of cooperating cord blood banks with join accessible database is necessary. In this paper the activity of Jose Carreras memorial Cord Blood Bank established in Warsaw was described. Since the middle of January the collection of cord blood units for clinical purposes was started. During first three months 80 samples of cord blood was collected. Collections were obtained from normal full-term deliveries after the third stage of labour. For the banking the collection over 60 ml or contain over 4 x 10(8) of mononuclear cells were qualified. Whole blood samples and plasma samples obtained following volume reduction were used for HLA and bacteriology tests. After volume reduction the number of nucleated cells (WBC), mononuclear cells (MNC) and hematopoietic cells (CD34+) were evaluated. After processing the cord blood samples were frozen using control freezer and were stored in liquid nitrogen storage tanks. According to results of cord blood transplantation hundred percent of banked samples are suitable for recipients weighing 10 kg and only 7 percent for these weighing 50 kg.
Asunto(s)
Bancos de Sangre/organización & administración , Humanos , PoloniaRESUMEN
Human umbilical cord blood (UCB) has been successfully used as a source of allogeneic hematopoietic cells for transplantation. Banking of the UCB requires its volume reduction to decrease storage space, costs and volume of infused DMSO. In order to select an optimal method for volume reduction we compared several methods of cord blood processing, namely buffy coat centrifugation, red cell lysis, hydroxyethyl starch (HES)-, methylcellulose- and gelatin-sedimentations. The viability of cells and the recoveries of total white blood cells, mononuclear cells and CD34+ cells was evaluated. We also compared the efficacy of red cells depletion from the original UCB sample. Buffy coat centrifugation, red cell lysis, HES, gelatin or methylcellulose resulted in high mononuclear cell recoveries, whereas high hematopoietic cell recovery was observed only after HES sedimentation and buffy coat processing. The HES sedimentation procedure compared to buffy coat processing is more time and labor consuming and resulted in higher red blood cell and platelets depletion. Both methods can be recommended as a method at choice for the umbilical cord blood processing before banking.