Enrichment of Human Bone Marrow Aspirates for Low-Density Mononuclear Cells Using a Haemonetics Discontinuous Blood Cell Separator

Abstract

Isopycnic density floatation centrifugation has been proven to be a suitable technique to enrich bone marrow aspirates for clonogenic cells on a small scale. We have tested a Haemonetics semicontinuous blood cell separator in order to process large volumes of bone marrow with minimal bone marrow manipulation. The efficacy of isopycnic density floatation was tested in a one and a two-step procedure. Both procedures showed a recovery of about 20% of the nucleated cells and 1-2% of the erythrocytes. The enrichment of clonogenic cells in the one-step procedure appeared superior to the two-step enrichment, first separating buffy coat cells. The recovery of clonogenic cells was 70 and 50%, respectively. Repopulation capacity of the low-density cell fraction containing the clonogenic cells was excellent after autologous reinfusion (6 cases) and allogeneic bone marrow transplantation (3 cases). Fast enrichment of large volumes of bone marrow aspirates with low-density cells containing the clonogenic cells by isopycnic density floatation centrifugation can be done safely using a Haemonetics blood cell separator.

Analysis for Recovery and Loss of Mononuclear Cells and Colony‐Forming Units Granulocyte‐Macrophage during ex vivo Processing of Autologous Bone Marrow

“…BM stem cell concentration with a cell separator was first described in I977 using the Haemonetics apheresis system [2 I]. In the following years attempts were made using Haemonetics models to improve the initial technique, resulting in recovery rates up to 90% for MNC and 50-90% for CFU-GM [7, 15,[22][23][24]. Other investigators worked with Cobe cell processors, a procedure originally described in 1982 [25].…”

Separation of Mononuclear Bone Marrow Cells using the Cobe 2997 Blood Cell Separator

“…The combination of a cell separator and the use of density gradients has been developed in the past few years. The first description of the combined use of the Cobe 299 1 blood processor and Ficoll-Hypaque [2] has been followed and confirmed by other reports [20, 211. Yet another technique involving the combination of the Haemonetics V 50 and Percoll has been recently described [22]. These two techniques give excellent results in all respects, with recoveries of approximately 20% of nucleated cells and 75% of MNC and CFU-GM.…”

“…Final RBC contamination is negligible, while remaining granulocytes rarely exceed 10%. However, the duration ofthese procedures can exceed 2 h, especially when a large volume of BM has been used for in vitro treatment with monoclonal antibodies and complement [5,[22][23][24]. The product obtained with the Cobe 2997 cell separator achieves virtually all the objectives of stem cell concentration.…”