A Simplified Method for Purification,Concentration and Freezing of Bone Marrow Cells for Autologous Transplantation

Abstract

Autologous bone marrow (BM) transplantation is being increasingly applied in hematological and oncological patients. However, because of the need to purify and preserve BM requiring high technology, such treatments are virtually concentrated in the “developed” countries. This paper examines methods of BM purification and freezing that could make the technique potentially applicable in developing countries. Hemapheresis is routinely applied for BM purification in our Dutch Centre, where the buffy coats obtained from routine blood donations were utilised in experimental settings. Using DMSO as cryoprotectant, semi-purified white cells were frozen in liquid N₂ (LN₂), by mechanical freezer or snap-frozen at -55° C. Different types of containers were compared including plastic tubes and ordinary blood bags. After thawing the results show that snap-freezing had a deleterious effect but the cell yields and viability were similar in LN₂ or the mechanical freezer where the tubes and the bag were equally effective as containers (86% cell recovery with 90% viability). In the purification/concentration stage, reduction of the volume of the material by extra centrifugation, thus requiring less DMSO, produced better results — 96% cell yield and 90% viability after thawing.

This simplified method was applied in a general hospital in Sao Paulo where four oncology patients underwent BM collection. BM was purified and concentrated within a blood bank facility. Hydroxyethyl starch sedimentation and centrifugation of the material in plastic blood bags gave 80% BM cell harvest. After thawing from the mechanical freezer 1 x 10⁸ BM cells/kg were available for reinfusion to patients. There was no immediate untoward reaction. Three of the patients showed signs of bone marrow regeneration by three weeks, but one patient died 16 days after transplantation, of septicemia. We conclude that certain high-technology procedures for ABMT can be adapted for existing facilities in developing countries.