Morphological, cytochemical, and culture study of normal human bone marrow frozen at −196 °C

Abstract

A morphological, cytochemical, and agar culture study was carried out on samples of bone marrow that had been taken from 13 normal individuals and frozen at −196 °C. The cryoprotective agent (DMSO) was removed by slow or rapid dilution. A large number of the thawed cells appeared to have been destroyed or exhibited vacuoles in the nucleus and cytoplasm, as well as numerous short cytoplasmic evaginations. The few mature cells of the neutrophilic line that had survived contained only rare, if any, specific granules; only the lymphocytes were apparently unaffected. There was a reduction in, or an irregular distribution of, the positive reactions to PAS, peroxidase, naphthol-ASD-chloro-acetate esterase, and Sudan black exhibited by cells of the neutrophilic line, and the lysozyme activity of the neutrophils and the monocytes was affected. Where the DMSO had been removed by slow dilution these changes were less severe and diffuse, the percentage of trypan blue-negative cells was higher, and a much larger number of the colony-forming cells were recovered, this number being constantly reproducible. Similar results were obtained by comparing the two dilution methods on thawed-out specimens of peripheral blood from five patients with chronic myeloid leukaemia. With both types of dilution very few cluster-forming cells were recovered and no spontaneous formation of clusters or colonies was observed. The results suggest that marrow frozen at −196 °C and treated after thawing by slow dilution is suitable for marrow-transplant experiments, as a control for the agar culture of fresh marrow samples and for the stimulating activity of the feeder layers of peripheral blood leucocytes.