Cell suspensions from collagenase digestion of bone marrow trephine biopsy specimens.

Abstract

SUMMARY A technique for the extraction of cells from bone marrow trephine core biopsy specimens using collagenase digestion was assessed in 39 cases (33 diagnostic and six normal). Diagnostically useful numbers ofcells were extracted from all marrows. Morphological assessment ofcytocentrifuge preparations of these cells gave a correct diagnosis in 23 (60%) of cases compared with 27 (70%) for the corresponding aspirated marrow smears. Phenotypic analysis using flow cytometry showed persistence of a range of surface membrane antigens following collagenase digestion. Increased autofluorescence was a problem in some cases.Cytochemistry, bone marrow culture, and cytogenetic analysis could also be carried out on these cells. It is concluded that this technique has useful diagnostic applications in cases of dry taps.Dry bone marrow taps may occur in many haematological disorders. These include the densely cell packed marrow of acute leukaemia and some malignant lymphomas, the fibrotic marrow of myeloproliferative disorders and some lymphoproliferative disorders such as hairy cell leukaemia, and the hypocellular marrow of aplastic anaemia. When this occurs diagnosis is impaired as marrow aspirate smears are not available for cell morphology and cytochemistry. Furthermore, karyotyping, bone marrow culture, and immunological phenotyping techniques using cell suspensions cannot be done.In these circumstances attempts to assess cell morphology by "roll imprints" are made but cell numbers and quality are poor. Alternatively, plastic embedded ultra thin histological section ofbone marrow trephine biopsy specimens may be used,' but this technique is difficult, labour intensive, and not available in every laboratory. Frozen section immunohistochemistry has been advocated for immunological phenotying of marrow in cases of dry taps,2 but this technique is also demanding and so not widely used.We present and assess a simple alternative technique for use in cases of dry taps in which cell suspensions suitable for morphological assessment, cytogenetics and immunological phenotyping are prepared by collagenase digestion of trephine bone marrow core biopsy specimens.

Failed or inadequate bone marrow aspiration: a fast, simple and cost-effective method to produce a cell suspension from a core biopsy specimen

“…This background was completely removed by the Ficoll gradient, which was introduced in the last four cases of this series. The condition of the cells on the cytospin stained with May‐Grünwald‐Giemsa was assessed on morphological grounds (Ades et al. , 1989).…”

“…While many haematological textbooks recommend imprints or roll imprints of unfixed BM biopsies, these do not work well in our hands. This view is shared by a study where roll imprints of good quality were obtained in only 10% (Ades et al. , 1989).…”

“…Collagenase digestion (Mononen & Jansson, 1986; Gibson et al. , 1988; Ades et al. , 1989; Maung, Bown & Hamilton, 1993) or physical disaggregation by manual ‘teasing’ (Pihan & Woda, 1990; Dunphy et al.…”

Trephine biopsies are enriched for activated T/NK cells and cytotoxic T cells

“…Thus, trephine biopsy yields a marrow sample that may more closely resemble that used for murine experiments. Few groups have examined trephine biopsies by means other than histology [14][15][16], and doing so was primarily regarded as an option in the case of dry-tap as- Table 2 Activation marker expression by NKR +/− T cell subsets and NK cells in paired (n = 7) bone marrow (BM) and trephine biopsy (TB) samples piration, i.e. where no readily stainable liquid sample could be obtained.…”

“…Such contamination can be largely avoided, and, perhaps, a more accurate representation of the immunological composition within the marrow obtained, by using an alternative sampling technique, the trephine biopsy, which yields a solid core of marrow, without blood contamination. Trephine biopsies are usually analysed as histological specimens, with only a few groups having processed them in order to extract cells from the bony matrix [14][15][16] and predominantly utilising the extracted cells in cytological preparations, regarding the processed trephine biopsy only as an alternative to be used in cases of "dry tap" aspiration [14][15][16].…”