The use of unfixed bone marrow trephines for multicolour flow cytometry.

An adequate bone marrow aspirate is essential for a rapid diagnosis of acute leukaemia by multicolour flow cytometry enabling the simultaneous assessment of multiple antigens on the cell surface as well as intracellular or nuclear ones. In the context of acute leukaemia, it is important to have a diagnosis of the blasts lineage as soon as possible to decide the appropriate treatment. This is sometimes delayed due to difficulties in obtaining a bone marrow aspirate due to a "dry tap". In this study we evaluated retrospectively cell markers results by flow cytometry of unfixed bone marrow trephines of 65 patients with leukaemia at diagnosis and including a few after treatment. Our aims were: 1) To compare cell markers results between bone marrow trephine (BMT) and bone marrow aspirate (BMA) 24 cases and BMT with peripheral blood (PB) 14 cases in paired samples to establish if they were reproducible with results of the unfixed bone marrow trephine biopsies. 2) To ascertain a precise diagnosis in 27 (42%) of the cases in which only a bone marrow trephine was available. We demonstrated that unfixed bone marrow trephine provides an adequate and representative cell suspension for flow cytometry and it is a powerful tool when no other material (bone marrow aspirate or peripheral blood) is available to make a rapid diagnosis. Furthermore when marrow aspirate or peripheral blood paired samples were available, flow cytometry results obtained were identical across all the sample types. Applicability to the clinical laboratory: We described a method to obtain a cell suspension from core biopsies that can easily be implemented routinely in a laboratory that performs diagnostic flow cytometry immunophenotyping. This method is simple, inexpensive and it doesn't require extra equipment.

Durable remissions of myelodysplastic syndrome and acute myeloid leukemia after reduced-intensity allografting.

PURPOSE: To evaluate the use of reduced-intensity (RI) conditioning with allogeneic hematopoietic stem cell transplantation (HSCT) from HLA-identical family donors in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). PATIENTS AND METHODS: Sixteen patients (median age, 54 years; range, 37 to 66 years) underwent RI-HSCT using a conditioning regimen of fludarabine 25 mg/m2 daily for 5 days and either cyclophosphamide 1 g/m2 daily for 2 days (14 patients) or melphalan 140 mg/m2 for 1 day (two patients). The median number of CD34+ cells and CD3+ cells infused per kilogram of recipient weight was 4.5 x 106 (range, 1.8 to 7.3 x 106 cells) and 2.9 x 108 (range, 0.1 to 9.6 x 108 cells), respectively. RESULTS: There was no transplant-related mortality (TRM) within 100 days of HSCT. Grade 1 to 2 acute graft-versus-host disease (GVHD) occurred in three patients, but neither grade 3 nor grade 4 disease was observed. Chronic GVHD occurred in 10 patients. One patient had cytomegalovirus (CMV) reactivation but did not develop CMV disease. With a median follow-up of 26 months (range, 15 to 45 months), 11 patients are alive (nine in continuous complete remission and one in complete remission after a second transplantation), and five have died (four from disease progression and one from bone-marrow aplasia induced by cyclosporine withdrawal). The 2-year actuarial overall and event-free survival rates were 69% (95% confidence interval [CI], 40% to 86%) and 56% (95% CI, 30% to 68%), respectively. CONCLUSION: This strategy of RI-HSCT resulted in reliable engraftment with low incidence of acute GVHD and TRM. Durable remissions were observed in patients with MDS and AML consistent with a graft-versus-leukemia effect.

Loss of CD20 expression following treatment with rituximab (chimaeric monoclonal anti-CD20): a retrospective cohort analysis.

A retrospective analysis of CD20 expression following rituximab for B-cell non-Hodgkin's lymphoma demonstrated a significant change in immunophenotype in 6/25 (24%) patients with persistent bone marrow (BM) infiltration. In three out of six patients, the B cells were uniformly CD20-/CD79alpha+, consistent with frank loss of CD20 expression. In the remaining three cases, the BM infiltrate was predominantly (> 80%) CD20-/CD79alpha+. Two of the former but none of the latter three cases achieved a clinical response. In three further cases, the post-treatment BM infiltrate was composed entirely of benign or reactive CD3+ T cells. Frank loss of CD20 was not seen in 25 post-treatment lymph node biopsies. Immunophenotyping is therefore an important adjunct in the diagnosis of BM infiltration following rituximab.