A new modification of our detergent technique for the preparation of nuclei for flow cytometric DNA analysis is described. The attainment of low coefficients of variation of the peaks and of quantitative staining of nuclei from different tissues was a problem with the original method. This was solved in the new modification by trypsinization of the unfixed nuclei. The nuclei were stabilized by spermine. A simple procedure for long-term storage of samples at -80°C was integrated into the method. The fluorescence of the nuclei was stable for at least 3 hours after staining. Light exposure protection of the samples was essential. No cell loss was caused by storage or staining. The method was successfully applied on samples including: (a) Normal tissues-human lymphocytes, granulocytes and spleen. Mouse lymphocytes, bone marrow, spleen, liver, kidney and thymus. (b) Human neoplasms-lung cancer, breast cancer, lymphoma, leukemia, bladder cancer and cancer of the oral cavity. Key terms: Flow cytometry, DNA, propidium iodide, solid tissues, preparation, storage A previously published method for the preparation of fineneedle aspirates from solid tissues for flow cytometric DNA analysis (9) has been useful for examination of human solid tumors (1,13,14). The method is based on detergent lysis of cell membranes and produces stained nuclei in monodisperse suspension in a single step. However, the low salt procedure of the technique, which has been used most extensively, has two major drawbacks: (a) Tissues rich in cytoplasm, for instance liver cells, are not prepared optimally because residual cytoplasm contaminates the nuclei, causing clumping and nonspecific staining. In the DNA distribution this produces asymmetrical peaks with high coefficients of variation (cv). ( b ) Staining of different types of cells is not perfectly quantitative. Thus a comparison of human lymphocytes and granulocytes for example showed a 6% difference in fluorescence.The original method was therefore modified with the aim of obtaining a preparation technique that would produce optimal results in a broad spectrum of tissues in regard to

Because information on management and outcome of AML relapse after allogeneic hematopoietic stem cell transplantation (HSCT) with reduced intensity conditioning (RIC) is scarce, a retrospective registry study was performed by the Acute Leukemia Working Party of EBMT. Among 2815 RIC transplants performed for AML in complete remission (CR) between 1999 and 2008, cumulative incidence of relapse was 32% ؎ 1%. Relapsed patients (263) were included into a detailed analysis of risk factors for overall survival (OS) and building of a prognostic score. CR was reinduced in 32%; remission duration after transplantation was the only prognostic factor for response (P ‫؍‬ .003).

Context A minimally toxic nonmyeloablative regimen was developed for allogeneic hematopoietic cell transplantation (HCT) to treat patients with advanced hematologic malignancies who are older or have comorbidities. Objective To describe outcomes of patients ≥ 60 years. Design, Setting, and Participants From 1998 to 2008, 372 patients, 60–75 years old were enrolled in prospective clinical HCT trials at 18 collaborating institutions using conditioning with low-dose total body irradiation alone or combined with fludarabine 90 mg/m2 before related (n=184) or unrelated (n=188) donor transplants. Post-grafting immunosuppression included mycophenolate mofetil and a calcineurin inhibitor. Main Outcome Measures Overall and progression-free survivals were estimated by Kaplan-Meier method. Cumulative incidence estimates were calculated for acute and chronic GVHD, toxicities, achievement of full donor chimerism, complete remission, relapse, and non-relapse mortality. Hazard ratios (HR) were estimated from Cox regression models. Results Overall, 5-year cumulative incidences of non-relapse mortality and relapse were 27% (95% CI, 22%–32%) and 41% (95% CI, 36%–46%), respectively, leading to overall and progression-free 5-year survivals of 35% (95% CI, 30%–40%) and 32% (95% CI, 27%–37%), respectively. These outcomes were not statistically significantly different when stratified by age groups. Furthermore, increasing age was not associated with increases in acute or chronic graft-versus-host disease (GVHD) or organ toxicities. In multivariate models, HCT-CI scores of 1–2 [HR, 1.58 (95% CI,1.08–2.31)] and ≥3 [HR, 1.97 (95% CI,1.38–2.80)] were associated with worse survival compared to HCT-CI score of 0 (overall P = 0.003). Similarly, standard relapse risk [HR, 1.67 (95% CI, 1.10–2.54)] and high relapse risk [HR, 2.22 (95% CI, 1.43–3.43)] were associated with worse survival compared to low relapse risk (overall P = 0.0008). Conclusion Among patients aged 60–75 years and treated with nonmyeloablative allogeneic HCT, 5-year overall and progression-free survivals were 35% (95% CI, 30%–40%) and 32% (95% CI, 27%–37%), respectively.

Determination of nuclear DNA content by flow cytometry requires comparison with a reference standard. The use of external standards such as lymphocytes or granulocytes is time-consuming and inaccurate.Chicken red blood cells (CRBC) have a DNA content of 35% of the human diploid value and have been widely used as internal standard. The ratio calculated on the basis of the peak channel numbers of the standard and

Allogeneic HCT relying on GVT effects is feasible and results in cures of an appreciable number of malignancies. Improved results could come from methods that control progression of malignancy early after HCT and effectively prevent GVHD.