Single-cell screening of multiple biophysical properties in leukemia diagnosis from peripheral blood by pure light scattering

Abstract Histology and histopathology are based on the morphometric observations of quiescent cells. Their diagnostic potential could largely benefit from a simultaneous screening of intrinsic biophysical properties at single-cell level. For such a purpose, we analyzed light scattering signatures of individual mononuclear blood cells in microfluidic flow. In particular, we extracted a set of biophysical properties including morphometric (dimension, shape and nucleus-to-cytosol ratio) and optical (optical density) ones to clearly discriminate different cell types and stages. By considering distinctive ranges of biophysical properties along with the obtained relative cell frequencies, we can identify unique cell classes corresponding to specific clinical conditions (p < 0.01). Based on such a straightforward approach, we are able to discriminate T-, B-lymphocytes, monocytes and beyond that first results on different stages of lymphoid and myeloid leukemia cells are presented. This work shows that the simultaneous screening of only three biophysical properties enables a clear distinction between pathological and physiological mononuclear blood stream cells. We believe our approach could represent a useful tool for a label-free analysis of biophysical single-cell signatures

Autoimmune hematological diseases after allogeneic hematopoietic stem cell transplantation in children: An Italian multicenter experience

AbstractAutoimmune hematological diseases (AHDs) may occur after allogeneic hematopoietic stem cell transplantation (HSCT), but reports on these complications in large cohorts of pediatric patients are lacking. Between 1998 and 2011, 1574 consecutive children underwent allogeneic HSCT in 9 Italian centers. Thirty-three children (2.1%) developed AHDs: 15 autoimmune hemolytic anemia (45%), 10 immune thrombocytopenia (30%), 5 Evans' syndrome (15%), 2 pure red cell aplasia (6%), and 1 immune neutropenia (3%). The 10-year cumulative incidence of AHDs was 2.5% (95% confidence interval, 1.7 to 3.6). In a multivariate analysis, the use of alternative donor and nonmalignant disease was statistically associated with AHDs. Most patients with AHDs (64%) did not respond to steroids. Sustained complete remission was achieved in 87% of cases with the anti-CD20 monoclonal antibody (rituximab). Four patients (9%) (1 autoimmune hemolytic anemia, 1 Evans' syndrome, 2 immune thrombocytopenia) died at a median of 87 days after AHD diagnosis as a direct or indirect consequence of their disorder. Our data suggest that AHDs are a relatively rare complication occurring after HSCT that usually respond to treatment with rituximab

Risk of Seizures in Children Receiving Busulphan-Containing Regimens for Stem Cell Transplantation

Busulphan (BU) is associated with neurotoxicity and risk of seizures. Hence, seizure prophylaxis is routinely utilized during BU administration for stem cell transplantation (SCT). We collected data on the incidence of seizures among children undergoing SCT in Italy. Fourteen pediatric transplantation centers agreed to report unselected data on children receiving BU as part of the conditioning regimen for SCT between 2005 and 2012. Data on 954 pediatric transplantation procedures were collected; of them, 66% of the patients received BU orally, and the remaining 34%, i.v. All the patients received prophylaxis of seizures, according to local protocols, consisting of different schedules and drugs. A total of 13 patients (1.3%) developed seizures; of them, 3 had a history of epilepsy (or other seizure-related pre-existing condition); 3 had documented brain lesions potentially causing seizures per se; 1 had febrile seizures, 1 severe hypo-osmolality. In the remaining 5 patients, seizures were considered not explained and, thus, potentially related to BU administration. The incidence of seizures in children receiving BU-containing regimen was very low (1.3%); furthermore, most of them had at least 1deither pre-existing or concurrentdassociated risk factor for seizures. 2014 American Society for Blood and Marrow Transplantation

A NEW PROTECTIVE AGENT AGAINST X-RAY DAMAGE OF HEMATOPOIETIC STEM CELLS

Aim of radiotherapy is the cancer cell damage destroying their ability to divide and grow, by using high-energy ionizing radiation (IR), although causing side effects on healthy cells. A limiting factor in the use of radiotherapy is the acute and long-term bone marrow injury: IR induces hematopoietic stem cell apoptosis and senescence. The direct ionization of cellular macromolecules generates the formation of reactive oxygen species (ROS), which lead to functional cell alterations1. A new isoform of human MnSOD, isolated from liposarcoma cells and obtained in a synthetic recombinant form (rMnSOD), exerts the radiosensitizing effect for tumor cells and meanwhile a radioprotective effect on healthy cells2. In our study, we analyzed the rMnSOD radioprotective effect on human umbilical cord hematopoietic stem cell (CB-HSCs). Mononuclear cells were isolated from cord blood after informed consent of healthy donors, received by the Cord Blood Bank (Ba.S.C.O) of “Santobono Pausilipon” hospital in Naples, by using Ficoll/Hypaque gradient. Cultured CB-HSCs were incubated with 0.5 μM MnSOD for 24h and then irradiated (2Gy) by using Mevatron (Siemens, Italia). After irradiation, an aliquot of cells was immediately stained with 0.4% Trypan Blue and cell viability and senescence was evaluated after 24h from irradiation, by using Guava Easy Cyte Flow Cytometer. Our preliminary results showed a viability increase of rMnSOD treated cells after IR exposure, compared to the only irradiated cells. Senescence evaluation confirmed the protective action of rMnSOD. HSC protection from IR should be a primary goal in the development of new medical countermeasures against radiation damages. Refernces 1. Shao L, et al. Antioxid Redox Signal 2014;20(9):1447-6 2. Borrelli A, et al. Free Radic Biol Med 2009;46(1):110-

KCTD15 Protein Expression in Peripheral Blood and Acute Myeloid Leukemia

Leukocytes are major cellular components of the inflammatory and immune response systems. After their generation in the bone marrow from hematopoietic stem cells, they maturate as granulocytes (neutrophils, eosinophils, and basophils), monocytes, and lymphocytes. The abnormal accumulation and proliferation of immature blood cells (blasts) lead to severe and widespread diseases such as leukemia. We have recently shown that KCTD15, a member of the potassium channel tetramerization domain containing protein family (KCTD), is remarkably upregulated in leukemic B-cells. Here, we extend our investigation by monitoring the KCTD15 expression levels in circulating lymphocytes, monocytes, and granulocytes, as well as in leukemia cells. Significant differences in the expression level of KCTD15 were detected in normal lymphocytes, monocytes, and granulocytes. Interestingly, we also found overexpression of the protein following leukemic transformation in the case of myeloid cell lineage. Indeed, KCTD15 was found to be upregulated in K562 and NB4 cells, as well as in HL-60 cell lines. This in vitro finding was corroborated by the analysis of KCTD15 mRNA of acute myeloid leukemia (AML) patients reported in the Microarray Innovations in Leukemia (MILE) dataset. Collectively, the present data open interesting perspectives for understanding the maturation process of leukocytes and for the diagnosis/therapy of acute leukemias

Outcome of children with ALL in second CR transplanted from an unrelated donor has significantly improved over time and is favourably influenced by the occurrence of grade I-II acute GVHD and limited chronic GVHD

Allogeneic HSCT from an unrelated volunteer is largely employed to treat children with ALL in second CR. We analyzed the outcome of patients transplanted in Centers belonging to the Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP) between 1995 and 2009. The outcome of children with 2nd CR ALL has significantly improved over time, due to a reduction of TRM. Limited severity acute and chronic GVHD protect from leukemia recurrence. BFM classification of 1st relapse predicts outcome