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BACKGROUND Acute myeloid leukaemia (AML) patients are at high risk of invasive aspergillosis (IA) after first induction chemotherapy (CHT). Although IA risk factors have been identified, few data are available on impact of IA, occurring during induction phase, on overall AML outcome. PATIENTS AND RESULTS The end point of this multicentre, case-control, study was to evaluate whether IA, occurring after first induction CHT, can affect treatment schedule and patient's outcome. We identified 40 AML patients (cases) who developed IA during first induction phase, 31 probable (77.5%) and 9 proven (22.5%). These cases were matched with a control group (80 AML) without IA, balanced according to age, type of CHT, AML characteristics and cytogenetic-molecular risk factors. The overall response rate to induction CHT was the same in the 2 groups. In the 40 cases with IA, the overall response rate to antifungal treatment was favourable (80%) but it was significantly affected by the achievement of leukaemia complete remission (CR) with induction CHT. In fact, in cases with AML responsive to induction CHT, responses of IA to antifungal therapy were 96% compared to 21% in cases of AML not responsive to induction treatment (P

Flow cytometry is a highly sensitive and specific approach for discriminating between normal and clonal plasma cells in multiple myeloma. Uniform response criteria after treatment have been established by the International Myeloma Working Group and the EuroFlow Group; however, the way in which flow cytometry data are reported has suffered from no collaborative or multicentre efforts. This study, involving 8 expert laboratories and 12 clinical hematology units of the Lazio region in Italy, aims to produce a uniform and shared report among the various Centres. From the pre-analytical phase to sample processing, data acquisition, analysis, and evaluation of the potential limitations and pitfalls of the entire process, the study reaches a final conclusion shared by laboratories and clinicians according to the most updated principles and recommendations. The aim was to identify the necessary data to be included in the clinical report by using multiple-choice questionnaires at every single stage of the process. An agreement of more than 75% of the laboratories was considered mandatory for the data to be included in the report. By ensuring the operational autonomy of each laboratory, this study provides a clear report that limits subjective interpretations and highlights possible bias in the process, better supporting clinical decision-making.

We tested the efficacy and safety of oral fludarabine and cyclophosphamide as front-line therapy in chronic lymphocytic leukemia (CLL) and assessed the influence of immunoglobulin variable region heavy chain (IgVH) gene mutation status, interphase cytogenetic abnormalities, and expression of ZAP-70 and CD38 on clinical outcome. Thirty-seven patients with previously untreated CLL received oral fludarabine (30 mg m(2)) and oral cyclophosphamide (250 mg m(2)) for three consecutive days every 4 weeks for six cycles. Eighteen patients had unmutated and 15 had mutated IgVH genes. Nine patients had the 'high risk' cytogenetic abnormality del(11q22.3) or del(17p13.1). Fifteen patients were ZAP-70-positive and eight patients were CD38-positive. Among the 35 valuable patients, 14 patients (40%) obtained a complete response and 13 (37%) a partial response. The median progression-free survival (PFS) was 23 months and median time to re-treatment (TTR) was 38 months. A significantly lower overall response rate (43% vs. 85%, p = 0.011), a shorter PFS (22 vs. 27 months, p = 0.015), and a shorter TTR (22 vs. 40 months, p = 0.031) were noticed in the 'high risk' cytogenetic abnormalities group; TTR was also shorter in IgVH-unmutated than in IgVH-mutated patients (26 vs. 41 months, p = 0.035). Hematologic toxicity included grade IV neutropenia (ten patients) and grade III/IV anemia (three patients). Gastrointestinal toxicity was mild and no patient required hospitalization. The oral combination of fludarabine and cyclophosphamide is an effective, safe, and well-tolerated regimen that, if confirmed with larger series, will be appropriate especially in patients with low risk biological parameters.

The development of a de novo lymphoma in patients affected by chronic myelogenous leukemia (CML) is a rare event. The introduction of new molecular cytogenetic techniques, such as fluorescence in situ hybridization (FISH), allows a correct differential diagnosis between lymphoid blastic crisis and a blastoid variant of mantle cell lymphoma (MCL), which shows an aggressive behavior and some molecular characteristics detectable by cytogenetics and immunohistochemistry. We report a case of a blastoid variant of MCL that developed in a patient with CML who achieved complete cytogenetic and molecular response to imatinib mesylate treatment.

Aims To assess changes in reticulocyte impedance volume, conductivity and light scatter (reticulocyte population data or RPD) obtained with the volume, conductivity and laser light scatter (VCS) technology in healthy subjects and in different erythropoietic states. Methods Blood samples were analysed with the Beckman–Coulter LH750 system, using the VCS method, from a group of 40 healthy subjects and three groups of patients with different types of untreated or treated anaemia: 24 cases of iron deficiency at the time of diagnosis, 16 patients with iron deficiency anaemia during intravenous iron administration, and 57 patients with chronic kidney disease undergoing dialysis and administration of rHu-erythropoietin and intravenous iron. Results RPD data were reproducible. Average mean channels for volume were 50.9 for controls and 49.2, 55.7 and 64.0 for the three patient groups, respectively. Average mean channels for conductivity were 52.0 for controls and 59.8, 55.7 and 56.1 for the three patient groups. Average mean channels for light scatter were 108.4 for controls and 113.3, 117.3 and 128.2 for the three patient groups. SD data indicated increased dispersion in the patient groups. Conclusion When values in the patient groups are compared with reference values obtained in healthy controls, the main differences are found in impedance volume and light scatter, which were both increased in patients with stimulated erythropoiesis. These data indicate the opportunity to further evaluate the clinical usefulness of RPD in haematological diseases.

To asses the real contribution of pre-transplantation treatment in the incidence of secondary neoplasia after autologous transplant for lymphoproliferative disorders, we used stringent inclusion/exclusion criteria. One hundred and forty-two patients out of 323 that underwent autologous transplantation for lymphoproliferative disorders were studied. The risk factors that were evaluated with univariate and multivariate analysis included: gender, sex, age, diagnosis, radiotherapy, and chemotherapy prior to conditioning regimen, disease status at peripheral blood stem-cell transplantation (PBSCT) and type of harvest. Three patients developed secondary myelodysplastic syndrome/acute myeloid leukemia (sMDS/AML) and three patients developed solid neoplasia. By univariate analysis diagnosis chronic lymphocytic leukemia and use of fludarabine and monoclonal antibodies were the only variables significantly associated with the development of sMDS/AML. By multivariate analysis, the variables associated with sMDS/AML were the use of fludarabine and disease status at PBSCT. By univariate analysis, we found that radiotherapy and the use of monoclonal antibodies were significantly associated with the development of secondary solid neoplasia. Multivariate analysis confirmed that the only two variables significantly associated with new cancers were radiotherapy and prior treatment with monoclonal antibodies. We report the lowest incidence of sMDS/AML after autologous stem-cell transplantation for lymphoproliferative malignancies. Major reasons could be ascribed to the stringent inclusion/exclusion criteria used to establish the real incidence of sMDS/AML because of chemo-radiotherapy used before transplant procedure. The low incidence of secondary solid tumors could be caused by the absence of total body irradiation as part of the conditioning regimen or the short follow-up.

An HIV-positive woman receiving antiretroviral therapy developed an opportunistic toxoplasma infection, detected by morphological examination of bone marrow aspirate in the absence of serological positivity. The intracellular presence of Toxoplasma gondii was confirmed by indirect immunofluorescence microscopy, using a polyclonal antitoxoplasma antiserum on marrow smears. This case report confirms the utility of morphological bone marrow examination for the diagnosis of parasitic infections in patients with impaired host defenses.

Anemia is a common complication of chronic kidney disease (CKD), particularly in dialysis patients. Correction of anemia in CKD patients includes the administration of both recombinant human erythropoietin and intravenous iron. An optimization of iron treatment requires obtaining a target hemoglobin level and avoiding an excessive body-iron overload. The reticulocyte hemoglobin content (CHr) has been shown to be an early indicator of iron-restricted erythropoiesis. The recent European guidelines for anemia treatment in CKD assessed the value for CHr >29 pg/cell as the reticulocyte parameter to evaluate a patient's iron needs. The reticulocyte hemoglobin equivalent (RET-He), recently introduced to determine the forward scatter of fluorescence-labeled reticulocytes, seems to be a sensitive indicator of iron-deficiency anemia. This study evaluates the concordance between the CHr parameter, used as a reference, and the new RET-He in a cohort of 57 dialysis patients referred to the Nephrology Unit of our hospital. All patients received erythropoietin, and iron was administered intravenously to maintain the hemoglobin level between 10 and 12 mg/dL. A total of 285 determinations were performed with both instruments. In the dialysis population, the 95% central range for CHr of 24.8 to 36.3 pg corresponds to a range for RET-He of 23.3 to 40.1 pg, with a mean bias of 1.12 pg between the 2 parameters. In comparison with CHr, the value of 30.5 pg for RET-He appeared to be the best cut-off point with a very good sensitivity and specificity to determine patients needing iron supplementation. Our study showed an excellent diagnostic efficiency of RET-He to evaluate patients needing iron support and demonstrated a strict correspondence between the classic CHr and the new Ret-He. This correspondence was independent of clinical changes, frequently occurring in dialysis patients. Both parameters could be used soon to guide and monitor iron treatment in dialysis patients.

Elevated levels of TNF-alpha have been associated with progressive disease in patients with chronic lymphocytic leukemia (CLL). Thalidomide has been shown to inhibit production of TNF-alpha. We investigated the effects of thalidomide on clinical outcome and TNF-alpha serum levels in five pre-treated CLL patients. The schedule consisted on daily thalidomide (Thal), oral fludarabine (Flu) and oral cyclophosphamide (CTX). Median duration of treatment was 60 days; four patients stopped treatment for disease progression and one patient for neurological toxicity. Serum TNF-alpha levels did not show any decrease during treatment. Low-dose thalidomide is not effective in CLL patients with refractory disease.

We report the efficacy and safety of intravenous low-dose alemtuzumab (10 mg three times weekly for 10 weeks) in 12 patients with relapsed or refractory chronic lymphocytic leukemia. Low-dose alemtuzumab induced significant responses in these patients (16% complete remission, 25% partial remission), with mild hematologic and extrahematologic side effects and a low rate of infections, even in the presence of long-lasting severe immunosuppression.

To evaluate the normalization of lymphocyte subsets several years after autologous peripheral blood stem cell transplantation (aPBSCT) and to detect any differences based on the underlying lymphoproliferative diseases, weanalyzed the immunological recovery of 149 patients with Non Hodgkin's Lymphoma (NHL), Hodgkin's Disease (HD), Multiple Myeloma (MM). Lymphocyte recovery was assessed before the transplant, on days 15, 30, 60, 90, 120 and on years 1, 2, 4, 6. Analysis of a total of 709 lymphocytes, including total lymphocyte count, CD3 + , CD4 + , CD8 + , CD4 + /CD8 + ratio, CD19 + , CD3 + HLA-DR + , CD16 + 56 + , was performed. The normalization of total lymphocyte counts was achieved between days 14 to 22 following PBSCT. CD3 + cells count showed a normalization after 2 years in the HD and NHL groups and after 4 years in MM group. CD4 + subset achieved normalization during the sixth year in the 3 groups. The CD8 + and CD19 + lymphocytes subsets achieved normal values in the 3 groups at day 60 and at day 120 respectively. CD16 + 56 + and CD3 + /HLA-DR + lymphocytes showed median values above the normal range starting from day 30. Immunological recovery was similar in all 3 groups. Moreover, the recovery of all subsets evaluated was similarly demonstrated within 6 years after aPBSCT.

Auteur(s) : Maria Clara D’Alessio1, Cinzia Mazzanti2, Nicoletta Di Simone1, Salvatore Mancuso1, Giovanni Reddiconto3, Mariagrazia Garzia3, Giuseppe Leone3, Tommaso Gobello2, Alessandro Caruso1 1Instituto di Ginecologia ed Ostetricia, Universita Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, Italy 2Istituto Dermopatico dell’Immacolata, IDI-IRCCS, via dei Monti di Creta 104, 00167 Roma, Italy 3Instituto di Ematologia, Universita Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 [...]

Secondary myelodysplastic syndromes following peripheral blood stem cell transplantation: morphological, cytogenetic and clonality evaluation and the limitation of FAB criteria

On July 28th 2005, a 53-year old woman was admitted to our Hematology Dpt. with fever (37.5 C), fatigue, marked gingiva hypertrophy, mild hepatomegaly and absence of lymphoadenopaty and splenomegaly. Laboratory tests showed high leukocyte count of 182 x109/L, moderately increased platelet count (438 x109/L), normocytic anemia (10,9 g/dl, MCV 84 fl). Biochemical parameters, including lysozyme, were in the normal range except for high level of lactate dehydrogenase (2524 UI/l). Peripheral blood (PB) smear showed the presence of 94% of blasts, without evident granules. At the cytochemical myeloperoxidase staining blast cells revealed the presence of single or multiple Auer rods, while cytoplasmic granules were positive to the α-naphtyl butyrate esterase. In the bone marrow (BM) aspirate blasts were 80% showing same morphological and cytochemical features as in peripheral blood, while the granulocytic maturating series was 14%. No evidence of morphologically identifiable monocytic series in PB neither in BM. Megakariocytes were slightly increased and there was trilineage dysplasia. Blast immunophenotyping in PB and in BM was as follow: CD45, CD33, CD13, CD7 and HLA-Dr positive, 36% MPO positive and 48% co-express CD11c an CD117. Moreover blasts were CD14, CD19 and CD56 negative. FAB diagnosis was AML-M4. Interphase Fluorescence in situ Hybridization (FISH) for detection of AML1/ETO and bcr/abl gene rearrangements was negative. After treatment with 2 g hydroxyurea and leucapheresis, the WBC count was still high (140 x109/L) while after 1g of aracytin, WBC count decreased to 31000/mm3. Patient is presently enrolled into the GIMEMA protocol AML-12. This case shows at the onset following abnormalities: - an increased platelet count is a rare evenience in an AML at the onset; - negativity for lysozyme; - absence of CD14; - presence of Auer rods in quite all the blast population, which is a very rare evenience associated with monocytic subtypes of acute myeloblastic leukemia (AML); - atypical homogenous cytochemical pattern of blasts, showing Auer rods MPO positive and granules ANBE positive; - absence of any morphological differentiation of monocytic series. S4479

Bone marrow (BM) analysis is conventionally performed by microscope examination on films of about 0.3ml of aspirated bone marrow fluid, stained with Romanowsky dyes. Until 1996 the simple automated screening of marrow composition was made very difficult by a number of factors, mainly the lack of the erythroblasts quantitation and the fat interference. From 1996 last generation automated hematology analyzers provide accurate and precise erythroblasts counts; moreover same systems have improved their software reducing the problem of fat interference. We have analyzed data from 100 normal BM samples from patients submitted for diagnostic and/or follow up purposes in our Hematology Day Hospital. BM fluid was harvested from the superior posterior iliac crest. The first 0,3–0.5 ml were used for smears, while the next 1–2 ml of BM, collected into K3-EDTA, were analysed with Coulter LH 750, a fully automated hematology analyzer which provides Complete Blood Count, White Blood Count Differential included Nucleated Red Blood Cells (NRBC) and Reticulocytes count. We used the microscope examination conventionally performed on films stained with Romanowsky dyes as reference method. Quantitative detection BM cellularity was obtained by semi quantitative evaluation based on the evaluation of hematopoietic cells in several marrow particles: physiological differences age related were also taken in account. If hematopoietic cells occupy less than 25% or more than 85% the sample is defined respectively hypocellular or hypercellular (none of our sample was as). Differential cell count was usually performed on two different slides counting 500 cells (1000 when hypercellular, but none of our sample was as). We found a strict correlation between microscope semi-quantitative cellularity evaluation and the instrumental cell count as sum of WBC plus NRBC, the Total Nucleted Cell Count (TNCC). The mean value of the TNCC in normal PM samples was 29,48 x109/L with a range 25,9–54,9 x109/L. These results are in good agreement with normal BM cell count reported in the literature using a cytofluorimetric method, which is 34,5 x109/L (SD28.0). The instrumental mean percentage of BM granulocytes corrected for TNNC was 62% (range: 23,5–93,7) versus a mean microscope percentage of 58,42% (range: 40–72). The automated NRBC BM count corrected for TNCC was 11,38% (range: 2,7 – 39,17) versus a microscopic mean value of 28% (range: 9–45). These results, including the slight NRBC underestimation probably due to partial mature cell lysis, are in line with the data of the literature. This study confirms the feasibility of routine automated cell count using a hematology in normal BM fluid samples. Automated methods will support morphologists quickly providing accurate and precise quantitative information such as TNCC and myeloid/erythroid ratio.

The hematology analyzer Coulter LH750 provides WBC differential counts using the VCS technology, which combines three simultaneous measurements (Volume, Conductivity, Scatter) to classify circulating blood cells. Cell volume and internal cell structure (opacity) are detected, respectively, by applying a low frequency direct current (DC) and a high frequency alternative (RF) current. The light scattered at different angles between 10° and 70°, on the other hand, is proportional to cell size, granularity, surface topography and reflectance. The system also provides for the main four WBC subtypes (neutrophils, lymphocytes, monocytes, eosinophils) a set of data called ‘positional parameters’ (PP). They express the mean value and the standard deviation (SD) of the three VCS measurements. We focused our study to the analysis of PP of the neutrophil population in normal and in pathological samples in order to: i) evaluate any correspondence with morphology and ii) determine the predictivity of changes in these parameters for the presence of morphological abnormalities. We analysed 100 samples from healthy subjects, 59 males and 41 females, age ranged from 2 years to 79 years, (25 children, 75 adults) to define our neutrophil reference PP ranges. We found a mean volume of 151,7 (SD ± 22,5), a mean opacity of 143,9 (SD ± 7,4) and a mean scatter of 142,19 (SD ± 13,1), without consistent differences due to age or sex. Our results do agree with those found in the literature. In normal samples we could not observe, as expected, any morphological abnormality at the microscope. Thereafter we also analyzed 16 samples from patients with bacterial pneumonitis. Their PP showed a moderate increase in the mean volume (168,9 with SD ± 24,1), a normal value of opacity and a slight decrease of the mean scatter (138,2 with SD ±14.4). Microscope analysis showed granulocytes with normal morphology, sometimes with toxic granules and the presence of band forms and metamyelocytes above 7%. The analysis of the PP of 11 patients with chronic myeloid leukemia at diagnosis showed an increase in the mean volume (160,1) with a consistently highly SD (± 33,4); opacity (142,8 with SD ± 11,7) and scatter (143,9 with SD ± 12,3) were normal. At the microscope the morphology of neutrophils was normal, with the presence of circulating immature granulocytes (range 22–28%). Finally we analyzed samples from N 28 patients with myelodysplastic syndromes (14 RA, 5 RC, 9 RAEB Type 1). Circulating granulocytes showed a great increase in the mean volume with a wide dispersion (178,2 with SD ± 35,6 ), a decreased mean opacity (132 with SD ± 8,3) and very low mean scatter value (119,9 with SD ± 12,4). At microscope neutrophils showed characteristic dysplastic features, such as abnormal nuclear segmentation, chromatin clumping and hypogranularity. Our study demonstrates the Coulter LH750 PP are highly predictive of the presence of dysplastic neutrophils in the peripheral blood. We have found a peculiar data set (high volume with increased SD, low opacity and scatter with small SD) which is easily distinguishable from normal as well as from other types of neutrophil abnormalities, such as myloproliferative and bacterial diseases. These parameters are automatically available during the routine load of hematology laboratories and can be usefully utilized to screen and to early identify patients with mylodysplasia.

The aims of the study were to analyze the clinical and epidemiological characteristics and treatments for patients who developed zygomycosis enrolled in Italy during the European Confederation of Medical Mycology of medical mycology survey. This prospective multicenter study was performed between 2004 and 2007 at 49 italian Departments. 60 cases of zygomycosis were enrolled: the median age was 59.5 years (range 1-87), with a prevalence of males (70%). The majority of cases were immunocompromised patients (42 cases, 70%), mainly hematological malignancies (37). Among non-immunocompromised (18 cases, 30%), the main category was represented by patients with penetrating trauma (7/18, 39%). The most common sites of infection were sinus (35%) with/without CNS involvement, lung alone (25%), skin (20%), but in 11 cases (18%) dissemination was observed. According to EORTC criteria, the diagnosis of zygomycosis was proven in 46 patients (77%) and in most of them it was made in vivo (40/46 patients, 87%); in the remaining 14 cases (23%) the diagnosis was probable. 51 patients received antifungal therapy and in 30 of them surgical debridement was also performed. The most commonly used antifungal drug was liposomal amphotericin B (L-AmB), administered in 44 patients: 36 of these patients (82%) responded to therapy. Altogether an attributable mortality rate of 32% (19/60) was registered, which was reduced to 18% in patients treated with L-AmB (8/44). Zygomycosis is a rare and aggressive filamentous fungal infection, still associated with a high mortality rate. This study indicates an inversion of this trend, with a better prognosis and significantly lower mortality than that reported in the literature. It is possible that new extensive, aggressive diagnostic and therapeutic procedures, such as the use of L-AmB and surgery, have improved the prognosis of these patients.

We test the efficacy and safety of the oral formulation of fludarabine combined with cyclophosphamide as front-line therapy of CLL. We studied the influence of IgVH gene mutation status, interphase cytogenetic abnormalities, and expression of ZAP-70 and CD38 on the overall response rate (OR), progression free survival (PFS), time to re-treatment (TTR) and overall survival (OS). Twenty seven patients were male and 10 were female, with a median age of 68 years (range, 52 to 75 years). Thirty patients were in stage B/II, 1 in stage C/III, and 6 in stage C/IV. The treatment schedule consisted of oral fludarabine (30 mg/m2) and oral cyclophosphamide (250 mg/m2) for 3 consecutive days every 4 weeks, for a maximum of 6 cycles. Eighteen patients had unmutated and 15 had mutated IgVH genes; in 4 patients the mutation status was not determined. Nine patients (24%) had the ‘high risk’ cytogenetic abnormality del(11q22.3) or del(17p13.1). Fifteen patients had more than 20% ZAP-70-positive and 8 patients had more than 30% CD38-positive CLL B-cells, respectively. Thirty-five patients were evaluable for response to treatment. Two patients discontinued the treatment protocol because of the development of immune thrombocytopenia after 2 and 4 cycles, respectively. Both of them belonged to the ‘high risk’ cytogenetic category. Fourteen patients (40%) obtained a complete response and 13 (37%) a partial response with an ORR of 77%. Four patients showed stable disease and four patients experienced progressive disease after 2, 3, 4 and 6 cycles, respectively. The median PFS was 23 months (range 2–42) and the median TTR was 38 months (range 2–42). The ‘high risk’ cytogenetic abnormalities del (17p13.1) and del (11q22.3) were predictive of a lower OR rate (43% vs 85%, p=0.011) as well as a shorter PFS (22 vs 27 months, p= 0.015) and shorter TTR (22 vs 40 months, p=0.031). In addition, unmutated IgVH genes were significantly associated with a reduced TTR (26 vs 41 months, p=0.035), whereas no significant impact was noticed in terms of OR, PFS and OS, even though all four deaths occurred in patients with unmutated IgVH genes. Expression of CD38 and ZAP-70 had no significant impact on clinical outcome. At present, median follow-up is 30 months, with an OS rate of 89%. The median time of OS has not been reached for any of the prognostic categories. In terms of haematological toxicity, ten patients developed grade IV neutropenia during treatment and received G-CSF. Three patients developed grade III and IV anemia that required red blood cell transfusions. Extra-hematological toxicity consisting of nausea was detected in 14 patients (40%) of whom 12 with grade I–II and 2 (6%) with grade III; half of these patients also experienced vomiting (6 patients grade I–II and one patient grade III). One patient developed fever of unknown origin after the first cycle of chemotherapy, which spontaneously resolved without hospitalization. The oral combination of fludarabine and cyclophosphamide is an effective, safe and well tolerated regimen that appears especially appropriate for a subset of previously untreated CLL patients characterized by mutated IgVH genes and the absence of “high risk” cytogenetic abnormalities.