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ABSTRACT: T acute lymphoblastic leukemia cell lines treated with hexamethylene bisacetamide (HMBA) undergo a delay in cell cycle progression and increase susceptibility to apoptosis, although they never overcome the differentiation block. In accordance with changes in cell cycle and apoptosis, transitory p53 pathway activation commonly occurs. Bcl-2 inhibition further favours the pro-apoptotic effect of HMBA. Notch1 expression is down regulated by reduction of its transcription level. Accordingly, Notch1 protein and transcriptional activity were affected. Even if HMBA generally reduces Notch1 level in T acute lymphoblastic leukemia (T-ALL) cell lines, this does not commonly influence the biological response; in fact all the analysed cell lines, except CEM cells, display no biological effect following DAPT-induced Notch inhibition.
Leukemia Research 06/2008; 32(5):791-7. · 2.92 Impact Factor
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ABSTRACT: A new method for measuring telomere length in a population of Down's syndrome patients aged 18-60 years old is presented. The method is based on flow cytometry and quantitative fluorescence in situ hybridization (flow-FISH) on whole cells. At least three methods for measuring the length of telomere repeats have been described: (i) Southern blot analysis, and quantitative FISH using either (ii) digital fluorescence microscopy (Q-FISH) or (iii) flow cytometry (flow-FISH). Both Southern blot analysis and Q-FISH have specific limitations and are time-consuming, whereas flow-FISH needed relatively few cells (1.5-2.5 x 106) and could be completed in 24-48 h. The method can be used to rapidly determine telomere length in subsets of nucleated blood cells from patients with age-related diseases such as Down's syndrome, Alzheimer's disease and Werner syndrome.
International journal of tissue reactions 02/2004; 26(1-2):61-4.
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M Montillo,
A Tedeschi,
V Rossi,
R Cairoli,
E Pungolino,
L Intropido,
A M Cafro,
G D'Avanzo,
R Farioli, B Brando,
B Scarpati,
S Veronese,
E Morra
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ABSTRACT: Chronic lymphocytic leukemia (CLL) cells could be undetectable by flow cytometry or polymerase chain reaction after sequential treatment with fludarabine and Campath-1H. Concern has been raised regarding the ability to mobilize sufficient peripheral blood progenitor cells (PBPCs) for autografting after purine analogues, and there are few data about PBPC collection after Campath-1H. In all, 16 CLL patients responding to sequential chemo-immunotherapy entered the study. In 10, mobilization regimen consisted of granulocyte colony-stimulating factor (G-CSF) 5-10 microg/kg/die. Patients failing mobilization or not achieving the target of 2.5 x 10(6) CD34+ cells/kg underwent a second attempt using intermediate-dose (ID) Ara-C, 800 mg/m(2) every 12 h for six doses+G-CSF. PBPC collection after G-CSF alone was successful in two out of 10 patients. An adequate number of CD34+ cells were collected after ID Ara-C+G-CSF in eight patients failing the mobilization with G-CSF alone and in five out of six who did not receive G-CSF before. Greater yields of PBPCs were collected with Ara-C+G-CSF compared with G-CSF alone (13.8 vs 3.3). The extrahematologic toxicity was manageable. In conclusion, PBPC collection is feasible in CLL patients treated with sequential therapy including fludarabine and Campath-1H. Excellent yields were obtained in 92.8% of patients primed with ID Ara-C+G-CSF.
Leukemia 01/2004; 18(1):57-62. · 9.56 Impact Factor
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ABSTRACT: Absolute counting of cells or cell subsets has a number of significant clinical applications: monitoring the disease status of HIV-infected patients, enumerating residual white blood cells in leukoreduced blood products, and assessing immunodeficiency in a variety of situations. The single-platform method (flow cytometry alone) has emerged as the method of choice for absolute cell enumeration. This technology counts only the cells of interest in a precisely determined blood volume. Exact cell identification is accomplished by a logical electronic gating algorithm capable of identifying lineage-specific immunofluorescent markers. Exclusion of unwanted cells is automatic. This extensive and detailed unit presents protocols for both volumetric and flow-rate determination of residual white blood cells and of leukocyte subsets.
Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 06/2001; Chapter 6:Unit 6.8.
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ABSTRACT: Using a single-platform protocol to count absolute CD34+ hematopoietic precursor cell (HPC) levels with different reference microbeads, we recorded occasionally artifactually high CD34+ HPC counts in some leukapheresis bags, whereas dual-platform calculations were always consistent. Abnormal countings were observed only when phosphate-buffered saline (PBS)-diluted leukapheresis samples were vortexed before analysis. A large series of blood samples analyzed similarly for CD34+ and CD4+ absolute counts did not show any sample or vortexing effect. With the volumetric absolute counting cytometer Partec-PAS, lower counts were also observed when different reference beads were vortexed before the instrument checking procedures. The counting abnormality was caused by a drop in microbead concentration (the "vanishing bead phenomenon"). This phenomenon reduced the total and relative bead event number in experimental and routine samples and in calibration procedures. This altered the bead denominator used to calculate absolute CD34+ HPC levels and it also reduced the concentration of standard calibration beads.
Using the Partec-PAS to measure volumetrically the actual bead concentration, we studied the vanishing bead phenomenon. Different types of counting and reference microbeads were resuspended in media with or without proteins or cells. Replicates were submitted either to gentle manual mixing or to vortexing before counting.
Vortex agitation almost invariably induced the vanishing bead phenomenon when beads were resuspended in saline media or when an insufficient protein concentration was present, such as in diluted leukapheresis samples. Different bead types showed various degrees of sensitivity to vortexing. The bead disappearance was not caused by bubble formation or disruption. The addition of small amounts of protein completely prevented the vanishing bead phenomenon. The causative effect of the electrostatic charging of tube induced by vortexing is hypothesized.
Sample suspensions containing counting beads for single-platform analysis must be resuspended in media with protein supplements to prevent the vanishing bead phenomenon and to ensure accurate counting.
Cytometry 03/2001; 43(2):154-60.
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B Brando,
D Barnett,
G Janossy,
F Mandy,
B Autran,
G Rothe,
B Scarpati,
G D'Avanzo,
J L D'Hautcourt,
R Lenkei,
G Schmitz,
A Kunkl,
R Chianese,
S Papa,
J W Gratama
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ABSTRACT: The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources.
Cytometry 01/2001; 42(6):327-46.
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G Muti,
A De Gasperi,
S Cantoni,
P Oreste,
G Gini,
G Civati,
G Busnach, B Brando,
M Frigerio,
M Mangiavacchi,
A Alberti,
L Decarus,
G Rondinara,
E De Giuli,
E Morra
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ABSTRACT: In the period 1973-1998, among 2139 allograft recipients treated with standard immunosuppression, posttransplant lymphoproliferative disorders (PTLD) developed in 19 patients (0.9%): one plasmacytic hyperplasia, two polymorphic PTLD, one myeloma, and 15 lymphomas. PTLD developed 1 year after transplantation (tx) in 14 patients. Five patients were diagnosed at autopsy, 2 were lost to follow up, 3 died before therapy could be instituted, and 1 patient has just started chemotherapy. Of the 8 evaluable patients, 2 received acyclovir and are alive in complete remission (CR) and 6 received chemotherapy +/- surgery. Of these 6, 4 died of lymphoma and/or infection, 1 died of unrelated causes in CR, and 1 is alive in CR. PTLD is a severe complication of tx, usually running an aggressive course which may preclude prompt diagnosis and treatment. Nevertheless, therapy is feasible and must be tailored on the histologic subtype. Seventy-four percent of patients were diagnosed with late-onset PTLD stressing the need for long-term follow up.
Transplant International 02/2000; 13 Suppl 1:S382-7. · 2.92 Impact Factor
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A Orfao,
G Schmitz, B Brando,
A Ruiz-Arguelles,
G Basso,
R Braylan,
G Rothe,
F Lacombe,
F Lanza,
S Papa,
P Lucio,
J F San Miguel
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ABSTRACT: At present, immunophenotyping of hematological malignancies represents one of the most relevant clinical applications of flow cytometry. In recent years, its use has extended from clinical research to diagnostic laboratories. The aim of this report is to critically review the type of information provided by the flow cytometric immunophenotyping of hematological malignancies and its clinical impact as well as to highlight its potential future applications.
The currently available information, including that provided by different international consensus groups on the phenotypic characterization of hematologic malignancies, was reviewed. Additionally, recent reports on the immunophenotypic analysis of hematological malignancies published in hematology, oncology, pathology, immunology, and cell biology journals were also analyzed.
A careful review of the literature showed that in spite of the well-established utility of immunophenotyping for the diagnosis, classification, prognostic stratification, and monitoring of hematological malignancies, only a small part of the information on the immunophenotypic characteristics of pathological hemopoietic cells has been used routinely. Specific and sensitive identification of neoplastic cells and their accurate enumeration and phenotypic characterization represent the major aims of these procedures. Similarities between leukemic and healthy cells allow the establishment of the lineage and maturation stage of the pathologic cells, this information being of great utility for the diagnosis, classification, and prognostic evaluation of different subtypes of hematological malignancies. On the other hand, the phenotypic aberrations displayed by leukemic cells could allow the selection of cases carrying specific genetic abnormalities in which further confirmatory molecular studies will be performed.
The information provided by the flow cytometric immunophenotyping of hematological malignancies is of great clinical utility, with a major challenge for the near future being the standardization of technical procedures, data interpretation, and reporting.
Clinical Chemistry 11/1999; 45(10):1708-17. · 7.91 Impact Factor
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Transplantation Proceedings 07/1998; 30(4):1343-5. · 1.00 Impact Factor
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J W Gratama,
A Orfao,
D Barnett, B Brando,
A Huber,
G Janossy,
H E Johnsen,
M Keeney,
G E Marti,
F Preijers,
G Rothe,
S Serke,
D R Sutherland,
C E Van der Schoot,
G Schmitz,
S Papa
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ABSTRACT: The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/microl) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34+ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34dim and CD34bright populations in the CD34+ cell count, 6) omission of the negative control staining, and 7) for apheresis products, enumeration of at least 100 CD34+ cells to ensure a 10% precision. Unresolved technical questions are 1) the replacement of conventional dual-platform by single-platform assay formats, i.e., derivation of absolute CD34+ cell counts from a single flow cytometric assessment instead of from combined flow cytometer (percent CD34+) and hematology analyzer (absolute leukocyte count) data, 2) the cross-calibration of the available single-platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and required numbers of HPCs responsible for short- and long-term recovery to optimize HPC transplant strategies.
Cytometry 07/1998; 34(3):128-42.
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European journal of histochemistry: EJH 02/1997; 41 Suppl 2:107-8. · 1.69 Impact Factor
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European journal of histochemistry: EJH 02/1996; 40 Suppl 1:53-62. · 1.69 Impact Factor
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European journal of histochemistry: EJH 02/1994; 38 Suppl 1:21-6. · 1.69 Impact Factor
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ABSTRACT: Plasmapheresis performance is improved in the treatment of hyperviscosity syndromes with one of the several cascade filtration techniques (CF), intended for plasma fractionation and reinfusion of albumin-enriched plasma filtrate to the patients, avoiding the need for exogenous reinfusion solutions. A retrospective open analysis of 103 CF, performed by dead-end mode, in 14 patients with macroglobulinemia, cryoglobulinemia, multiple myeloma and other diseases, has been performed. Protein fractions removals have been calculated, normalized to the treatment of one plasma volume, compared in different macromolecular diseases and according to the different plasma fractionators employed. [table: see text] Protein removal is partially dependent of the surface area of the fractionator, but a wide variability has been reported, mainly depending on the underlying macromolecular disease.
The International journal of artificial organs 01/1994; 16 Suppl 5:155-9. · 1.86 Impact Factor
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Transplantation Proceedings 03/1993; 25(1 Pt 2):1389-90. · 1.00 Impact Factor
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ABSTRACT: The Italian Society for Cytometry (Gruppo Italiano di Citometria, GIC) promoted this nationwide large-scale quality control trial on cellular immunophenotyping and flow cytometer performance in 1991. The aim of this independent study was to evaluate instrument performance with calibrated fluorescence microbeads (minimum threshold for FITC, linearity, coefficients of variation and resolution indexes for fluorescence), and to correlate it to the measurement of lyophilized lymphocyte surface immunofluorescence staining with a wide spectrum of antibodies (percentage of positive cells and fluorescence mean intensity). A single send-out was made to 306 laboratories with 350 instruments throughout Italy. Each kit included anonymous vials containing premixed calibrated microbeads, lyophilized human lymphocytes and small aliquots of conjugated monoclonal antibodies, for both single FITC and double FITC/PE staining. Participants were also asked to use their own anti-CD4 monoclonal. A valid answer was returned by 209 laboratories with 221 instruments. Gating was not an assay variable. The minimum sensitivity threshold for FITC ranged from 26.6 to 11,293 MESF, with marked instrument heterogeneity as far as the relationship between FITC threshold and coefficients of variation for fluorescence was concerned. A low FITC threshold also correlated with a good resolution index for low intensity stainings. No performance comparisons among instrument brands and models were made. The lyophilized lymphocyte analysis showed an overall frequency of outliers ranging from 3.2% to 19.7%, with a maximum for CD2 FITC (19.7%) and CD3 + HLA-DR + (12.1%). A strongly negative relationship was evident between the FITC threshold and the number of CD2+ cells, whereas the sensitivity threshold had virtually no effects on the measured level of higher antigen density markers like CD4 and many others. Absolute fluorescence intensity measurements of CD4+ and CD19+ were also made. This study design proved valid and suitable for a large-scale evaluation of both instrument performance and cytometer operators' skill. A number of practical problems were identified among participants, thus stressing the need for more effective educational programmes and technical guidelines.
Cytometry 02/1993; 14(3):294-306.
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ABSTRACT: Low-density lipoprotein apheresis (LDL-apheresis) was done with either cascade filtration (DF) or dextran sulfate cellulose adsorption (DSC) in a patient with primary biliary cirrhosis who developed severe dyslipidemia associated with cholestasis and accumulation of lipoprotein-X (LP-X). The extracorporeal treatment was initially performed weekly, and resulted in a sharp drop in total cholesterol from 1038 to 430 mg/dl. During the next four months the patient was treated every 10-15 days, and pre-apheresis cholesterol levels were maintained between 438 and 505 mg/dl, until an orthotopic liver transplantation was successfully performed. With semi-selective DF a mean 47.1% of total cholesterol was removed per procedure compared to 30.0% with DSC, although the volume of treated plasma was 38.0 vs 49.9 ml/kg body weight. The changes in plasma cholesterol levels during DSC and DF showed that the kinetics of cholesterol removal were similar with both techniques, but the efficacy differed; DF removed both LDL and LP-X from plasma, whereas DSC selectively lowered the LDL content. Cascade filtration may therefore be considered as a first-choice treatment for patients with LP-X accumulation due to cholestasis.
The International journal of artificial organs 05/1991; 14(4):246-50. · 1.86 Impact Factor
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ABSTRACT: Optimal criteria for harvesting circulating hematopoietic progenitors (CHP) for autologous transplantation to support myeloablative cancer therapy are still uncertain mostly because the CFU-GM assay, the commonly used indirect indicator of the hematopoietic recovery of the graft, is poorly standardized and provides information evaluable only retrospectively. Based on the knowledge that CHP express CD34 and CD33 differentiation antigens and facilitated by the availability of a very efficient fluorescein-conjugated CD34 antibody (8G12), we developed a direct immunofluorescence flow cytometry assay with the aim of replacing the CFU-GM assay advantageously. Recently, in a comparative study, both assays were applied to 157 blood samples obtained daily throughout 20 different recoveries from pancytopenia induced by high-dose cyclophosphamide (7 g/m2) cancer therapy w/ or w/o rhGM-CSF. Results showed that: a) detectability of CD34+ CHP indicated an increase to greater than 500 CFU-GM/mL, a level clinically adequate for harvesting CHP; b) CD34+ cells correlated well with CFU-GM (R=0.89) and data fitted a linear regression line (y=388.3 + 64.0x; y=CFU-GM/mL and x=CD34+/uL); c) in a series of 8 patients treated with myeloablative chemoradiotherapy, early recovery of marrow functions was predicted more accurately by the number of transplanted blood CD34+/CD33+ cells than by nucleated cells, CFU-GM, CD34+/CD33-cells, or CD34-/CD33+ cells. As a guideline, provided platelets are greater than 70,000/uL, harvest of CHP by leukapheresis during recovery from chemotherapy induced pancytopenia should be started as soon as CD34+ cells appear in the circulation and continued until the threshold dose of 7.8x10(6) CD34+ cells/kg, equivalent to 50 x 10(4) CFU-GM/kg, is achieved.(ABSTRACT TRUNCATED AT 250 WORDS)
Haematologica 04/1991; 76 Suppl 1:46-9. · 6.42 Impact Factor
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ABSTRACT: Optimum methods of harvesting circulating hematopoietic progenitors for autologous transplantation to support myeloablative cancer therapy are still uncertain, mostly because of the lack of an assay for marrow-repopulating stem cells. The CFU-GM assay, the commonly used indirect indicator of the quality of the graft, is poorly standardized and provides results evaluable only retrospectively. Based on the knowledge that hematopoietic progenitors express CD34 and CD33 differentiation antigens, we developed a dual-color direct immunofluorescence flow cytometry assay with the aim of replacing the CFU-GM assay advantageously. For this purpose, we applied both assays to 157 blood samples obtained daily throughout 20 different recoveries from pancytopenia induced by high-dose cyclophosphamide or etoposide cancer therapy with or without recombinant human GM colony-stimulating factor (rhGM-CSF). The appearance of CD34+ cells in the circulation indicated that hematopoietic progenitors had increased to more than 500 CFU-GM/mL, a level clinically adequate for large-scale harvest by leukapheresis. Total CD34+ cells correlated well with CFU-GM (r = .89), and data could be fitted by a linear regression line described by the equation y = 388.3 + 64.0x, where y = CFU-GM/mL and x = CD34+ cells per microliter. Moreover, in a series of six patients treated with myeloablative chemoradiotherapy, early hematopoietic recovery of marrow functions was predicted more accurately by the number of transplanted CD34+/CD33+ cells than by either total nucleated cells, CFU-GM, CD34+/CD33- cells, or CD34-/CD33+ cells. Data presented in this article favor clinical use of the CD34/CD33 flow cytometry assay to guide harvesting of circulating hematopoietic progenitors for autologous transplantation and contribute to better understanding of the role played by circulating hematopoietic progenitor cell subsets in marrow recovery after myeloablative cancer therapy.
Blood 02/1991; 77(2):400-9. · 9.90 Impact Factor
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Contributions to nephrology 02/1991; 94:115-22. · 1.49 Impact Factor
