L’applicazione della citogenetica convenzionale e molecolare può identificare: Ph-negatività, traslocazioni t(9;22) varianti e alterazioni citogenetiche addizionali (ACA) al cromsoma Ph in pazienti con LMC alla diagnosi. Prima dell’introduzione della terapia con Imatinib, esse mostravano un impatto prognostico negativo o non chiaro. Nel nostro studio, 6 casi di LMC Ph- erano trattati con Imatinib. La FISH identificava 4 casi con riarrangiamento BCR/ABL sul der(9q), 1 sul der(22q) e 1 su entrambi i derivativi. Quattro pazienti (66,7%) raggiungevano la RCgC, 2 fallivano il trattamento e 1 sottoposto a TMO. A causa dello scarso numero di casi, non era possibile nessuna correlazione con la prognosi. Nell’ambito di studi prospettici multicentrici del GIMEMA-WP, abbiamo valutato: traslocazioni varianti e ACA. Dei 559 pazienti arruolati, 30(5%) mostravano traslocazioni varianti, 24 valutabili in FISH: 18(75%) mostravano meccanismo 1-step, 4(16,7%) meccanismo 2-step e 2(8,3%) meccanismo complesso. Abbiamo confermato che le varianti non influenzano la risposta e la sopravvivenza dei pazienti trattati con Imatinib. Dei 378 pazienti valutabili alla diagnosi con citogenetica convenzionale, 21(5,6%) mostravano ACA: 9(43%) avevano la perdita del cromosoma Y, 3(14%) trisomia 8, 2(10%) trisomia 19, 6(28%) altre singole anomalie e 1 cariotipo complesso. La presenza di ACA influenzava la risposta: le RCgC e RMolM erano significativamente più basse rispetto al gruppo senza ACA e le curve di sopravvivenza EFS e FFS non erano significativamente diverse. Le curve di PFS e OS erano sovrapponibili nei due gruppi, per il basso numero di eventi avversi oppure perché alcuni raggiungevano la risposta con TKI di seconda generazione. Le anomalie “major route” mostravano decorso clinico peggiore, ma non è stato possibile determinare l’impatto prognostico in relazione al tipo di alterazione. Pertanto, le ACAs alla diagnosi rivestono un ruolo negativo nella prognosi dei pazienti trattati con Imatinib, che quindi rappresentano una categoria più a rischio per la risposta. / At diagnosis in chronic myeloid leukemia (CML) patients, conventional cytogenetics and FISH analysis can identify: Ph- CML cases, variant t(9;22) translocations and additional chromosomal abnormalities (ACAs) in Ph-positive clone. Before the introduction of the Imatinib therapy, these characteristics showed a negative or not clear prognostic role. We analysed 6 Ph- cases treated with Imatinib. FISH analysis showed 4 cases with BCR/ABL rearrangement on der(9q), 1 on der(22q) and 1 on both derivatives chromosomes. Four patients (66,7%) reached CCgR, 2 failed treatment and 1 died after stem cell transplantation. Beacause of small of number of cases, we cannot stated that Ph-masked cases have a worse prognosis. Within the clinical trial of GIMEMA WP, we have analysed variant t(9;22) translocations and ACAs. Five-hundred-fifty-nine patients were enrolled; 30(5%) had variant translocations of which 24 could be evaluated by FISH: 18(75%) showed 1-step mechanism, 4(16,7%) 2-step mechanism and 2(8,3%) complex mechanism. We observed that variant translocations didn’t influence the responses and the outcome in patients treated with Imatinib. At diagnosis, 378 patients were evaluable by cytogenetics. Twenty-one (5,6%) showed ACAs: 9 (43%) had loss of Y chromosome, 3(14%) trisomy 8, 2(10%) trisomy 19, 6(28%) other isolated abnormalities and 1(15%) complex karyotype. We reported that the presence of ACAs influenced the responses: the CCgR and MMolR were significantly lower compared with those of the group without ACAs, but EFS and FFS were not significantly different. PFS and OS curves were overlapped, because of the low number of negative events or because some patients reached response with second generation TKI treatment. Cases with “major route” abnormalities showed worse outcome, but we cannot established the impact related to the kind of abnormality. However, ACAs at diagnosis had a negative role on the response to the therapy in CML patients, which constitute a “warning” category for Imatinib treatment.

Understanding the biology of Multiple Myeloma (MM) is of primary importance in the struggle to achieve a cure for this yet incurable neoplasm. A better knowledge of the mechanism underlying the development of MM can guide us in the development of new treatment strategies. Studies both on solid and haematological tumours have shown that cancer comprises a collection of related but subtly different clones, a feature that has been termed “intra-clonal heterogeneity”. This intra-clonal heterogeneity is likely, from a “Darwinian” natural selection perspective, to be the essential substrate for cancer evolution, disease progression and relapse. In this context the critical mechanism for tumour progression is competition between individual clones (and cancer stem cells) for the same microenvironmental “niche”, combined with the process of adaptation and natural selection. The Darwinian behavioural characteristics of cancer stem cells are applicable to MM. The knowledge that intra-clonal heterogeneity is an important feature of tumours’ biology has changed our way to addressing cancer, now considered as a composite mixture of clones and not as a linear evolving disease. In this variable therapeutic landscape it is important for clinicians and researchers to consider the impact that evolutionary biology and intra-clonal heterogeneity have on the treatment of myeloma and the emergence of treatment resistance. It is clear that if we want to effectively cure myeloma it is of primarily importance to understand disease biology and evolution. Only by doing so will we be able to effectively use all of the new tools we have at our disposal to cure myeloma and to use treatment in the most effective way possible. The aim of the present research project was to investigate at different levels the presence of intra-clonal heterogeneity in MM patients, and to evaluate the impact of treatment on clonal evolution and on patients’ outcomes.

I nucleotidi trifosfato sono, dal punto di vista evoluzionistico, tra le molecole più antiche e conservate tra le specie. Oltre al ruolo che ricoprono nella sintesi degli acidi nucleici e nel metabolismo energetico della cellula, negli ultimi anni è emerso sempre di più il loro coinvolgimento nella regolazione di numerose funzioni cellulari. Questi importanti mediatori cellulari sono presenti nel microambiente e cambiamenti nella loro concentrazione extracellulare possono modulare la funzionalità cellulare. I nucleotidi trifosfato ATP e UTP, presenti nel microambiente midollare, sono dei potenti stimolatori dei progenitori emopoietici. Essi stimolano la proliferazione e l’attecchimento delle cellule staminali emopoietiche, così come la loro capacità migratoria, attraverso l’attivazione di specifici recettori di membrana, i recettori purinergici (P2R). In questo studio abbiamo dimostrato che ATP e UTP esercitano un effetto opposto sul compartimento staminale leucemico di leucemia acuta mieloide (LAM). Abbiamo dimostrato che le cellule leucemiche esprimono i recettori P2 funzionalmente attivi. Studi di microarray hanno evidenziato che, a differenza di ciò che avviene nelle CD34+, la stimolazione di cellule leucemiche con ATP induce la down-regolazione dei geni coinvolti nella proliferazione e nella migrazione, mentre up-regola geni inibitori del ciclo cellulare. Abbiamo poi confermato a livello funzionale, mediante test in vitro, gli effetti osservati a livello molecolare. Studi di inibizione farmacologica, ci hanno permesso di capire che l’attività inibitoria dell’ATP sulla proliferazione si esplica attraverso l’attivazione del recettore P2X7, mentre i sottotipi recettoriali P2 prevalentemente coinvolti nella regolazione della migrazione sono i recettori P2Y2 e P2Y4. Esperimenti di xenotrapianto, hanno evidenziato che l’esposizione ad ATP e UTP sia dei blasti leucemici sia delle cellule staminali leucemiche CD38-CD34+ diminuisce la loro capacità di homing e di engraftment in vivo. Inoltre, il trattamento farmacologico con ATP, di topi ai quali è stata indotta una leucemia umana, ha diminuito lo sviluppo della leucemia in vivo. / Extracellular adenosine-triphosphate (ATP) and uridine-triphosphate (UTP) nucleotides increase the proliferation and engraftment potential of normal human hematopoietic stem cells via the engagement of purinergic receptors (P2Rs). Here, we show that ATP or UTP have strikingly opposite effects on human acute myeloblastic leukemia (AML) cells. Leukemic cells express P2Rs. ATP-stimulated leukemic cells, but not normal CD34+ cells, undergo down-regulation of genes involved in cell proliferation and migration whereas cell cycle inhibitors are up-regulated. Functionally, ATP induced the inhibition of proliferation and accumulation of AML cells, but not of normal cells, in the G0 phase of cell cycle. Exposure to ATP or UTP inhibited AML cell migration in vitro. In vivo, xenotransplant experiments demonstrated that the homing and the engraftment capacity of AML blasts and CD34+CD38- cells to immunodeficient mice bone marrow was significantly inhibited by pre-treatment with nucleotides. P2Rs expression analysis and pharmacological profile suggest that the inhibition of proliferation by ATP was mediated by the down-regulation of the P2X7R, which is up-regulated on untreated blasts, while the inhibition of chemotaxis was mainly mediated via P2Y2R and P2Y4R subtypes. Thus, conversely to normal cells, P2Rs signaling inhibits leukemic cells and its pharmacologic modulation may represent a novel therapeutic strategy.

The existence of Multiple Myeloma Stem cells (MMSCs)is supposed to be one of the major causes of MM drug-resistance. However, very little is known about the molecular characteristics of MMSCs, even if some studies suggested that these cells resembles the memory B cells. In order to molecularly characterize MMSCs, we isolated the 138+138- population. For each cell fraction we performed a VDJ rearrangement analysis. The complete set of aberrations were performed by SNP Array 6.0 and HG-U133 Plus 2.0 microarray analyses (Affymetrix). The VDJ rearrangement analyses confirmed the clonal relationship between the 138+ clone and the immature clone. Both BM and PBL 138+ clones showed exactly the same genomic macroalterations. In the BM and PBL 138-19+27+ cell fractions several micro-alterations (range: 1-350 Kb) unique of the memory B cells clone were highlighted. Any micro-alterations detected were located out of any genomic variants region and are presumably associated to the MM pathogenesis, as confirmed by the presence of KRAS, WWOX and XIAP genes among the amplified regions. To get insight into the biology of the clonotypic B cell population, we compared the gene expression profile of 8 MM B cells samples 5 donor B cells vs, thus showing a differential expression of 11480 probes (p-value: <0,05). Among the self-renewal mechanisms, we observed the down-regulation of Hedgehog pathway and the iperactivation of Notch and Wnt signaling. Moreover, these immature cells showed a particular phenotype correlated to resistance to proteasome inhibitors (IRE1α-XBP1: -18.0; -19.96. P<0,05). Data suggested that the MM 138+ clone might resume the end of the complex process of myelomagenesis, whereas the memory B cells have some intriguing micro-alterations and a specific transcriptional program, supporting the idea that these post germinal center cells might be involved in the transforming event that originate and sustain the neoplastic clone.

The human p53 tumor suppressor, known as the “guardian of the genome”, is one of the most important molecules in human cancers. One mechanism for suppressing p53 uses its negative regulator, MDM2, which modulates p53 by binding directly to and decreasing p53 stability. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph-) leukemic cell line models, and primary B-Acute lymphoblastic leukemia (ALL) patient samples. In this study we demonstrated that treatment with Nutlin-3a induced grow arrest and apoptosis mediated by p53 pathway in ALL cells with wild-type p53, in time and dose-dependent manner. Consequently, MDM2 inhibitor caused an increase of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from Ph+ ALL patients with the T315I Bcr-Abl kinase domain mutation. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis in sensitive cell lines was performed. A total of 621 genes were differentially expressed (p < 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change -1.35 and -1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21. Given the importance of BMI in the control of apoptosis, we investigated its pattern in treated and untreated cells, confirming a marked decrease after exposure to MDM2 inhibitor in ALL cells. Noteworthy, the BMI-1 levels remained constant in resistant cells. Therefore, BMI-1 may be used as a biomarker of response. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph-ALL.

In chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL kinase domain mutation status is an essential component of the therapeutic decision algorithm. The recent development of Ultra-Deep Sequencing approach (UDS) has opened the way to a more accurate characterization of the mutant clones surviving TKIs conjugating assay sensitivity and throughput. We decided to set-up and validated an UDS-based for BCR-ABL KD mutation screening in order to i) resolve qualitatively and quantitatively the complexity and the clonal structure of mutated populations surviving TKIs, ii) study the dynamic of expansion of mutated clones in relation to TKIs therapy, iii) assess whether UDS may allow more sensitive detection of emerging clones, harboring critical 2GTKIs-resistant mutations predicting for an impending relapse, earlier than SS. UDS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II (Interlaboratory Robustness of Next-Generation Sequencing) International consortium.Samples from CML and Ph+ ALL patients who had developed resistance to one or multiple TKIs and collected at regular time-points during treatment were selected for this study. Our results indicate the technical feasibility, accuracy and robustness of our UDS-based BCR-ABL KD mutation screening approach. UDS was found to provide a more accurate picture of BCR-ABL KD mutation status, both in terms of presence/absence of mutations and in terms of clonal complexity and showed that BCR-ABL KD mutations detected by SS are only the “tip of iceberg”. In addition UDS may reliably pick 2GTKIs-resistant mutations earlier than SS in a significantly greater proportion of patients.The enhanced sensitivity as well as the possibility to identify low level mutations point the UDS-based approach as an ideal alternative to conventional sequencing for BCR-ABL KD mutation screening in TKIs-resistant Ph+ leukemia patients

Backgrounds:Treatment of patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) not eligible to high dose therapy represents an unmet medical need. Panobinostat showed encouraging therapeutic activity in studies conducted in lymphoma cell lines and in vivo in patients with advanced hematologic malignancies.Purpose:FIL-PanAL10 (NCT01523834) is a phase II, prospective multicenter trial of the Fondazione Italiana Linfomi (FIL) to evaluate safety and efficacy of single agent Panobinostat as salvage therapy for R/R DLBCL patients and to evaluate a possible relationships between response and any biological features. Patients and Methods:Patients with R/R DLBCL were included. The treatment plan included 6 induction courses with Panobinostat monotherapy followed by other 6 courses of consolidation. The primary objective was to evaluate Panobinostat activity in terms of overall response (OR); secondary objectives were: CR rate, time to response (TTR), progression-free survival (PFS), safety and feasibility of Panobinostat. We included evaluation of the impact of pharmacogenetics, immunohistochemical patterns and patient’s specific gene expression and mutations as potential predictors of response to Panobinostat as explorative objectives. To this aim a pre-enrollment new tissue biopsy was mandatory. ResultsThirty-five patients, 21 males (60%), were enrolled between June 2011 and March 2014. At the end of induction phase, 7 responses (20%) were observed, including 4 CR (11%), while 28 patients (80%) discontinued treatment due to progressive disease (PD) in 21 (60%) or adverse events in 7 (20%). Median TTR in 9 responders was 2.6 months (range 1.8-12). With a median follow up of 6 months (range 1-34), the estimated 12 months PFS and OS were 27% and 30.5%, respectively. Grade 3-4 thrombocytopenia and neutropenia were the most common toxicities (in 29 (83%) and 12 (34%) patients, respectively. Conclusions The results of this study indicate that Panobinostat might be remarkably active in some patients with R/R DLBCL, showing durable CR

L’enzima IDO interviene nella via di degradazione del triptofano, essenziale per la vita cellulare; l’iperespressione di IDO favorisce la creazione di un microambiente immunotollerante. Nelle LAM IDO è funzionalmente attivo nelle cellule blastiche e determina l’acquisizione di un fenotipo regolatorio da parte delle cellule T alloreattive; l’espressione della proteina aumenta in modo consensuale con l’evoluzione clinica della patologia. Scopo della Tesi è indagare l’esistenza di una correlazione tra l’iperespressione di IDO da parte delle cellule leucemiche, le caratteristiche di rischio alla diagnosi e l’outcome dei pazienti. Sono stati esaminati 45 pazienti adulti affetti da LAM afferiti all’Istituto di Ematologia di Bologna. I pazienti sono stati stratificati a seconda di: età di insorgenza della leucemia, secondarietà a Mielodisplasia o radio chemioterapia, iperleucocitosi, citogenetica, biologia molecolare (sono state valutate le alterazioni a carico dei geni FLT3 ed NPM). I pazienti sono stati analizzati per l’espressione del gene IDO mediante RT-PCR, seguita da Western Blot, allo scopo di stabilire la presenza di una proteina attiva; successivamente si è proceduto a verificare l’esistenza di una correlazione tra l’espressione di IDO e le caratteristiche di rischio alla diagnosi per identificare una relazione tra l’espressione del gene ed un subset di pazienti a prognosi favorevole o sfavorevole. Dei 45 pazienti adulti affetti da LAM il 28,9% è risultato negativo per l’espressione di IDO, mentre il rimanente 71,1% è risultato positivo ed è stato suddiviso in tre ulteriori categorie, in base ai livelli di espressione. I dati non sembrano al momento suggerire l’esistenza di una correlazione tra l’espressione di IDO e le caratteristiche di rischio alla diagnosi. Nel gruppo di pazienti ad elevata espressione di IDO si riscontra un rate di resistenza alla chemioterapia di induzione più elevato, con una quota di pazienti resistenti pari al 71,4%, contro il 23,1% nel gruppo di pazienti IDO-negativi. / Tryptophan is an essential amino-acid required for protein synthesis and for cell proliferation. IDO is an eme-containing enzyme that catalyzes the rate-limiting step in tryptophan degradation along the kynurenine pathway and its overexpression is known to be involved in immune tolerance induction. IDO is functionally active in Acute Myeloid Leukemia tumor cells and it acts by inducing the transformation of T-cells into regulatory T-cells. Several observations demonstrated that IDO expression increases along with disease progression. The object of the work is to establish a correlation between IDO expression by leukemic cells, risk factors at diagnosis and patients’ outcome. Adult AML patients from the Hematology Institute “L. and A. Seràgnoli” in Bologna were analyzed for risk characteristics at diagnosis and for IDO expression. Patients were stratified according to: age at diagnosis, de novo or secondary disease (pre-existing myelodysplastic syndrome or radio-chemotherapy), hyperleucocytosis, cytogenetics (on the basis of cytogenetic characteristics patients were divided into low, intermediate or high risk group), molecular biology (the presence of FLT3 and NPM alterations was studied). These patients were analyzed for IDO expression by RT-PCR and the presence of a functionally active IDO protein was established by Western Blotting analysis. Eventually the correlation between IDO expression, risk factors at diagnosis and patients’ outcome was evaluated. Thirteen out of the 45 analyzed patients (28,9%) resulted to be IDO-negative, 71,1% being IDO-positive. Positive patients were divided into three different subgroups according to the IDO level. No significant differences in the recurrence of prognostic characteristics at diagnosis between IDO-negative and IDO-positive patients were experienced. Intriguingly, it has been found that refractory patients were 71,4% among patients who express IDO at high level and 23,1% among IDO-negative patients.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Enfermagem. / Made available in DSpace on 2012-10-23T04:35:56Z (GMT). No. of bitstreams: 1 239438.pdf: 2841814 bytes, checksum: 6b05267aeb3ee43f4336fe45221ce552 (MD5) / Trata-se de um estudo realizado em um hemocentro do Estado de Santa Catarina, no qual abordamos o tema Triagem Clínica de Doadores de Sangue, a partir de algumas inquietações pessoais referentes a esta prática profissional. Teve como objetivo geral: conhecer o processo da triagem clínica deste hemocentro, a partir da percepção dos doadores e enfermeiras triagistas, a fim de refletir acerca da triagem clínica como um espaço de cuidado e educação para a doação de sangue. Optamos por uma pesquisa qualitativa, utilizando para coleta de dados a entrevista semi-estruturada aplicada a dez doadores e seis enfermeiras triagistas. Para análise dos dados, utilizamos a Análise de Conteúdo de Bardin, a partir da qual surgiram as seguintes categorias analíticas: - Doação de sangue como um ato de solidariedade; - O significado da triagem clínica de doadores de sangue; - Facilidades, dificuldades e alternativas para o processo de cuidar e educar o doador de sangue. Os dados apontam que os doadores e enfermeiras percebem que a triagem clínica é importante etapa para o processo da doação, a qual assegura a qualidade do sangue para o receptor e visa a proteção do doador. Os doadores sentem-se cuidados e educados pelas enfermeiras. As enfermeiras afirmam que o processo de cuidar e educar o doador já acontece, porém não de um modo integral e equânime, atribuindo a vários fatores colocados na categoria das dificuldades; referem serem necessários momentos de reflexão para repensar a prática da triagem clínica, pois salientam que esta caracteriza-se em uma consulta de enfermagem e não é percebida como tal e que realmente deve ser um momento de cuidar e educar o doador. Atualmente a educação ao doador acontece aos moldes de uma educação tradicional. A triagem clínica enquanto uma consulta de enfermagem é realizada apenas com caráter investigativo, porém deve favorecer a interação doador e enfermeira, propiciar a relação dialógica horizontal com acolhimento, escuta sensível e respeito, deste modo possibilitando o cuidado e educação em saúde aos moldes de uma educação problematizadora, onde haja troca de saberes e crescimento mútuo, valorizando sempre o doador enquanto "ser humano" e não como "repositor de sangue". É necessário um trabalho perseverante, sensibilizando todos os profissionais para possibilitar esta mudança, a qual repercutirá na conscientização dos doadores de sangue como protagonistas do processo, bem como a importância da doação como ato de solidariedade e cidadania. This is about a study carried through in one Blood bank of Santa Catarina State, in which we approach the subject Clinical Selection of Blood Givers, from some referring personal fidgets to this professional practice. Having as general objective,in order to reflect about the clinical selection as a space of care and education for the blood donation, one needs to know the process of the clinical selection of this hemocenter, from the perception of the givers of blood and selector nurses. We opt to a qualitative research, using for data collection the interview half-structuralized applied to ten givers and six selector nurses. We also pcked up some data collected from a reflection moment of a clinical selection experience from nurses before this research. For analysis of the data, we use the Analysis of Content of Bardin, from where the following analytical categories had appeared:- Donation of blood as a solidarity act;- The meaning of the clinical selection of blood givers;-Facilities, difficulties and alternatives for the process to take care of and to educate the blood givers. The data point that the gives and nurses perceive that the clinical selection is a important stage for the process of donation, which assure the quality of the blood for the receiver and aim the protection of the giver. The givers felt well-taken care and educated by nurses. The nurses declare that process of taking care and educate the giver already happens, however not as an integral and impartial way, cause by several factors, they relate to be necessary moments of reflection rethink about this practical, therefore they point out that the selection is characterized in a nursing consultation and is not perceived as such and that the selection must be a moment to take care of and really to educate the giver. Now a days the education to the giver happens as the molds of a traditional education. The clinical selection, while a nursing consultation, is carried through only as a investigable character, however is must help the interaction between giver and nurse, to propitiate the horizontal dialogic relation with shelter, sensible listening and respect, in this way making possible the care of and education in health in molds of a problematical education, where is has exchange of knowledge and mutual growth, valuing always the giver while "human being" and not as "repositor of blood". It is necessary a persistent work, sensitizing all the professionals to make possible this change, which will re-echo in the awareness of the blood givers as protagonists of the process, as well as the importance of the donation as act of solidarity and citizenship.

Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2013-04-23T11:23:46Z No. of bitstreams: 1 Macedo C.pdf: 387637 bytes, checksum: 6df0bb17d013bb905008e47b9c3ca31b (MD5) / Made available in DSpace on 2013-04-23T11:23:46Z (GMT). No. of bitstreams: 1 Macedo C.pdf: 387637 bytes, checksum: 6df0bb17d013bb905008e47b9c3ca31b (MD5) Previous issue date: 1996 / No presente trabalho desenvolveu-se uma nova metodologia analítica para a determinação de chumbo em sangue através da espectrofotometria molecular. O método utiliza o quelante 2-(5-bromo-2-piridilazo)-5-dietilamonofenol (5-Br-PADAP). A composição da solução final de medida é a seguinte: 5-Br-PADAP 1,2x10-4 M; álcool etílico 50 % em volume; Triton X-100 0,15 %; ácido nítrico 0,5 M e hidróxido de amônia 0,5 M. As medidas espectrofotométricas foram realizadas a 575 nm, com abertura de fenda de 1 nm e cubeta com caminho ótico de 1 cm. A eliminação preliminar de interferências de metais foi obtida utilizando-se uma resina aniônica quaternária fortemente básica (AG1-X8, 200-400 mesh) empregando uma mistura a 90 % de metanol e 10 % v/v de ácido nítrico 5 N, em coluna de 3 cm de comprimento e diâmetro interno de 0,5 cm. O fluxo de eluição dos metais interferentes foi 0,5-0,6 mL/min e do chumbo, com ácido nítrico 1 N, 0,2 mL/min. As características analíticas da metodologia são: limite de quantificação (L.Q.) em 500 µL de amostra de sangue, 112 ppb; exatidão ao nível de 250 ppb ³ 96,5 %, as quais permitem uma ampla aplicação a estudos epidemiológicos de populações e controle de exposição profissional. / Salvador