Volumetric dried blood microsampling for monitoring imatinib mesylate therapy: Method development and clinical application in patients with chronic myeloid leukemia.

Chronic Myeloid Leukemia (CML) is a hematologic neoplasia, characterized as a proliferative disease of the hematopoietic system. Imatinib mesylate (IM), a selective tyrosine kinase inhibitor, is considered a first-line therapy for CML, indicated for both adult and pediatric patients presenting the Philadelphia chromosome (Ph+). However, patients in treatment with IM may show different responses due to interindividual pharmacokinetic variability. Therapeutic drug monitoring should be routinely performed to identify treatment response profile, adherence to treatment, or possible drug interactions, thus supporting better treatment management. Volumetric absorptive microsampling (VAMS) are innovative devices for blood collection whose advantages include the possibility of home collection by the patient or at the physician's office. The assay was fully validated according to bioanalytical validation guidelines. Estimated plasma concentrations of IM were not statistically different between groups according to adherence (p = 0.15), with median of 789 ng ml-1 in the group with some level of non-adherence versus 1141.9 ng ml-1 in the group with adherence, classified with the Morisky-Green questionnaire. This study included 33 patients with CML in treatment with IM. These patients answered socioeconomic, sociodemographic, and adherence profile (Morisky-Green) questionnaires. Patients also received instructions for home blood collection with VAMS devices. Afterwards, the samples were analyzed by LC-MS/MS. The mean age of the patients was 52 years, 84.8% were ingesting doses of 400 mg/day and the majority were male (69.7%). IM and its metabolite NIM were extracted from VAMS with an aqueous solution with 0.1% formic acid, followed by protein precipitation with acetonitrile. The methodology developed in this study was satisfactory for the determination of IM and NIM in VAMS and can be used in hospital and office routines for the therapeutic monitoring of patients with CML.

Quantification of peripheral blood CD34+ cells prior to stem cell harvesting by leukapheresis: a single center experience.

BACKGROUND: Due to laboratory logistic issues, our center has traditionally scheduled peripheral blood stem cell harvests based on timing from the start of mobilization. This has proved to be useful in some cases, but also resulted in many fruitless harvests due to poor mobilization. In order to improve the efficiency of collections and compare the effectiveness of peripheral blood CD34+ cells as a predictor with data from other reports, this study analyzed the implementation of this routine. METHODS: Peripheral blood and leukapheresis samples were quantified by flow cytometry and the association between these parameters was assessed. RESULTS: Sixty-six consecutive leukapheresis samples were collected from 34 patients after the collection of peripheral blood samples for CD34+ quantification. A moderate positive correlation was observed between peripheral blood CD34+ cell count and total CD34+ cell count/kg (r = 0.596; p-value < 0.001). A multivariable regression model also confirmed this association and allowed the estimation that for every increase in five CD34+ cells/µL in the peripheral blood, a mean increase of 0.38 × 106 CD34+ cells/kg could be predicted. Demographic characteristics, baseline comorbidities and mobilization regimen did not influence final CD34+ cell count in this sample. CONCLUSIONS: As observed in other centers, quantification of peripheral blood CD34+ progenitor cells is a strong predictor of effectiveness to guide stem cell harvesting. Due to the results of this study, a modification in the peripheral blood stem cell harvesting logistics was implemented at our center in order to incorporate this routine.

Do Alpha Thalassemia, Fetal Hemoglobin, and the UGT1A1 Polymorphism have an Influence on Serum Bilirubin Levels and Cholelithiasis in Patients with Sickle Cell Disease?

BACKGROUND: Increased destruction of erythrocytes in patients with sickle cell disease results in chronic hyperbilirubinemia and leads to the formation of gallstones. OBJECTIVES: The objective of this study was to determine the combined influence of alpha thalassemia, fetal hemoglobin, and the UGT1A1 polymorphism on serum bilirubin levels and cholelithiasis in patients with sickle cell disease. METHODS: We analyzed 72 patients treated in the outpatient hematology unit of the Clinical Hospital of Porto Alegre. The alpha thalassemia trait was determined by multiplex polymerase chain reaction and the polymorphisms of UGT1A1 by capillary electrophoresis with tagged primers. RESULTS: Total and indirect bilirubin levels differed significantly between genotypes TA7/TA7 and TA6/TA6 (p < 0.05). Bilirubin levels were influenced by the UGT1A1 polymorphism but not by alpha thalassemia and fetal hemoglobin. There was no association between cholelithiasis and any of the variables studied. CONCLUSION: These preliminary findings suggest that the UGT1A1 gene can influence serum bilirubin levels in sickle cell anemia and serve as a tool to differentiate an acute hemolytic condition from a pre-existing condition of hyperbilirubinemia.

Association of Hb Shelby with Hb S in the south of Brazil.

We, herein, report the first observation of compound heterozygosity for hemoglobin Shelby and hemoglobin S identified in the south of Brazil. The variant hemoglobin was identified by isoelectric focusing (IEF), high performance liquid chromatography (HPLC) and DNA sequence analysis.

First report of imatinib measurement in hair: Method development and preliminary evaluation of the relation between hair and plasma concentrations with therapeutic response in chronic myeloid leukemia.

BACKGROUND: Imatinib (IM) is a first choice drug for treatment of chronic myeloid leukemia (CML), with a widely accepted concentration threshold of 1000ng/ml being used as a target for therapeutic drug monitoring. Once adherence to the pharmacotherapeutic regimen is of paramount importance during the long treatment course of CML, the measurement of hair IM concentrations could be a surrogate of the patient's exposure to the drug. METHODS: IM was extracted from a 5mg hair sample by a liquid-liquid extraction with ethyl acetate, and IM-d8 was used as internal standard (IS). After evaporation, and reconstitution in acetonitrile, the extract was injected into a LC-MS/MS system. Compounds were eluted on a C8 column in isocratic mode. IM and IS were identified in positive electrospray ionization mode using ion transitions of m/z 494.5>394.5 and 503.0>394.3 respectively. The method was applied to 102 paired hair and samples obtained from CML patients. Treatment response was evaluated according to the European LeukemiaNet recommendations. RESULTS: The assay was validated in the concentration range of 0.5-25ng/mg, with intra- and inter-assay imprecisions of <13.1% and <9.3%, respectively. The limits of quantification and detection were 0.5 and 0.15ng/mg, respectively. Median hair IM concentrations are significantly smaller in patients with therapeutic failure when compared with patients with partial or optimal response (4.63 vs. 7.93, p=0.040), the same trend presented by median plasma IM concentrations (629.5 vs. 1084.8, p=0.009). An IM hair concentration below 5.8ng/mg has 83% sensibility and 70% specificity to identify patients with therapeutic failure. CONCLUSIONS: A fast, sensitive, and selective LC-MS/MS method allowing quantification of IM in hair samples was developed and validated. CML patients with therapeutic failure had significantly lower hair IM concentrations when compared with patients with optimal response. These preliminary findings may support the use of hair as a matrix for IM monitoring in clinical settings, with significant logistic advantages over the collection of venous blood, particularly in developing countries.

DBS sampling in imatinib therapeutic drug monitoring: from method development to clinical application.

BACKGROUND: Imatinib (IM) is widely used in treatment of chronic myeloid leukemia with target trough plasma concentrations above 1000 ng ml(-1). DBS can increase access to IM therapeutic drug monitoring. RESULTS: IM was measured in the range 50-4000 ng ml(-1) by UHPLC-MS/MS using one 6 mm DBS in a fully validated method. IM was stable at DBS maintained at 40°C for 36 days. Plasma and DBS concentrations were highly correlated (r > 0.96). The use of a IM concentration target of 765 ng ml(-1) in DBS identified 93% of patients with plasma concentration below 1000 ng ml(-1). CONCLUSION: IM can be measured in DBS using UHPLC-MS/MS with results comparable to those obtained in blood plasma.

Quantification of peripheral blood CD34+ cells prior to stem cell harvesting by leukapheresis: a single center experience

ABSTRACT Background: Due to laboratory logistic issues, our center has traditionally scheduled peripheral blood stem cell harvests based on timing from the start of mobilization. This has proved to be useful in some cases, but also resulted in many fruitless harvests due to poor mobilization. In order to improve the efficiency of collections and compare the effectiveness of peripheral blood CD34+ cells as a predictor with data from other reports, this study analyzed the implementation of this routine. Methods: Peripheral blood and leukapheresis samples were quantified by flow cytometry and the association between these parameters was assessed. Results: Sixty-six consecutive leukapheresis samples were collected from 34 patients after the collection of peripheral blood samples for CD34+ quantification. A moderate positive correlation was observed between peripheral blood CD34+ cell count and total CD34+ cell count/kg (r = 0.596; p-value < 0.001). A multivariable regression model also confirmed this association and allowed the estimation that for every increase in five CD34+ cells/µL in the peripheral blood, a mean increase of 0.38 × 106 CD34+ cells/kg could be predicted. Demographic characteristics, baseline comorbidities and mobilization regimen did not influence final CD34+ cell count in this sample. Conclusions: As observed in other centers, quantification of peripheral blood CD34+ progenitor cells is a strong predictor of effectiveness to guide stem cell harvesting. Due to the results of this study, a modification in the peripheral blood stem cell harvesting logistics was implemented at our center in order to incorporate this routine.

Prevalence of UGT1A1 gene polymorphism in patients with hemolytic anemia in southern Brazil.

The aim of the work was to determine the variation of UGT1A1 genotypes in patients with hemolytic anemia in the southern Brazil. Three hundred twenty-three patients with hemolytic anemia were genotyped for UGT1A1 along with 232 controls. Allelic and genotypic distribution did not differ among studied groups. The TA7/TA7 genotype presented a frequency that ranged from 3.2% to 18.0% (nonsignificant). Alleles TA5 and TA8 were also found in the sample, even though southern Brazil is a major Caucasoid region. Genotype prevalence was very similar to those of African origins, reflecting the diversity of ethnic origins and the high degree of admixture in southern Brazil. Further studies should be conducted to correlate the modulating role of UGT1A1 polymorphism with the clinical conditions of each patient with hemolytic anemia.

Monitoring imatinib plasma concentrations in chronic myeloid leukemia.

Imatinib has proved to be effective in the treatment of chronic myeloid leukemia, but plasma levels above 1,000 ng/mL must be achieved to optimize activity. Therapeutic drug monitoring of imatinib is useful for patients that do not present clinical response. There are several analytical methods to measure imatinib in biosamples, which are mainly based on liquid chromatography with mass spectrometric or diode array spectrophotometric detection. The former is preferred due to its lower cost and wider availability. The present manuscript presents a review of the clinical and analytical aspects of the therapeutic drug monitoring of imatinib in the treatment of chronic myeloid leukemia. The review includes references published over the last 10 years. There is evidence that the monitoring of plasmatic levels of imatinib is an useful alternative, especially considering the wide pharmacokinetic variability of this drug.

Prevalence of hepatitis C in patients with renal disease undergoing hemodialysis treatment / Prevalência de hepatite C em pacientes com doença renal submetidos a tratamento hemodialítico

Introduction and Objective: This study aimed at determining the prevalence of hepatitis C among 649 patients diagnosed with chronic or acute kidney disease − patients were undergoing hemodialysis treatment at a large hemodialysis center in Porto Alegre-RS, from January through December, 2012 –, as well as relating our data to that presented in the national census, reporting cases of coinfection by hepatitis C and human immunodeficiency virus (HIV), and defining the demographic profile of these patients. Method: An observational cross-sectional study was conducted and data was obtained from information in patients’ electronic medical records. Result and conclusion: The prevalence of hepatitis C in this study was 10.17% of the sampled population. However, further analysis of other liver centers would be required to estimate an accurate prevalence rate of infection caused by the hepatitis C virus in patients undergoing hemodialysis in Porto Alegre. .

Introdução e objetivo: Este trabalho teve como objetivo determinar a prevalência de hepatite C em 649 pacientes diagnosticados com doença renal aguda ou crônica − eles se submeteram a tratamento hemodialítico em uma unidade de hemodiálise de um hospital de grande porte de Porto Alegre-RS, de janeiro a dezembro de 2012 −, bem como relacionar os dados encontrados com os apresentados no censo nacional, relatar casos de coinfecção de hepatite C e vírus da imunodeficiência humana (HIV) e, finalmente, conhecer o perfil demográfico dos pacientes. Método: Realizou-se um estudo observacional do tipo transversal cujos dados foram obtidos por meio de informações dos prontuários eletrônicos dos pacientes. Resultado e Conclusão: A prevalência de hepatite C encontrada no presente estudo foi de 10,17% da população amostrada. Entretanto, seriam necessárias mais análises em outros centros a fim de estimar a real prevalência para infecção pelo vírus da hepatite C em pacientes submetidos a hemodiálise em Porto Alegre. .

Individualização farmacocinética das doses de 5-Fluoruracil nocâncer colorretal / Pharmacokinetic Individualization of 5-Fluorouracil Doses in Colorectal Cancer / Individualización Farmacocinética de las Dosis de 5-Fluorouracil en el Cáncer Colorrectal

Introdução: O 5-Fluoruracil (5-FU) é um antimetabólito amplamente utilizado no tratamento do câncer colorretal,com significativa variabilidade na resposta terapêutica e na ocorrência de toxicidade associada à sua farmacocinética variável. Objetivo: Revisar os aspectos clínicos e laboratoriais da individualização farmacocinética dos tratamentos com5-FU em pacientes com câncer colorretal. Método: Realizou-se uma revisão de literatura nas bases de dados PubMed, Periódicos Capes, SciElo, Medline e Bireme. Foram incluídos estudos publicados a partir de 1997, com as palavraschave: 5-Fluoruracil; Individualização farmacocinética; Câncer colorretal; Monitoramento terapêutico de fármacos. Resultados: Existem evidências de uma relação entre a exposição sistêmica ao 5-FU e as respostas terapêuticas no tratamento, sendo que áreas sob a curva concentração-tempo entre 20 a 25 mg. h/L permitem um adequado balançoentre maximização da eficácia e redução da toxicidade. Também é possível avaliar a atividade da enzima diidropirimidina desidrogenase (DPD) previamente ao tratamento com 5-FU através de métodos fenotípicos, identificando os pacientesem risco de toxicidade grave. Métodos analíticos baseados em cromatografia líquida de alta eficiência estão disponíveis para determinar 5-FU em plasma de forma confiável, permitindo a disseminação do monitoramento terapêutico desse fármaco. Conclusão: Através da individualização farmacocinética é possível desenvolver regimes terapêuticos de 5-FUpersonalizados no câncer colorretal, com impactos significativos nos resultados terapêuticos.

Monitoring imatinib plasma concentrations in chronic myeloid leukemia

Imatinib has proved to be effective in the treatment of chronic myeloid leukemia, but plasma levels above 1,000 ng/mL must be achieved to optimize activity. Therapeutic drug monitoring of imatinib is useful for patients that do not present clinical response. There are several analytical methods to measure imatinib in biosamples, which are mainly based on liquid chromatography with mass spectrometric or diode array spectrophotometric detection. The former is preferred due to its lower cost and wider availability. The present manuscript presents a review of the clinical and analytical aspects of the therapeutic drug monitoring of imatinib in the treatment of chronic myeloid leukemia. The review includes references published over the last 10 years. There is evidence that the monitoring of plasmatic levels of imatinib is an useful alternative, especially considering the wide pharmacokinetic variability of this drug.

Standardization of method for determining glycosylated hemoglobin (Hb A1c) by cation exchange high performance liquid chromatography / Padronização de método para determinação de hemoglobina glicosilada (Hb A1c) por troca de cátions de alto desempenho cromatografia líquida

Hemoblobin A1c is the most important parameter for the monitoring of metabolic control of patients with diabetes mellitus. The purpose of this study was to adapt the Mono S method to a conventional HPLC system, allowing highly selective HbA1c determination without the acquisition of kits or the use of dedicated systems The results obtained were compared to the Tinaquant® immune turbidimetric method and the Bio-Rad Variant® chromatographic method. The developed method presented intra-study precision (C.V. percent) of 1.39-3.65 and inter-study precision (C.V. percent) of 2.80-3.02 percent. The determination coefficients among methods were: HPLC Mono S x Tinaquant®: r²: 0.9856 (n=60) and HPLC Mono S x HPLC Bio-Rad Variant®: r²: 0.9806 (n=16). A conversion equation between HPLC Mono S and Bio-Rad Variant® was calculated allowing yielding comparable and interchangeable values. The HPLC Mono-S is a precise, low-cost method which yields similar values to the Bio-Rad Variant® method on conventional HPLC equipment.

A hemoglobina A1c é o parâmetro laboratorial mais importante no monitoramento do controle metabólico de pacientes portadores de diabetes melito. Dentre as metodologias existentes para a quantificação desta fração de hemoglobina, a cromatografia líquida de alta eficiência (CLAE) baseada em troca catiônica apresenta a melhor precisão, sendo o método de escolha. O objetivo deste trabalho foi adaptar o método Mono S a um sistema de CLAE convencional permitindo a disponibilidade da determinação altamente seletiva de Hb A1c sem a aquisição de kits e comparar os resultados obtidos com o método imunoturbidimétrico Tinaquant® (Roche®) e com o método de cromatografia líquida Bio-Rad Variant®. O método desenvolvido apresentou precisão intra-ensaio de 1,39-3,65 por cento e inter-ensaio de 2,80-3,02 por cento. Os coeficientes de determinação entre os métodos foram: CLAE Mono S x Tinaquant®: r² = 0,9856 (n=60) e CLAE Mono S x Bio-Rad Variant®: r² = 0,9806 (n=16). Não foram observadas diferenças entre CLAE Mono S e Bio-Rad Variant® através de gráfico de Bland-Altman e regressão de Passing-Bablok. Foi obtida uma equação de conversão entre os valores do método teste e os valores obtidos por métodos rastreáveis ao National Glycohemoglobin Standardization Program (NGSP), permitindo a obtenção de valores comparáveis e intercambiáveis entre as metodologias com o uso de instrumentos convencionais de CLAE e com custo reduzido.

Avaliação laboratorial da doença residual mínima na leucemia mielóide crônica por Real-Time PCR: [revisão] / Evaluation diagnosis of minimal residual disease in chronic myeloid leukemia by Real-Time PCR: [review]

A leucemia mielóide crônica (LMC) representa 15 por cento das leucemias e apresenta três fases: crônica, acelerada e crise blástica. A partir da análise citogenética, pode ser identificado o cromossomo Philadelphia, característico da LMC. O transplante de células-tronco é o único tratamento curativo, mas é acompanhado de altas taxas de morbimortalidade, dificultando sua aplicação. A doença residual mínima é de grande importância para avaliar a resposta ao tratamento, tanto na verificação de doença residual, quanto na identificação de pacientes com alto risco de recaída. Muitas técnicas específicas têm sido introduzidas para detectar as translocações ou os produtos do cromossomo Philadelphia. A mais sensível é a Real-Time PCR, que detecta uma célula leucêmica em 10(5) células normais. O objetivo deste trabalho foi realizar uma revisão bibliográfica sobre a LMC, dando ênfase à utilização da técnica por Real-Time PCR.

Chronic myeloid leukemia (CML) represents about 15 percent of all leukemias and has three phases: the chronic phase, accelerated phase and blast crisis. After cytogenetic analysis, the Philadelphia chromosome, characteristic of CML, can be identificated. Stem cell transplantation is the only curative treatment for CML, but it is accompanied by high levels of morbimortality, difficulting its application. The minimal residual disease is very important for the evaluation of the response to treatment, to verify the residual disease and also to identify patients with a high risk of relapse. Many specific techniques have been introduced for the detection of translocations or products of the Philadelphia chromosome; the most sensitive being Real-Time PCR which detects 1 leukemia cell in 10(5) normal cells. The aim of this study was to perform a bibliographic review of CML, with emphasis on the utilization of the Real-Time PCR technique.