Validation of the single-platform ISHAGE protocol for enumeration of CD34+ hematopoietic stem cells in umbilical cord blood in a Brazilian center

Abstract Background This study aims to validate the single-platform method for enumeration of CD34+ cells, by comparing the performance of two different commercial kits, as well as to evaluate the efficiency of the AccuriTM C6 cytometer in providing direct counts of absolute cell numbers. Method We evaluated 20 samples from umbilical cord blood (UCB), comparing the two different methodologies for enumeration of CD34+ cells: single and dual-platform. For the assessment of the single-platform, Procount and SCE kits were used, both of which use fluorescent beads as a counting reference to obtain absolute CD34+ cells numbers. Moreover, after the acquisition of samples in flow cytometer AccuriTM C6, following the protocol established for each kit, the number of CD34+ cells was recalculated, considering the cell count provided by the AccuriTM C6. Main Results In our analysis, the results showed a strong correlation between the number of CD34+ cells/μL (r2 = 0.77) when comparing the SCE kit and the current dual-platform method. On the other hand, the comparison between Procount kit and dual-platform results showed a moderate correlation for the number of CD34+/μL cells (r2 = 0.64). Conclusion Our results showed that the AccuriTM C6 flow cytometer can be used safely, applying both the dual and single platform analysis strategy. Considering the ISHAGE protocol-based single-platform approach, as the most appropriate methodology for CD34+ cells enumeration, our results demonstrated that the SCE kit has great potential for national standardization of UCB samples analysis methodology.

Nutritional status of patients submitted to transplantation of allogeneic hematopoietic stem cells: a retrospective study

Objective: This study aimed to describe and compare the nutritional status of adult patients submitted to allogeneic hematopoietic stem cell transplantation at two different time points (admission and discharge). Methods: A retrospective, descriptive and quantitative study was performed based on clinical, laboratory and nutritional data obtained from medical records of adult patients of both genders submitted to allogeneic hematopoietic stem cell transplantation in a bone marrow transplantation reference center in Rio de Janeiro in the period from 2010 to 2013. Statistical analysis was performed using the SPSS software (version 22.0). Results: Sixty-four patients were evaluated. The mean age was 42.1 ± 3.2 years and the most prevalent disease was acute myeloid leukemia (39%). There was a high prevalence of gastrointestinal symptoms including nausea (100%), vomiting (97%) and mucositis (93%). Between admission and discharge there was a significant decrease in the median weight (−2.5 kg; 71.5 vs. 68.75 kg; p-value < 0.001), body mass index (−0.9kg/m2; 24.8 vs. 24.4kg/m2; p-value < 0.001), and serum albumin levels (−0.2g/dL; 3.7 vs. 3.6g/dL; p-value = 0.024). The survival time after hematopoietic stem cell transplantation correlated negatively with C-reactive protein at discharge (CC = −0.72; p-value < 0.001) and positively with serum albumin levels (CC = 0.56; p-value = 0.004) and with high total protein level at discharge (CC = 0.53; p-value = 0.006). Conclusion: Our results suggest that patients submitted to allogeneic hematopoietic stem cell transplantation have compromised nutritional status during the hospital stay for transplantation...

Brazilian workshop model to train investigators in chronic graft-versus-host disease clinical trials according to the 2005-2006 National Institutes of Health recommendations

BACKGROUND: The lack of standardization of clinical diagnostic criteria, classification and severity scores of chronic graft-versus-host disease led the National Institutes of Health to propose consensus criteria for the purpose of clinical trials. METHODS: Here we describe a one-day workshop model conducted by the Chronic Graft-versus-Host Disease Brazil-Seattle Consortium Study Group to train investigators interested in participating in multicenter clinical trials in Brazil. Workshop participants included eight transplant physicians, one dermatologist, two dentists, three physical therapists and one psychologist from five institutions. Workshop participants evaluated nine patients with varying degrees of severity of mucocutaneous lesions and other manifestations of the disease followed by a training session to review and discuss the issues encountered with the evaluation and scoring of patients and in the methods used to evaluate grip strength and the 2-minute walk test. RESULTS: Most participants had difficulties in rating the percentage of each type of mucocutaneous lesion and thought 20 minutes was insufficient to evaluate and record the scores of each patient using the National Institutes of Health criteria and other cutaneous assessments. Several specific areas of difficulties encountered by the evaluators were: 1) determining the percentage of erythema in movable and non-movable sclerosis, 2) whether to score all cutaneous findings in a particular area or just the dominant lesion; 3) clarification of the definition of poikiloderma in chronic graft-versus-host disease; 4) discrepant interpretation of the mouth score and 5) clarification on the methodology used for the evaluation of grip strength and the 2-minute walk tests. CONCLUSIONS: Results of this workshop support the need to train investigators participating in clinical trials on chronic graft-versus-host disease.

A multicenter feasibility study of chronic graft-versus-host disease according to the National Institute of Health criteria: efforts to establish a Brazil-Seattle consortium as a platform for future collaboration in clinical trials

BACKGROUND: New criteria for the diagnosis and classification of chronic graft-versus-host disease were developed in 2005 for the purpose of clinical trials with a consensus sponsored by the National Institute of Health. OBJECTIVES: The aim of this study is to present the results of a multicenter pilot study performed by the Brazil-Seattle chronic graft-versus-host disease consortium to determine the feasibility of using these criteria in five Brazilian centers. METHODS: The study was performed after translation of the consensus criteria into Portuguese and training. A total of 34 patients with National Institute of Health chronic graft-versus-host disease were enrolled in the pilot study between June 2006 and May 2009. RESULTS: Of the 34 patients, 26 (76 percent) met the criteria of overlap syndrome and eight (24 percent) the classic subcategory. The overall severity of disease was moderate in 21 (62 percent) and severe in 13 (38 percent) patients. The median time from transplant to onset of chronic graft-versus-host disease was 5.9 months (Range: 3 - 16 months); the median time for the overlap syndrome subcategory was 5.9 months (Range: 3 - 10 months) and for the classic subcategory, it was 7.3 months (Range: 3 - 16 months). At a median follow up of 16.5 months (Range: 4 - 39 months), overall survival was 75 percent. CONCLUSIONS: It was feasible to use the National Institute of Health consensus criteria for the diagnosis and scoring of chronic graft-versus-host disease in a Brazilian prospective multicenter study. More importantly, a collaborative hematopoietic cell transplantation network was established in Brazil offering new opportunities for future clinical trials in chronic graft-versus-host disease and in other areas of research involving hematopoietic stem cell transplantation.

Análise da Capacidade do REDOME/RENACORD em suprir as necessidades dos pacientes registrados no REREME

Classical Hodgkin lymphoma (cHL) is characterized by a small number of neoplastic, Hodgkin and Reed-Sternberg (H-RS) cells in a background of non-neoplastic, mainly B and T cells. This tumor microenvironment has been considered to be a manifestation of host immune reactions to malignant cells. Since children and adults have difference in the constitution and functionality of the immune system, and pathogenic events differ in pediatric and adult cHL, it is likely that the tumor microenvironment in cHL may be distinct in the pediatric setting. In this study, we analyzed a series of pediatric cHL (100 cases) regarding the clinical and histological characteristics, as well as composition of the tumor microenvironment by CD3, CD4, C-maf, T-bet, FoxP3, CD8, Tia-1, Granzyme B (GrB) and CD20 immunostain and its prognostic impact. Proliferation index of neoplastic and benign infiltrating cells was determined by Ki67 immunostain. We determined also the IL10 -1082A/G, -819C/T and -592C/A genotype of single nucleotide polymorphisms (SNPs) and haplotypes by Allele-specific (AS) PCR and CTLA4 -1722 A/G, +49 A/G and CT60 A/G SNPs by allele discrimination with fluorogenic hydrolysis probes (Taqman® Applied Biosystems). Epstein-Barr virus (EBV) was determined by in situ hybridization (EBERs-ISH) and LMP1 immunostain. Age at diagnosis ranged from 3 to 18 years (median 14) with 27% of cases <10 years. Age and histologic subtype did not A prática clínica do transplante de células tronco hematopoéticas (TCTH) somente foi possível com a melhoria dos conhecimentos na área de imunologia e histocompatibilidade (HLA). Entre os fatores facilitadores está a obtenção de um doador HLA compatível que idealmente é um irmão, mas na maioria das vezes (70%) é um doador não aparentado. As alternativas viáveis para populações como a brasileira, com grande miscigenação, foram a expansão do Registro Brasileiro de Doadores de Medula Óssea – REDOME e da Rede de Bancos de Sangue de Cordão Umbilical e Placentário (SCUP) - BRASILCORD. O presente estudo teve por objetivo avaliar as frequências dos alelos e haplótipos de HLA identificados no REDOME, no Registro de Receptores – REREME e nas unidades de SCUP armazenadas no BRASILCORD e registradas no Registro Nacional de Sangue de Cordão - RENACORD. Trata-se de uma amostragem significativa da população brasileira, com cadastros oriundos de todas as regiões do país. As frequências de grupos alélicos HLA foram obtidas dos três bancos de dados e através do teste estatístico do qui-quadrado foram realizadas comparações das freqüências observadas entre os registros. Para determinação das frequências dos haplótipos utilizou-se o programa Arlequim 3.01. A constituição genética da população brasileira de doadores de medula óssea, calculada em janeiro de 2009 (887.616 doadores) foi composta de 21 grupos alélicos HLA-A*, 36 grupos alélicos HLA-B* e 13 grupos alélicos HLA-DRB1*. Esta constituição foi a mesma encontrada nos registros do REREME e RENACORD. A identificação de haplótipos presentes no cadastro do REREME e ausentes ou pouco frequentes no REDOME e no RENACORD assim como a análise da origem destes haplótipos mostrou a presença de haplótipos nunca descritos em outras populações. A análise dos alelos que compõem estes haplótipos mostra uma miscigenação entre grupos antropológicos distintos como asiáticos, negróides, indígenas e caucasianos. Os dados obtidos neste estudo contribuem para o estabelecimento dos critérios necessários para o planejamento estratégico de expansão do BRASILCORD e do próprio REDOME. Além desta contribuição, em função do número de indivíduos incluídos nos cadastros dos registros, trata-se da maior e mais diversificada fonte de informação sobre a constituição genética da população brasileira

Stem cell transplantation clinical practice was only possible with knowledge improvement in Immunology and Histocompatibility (HLA). Whereas transplants between HLA identical siblings ideally produce the best outcomes, the majority (70%) does not have one family donor and will depend on an unrelated donor. Possible alternatives for mixed populations as the Brazilian, include the expansion of the Volunteer Marrow Donor Brazilian Registry - REDOME and the Cord Blood Bank Network – BRASILCORD. The aim of the present study was to evaluate the HLA alleles and haplotypes frequencies in REDOME, REREME – Brazilian Receptor Registry and Brazilian Cord Blood Registry - RENACORD. This database can be considered a representative sample of the Brazilian population originated from all regions of the country. HLA alleles groups frequencies were obtained from the three Registries and were compared using qui square test. To determine haplotype frequencies, Arlequim 3,01 software was used. In this study we included, from 2004 till January 2009, 887616 donors and 21 HLA-A*, 36 HLA-B* and 13 HLA-DRB1* alleles groups were identified. Haplotypes existing in REREME database but absent or less frequent in REDOME and in RENACORD, as well as the analysis of haplotypes origins, revealed haplotypes identification never described before in other populations. The allele analysis of the haplotypes showed miscegenation among different anthropological groups such as Asians, Blacks, Indians and Caucasians. Data obtained in this study contributed to establish the necessary criteria for a strategic plan to expand BRASILCORD and REDOME as well. Moreover, considering the huge number of individuals evaluated, this study is one of the biggest and the most diverse source of information about the genetic profile of the Brazilian population.

Doença enxerto contra hospedeiro crônica em transplante de medula óssea: tratamento com psoraleno e ultravioleta A

A doença enxerto-contra-hospedeiro crônica (DECH-cr) é uma afecção de origem imunológica que ocorre como complicação tardia nos pacientes submetidos a transplante de medula óssea (TMO) alogênico, caracterizada na pele e mucosa por lesões do tipo liquenóide, vitiligóide e esclerodermóide. O tratamento consiste em imunossupressão com drogas isoladas ou combinadas, que controlam a doença porém levam a um aumento da mortalidade, principalmente relacionada às complicações infecciosas. A terapia com 8metoxipsoraleno e ultravioleta A (Puvaferapia) para o controle da DECH-cr resistente às drogas imunossupressoras, baseia-se na indução de alterações funcionais nos efetores da doença (Iinfócitos T e citoquinas). Devido a esta ação imunológica, este método tem sido utilizado no tratamento de afecções cutâneas como a psoríase, o vitiligo e o líquen plano, que são mimetizadas em suas manifestações clínicas e histopatológicas pela DECH-cr. Foram avaliados entre fevereiro de 1991 e maio de 1995, 149 pacientes submetidos a TMO alogênico no CEMO-HC-INCA desde outubro de 1984, quanto ao diagnóstico de DECH-cr: Quarenta e nove pacientes (32,88%) apresentavam DECH-cr, dos quais sete (14,3%) foram selecionados de acordo com os critérios de inclusão do protocolo de Puvaferapia. Um paciente se recusou a receber o tratamento e um paciente iniciou o tratamento após o término do estudo em maio de 1995. Quanto à classificação da DECH-cr, 2 pacientes apresentaram doença de Novo e 3 pacientes a forma progressiva. Quatro dos cinco pacientes desenvolveram DECH-cr extensa (incluindo lesões esclerodermóides) e 1 paciente doença limitada. Nesta casuística, o acometimento cutâneo-mucoso ocorreu nos 5 pacientes e o envolvimento de outros orgãos (doença sistêmica) esteve presente em 3 pacientes: 1 paciente com bronquiolite obliterante (pulmões); 3 pacientes com a sindrome ceratoconjuntivite seca ou S/CCA (olhos, mucosas, vias aéreas). A dose de psoraleno (8-MOP) utilizada foi de 0,6 mg/kg/vez administrada por via oral com alimentos, duas horas antes da irradiação com UVA, cuja dose inicial foi determinada pela dose eritematosa mínima e o tipo de pele. Os pacientes receberam irradiação sistêmica, não havendo exposição proposital da mucosa oral. Os cinco pacientes receberam 142, 228, 100, 150 e 64 aplicações de Puvaterapia com duração total de 38, 31, 40, 25 e 11 meses, respectivamente. Os principais efeitos colaterais imediatos foram: intolerância digestiva (náuseas, vômitos epigastralgia) em 03 pacientes e fotofobia em 01 paciente, controlados com medicação sintomática e orientação. Nenhum paciente desenvolveu tumores secundários ao tratamento, como complicação tardia. A imunossupressão por drogas foi reduzida em 01 paciente e suspensa em 04 pacientes pós-Puvaterapia...

Chronic graft-versus-host disease (c-GVHD) is an immunological disorder that occurs as a late effect in patients submitted to allogeneic bone marrow transplantation (BMT), caracterized by lichenoid, vitiligo-like and sclerodermatous lesions on mucosae and skin. Treatment consists of immunosuppressive drug combinations which control the disease, although leading to an increased mortality rate, mostly related to infectious complications. Therapy with 8-methoxypsoralen (8-MOP) and ultraviolet A (PUVA) to control drug resistant c-GVHD was based on the induction of functional changes in the effectors of the disease (T Iymphocytes and cytokines). Based on these immunological effects, it has been utilized to treat cutaneous disorders such as psoriasis, vitiligo and lichen planus. Chronic GVHD mimics these disorders, suggesting the use of Puvatherapy for disease control. From February 1991 to May 1995, 149 patients who underwent to allogeneic BMT in CEMO-HC-INCA since october 1984, were evaluated regarding c-GVHD diagnosis. Fourty-nine patients (32,88%) were c-GVHD affected and 7/49 (14,3%) were selected to receive Puvatherapy. One patient refused the treatment and another started the protocol after may 1995. Two patients were classified as de Novo c-GVHD and three had progressive disease. Extensive c-GVHD was diagnosed in 4/5 patients (including sclerodermatous lesions) and 1/5 patient had limited disease. In this study, cutaneous and mucosal involvement was presented in ali patients and 3 patients had systemic disease evidence as noted by obliterative bronchiolitis in 1 patient and S/CCA syndrome in 3 patients (mouth, sinuses, eyes, respiratory system). 8-MOP dose was 0.6mg/Kg given two hours before UVA irradiation. UVA initial dose was determined by minimal erytematous dose and skin type. Patients received systemic UVA irradiation, without oral mucosae direct exposure. Five patients received 142, 228, 100, 150 and 64 PUVA treatments, during 38, 31, 40, 25 and 11 months, respectively. Early side effects were: nauseas, vomiting and gastric intolerance in 03 patients and photofobia in 01 patient, that responded well to drugs and orientation. None presented with secondary cancer as a late si de effect. Immunosuppressive therapy could be reduced in 1 patient and discontinued in 4 post-puvatherapy. Four patients had complete response and one patient a partial response during PUVA treatment and maintenance. Vitiligo-like lesions had complete resolution in ali patients and scleroderma improved considerably in 3 patients. Evidence of systemic effect of Puvatherapy could be observed by: oral lesions healed in ali patients without direct exposure, significant improvement in a patient with obliterative bronchiolitis and in 3 patients with S/CCA symptoms...

Experiência do Centro Nacional de Transplante de medula óssea (CEMO) com uso de cateteres tipo Heckman-Broviac / Experience of the National Center of Bone Marrow Transplantation - CEMO with the use of Hickman-Broviac catheters

Quarenta e sete pacientes foram admitidos no Centro Nacional de Transplante de Medula Ossea - CEMO - do Rio de Janeiro, tendo sido introduzidos 57 cateteres de Hickman-Broviac. O período mediano de permanência dos cateteres foi de 38 dias, com uma variaçäo de um a 209 dias e um total de 3.104 dias de catéter. Vinte e oito cateteres (49,1%) foram removidos devido a complicaçöes relacionadas ao catéter. Vinte e quatro cateteres apresentaram complicaçöes infecciosas, com uma taxa de infecçäo de 42,1% e uma taxa de mortalidade de 4,2%. Após a introduçäo do manuseio e heparinizaçäo semanal dos cateteres pela equipe de enfermagem, a taxa inicial de infecçäo (45,2%) foi reduzida para 20% nos últimos 15 cateteres avaliados. A neutropenia näo constituiu um fator de risco para o desenvolvimento de sepse relacionada ao catéter. Os microorganismos Gram-negativos predominaram como agentes etiológicos durante aos episódios infeciosos (52%). Isoladamente, a Enterobacter sp. e o S. epidermidis foram responsáveis por 48% das infecçöes relacionadas ao catéter. Seis microorganismos (24%) foram resistentes aos antibióticos em uso durante o episódio infeccioso, sendo quatro bactérias Gram-negativas resistentes à amicacinam, e duas Gram-positivas resistentes à oxacilina