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.

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.

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.

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%) met the criteria of overlap syndrome and eight (24%) the classic subcategory. The overall severity of disease was moderate in 21 (62%) and severe in 13 (38%) 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%. 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.

A compatibilidade HLA é o fator mais valorizado na escolha do doador de medula óssea voluntário, preconizando-se a realização de HLA de alta resolução nos locos HLA-A,B,C, DRB1 e DQB1. Tem sido dado preferência para o doador com consanguinidade alélica 8x8 (A,B,C, DRB1). Na presença de incompatibilidade na classe-I sugere-se a busca de doador com compatibilidade DQB1 (9x10). Já as incompatibilidades dos locos DPB1 não constituem critério de exclusão de doador, exceto quando existir presença de anticorpo contra o loco HLA-DP do doador.

The HLA system is considered the most important factor in choosing a volunteer bone marrow donor with the recommendation of performing high resolution HLA tests for the HLA-A, B, C, DRB1 and DQB1 loci. A preference has been given for donor 8x8 (A, B, C, DRB1) allele matching. In the presence of class-I incompatibility a search for DQB1 (9x10) donor compatibility is suggested. The incompatibility of the DPB1 locus does not constitute exclusion of the donor, except when there is the presence of antibodies against the HLA-DP locus of the donor.

Apesar da presença de anticorpos anti-HLA em transplantes de órgãos sólidos estar associada à rejeição, essa correlação não havia sido pesquisada em transplante alogênico de células progenitoras hematopoéticas (TCPH). Estudos mais recentes na literatura têm demonstrado que a falência da enxertia no TCPH pode ser mediada por aloanticorpos anti-HLA doador especifico (DSA). A especificidade desses anticorpos pode ser evidenciada pelas técnicas de fase sólida, onde os antígenos HLA únicos são aderidos a pérolas de poliestireno, que permite a realização da prova cruzada virtual. Na presença de DSA, é recomendável selecionar outro doador ou realizar as estratégias de remoção dos anticorpos.

In spite of Anti-HLA antibodies being associated to rejection in solid organs transplantation, this correlation has not been well established yet in allogenic bone marrow transplantation.Recent studies in the literature have demonstrated that engraftment failure in hematopoietic cell transplantation (HCT) can be mediated by donor specific anti-HLA antibodies (DSA). These antibodies specificity can be detected by solid-phase techniques, where single HLA antigens are adhered to microbeads, which allows the interpretation of host reactivity by "virtual crossmatch". In the presence of DSA, it is advisable to either search for another donor or remove the antibodies prior to transplantation.

A frequente utilização de sangue de cordão umbilical - SCU como fonte de células- tronco hematopoéticas - CTH, tanto em crianças, como em adultos, que não dispõem de doador na família, tem levado ao estabelecimento da padronização de critérios em sua seleção, objetivando a obtenção de melhores resultados. A escolha da unidade de SCU deve basear-se no número total de células nucleadas e no número de diferenças de antígenos leucocitários humanos (HLA). Diante de uma unidade com celularidade mínima, deve-se considerar a possibilidade da utilização de duplo cordão. Frente a mais de uma unidade com características semelhantes, a realização da contagem de células CD34 e da compatibilidade ABO, assim como a qualidade e a rapidez para obtenção da unidade, podem definir a escolha.

The frequent use of umbilical cord blood as the source of hematopoietic stem cells, both in children and adults who do not have related donors, has led to the establishment of a better standardization of selection criteria aiming at improving the results. The choice of the umbilical cord blood unit should be based on the total number of nucleated cells and the number of differences in the human leukocyte antigen (HLA) system. When a unit has minimal cellularity, the use of a double cord blood transplant should be considered. When two or more units have similar characteristics, the choice may be determined by the CD34 count, ABO compatibility and the quality and speed to obtain the unit.

A célula-tronco hematopoética (CTH) tem um enorme potencial para reconstituir o sistema hematopoético, o que permitiu o desenvolvimento de estratégias de terapias celulares para doenças neoplásicas ou não. Em paralelo com os avanços clínicos, estudos sobre os mecanismos moleculares que levam as células-tronco hematopoéticas a decidir pela autorrenovação, diferenciação ou apoptose têm contribuído para o conhecimento de como controlar a cinética da CTH. Esta revisão tenta descrever como estes novos avanços podem ser utilizados no desenvolvimento de estratégias de expansão das CTHs para uso terapêutico.

Hematopoietic stem cells (HSCs) have the potential for reconstituting the hematopoietic system a characteristic that has enabled the development of cell based therapies for neoplastic and non-malignant diseases. In parallel with these clinical advances, elucidation of molecular mechanisms controlling self-renewal, differentiation or apoptosis have contributed to our understanding of the molecular events that control HSC kinetics. This review focuses on how these advances can be translated in new strategies for HSC expansion and their use in therapies.

Fotoferese extracorpórea é uma modalidade de terapia celular que tem demonstrado eficácia e segurança em pacientes portadores de doenças auto-imunes e mediadas por células T, incluindo esclerodermia, linfoma de células T cutâneo/ Síndrome de Sézary e doença enxerto-contra-hospedeiro após transplante de células-tronco hematopoiéticas. Tem sido empregada, ainda, com o intuito de induzir tolerância em transplantes de órgãos sólidos. O tratamento se baseia no efeito biológico de uma substância fotossensibilizante, o 8-metoxipsoraleno, e da radiação ultravioleta A, nas células mononucleares coletadas por aférese e reinfundidas ao paciente. O mecanismo de ação parece ser devido à indução de imunidade anticlonotípica direcionada a clones patogênicos de células T. O tratamento induz a apoptose de células T patogênicas, e a ativação de células apresentadoras de antígenos desempenha papel importante no processo imunomodulador.

Extracorporeal photopheresis is a method of cell therapy that has demonstrated efficacy in autoimmune diseases, cutaneous T-cell lymphoma/Sézary syndrome and graft-versus-host disease after hematopoietic stem-cell transplantation. This technique has also been employed as a treatment strategy to induce tolerance after solid organ transplants. Treatment is based on the biological effect of ultraviolet A radiation and a photosensitizing drug, 8-methoxypsoralen, on mononuclear cells collected by apheresis and reinfused into the patient. The suggested mechanism of action is related to the induction of anticlonotypic immunity directed against pathogenic clones of T lymphocytes. Treatment induces apoptosis of pathogenic T-cells, and it appears that activation of antigen-presenting cells has an important role in this process.

RESUMO CONTEXTO E OBJETIVO: Após o transplante de células tronco-hematopoéticas (TCTH), o cariótipo é uma ferramenta valiosa para monitorar o status do enxerto e da doença. Poucos estudos investigaram o significado prognóstico do cariótipo nos pacientes que se submeteram ao TCTH para leucemia mielóide crônica (LMC). O objetivo desse estudo foi verificar o significado dos achados citogenéticos pré-TCTH em pacientes portadores de LMC. TIPO DE ESTUDO E LOCAL: Série de casos. Instituto Nacional do Câncer (INCA), Rio de Janeiro, Brasil. METODOLOGIA: Foram realizados estudos citogenéticos por bandeamento G em 39 pacientes submetidos ao TCTH. RESULTADOS: Trinta e um pacientes estavam em fase crônica e oito em fase acelerada. Pré-TCTH, alterações cromossômicas adicionais ao cromossomo Philadelphia (Ph) foram observadas em 11 pacientes. A mais freqüente foi o duplo Ph observado em quatro casos. Após o TCTH, quimerismo total foi observado em 31 pacientes (79,5%). Desses, 23 (82,3%) apresentavam somente o cromossomo Ph. Quimerismo misto foi observado em sete pacientes, sendo três com alterações adicionais ao Ph. Um caso não apresentou resposta ao TCTH. Recaída citogenética associada com recaída clínica foi observada em cinco pacientes. Após o TCTH, 27 pacientes permanecem vivos e com remissão clínica e citogenética. CONCLUSÃO: Em nosso estudo a presença de alterações cromossômicas adicionais ao Ph, prévias ao TCTH, não foi associada com pior evolução, com risco de recaída, bem como não foi observada diferença entre as taxas de sobrevida. Nosso estudo sugere que a citogenética clássica permanece uma grande ferramenta no monitoramento do TCTH.

CONTEXT AND OBJECTIVE: Following hematopoietic stem cell transplantation (HSCT), karyotyping is a valuable tool for monitoring engraftment and disease status. Few studies have examined the prognostic significance of karyotypes in patients who underwent HSCT for chronic myeloid leukemia (CML). The objective of this study was to evaluate the significance of pretransplantation cytogenetic status in relation to outcomes following HSCT in CML patients. DESIGN AND SETTING: Case series study at Instituto Nacional do Câncer (INCA), Rio de Janeiro, Brazil. METHODS: Cytogenetic analysis was performed by G banding on 39 patients treated with HSCT. RESULTS: Thirty-one patients were in the chronic phase and eight were in the accelerated phase. Prior to HSCT, additional chromosomal abnormalities on the Philadelphia (Ph) chromosome were found in 11 patients. The most frequent additional abnormality was a double Ph, which was observed in four cases. Following HSCT, full chimeras were observed in 31 patients (79.5%). Among these, 23 (82.3%) had presented Ph as the sole abnormality. Mixed chimeras were observed in seven patients, of which three had additional abnormalities. Only one case did not present any cytogenetic response. Five patients presented cytogenetic relapse associated with clinical relapse following HSCT. Twenty-seven patients are still alive and present complete hematological and cytogenetic remission. CONCLUSION: In our study, the presence of additional abnormalities was not associated with worse outcome and relapse risk. Also, no differences in survival rates were observed. Our study supports the view that classical cytogenetic analysis remains an important tool regarding HSCT outcome.

A doença enxerto contra hospedeiro (DECH) é uma síndrome sistêmica que ocorre em pacientes que recebem linfócitos imunocompetentes. A fisiopatologia envolve uma reação imunológica entre linfócitos transplantados e tecidos do hospedeiro, e ocorre por ataque imune das células T do doador às células do hospedeiro, as quais diferem daquelas pelos antígenos de histocompatibilidade. É, assim, uma complicação primária do transplante de medula óssea (TMO) alogênico. O envolvimento cutâneo é freqüente na DECH e contribui para a morbidade e mortalidade do TMO. O dermatologista tem papel importante na avaliação dos pacientes auxiliando no reconhecimento precoce da DECH e suas complicações e no acompanhamento clínico desses pacientes. Nesta revisão os autores enfatizam as manifestações cutâneas da DECH, tendo como base sua experiência pessoal no acompanhamento de pacientes portadores de DECH transplantados de medula óssea no Centro Nacional de Transplante de Medula Óssea/Inca/MS, no Rio de Janeiro, nos últimos 14 anos.

Graft-versus-host disease (GVHD) is a systemic disease that occurs in patients that receive immunocompetent lymphocytes. Pathophysiology involves an immunologic reaction between transplanted lymphocytes and tissues of the host, through an immune attack of donor T cells against recipient cells that differ from the donor's by histocompatibility antigens. It is a major complication of allogeneic hematopoietic stem cell transplantation. GVHD skin involvement is frequent and contributes to morbidity and mortality of bone marrow transplantation. Dermatologists have an important role on patient's evaluation, providing early diagnosis of GVHD disease and its complications, so as to follow-up these patients. In this review, we emphasize the skin manifestations of GVHD, taking into account our 14-year personal experience at Centro Nacional de Transplante de Medula Óssea/INCA/MS.