Abstract Introduction: The minimal residual disease (MRD) status plays a crucial role in the treatment of acute lymphoblastic leukemia (ALL) and is currently used in most therapeutic protocols to guide the appropriate therapeutic decision. Therefore, it is imperative that laboratories offer accurate and reliable results through well standardized technical processes by establishing rigorous operating procedures. Method: Our goal is to propose a monoclonal antibody (MoAb) panel for MRD detection in ALL and provide recommendations intended for flow cytometry laboratories that work on 4-color flow cytometry platforms. Results and conclusion: The document includes pre-analytical and analytical procedures, quality control assurance, technical procedures, as well as the information that needs to be included in the reports for clinicians.

Abstract Background: There is great interest in reducing the number of automated complete blood counts requiring manual blood smear reviews without sacrificing the quality of patient care. This study was aimed at evaluating and establishing appropriate screening criteria for manual blood smear reviews to improve the performance in a hematology laboratory. Method: A total of 1977 consecutive samples from the daily workload were used to evaluate four sets of screening criteria for manual blood smear reviews to identify samples with positive smear findings. Three sets of screening criteria were arbitrarily proposed in this study: Group 1 (narrow ranges), Group 2 (intermediate ranges), and Group 3 (wide limits) and one set (Group 4) was adapted from the International Society for Laboratory Hematology. All samples were run on Sysmex hematology analyzers and were investigated using manual blood smear reviews. Diagnostic accuracy and agreement were performed for each set of screening criteria, including an investigation of microscopic review rate and efficiency. Results: The microscopic review rates for Groups 1, 2, 3 and 4 were 73.85%, 54.52%, 46.33% and 46.38%, respectively; the false-negative rates were 0.50%, 1.97%, 2.73% and 3.95%, respectively. The efficiency and negative predictive values of Group 3 were 73.04% and 4.91%, respectively. Conclusions: Group 3 had the best relationship between safety (false-negative rate: ≤3%) and efficiency to estimate the limits of automation in performing complete blood counts. This study strengthens the importance of establishing screening criteria for manual blood smear reviews, which account for the different contexts in which hematological determinations are performed. Each laboratory should optimize the screening criteria for manual blood smear reviews in order to maximize their efficiency and safety.

ABSTRACT Minimal residual disease is the most powerful predictor of outcome in acute leukemia and is useful in therapeutic stratification for acute lymphoblastic leukemia protocols. Nowadays, the most reliable methods for studying minimal residual disease in acute lymphoblastic leukemia are multiparametric flow cytometry and polymerase chain reaction. Both provide similar results at a minimal residual disease level of 0.01% of normal cells, that is, detection of one leukemic cell in up to 10,000 normal nucleated cells. Currently, therapeutic protocols establish the minimal residual disease threshold value at the most informative time points according to the appropriate methodology employed. The expertise of the laboratory in a cancer center or a cooperative group could be the most important factor in determining which method should be used. In Brazil, multiparametric flow cytometry laboratories are available in most leukemia treatment centers, but multiparametric flow cytometry processes must be standardized for minimal residual disease investigations in order to offer reliable and reproducible results that ensure quality in the clinical application of the method. The Minimal Residual Disease Working Group of the Brazilian Society of Bone Marrow Transplantation (SBTMO) was created with that aim. This paper presents recommendations for the detection of minimal residual disease in acute lymphoblastic leukemia based on the literature and expertise of the laboratories who participated in this consensus, including pre-analytical and analytical methods. This paper also recommends that both multiparametric flow cytometry and polymerase chain reaction are complementary methods, and so more laboratories with expertise in immunoglobulin/T cell receptor (Ig/TCR) gene assays are necessary in Brazil.

Background: Paroxysmal nocturnal hemoglobinuria is an acquired chronic hemolytic ane- mia, which often manifests as peripheral blood cytopenias and thrombosis. Objective: The aim of this study is to describe a Brazilian population of paroxysmal nocturnal hemoglobinuria patients. Methods: One hundred and three paroxysmal nocturnal hemoglobinuria cases were retrospectively reviewed and the clinical presentation, thrombosis, survival, and clone size were assessed. Diagnosis was established by flow cytometry. Results: Fifty-two male and 51 female patients with a median age of 24.1 years (5.5-62 years) were studied. Clinical symptoms included hemoglobinuria (18.4%), infection (46.6%) and thrombosis (16.5%), and 80.6% had pancytopenia. Patients were classified as classic parox- ysmal nocturnal hemoglobinuria (10), paroxysmal nocturnal hemoglobinuria with aplastic anemia (39), and paroxysmal nocturnal hemoglobinuria with subclinical features and aplas- tic anemia (54). There were significant differences in terms of median age, size of clone, clinical symptoms, and peripheral blood cell counts between the three subcategories. The clone size in erythrocytes and granulocytes were respectively 0.04% (range: 0-18%) and 7.3% (range: 0.3-68.7%) in patients with subclinical features and aplastic anemia, 15.8% (range: 0-99.7%) and 63.0% (range: 1.7-99.8%) in patients with aplastic anemia alone, and 82.2% (range: 0-99.85%) and 98.0% (81.3-100.0%) in Classic disease. Statistical differences were identified for platelets (p-value = 0.001), lactate dehydrogenase (p-value = 0.002) and the clone size (p-value < 0.001) in patients who suffered thrombotic events compared to those who did not. Overall survival was 81.7%, with patients with subclinical features and aplastic anemia having lower overall survival (76.5%). Conclusion: This retrospective review of 103 patients over an 11-year period represents the largest collection of paroxysmal nocturnal hemoglobinuria cases from a single center in Brazil. Flow cytometry showed that a larger clone was associated with classical symptoms and increased risk of thrombosis, even in patients with bone marrow failure, whereas a smaller clone was associated with bone marrow aplasia.

A esclerose sistêmica (ES) é uma doença inflamatória crônica do tecido conjuntivo, de etiologia desconhecida, caracterizada por fibrose e dano micro vascular dos órgãos afetados. Há várias evidências de que a ativação do sistema imune celular participa de maneira expressiva na sua patogênese. OBJETIVO: Analisar numericamente as subpopulações linfocitárias no sangue periférico de pacientes com ES e sua possível relação com fatores clínicos e laboratoriais. MÉTODOS: Foram analisadas as subpopulações linfocitárias de 42 pacientes com ES e 28 controles normais, através da citometria de fluxo utilizando os antígenos linfocitários: CD2, CD3, CD4, CD8, CD19, CD25, CD45RA, CD56, CD71, HLA-DR , TCRα/β e TCRγ/δ. RESULTADOS: Nos pacientes com ES comparados com o grupo-controle, foram encontrados valores porcentuais normais para os linfócitos T CD2+, CD3+, CD3+CD4+, CD3+CD8+, CD25+, CD4+CD45RA+, CD8+CD45RA+, CD71 + e para a relação CD4+/CD8+. Em contraste, os linfócitos T CD3+TCRγ/δ mostraram-se com porcentuais diminuídos nos pacientes com ES difusa, de longa duração, com envolvimento pulmonar caracterizado por doença pulmonar restritiva, envolvimento muscular e presença de antilcorpo anti-Scl-70. Os pacientes com a forma difusa de ES apresentaram, tanto na fase recente como na tardia, aumento na porcentagem de linfócitos T CD4+ e CD8+ expressando moléculas de ativação HLA-DR. Na fase tardia da doença, não importando a forma clínica, foi encontrado aumento na porcentagem de linfócitos T CD4+CD45RA+. Os pacientes com ES limitada e fase recente mostraram diminuição na porcentagem dos linfócitos B CD19+. Os pacientes com doença difusa e fase tardia apresentaram diminuição na porcentagem dos linfócitos NK CD56+. CONCLUSÕES: Estes resultados sugerem que as alterações dos linfócitos T, B e NK possam estar envolvidas no processo de desencadeamento e/ ou perpetuação da ES, havendo a possibilidade de ter utilidade prognóstica em alguns grupos de pacientes.

Systemic sclerosis (SSc) is a chronic inflammatory connective tissue disease of unknown etioiogy characterized by fibrosis and microvascuiar injury in affected organs. It has become clear that th e activation of celluiar immune system piays a central roie in the pathogenesis of SSc. OBJECTIVE: The purpose oE this study was to anaiyze numerically iymphocytic subpopuiations in the biood of systemic sclerosis patients and their relations with clinicai and iaboratory mani[estations. METHODS: We studied a group of 42 patients with SSc and a group oE 28 matched normai controis by flow cytometry using the following iymphocyte celi-surrace markers: CD2, CD3, CD4, CDS, CD19, CD25, CD45RA, CD56, CD71, HLADR, TCRα/β and TCRγ/δ. RESULTS: SSc patients had similar percentages of CD2+, CD3+, CD3+CD4+, CD3+CDS+, CD25+, CD4+CD45RA+, CDS+CD45RA+, CD71+ cells and CD4+/ CDS+ celi ratio when compared to . normal controis. ln contrast, the percentages of TCRγ/δ cells were significantiy iower in SSc patients with diffuse and iate-stage disease with puimonary and muscle invoivement and the presence of anti-Scl-70 antibodies. Patients with diffuse SSc in early and iate-stage disease had significantly increased percentages of HLA-DR in CD4+ and CDS+ celis; patients with iate-stage disease had increased percentages of CD4+CD45RA+ T celis; patients with iimited and eariy-stage disease had small pereentages of B eelis (CD19+): and patients with diffuse and iate-stage disease had small pereentages of NK eells (CD56 +). CONCLUSIONS: These results suggest that T, B and NK eell alterations may be invoived in the onset of the disease and/ or in the perpetuation of disease, and may eventuaJJy be usefui as a prognostie indieator in seleeted patient subgroups.