Immune recovery and the role of recent thymic emigrated T lymphocytes after pediatric hematopoietic stem cell transplantation.

BACKGROUND AIMS: Adequate re-establishment of thymopoiesis is critical for long-term immune reconstitution after hematopoietic cell transplantation (HCT), potentially impacting patient survival rates. This study aimed to evaluate immune reconstitution in pediatric HCT recipients by quantifying recent thymic emigrants (RTEs), specifically CD3+CD31+CD45RA+ cells. METHODS: We conducted a retrospective analysis of 186 pediatric patients transplanted between 2013 and 2020, undergoing their first allogeneic HCT, who were alive in the first 100 days after transplantation with immune recovery evaluation at three time points: day 100, day 180 and day 360 after HCT. We analyzed the distribution of peripheral blood subsets of T, B and natural killer lymphocytes and assessed the impact of underlying disease, HCT type, stem cell source, recipient age, conditioning regimen, graft-versus-host disease (GVHD) occurrence and cytomegalovirus (CMV) reactivation on immune recovery. RESULTS: At day 100, patients under 10 years exhibited higher RTE CD4+ and CD8+CD31+CD45RA+ counts compared with older patients (5.3 versus 2.2 cells/µL, P = 0.022 and 48 versus 72.8 cells/µL, P = 0.049, respectively). Patients with haploidentical HCT had lower RTE CD4+ counts compared with those with unrelated or related donors (2.4 versus 4.4 versus 7.9 cells/µL, P = 0.024). Administration of rabbit anti-thymocyte globulin negatively impacted RTE CD4+ production (median, 6.5 versus 2.4 cells/µL, P = 0.007). At day 180, the presence of GVHD had a negative influence on RTE production (11.7 versus 56.8 cells/µL, P < 0.001), particularly higher-grade acute GVHD (without, 56.8 cells/µL, grade 1-2, 28.1 cells/µL, grade 3-4, 6.0 cells/µL, P < 0.001). Patients with CMV reactivation had higher CD8+CD31+CD45RA+ compared with those without reactivation (median, 204.6 versus 100.2 cells/µL, P = 0.022). At day 360, no variables significantly affected RTE recovery. Overall survival at 5-year follow-up was 87.7%, with a median of 1170 days (range, 122-3316). Multivariate analysis showed that age >10 years (P = 0.038), negative CMV donor serology (P = 0.0029) and acute GVHD (P = 0.0026) had a negative impact on survival. CONCLUSIONS: This study highlights variations in RTE production based on patient age, donor type and immunosuppression regimen employed.

The Screening of microRNAs in Chronic Myeloid Leukemia: A Clinical Evaluation.

Chronic myeloid leukemia (CML) is a type of leukemia whose main genetic marker is the reciprocal translocation that leads to the production of the BCR::ABL1 oncoprotein. The expression of some genes may interfere with the progression and development of leukemias. MicroRNAs are small non-coding RNAs that have the potential to alter the expression of some genes and may be correlated with some types of leukemia and could be used as biomarkers in the diagnosis and prognosis of patients. Therefore, this project carried out an analysis of microRNA-type plasma biomarkers in patients with chronic myeloid leukemia at unique points, including follow-up analysis of patients from the Erasto Gaertner Hospital. 35 microRNAs were analyzed in different cohorts. Inside those groups, 70 samples were analyzed at unique points and 11 patients in a follow-up analysis. Statistically different results were found for microRNA-7-5p, which was found to be upregulated in patients with high expression of the BCR::ABL1 transcript when compared to healthy controls. This microRNA also had evidence of behavior related to BCR::ABL1 when analyzed in follow-up, but strong evidence was not found. In this way, this work obtained results that may lead to manifestations of a relationship between miR-7-5p and chronic myeloid leukemia, and evaluations of possible microRNAs that are not related to this pathology.

Cerebrospinal fluid CD14++CD16+ monocytes in HIV-1 subtype C compared with subtype B.

CD14++CD16+ monocytes are susceptible to HIV-1 infection, and cross the blood-brain barrier. HIV-1 subtype C (HIV-1C) shows reduced Tat protein chemoattractant activity compared to HIV-1B, which might influence monocyte trafficking into the CNS. We hypothesized that the proportion of monocytes in CSF in HIV-1C is lower than HIV-1B group. We sought to assess differences in monocyte proportions in cerebrospinal fluid (CSF) and peripheral blood (PB) between people with HIV (PWH) and without HIV (PWoH), and by HIV-1B and -C subtypes. Immunophenotyping was performed by flow cytometry, monocytes were analyzed within CD45 + and CD64 + gated regions and classified in classical (CD14++CD16-), intermediate (CD14++CD16+), and non-classical (CD14lowCD16+). Among PWH, the median [IQR] CD4 nadir was 219 [32-531] cell/mm3; plasma HIV RNA (log10) was 1.60 [1.60-3.21], and 68% were on antiretroviral therapy (ART). Participants with HIV-1C and -B were comparable in terms of age, duration of infection, CD4 nadir, plasma HIV RNA, and ART. The proportion of CSF CD14++CD16+ monocytes was higher in participants with HIV-1C than those with HIV-1B [2.00(0.00-2.80) vs. 0.00(0.00-0.60) respectively, p = 0.03 after BH correction p = 0.10]. Despite viral suppression, the proportion of total monocytes in PB increased in PWH, due to the increase in CD14++CD16+ and CD14lowCD16+ monocytes. The HIV-1C Tat substitution (C30S31) did not interfere with the migration of CD14++CD16+ monocytes to the CNS. This is the first study to evaluate these monocytes in the CSF and PB and compare their proportions according to HIV subtype.

Main lymphocyte subpopulations in cerebrospinal fluid and peripheral blood in HIV-1 subtypes C and B.

HIV-1 subtype C (HIV-1C) shows reduced Tat protein chemoattractant activity compared with HIV-1B. The impact of HIV-1C Tat on the chemotaxis of the main lymphocyte subpopulations in the cerebrospinal fluid (CSF) and the peripheral blood (PB) is unclear. We hypothesized that there would be a lower frequency of specific lymphocyte subpopulations CD3+ or CD19+ in CSF in HIV-1C than in HIV-1B. The objectives were to detect the differences in the proportions of main lymphocyte subpopulations in CSF and PB, between people with HIV (PWH) and HIV-1-uninfected volunteers (PWoH) and in HIV-1B and HIV-1C. Lymphocyte immunophenotyping was studied in CSF and paired PB samples of PWH (n = 22) and PWoH (n = 14). Lymphocytes were analyzed within the CD45+ gated region. The proportions of CSF CD3+CD4+, CD3+CD8+, and CD3-CD19+ lymphocytes in CSF were comparable in HIV-1B and C. There was an increase in the proportion of CD3+CD8+ cells and a decrease in CD3+CD4+ T cells (ps = 0.016) in the CSF samples of the PWH compared with the PWoH group. In the PWH group, both CD3+CD4+ and CD3+CD8+ lymphocytes were significantly higher in the CSF than in the PB (p = 0.047 and 0.005). The proportion of CD3+CD4+ was lower and that of CD3+CD8+ was higher in the CSF samples of the aviremic group than that of HIV-negative control (p = 0.0008 and < 0.0001, respectively). HIV-1C Tat substitution (C30S) did not interfere with the CNS migration of the main lymphocyte subpopulations. This is the first study to evaluate these lymphocytes in CSF and PB of HIV-1C compared with HIV-1B.

Endoglin: a novel target for therapeutic intervention in acute leukemias revealed in xenograft mouse models.

Endoglin (CD105), a receptor of the transforming growth factor-ß superfamily, has been reported to identify functional long-term repopulating hematopoietic stem cells, and has been detected in certain subtypes of acute leukemias. Whether this receptor plays a functional role in leukemogenesis remains unknown. We identified endoglin expression on the majority of blasts from patients with acute myeloid leukemia (AML) and acute B-lymphoblastic leukemia (B-ALL). Using a xenograft model, we find that CD105+ blasts are endowed with superior leukemogenic activity compared with the CD105- population. We test the effect of targeting this receptor using the monoclonal antibody TRC105, and find that in AML, TRC105 prevented the engraftment of primary AML blasts and inhibited leukemia progression following disease establishment, but in B-ALL, TRC105 alone was ineffective due to the shedding of soluble CD105. However, in both B-ALL and AML, TRC105 synergized with reduced intensity myeloablation to inhibit leukemogenesis, indicating that TRC105 may represent a novel therapeutic option for B-ALL and AML.

Bone Marrow-Derived Stem Cell Populations Are Differentially Regulated by Thyroid or/and Ovarian Hormone Loss.

Bone marrow-derived stem cells (BMDSCs) play an essential role in organ repair and regeneration. The molecular mechanisms by which hormones control BMDSCs proliferation and differentiation are unclear. Our aim in this study was to investigate how a lack of ovarian or/and thyroid hormones affects stem cell number in bone marrow lineage. To examine the effect of thyroid or/and ovarian hormones on the proliferative activity of BMDSCs, we removed the thyroid or/and the ovaries of adult female rats. An absence of ovarian and thyroid hormones was confirmed by Pap staining and Thyroid Stimulating Hormone (TSH) measurement, respectively. To obtain the stem cells from the bone marrow, we punctured the iliac crest, and aspirated and isolated cells by using a density gradient. Specific markers were used by cytometry to identify the different BMDSCs types: endothelial progenitor cells (EPCs), precursor B cells/pro-B cells, and mesenchymal stem cells (MSCs). Interestingly, our results showed that hypothyroidism caused a significant increase in the percentage of EPCs, whereas a lack of ovarian hormones significantly increased the precursor B cells/pro-B cells. Moreover, the removal of both glands led to increased MSCs. In conclusion, both ovarian and thyroid hormones appear to have key and diverse roles in regulating the proliferation of cells populations of the bone marrow.

Immune reconstitution in patients with Fanconi anemia after allogeneic bone marrow transplantation.

BACKGROUND AIMS: Fanconi anemia is an autosomal recessive or X-linked genetic disorder characterized by bone marrow (BM) failure/aplasia. Failure of hematopoiesis results in depletion of the BM stem cell reservoir, which leads to severe anemia, neutropenia and thrombocytopenia, frequently requiring therapeutic interventions, including hematopoietic stem cell transplantation (HSCT). Successful BM transplantation (BMT) requires reconstitution of normal immunity. METHODS: In the present study, we performed a detailed analysis of the distribution of peripheral blood subsets of T, B and natural killer (NK) lymphocytes in 23 patients with Fanconi anemia before and after BMT on days +30, +60, +100, +180, +270 and +360. In parallel, we evaluated the effect of related versus unrelated donor marrow as well as the presence of graft-versus-host disease (GVHD). RESULTS: After transplantation, we found different kinetics of recovery for the distinct major subsets of lymphocytes. NK cells were the first to recover, followed by cytotoxic CD8(+) T cells and B cells, and finally CD4(+) helper T cells. Early lymphocyte recovery was at the expense of memory cells, potentially derived from the graft, whereas recent thymic emigrant (CD31(+) CD45RA(+)) and naive CD4(+) or CD8(+) T cells rose only at 6 months after HSCT, in the presence of immunosuppressive GVHD prophylactic agents. Only slight differences were observed in the early recovery of cytotoxic CD8(+) T cells among those cases receiving a graft from a related donor versus an unrelated donor. Patients with GVHD displayed a markedly delayed recovery of NK cells and B cells as well as of regulatory T cells and both early thymic emigrant and total CD4(+) T cells. CONCLUSIONS: Our results support the utility of post-transplant monitoring of a peripheral blood lymphocyte subset for improved follow-up of patients with Fanconi anemia undergoing BMT.

Circulating progenitor and mature endothelial cells in deep vein thrombosis.

INTRODUCTION: Mature circulating endothelial cells (CEC) and circulating endothelial progenitor cells (EPC) have been described in several conditions associated with endothelial injury. Their role in deep vein thrombosis (DVT) has not been previously evaluated. PATIENTS AND METHODS: In this pilot study we evaluated the time course of CEC and EPC release after vena cava experimental DVT in mice, using the FeCl3 model. We also evaluated their presence in patients with DVT at different phases of the disease (acute and chronic phase). CEC and EPC were evaluated by Flow Cytometry. RESULTS: In mice, both CEC and EPC were increased 24 hours after DVT induction, peaking 48 hours thereafter. After 72 hours, CEC counts decreased sharply, whereas EPC counts decreased less substantially. In DVT patients we observed a significant increase in CEC counts immediately after DVT compared to healthy individuals. Patients with chronic disease also presented a significant elevation of these cell count. In a subgroup of patients for whom serial samples were available, CEC counts decreased significantly after 9-15 months of the acute event. CONCLUSIONS: Our results suggest the participation of these cells in the reparative processes that follows DVT, both at immediate and late time-points. The different kinetics of CEC and EPC release in experimental DVT suggests a heterogeneous role for these cells in the reparative events after DVT.

CD3+CD56+ and CD3-CD56+ lymphocytes in the cerebrospinal fluid of persons with HIV-1 subtypes B and C.

The transactivator of transcription (Tat) is a HIV regulatory protein which promotes viral replication and chemotaxis. HIV-1 shows extensive genetic diversity, HIV-1 subtype C being the most dominant subtype in the world. Our hypothesis is the frequency of CSF CD3+CD56+ and CD3-CD56dim is reduced in HIV-1C compared to HIV-1B due to the Tat C30S31 substitution in HIV-1C. 34 CSF and paired blood samples (PWH, n = 20; PWoH, n = 14) were studied. In PWH, the percentage of CD3+CD56+ was higher in CSF than in blood (p < 0.001), comparable in both compartments in PWoH (p = 0.20). The proportion of CD3-CD56dim in CSF in PWH was higher than PWoH (p = 0.008). There was no subtype differences. These results showed CNS compartmentalization of NKT cell response in PWH.

Minimal residual disease assessment in acute lymphoblastic leukemia by 4-color flow cytometry: Recommendations from the MRD Working Group of the Brazilian Society of Bone Marrow Transplantation.

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.

Updating recommendations of the Brazilian Group of Flow Cytometry (GBCFLUX) for diagnosis of acute leukemias using four-color flow cytometry panels.

INTRODUCTION: Flow cytometry has become an increasingly important tool in the clinical laboratory for the diagnosis and monitoring of many hematopoietic neoplasms. This method is ideal for immunophenotypic identification of cellular subpopulations in complex samples, such as bone marrow and peripheral blood. In general, 4-color panels appear to be adequate, depending on the assay. In acute leukemias (ALs), it is necessary identify and characterize the population of abnormal cells in order to recognize the compromised lineage and classify leukemia according to the WHO criteria. Although the use of eight- to ten-color immunophenotyping panels is wellestablished, many laboratories do not have access to this technology. OBJECTIVE AND METHOD: In 2015, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) proposed antibody panels designed to allow the precise diagnosis and characterization of AL within available resources. As many Brazilian flow cytometry laboratories use four-color immunophenotyping, the GBCFLUX has updated that document, according to current leukemia knowledge and after a forum of discussion and validation of antibody panels. RESULTS: Recommendations for morphological analysis of bone marrow smears and performing screening panel for lineage (s) identification of AL were maintained from the previous publication. The lineage-oriented proposed panels for B and T cell acute lymphoblastic leukemia (ALL) and for acute myeloid leukemia (AML) were constructed for an appropriate leukemia classification. CONCLUSION: Three levels of recommendations (i.e., mandatory, recommended, and optional) were established to enable an accurate diagnosis with some flexibility, considering local laboratory resources and patient-specific needs.

Flow cytometry as a tool in the diagnosis of Bernard-Soulier syndrome in Brazilian patients.

Bernard-Soulier Syndrome (BSS) is an inherited recessive bleeding disorder. In some instances, diagnosis might be restricted to routine blood exams, including bleeding time, prothrombin time (PT), and partial thromboplastin time (APTT). Exams such as platelet aggregation, and testing for expression of ristocetin cofactor, or von Willebrand factor may not be commonly performed. This leads to misdiagnosis in a number of patients, which are subsequently treated erroneously. Flow cytometry has been used widely as a tool in the diagnosis of leukemias, lymphomas, and many other immuno-hematological diseases. The purpose of this study was to assess whether flow cytometry could be helpful in the diagnosis of Bernard-Soulier Syndrome in Brazilian patients. For this, we examined a selected group of 15 patients with suspected BSS based on classical diagnosis. We used a panel of antibodies to detect the expression of glycoproteins GPIbalpha, GPIIb, GPIIIa, GPIX, as well as CD9. Abnormalities of GPIb and GPIX were observed in nine of the 15 patients, which included severe reduction of both antigens, of one or the other, or normal levels but weak expression. Strikingly, this abnormality correlated with severely reduced expression of CD9 in all cases. We discuss the implications for flow cytometric diagnosis of BSS.

Minimal residual disease assessment in acute lymphoblastic leukemia by 4-color flow cytometry: Recommendations from the MRD Working Group of the Brazilian Society of Bone Marrow Transplantation

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.

Updating recommendations of the Brazilian Group of Flow Cytometry (GBCFLUX) for diagnosis of acute leukemias using four-color flow cytometry panels

ABSTRACT Introduction: Flow cytometry has become an increasingly important tool in the clinical laboratory for the diagnosis and monitoring of many hematopoietic neoplasms. This method is ideal for immunophenotypic identification of cellular subpopulations in complex samples, such as bone marrow and peripheral blood. In general, 4-color panels appear to be adequate, depending on the assay. In acute leukemias (ALs), it is necessary identify and characterize the population of abnormal cells in order to recognize the compromised lineage and classify leukemia according to the WHO criteria. Although the use of eightto ten-color immunophenotyping panels is wellestablished, many laboratories do not have access to this technology. Objective and Method: In 2015, the Brazilian Group of Flow Cytometry (Grupo Brasileiro de Citometria de Fluxo, GBCFLUX) proposed antibody panels designed to allow the precise diagnosis and characterization of AL within available resources. As many Brazilian flow cytometry laboratories use four-color immunophenotyping, the GBCFLUX has updated that document, according to current leukemia knowledge and after a forum of discussion and validation of antibody panels. Results: Recommendations for morphological analysis of bone marrow smears and performing screening panel for lineage (s) identification of AL were maintained from the previous publication. The lineage-oriented proposed panels for B and T cell acute lymphoblastic leukemia (ALL) and for acute myeloid leukemia (AML) were constructed for an appropriate leukemia classification. Conclusion: Three levels of recommendations (i.e., mandatory, recommended, and optional) were established to enable an accurate diagnosis with some flexibility, considering local laboratory resources and patient-specific needs.

Proposal for the standardization of flow cytometry protocols to detect minimal residual disease in acute lymphoblastic leukemia.

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.

Proposal for the standardization of flow cytometry protocols to detect minimal residual disease in acute lymphoblastic leukemia

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.

Avaliação do desempenho analítico do analisador hematológico Sysmex® XE-2100D / Performance evaluation of the Sysmex XE-2100D automated hematology analyzer

Introduction: The Sysmex® XE-2100D is a multiparameter hematology analyzer designed for hematology testing in samples with ethylenediamine tetraacetic acid (EDTA). Objectives: Considering the importance of this hematology analyzer for clinical and laboratory practice, the objective of this study was to evaluate its analytical performance, comparing the obtained results with quality specifications described in literature. Material and method: In the evaluation of analytical performance, according to recommendations of the document H26-A2 of the Clinical and Laboratory Standards Institute (CLSI), intra-run imprecision, inter-run imprecision, linearity, carryover, autosampler evaluation, clinical sensitivity of the atypical lymphocytes flag (n = 400 samples) were included, as well as the comparison between automated and manual leukocyte differential count (n = 400 samples), based on an adaptation of the document H20-A2 of CLSI. Results: Repeatability, reproducibility, linearity and carryover were satisfactory according to the manufacturer's specifications. The clinical sensitivity of the atypical lymphocytes flag showed efficiency, sensitivity and specificity of 92.5%, 65.2% and 94.1% respectively. The correlation coefficients between the automated and manual differential counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils were 0.991, 0.99, 0.872, 0.974 and 0.557, respectively. Conclusions: The results were in accordance with quality specifications described in literature, indicating reliability in Sysmex® XE-2100D. This fact ensures certainty to both laboratory professionals and medical staff. We conclude that the Sysmex® XE-2100D showed excellent analytical performance, and is useful to provide reliable hematology data...

Introdução: O Sysmex® XE-2100D é um analisador hematológico multiparamétrico destinado à realização de testes hematológicos em sangue anticoagulado com ácido etilenodiamino tetra-acético (EDTA). Objetivos: Considerando a sua importância na prática clínica e laboratorial, o objetivo deste estudo foi avaliar seu desempenho analítico, comparando os resultados obtidos com especificações de qualidade descritas na literatura. Material e método: Na avaliação de desempenho analítico, conforme recomendações do documento H26-A2 do Clinical and Laboratory Standards Institute (CLSI), foram incluídos ensaios de verificação da imprecisão intraensaio ou repetitividade, imprecisão entre ensaios ou reprodutibilidade, linearidade, carryover (arraste), avaliação do mecanismo homogeneizador de amostras, sensibilidade clínica do alerta morfológico (flag) de linfócitos atípicos (n = 400 amostras) e a comparação entre a contagem diferencial de leucócitos automatizada e a manual (n = 400 amostras), baseada em uma adaptação do documento H20-A2 do CLSI. Resultados: Os ensaios de verificação da repetitividade, reprodutibilidade, linearidade, carryover (arraste) foram satisfatórios conforme especificações do fabricante. O ensaio de sensibilidade clínica do alerta morfológico (flag) de linfócitos atípicos mostrou eficiência, sensibilidade e especificidade de 92,5%; 65,2% e 94,1% respectivamente. Os coeficientes de correlação entre as contagens diferenciais automatizadas e manuais de neutrófilos, linfócitos, monócitos, eosinófilos e basófilos foram de 0,991; 0,99; 0,872; 0,974 e 0,557 respectivamente...