Flow cytometry immunophenotyping of healthy platelets and hospitalized patients with suspected platelet dysfunction: Challenges for establishing a cutoff value.

INTRODUCTION AND OBJECTIVE: Flow Cytometry (FC) is one of the techniques, which allows the identification and characterization of platelets. The detection of absent or reduced expression of the glycoproteins is the main objective of this technique. Abnormalities of glycoproteins lead to hemorrhagic syndromes. Among the main diseases, the Bernard-Soulier syndrome (BSS) and Glanzmann thrombasthenia (GT) stand out. We aimed to show a FC-based platelet assessment test for diagnostic use, which measures the expression of markers in normal patients, and evaluate these markers in patients with platelet disorders. METHODS: We examined a control group of 41 healthy adults to establish reference values and assess the variability of the relative expression of platelet markers and subsequently compared these findings to those of 30 patients with suspected platelet dysfunctions. We determined the mean fluorescent intensity (MFI) of the expressed parameters by FC using CD41, CD42a, CD42b and CD61 and SSC/FSC platelet-gated cells. RESULTS: We determined our baseline panel of markers and compared them to suspected platelet dysfunctions. Patients with suspected BSS presented increased levels of the MFI for the GPIIIa (CD61) and GPIIb (CD41). They showed significantly reduced levels of the GPIb (CD42b) and GPIX (CD42a). Patients with suspected GT showed normal expression of the GPIX (CD42a), increased expression of the GPIb (CD42b) and reduced levels of the GPIIIa (CD61). In this case, with reduced levels of only one marker, the GPIIb (CD41), values showed normal expression. CONCLUSIONS: We describe the FC assay to support the diagnosis of different platelet disorders. Our study made it possible to implement a technique that brought benefits to care.

Comparison of Nonsequencing Techniques for Identification of NPM1 Mutations and Associated Blast Morphology in Patients With Acute Myeloid Leukemia.

CONTEXT.­: Nucleophosmin 1 (NPM1) mutations affect 20% to 30% of all acute myeloid leukemia (AML) patients; several methods are employed to analyze NPM1 mutations, each of them with its advantages and limitations. OBJECTIVE.­: To compare 3 nonsequencing protocols capable of detecting the main NPM1 mutations and to evaluate nuclear morphometric analysis (NMA) as an alternative to cuplike blast detection. DESIGN.­: We selected multiparameter flow cytometry (MFC), amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), and a quantitative PCR (qPCR) kit to identify NPM1 mutations in AML patients at diagnosis. We also evaluated the presence of cuplike blasts and assessed nuclear morphometry using NMA. RESULTS.­: MFC appears as a screening method for NPM1 mutations because of its lower specificity. ARMS-PCR demonstrated specificity similar to that of the qPCR kit, although it was more laborious. qPCR testing, conversely, is relatively fast and easy to standardize. Of these methods, qPCR was the only one capable of identifying the type of NPM1 mutation. With regard to morphology, NMA could be used as an alternative for the evaluation of cuplike blasts in AML smears. CONCLUSIONS.­: qPCR appears to be the best option to identify NPM1 mutations, with ARMS-PCR representing a cheaper alternative. MFC may be used as a screening method, in which results falling within and above the gray zone should be confirmed by molecular testing.

NT5E gene and CD38 protein as potential prognostic biomarkers for childhood B-acute lymphoblastic leukemia.

The risk stratification of B-acute lymphoblastic leukemia (B-ALL) is based on clinical and biological factors. However, B-ALL has significant biological and clinical heterogeneity and 50% of B-ALL patients do not have defined prognostic markers. In this sense, the identification of new prognostic biomarkers is necessary. Considering different cohorts of childhood B-ALL patients, gene (DPP4/CD38/ENTPD1/NT5E) and protein (CD38/CD39/CD73) expressions of ectonucleotidases were analyzed in silico and ex vivo and the association with prognosis was established. In univariate analyses, expression of NT5E was significantly associated with worse progression-free survival (PFS) in bone marrow (BM) samples. In multivariate analyses, Kaplan-Meier analysis, and log-rank test, higher NT5E expression predicted unfavorable PFS in BM samples. Considering minimal residual disease (MRD), higher levels of cellularity were associated with the high NT5E expression at day 8 of induction therapy. In addition, we observed that white blood cells (WBC) of childhood B-ALL patients had more CD38 compared to the same cell population of healthy donors (HD). In fact, MRD > 0.1% patients had higher CD38 protein expression on WBC in comparison to HD. Noteworthy, we observed higher CD38 expression on WBC than blasts in MRD > 0.1% patients. We suggest that NT5E gene and CD38 protein expression, of the ectonucleotidases family, could provide interesting prognostic biomarkers for childhood B-ALL.

Flow cytometry as a diagnostic tool in neuroblastoma.

In recent years, there has been an expansion in the use of flow cytometry (FC) immunophenotyping in the diagnosis and monitoring of childhood solid neoplasms. Neuroblastoma (NB), in turn, is the most common extracranial solid tumor in childhood. In the present study, we sought to compare FC and anatomopathological examination (PA) / immunohistochemistry (IHC) of children diagnosed or suspected with NB. The median age was 59 months (minimum 0; maximum 325 months), of these 12 were male (57.1%, 12/21). Forty-eight samples (27 bone marrow (BM), 10 peripheral blood (PB), 8 primary tumors (PT) and 2 liver nodules (HN) and 1 rib fragment (RF)) from 21 patients were evaluated. Twenty-nine samples were from patients with clinical suspicion while 19 samples were from patients with previously confirmed diagnosis. Thirteen samples (7 BM, 5 PT and 1 HN) presented NB when analyzed in FC while 8 (3 BM and 5 PT) samples were positive for NB in the PA/IHC. They were concordant in 88.9% of the cases. No NB cells were identified in any PB. Considering the PA as the gold standard, the FC obtained a sensitivity of 100%, a specificity of 86%, a positive predictive value of 67% and a negative predictive value of 100%. This study demonstrates that FC can be used as a methodology for diagnosis and assessment of NB involvement. In addition, FC has the advantage of allowing a quick diagnosis and accurate classification of the disease, and can also assist in monitoring the treatment.

Individuals with controlled hypertension show endothelial integrity following a bout of moderate-intensity exercise: randomized clinical trial.

To examine the acute effects of aerobic exercise (AE), resistance exercise (RE) or combined exercise (CE) on flow-mediated dilation (FMD), progenitor cells (PCs), endothelial progenitor cells (EPCs), oxidative stress markers and endothelial-cell derived microvesicles (EMVs) in patients with hypertension. This is a randomized, parallel-group clinical trial involving an intervention of one session of three different modalities of exercise. Thirty-three males (43 ± 2y) were randomly divided into three groups: a session of AE (n = 11, 40 min, cycle ergometer, 60% HRR); a session of RE (n = 11, 40 min, 4 × 12 lower limb repetitions, 60% 1-RM); or a session of CE (n = 11, 20-min RE + 20-min AE). FMD was assessed 10 min before and 10, 40 and 70 min post-intervention. Blood samples were collected at the same time points (except 40 min). FMD were similar in all groups and from baseline (within each group) after a single exercise bout (AE, RE or CE). At 70 min, RE group showed higher levels of PCs compared to the AE (81%) and CE group (60%). PC levels were reduced from baseline in all groups (AE: 32%, p = 0.037; RE: 15%, p = 0.003; CE: 17%, p = 0.048). The levels of EPCs, EMVs and oxidative stress were unchanged. There were no acute effects of moderate-intensity exercise on FMD, EPCs, EMVs and oxidative stress, but PCs decreased regardless of the exercise modality. Individuals with controlled hypertension do not seem to have impaired vascular function in response to a single exercise bout.

HEMOPHAGOCYTOSIS BY BLASTS IN A CHILD WITH ACUTE MONOCYTIC LEUKEMIA AFTER CHEMOTHERAPY.

OBJECTIVE: To describe the case of a child who presented hemophagocytic lymphohistiocytosis (HLH) associated with acute monocytic leukemia after chemotherapy, with hemophagocytosis caused by leukemic cells. CASE DESCRIPTION: In a university hospital in Southern Brazil, a 3-year-old female was diagnosed with acute monocytic leukemia with normal karyotype. The chemotherapy regimen was initiated, and she achieved complete remission six months later, relapsing after four months with a complex karyotype involving chromosomes 8p and 16q. The bone marrow showed vacuolated blasts with a monocytic aspect and evidence of hemophagocytosis. The child presented progressive clinical deterioration and died two months after the relapse. COMMENTS: HLH is a rare and aggressive inflammatory condition characterized by cytopenias, hepatosplenomegaly, fever, and hemophagocytosis in the bone marrow, lymph nodes, spleen, and liver. Although rare, malignancy-associated HLH (M-HLH) is fatal. The patient in this case report met five out of the eight established criteria for HLH. The evolution of the patient's karyotype, regardless of the diagnostic profile, seemed secondary to the treatment for acute monocytic leukemia. In this case, the cytogenetic instability might have influenced the abnormal behavior of leukemic cells. This is a rare case of HLH in a child with acute monocytic leukemia.

Hemophagocytosis by blasts in a child with acute monocytic leukemia after chemotherapy / Hemofagocitose por células blásticas em paciente pediátrico com leucemia monocítica aguda após quimioterapia

ABSTRACT Objective: To describe the case of a child who presented hemophagocytic lymphohistiocytosis (HLH) associated with acute monocytic leukemia after chemotherapy, with hemophagocytosis caused by leukemic cells. Case description: In a university hospital in Southern Brazil, a 3-year-old female was diagnosed with acute monocytic leukemia with normal karyotype. The chemotherapy regimen was initiated, and she achieved complete remission six months later, relapsing after four months with a complex karyotype involving chromosomes 8p and 16q. The bone marrow showed vacuolated blasts with a monocytic aspect and evidence of hemophagocytosis. The child presented progressive clinical deterioration and died two months after the relapse. Comments: HLH is a rare and aggressive inflammatory condition characterized by cytopenias, hepatosplenomegaly, fever, and hemophagocytosis in the bone marrow, lymph nodes, spleen, and liver. Although rare, malignancy-associated HLH (M-HLH) is fatal. The patient in this case report met five out of the eight established criteria for HLH. The evolution of the patient's karyotype, regardless of the diagnostic profile, seemed secondary to the treatment for acute monocytic leukemia. In this case, the cytogenetic instability might have influenced the abnormal behavior of leukemic cells. This is a rare case of HLH in a child with acute monocytic leukemia.

RESUMO Objetivo: Descrever um caso de um paciente pediátrico que apresentou linfo-histiocitose hemofagocítica (LHH) associada à leucemia monocítica aguda pós-quimioterapia, com hemofagocitose causada pelas próprias células leucêmicas. Descrição do caso: Em um hospital universitário do Sul do Brasil, uma menina de três anos foi diagnosticada com leucemia monocítica aguda com cariótipo normal. Após receber protocolo quimioterápico, atingiu remissão seis meses depois do início do tratamento, recaíndo quatro meses após com um cariótipo complexo envolvendo ambos os cromossomos, 8p e 16q. A medula óssea mostrava-se infiltrada por células blásticas vacuolizadas com aspecto monocítico, com evidências de hemofagocitose. A criança apresentou um declínio clínico progressivo e dois meses após a recaída foi a óbito. Comentários: A LHH é uma condição inflamatória rara e agressiva caracterizada por citopenias, hepatoesplenomegalia, febre e hemofagocitose na medula óssea, linfonodos, baço e fígado. A LHH associada a doenças malignas, embora seja uma condição rara, é potencialmente fatal. A paciente deste caso apresentou cinco dos oito critérios estabelecidos para o diagnóstico de LHH. A evolução do cariótipo do paciente, independentemente do perfil do diagnóstico, pareceu ser secundária ao tratamento da leucemia monocítica aguda, sendo que a instabilidade citogenética pode ter influenciado o comportamento atípico observado nas células leucêmicas. Este é um dos raros casos de LHH em uma criança com leucemia monocítica aguda.

Flow cytometry as a diagnostic tool in childhood solid tumors / Citometria de fluxo como ferramenta diagnóstica em tumores sólidos da infância

RESUMEN Los tumores sólidos infantiles representan aproximadamente el 30% de todos los cánceres pediátricos. En los últimos años se ha incrementado el uso de la citometría de flujo (CF) en el diagnóstico y seguimiento de estas patologías, ya que es un método que permite obtener resultados rápidos y precisos, posibilitando un manejo más precoz. Realizamos esta revisión sistemática para la búsqueda bibliográfica de los siguientes términos en las plataformas de datos Lilacs, PubMed y Scielo: neoplasma, oncología, pediatría, inmunofenotipificación y citometría de flujo. Así, describimos los principales hallazgos hasta la fecha sobre el uso de CF en el diagnóstico diferencial de los cinco principales tumores de células pequeñas, redondas y azules de la infancia: neuroblastoma, sarcoma de Ewing, tumor neuroectodérmico primitivo, tumor de Wilms y rabdomiosarcoma. Además, describimos las principales ventajas y desventajas del método y paneles que se proponen en el diagnóstico diferencial de estas patologías a través de la literatura internacional. A través de esta revisión, observamos que el uso de CF en el diagnóstico de tumores sólidos puede ser útil para la identificación rápida y precisa de la efermedade, así como para el inicio más temprano del tratamiento.

Influence of minimal residual disease by multiparametric flow cytometry at day 15 of induction in risk stratification of children with B-cell acute lymphoblastic leukemia treated at a referral hospital in southern Brazil

ABSTRACT Background: The minimal residual disease (MRD) is the most important prognostic factor for acute lymphoblastic leukemia (ALL) in children. This study aimed to investigate the influence of detecting the MRD by the multiparametric flow cytometry (MFC) at day 15 (D15) of the induction on the analysis of the risk group classifications of the different childhood ALL treatment protocols used in a referral hospital in southern Brazil. Method: We retrospectively reviewed the medical records of patients with B-cell ALL, aged 1 to 18 years, treated at a hospital from January 2013 to April 2017. Main results: Seventy-five patients were analyzed. Regarding the MRD by the MFC at D15, the analyses showed statistical significance when the MRD was grouped into three categories, < 0.1%, 0.1-10%, and > 10%, with the following distribution: 30.7%, 52.0%, and 17.3%, respectively. There was a significant association between D15 MRD-MFC < 0.1% and the likelihood of dying or relapsing and between D15 MRD-MFC > 10% and the likelihood of dying or relapsing. The cumulative hazard ratio for the relapse of patients with D15 MRD-MFC < 0.1%, 0.1-10%, and > 10% was 19.2%, 59.8%, and 80.1%, respectively. Conclusion: Our analysis suggests D15 MRD-MFC < 0.1% as a cut-off point for patients with more favorable outcomes and that the MRD at D15 in risk classifications is particularly useful for the stratification of patients with a more favorable prognosis.

Influence of minimal residual disease by multiparametric flow cytometry at day 15 of induction in risk stratification of children with B-cell acute lymphoblastic leukemia treated at a referral hospital in southern Brazil.

BACKGROUND: The minimal residual disease (MRD) is the most important prognostic factor for acute lymphoblastic leukemia (ALL) in children. This study aimed to investigate the influence of detecting the MRD by the multiparametric flow cytometry (MFC) at day 15 (D15) of the induction on the analysis of the risk group classifications of the different childhood ALL treatment protocols used in a referral hospital in southern Brazil. METHOD: We retrospectively reviewed the medical records of patients with B-cell ALL, aged 1 to 18 years, treated at a hospital from January 2013 to April 2017. MAIN RESULTS: Seventy-five patients were analyzed. Regarding the MRD by the MFC at D15, the analyses showed statistical significance when the MRD was grouped into three categories, < 0.1%, 0.1-10%, and > 10%, with the following distribution: 30.7%, 52.0%, and 17.3%, respectively. There was a significant association between D15 MRD-MFC < 0.1% and the likelihood of dying or relapsing and between D15 MRD-MFC > 10% and the likelihood of dying or relapsing. The cumulative hazard ratio for the relapse of patients with D15 MRD-MFC < 0.1%, 0.1-10%, and > 10% was 19.2%, 59.8%, and 80.1%, respectively. CONCLUSION: Our analysis suggests D15 MRD-MFC < 0.1% as a cut-off point for patients with more favorable outcomes and that the MRD at D15 in risk classifications is particularly useful for the stratification of patients with a more favorable prognosis.