The potential of circHIPK3 as a biomarker in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by leukocytosis and left shift. The primary molecular alteration is the BCR::ABL1, chimeric oncoprotein with tyrosine kinase activity, responsible for the initial oncogenesis of the disease. Therapy of CML was revolutionized with the advent of tyrosine kinase inhibitors, but it is still not considered curative and may present resistance and serious adverse effects. Discoveries in CML inaugurated a new era in cancer treatment and despite all the advances, a new biomarker is needed to detect resistance and adverse effects. Circular RNAs (circRNAs) are a special type of non-coding RNA formed through a process called backsplicing. The majority of circRNAs are derived from protein-coding genes. CircHIPK3 is formed from the second exon of the HIPK3 gene and has been found in various pathologies, including different types of cancer. New approaches have demonstrated the potential of circular RNAs in cancer research, and circHIPK3 has shown promising results. It is often associated with cellular regulatory pathways, suggesting an important role in the molecular dynamics of tumors. The identification of biomarkers is an important tool for therapeutic improvement; thus we review the role of circHIPK3 and its potential as a biomarker in CML.

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.

Lymphocytes from B-acute lymphoblastic leukemia patients present differential regulation of the adenosinergic axis depending on risk stratification.

PURPOSE: Although risk-stratified chemotherapy regimens improve B-cell acute lymphoblastic leukemia (B-ALL) clinical outcome, relapse occurs in a significant number of cases. The identification of new therapeutic targets as well as prognostic and diagnostic biomarkers can improve B-ALL patients' clinical outcomes. Purinergic signaling is an important pathway in cancer progression, however the expression of ectonucleotidases and their impact on immune cells in B-ALL lacks exploration. We aimed to analyze the expression of ectonucleotidases in B-ALL patients' lymphocyte subpopulations. METHODS: Peripheral blood samples from 15 patients diagnosed with B-ALL were analyzed. Flow cytometry was used to analyze cellularity, expression level of CD38, CD39, and CD73, and frequency of [Formula: see text], and [Formula: see text] in lymphocyte subpopulations. Plasma was used for cytokines (by CBA kit) and adenine nucleosides/nucleotides detection (by HPLC). RESULTS: Comparing B-ALL patients to health donors, we observed an increase of CD4 + and CD8 + T-cells. In addition, a decrease in CD38 expression in B and Treg subpopulations and an increase in CD39+ CD73+ frequency in Breg and CD8+ T-cells. Analyzing cytokines and adenine nucleosides/nucleotides, we found a decrease in TNF, IL-1ß, and ADO concentrations, together with an increase in AMP in B-ALL patients' plasma. CONCLUSION: As immunomodulators, the expression of ectonucleotidases might be associated with activation states, as well as the abundance of different cellular subsets. We observed a pro-tumor immunity expression profile in B-ALL patients at diagnosis, being associated with cell exhaustion and immune evasion in B-ALL.

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.

First and second COVID-19 waves in Brazil: A cross-sectional study of patients' characteristics related to hospitalization and in-hospital mortality.

BACKGROUND: Background The second wave of the COVID-19 pandemic was more aggressive in Brazil compared to other countries around the globe. Considering the Brazilian peculiarities, we analyze the in-hospital mortality concerning socio-epidemiological characteristics of patients and the health system of all states during the first and second waves of COVID-19. METHODS: We performed a cross-sectional study of hospitalized patients with positive RT-PCR for SARS-CoV-2 in Brazil. Data was obtained from the Influenza Epidemiological Surveillance Information System (SIVEP-Gripe) and comprised the period from February 25, 2020, to April 30, 2021, separated in two waves on November 5, 2020. We performed a descriptive study of patients analyzing socio-demographic characteristics, symptoms, comorbidities, and risk factors stratified by age. In addition, we analyzed in-hospital and intensive care unit (ICU) mortality in both waves and how it varies in each Brazilian state. FINDINGS: Between February 25, 2020 and April 30, 2021, 678 235 patients were admitted with a positive RT-PCR for SARS-CoV-2, with 325 903 and 352 332 patients for the first and second wave, respectively. The mean age of patients was 59 · 65 (IQR 48 · 0 - 72 · 0). In total, 379 817 (56 · 00%) patients had a risk factor or comorbidity. In-hospital mortality increased from 34 · 81% in the first to 39 · 30% in the second wave. In the second wave, there were more ICU admissions, use of non-invasive and invasive ventilation, and increased mortality for younger age groups. The southern and southeastern regions of Brazil had the highest hospitalization rates per 100 000 inhabitants. However, the in-hospital mortality rate was higher in the northern and northeastern states of the country. Racial differences were observed in clinical outcomes, with White being the most prevalent hospitalized population, but with Blacks/Browns (Pardos) having higher mortality rates. Younger age groups had more considerable differences in mortality as compared to groups with and without comorbidities in both waves. INTERPRETATION: We observed a more considerable burden on the Brazilian hospital system throughout the second wave. Furthermore, the north and northeast of Brazil, which present lower Human Development Indexes, concentrated the worst in-hospital mortality rates. The highest mortality rates are also shown among vulnerable social groups. Finally, we believe that the results can help to understand the behavior of the COVID-19 pandemic in Brazil, helping to define public policies, allocate resources, and improve strategies for vaccination of priority groups. FUNDING: Coordinating Agency for Advanced Training of Graduate Personnel (CAPES) (C.F. 001), and National Council for Scientific and Technological Development (CNPq) (No. 309537/2020-7).

Clinical validation of an in-house quantitative real time PCR assay for cytomegalovirus infection using the 1st WHO International Standard in kidney transplant patients / Validação clínica de um ensaio de PCR in house quantitativo em tempo real para infecção por citomegalovírus usando o 1º Padrão Internacional da OMS em pacientes com transplantados renais

Abstract Introduction: Cytomegalovirus (CMV) is one of the most common agents of infection in solid organ transplant patients, with significant morbidity and mortality. Objective: This study aimed to establish a threshold for initiation of preemptive treatment. In addition, the study compared the performance of antigenemia with qPCR results. Study design: This was a prospective cohort study conducted in 2017 in a single kidney transplant center in Brazil. Clinical validation was performed by comparing in-house qPCR results, against standard of care at that time (Pp65 CMV Antigenemia). ROC curve analysis was performed to determine the ideal threshold for initiation of preemptive therapy based on the qPCR test results. Results: Two hundred and thirty two samples from 30 patients were tested with both antigenemia and qPCR, from which 163 (70.26%) were concordant (Kappa coefficient: 0.435, p<0.001; Spearman correlation: 0.663). PCR allowed for early diagnoses. The median number of days for the first positive result was 50 (range, 24-105) for antigenemia and 42 (range, 24-74) for qPCR (p<0.001). ROC curve analysis revealed that at a threshold of 3,430 IU/mL (Log 3.54), qPCR had a sensitivity of 97.06% and a specificity of 74.24% (AUC 0.92617 ± 0.0185, p<0.001), in the prediction of 10 cells/105 leukocytes by antigenemia and physician's decision to treat. Conclusions: CMV Pp65 antigenemia and CMV qPCR showed fair agreement and a moderate correlation in this study. The in-house qPCR was revealed to be an accurate method to determine CMV DNAemia in kidney transplant patients, resulting in positive results weeks before antigenemia.

Resumo Introdução: Citomegalovírus (CMV) é um dos agentes infecciosos mais comuns em pacientes com transplante de órgãos sólidos, com morbidade e mortalidade significativas. Objetivo: Este estudo visou estabelecer um limite para o início do tratamento preemptivo. Além disso, comparou o desempenho da antigenemia com os resultados da qPCR in house. Desenho do estudo: Este foi um estudo de coorte prospectivo realizado em 2017 em um centro único de transplante renal no Brasil. A validação clínica foi realizada comparando resultados de qPCR in house, com o padrão de atendimento na época (Antigenemia para CMV Pp65). A análise da curva ROC foi realizada para determinar o limite ideal para o início da terapia preemptiva baseado nos resultados do teste qPCR in house. Resultados: 232 amostras de 30 pacientes foram testadas com antigenemia e qPCR, das quais 163 (70,26%) foram concordantes (Coeficiente Kappa: 0,435, p<0,001; Correlação Spearman: 0,663). PCR permitiu diagnósticos precoces. O número médio de dias para o primeiro resultado positivo foi 50 (intervalo, 24-105) para antigenemia e 42 (intervalo, 24-74) para qPCR (p<0,001). A análise da curva ROC revelou que em um limite de 3.430 UI/mL (Log 3,54), qPCR teve sensibilidade de 97,06% e especificidade de 74,24% (AUC 0,92617 ± 0,0185, p<0,001), na previsão de 10 células/10(5) leucócitos por antigenemia e na decisão do médico de tratar. Conclusões: Antigenemia para CMV Pp65 e qPCR para CMV mostraram uma concordância aceitável e uma correlação moderada neste estudo. qPCR in house revelou-se um método preciso para determinar DNAemia do CMV em pacientes transplantados renais, obtendo resultados positivos semanas antes da antigenemia.

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.

Influence of different asparaginase formulations in the prognosis of children with acute lymphocytic leukaemia in Brazil: a multicentre, retrospective controlled study.

Our group recently showed that the (ASNase) formulation available in Brazil from 2017 to 2018 when used at the same dose and frequency as the formulation provided previously did not reach the activity considered therapeutic. Based on these, our goal was to assess the impact of these facts on the prognosis of children with ALL at different oncology centers. A multicentre retrospective observational study followed by a prospective follow-up. Patients aged >1 and <18 years in first-line treatment followed up at 10 referral centres, between 2014 and 2018 who received the formulation Leuginase® were identified (Group B). For each patient, the centre registered 2 patients who received ASNase in the presentation of Aginasa® exclusively (Group A). Data collection was registered using (Redcap® ). A total of 419 patients were included; 282 in Group A and 137 in B. Group A had a 3-year OS and EFS of 91·8% and 84·8% respectively, while Group B had a 3-year OS of 83·8% (P = 0·003) and EFS of 76·1% (P = 0·008). There was an impact on 3-year OS and EFS of children who received a formulation. This result highlights the importance of evaluating ASNase and monitoring its activity.

Clinical validation of an in-house quantitative real time PCR assay for cytomegalovirus infection using the 1st WHO International Standard in kidney transplant patients.

INTRODUCTION: Cytomegalovirus (CMV) is one of the most common agents of infection in solid organ transplant patients, with significant morbidity and mortality. OBJECTIVE: This study aimed to establish a threshold for initiation of preemptive treatment. In addition, the study compared the performance of antigenemia with qPCR results. STUDY DESIGN: This was a prospective cohort study conducted in 2017 in a single kidney transplant center in Brazil. Clinical validation was performed by comparing in-house qPCR results, against standard of care at that time (Pp65 CMV Antigenemia). ROC curve analysis was performed to determine the ideal threshold for initiation of preemptive therapy based on the qPCR test results. RESULTS: Two hundred and thirty two samples from 30 patients were tested with both antigenemia and qPCR, from which 163 (70.26%) were concordant (Kappa coefficient: 0.435, p<0.001; Spearman correlation: 0.663). PCR allowed for early diagnoses. The median number of days for the first positive result was 50 (range, 24-105) for antigenemia and 42 (range, 24-74) for qPCR (p<0.001). ROC curve analysis revealed that at a threshold of 3,430 IU/mL (Log 3.54), qPCR had a sensitivity of 97.06% and a specificity of 74.24% (AUC 0.92617 ± 0.0185, p<0.001), in the prediction of 10 cells/105 leukocytes by antigenemia and physician's decision to treat. CONCLUSIONS: CMV Pp65 antigenemia and CMV qPCR showed fair agreement and a moderate correlation in this study. The in-house qPCR was revealed to be an accurate method to determine CMV DNAemia in kidney transplant patients, resulting in positive results weeks before antigenemia.

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.

Implementation of the asparaginase activity assessment technique for clinical use: experience of a Brazilian Center.

Acute lymphoid leukemia is a childhood cancer that in high-income countries has event-free survival rates of 80% and global survival rates of 90%. In Brazil these rates are under 70%. This difference may be due to the implementation of supportive care, including the assessment of asparaginase (ASNase) activity. ASNase may cause hypersensitivity reactions and silent drug inactivation. For this reason, ASNase activity monitoring is an essential tool to ensure an effective treatment. Our aim was to implement an ASNase activity measurement technique at a hospital setting. samples from children who were given Escherichia coli-derived ASNase were collected. The results of the analyses conducted in our laboratory Hospital de Clínicas de Porto Alegre were compared to those of two institutions: Centro Infantil Boldrini and University of Munster. 262 samples were assessed. The results of the first analyses were compared with those obtained at Centro Infantil Boldrini and showed an ICC of 0.954. Thirty samples were sent to the University of Munster and presented an ICC was 0.960. Our results, when compared to those of national and international centers, showed an excellent agreement. The study was able to implement an ASNase activity test to monitor the treatment.

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.

Comparison of a direct immunofluorescence assay (Oxoid IMAGEN®) and a multiplex RT-PCR DNA microarray assay (CLART® PneumoVir) for the detection of respiratory viruses in hospitalized children.

The objective of this study was to compare the positive detection rates obtained using the Oxoid IMAGEN® direct immunofluorescence assay (designated as IF) with those obtained using the CLART® PneumoVir multiplex RT-PCR DNA microarray assay (designated as RT-PCR) in the diagnosis of respiratory viruses in hospitalized children. This was a retrospective study of 62 individuals < 18 years old who had nasopharyngeal aspirates collected for virus identification in a tertiary university hospital in south Brazil between January 1st, 2014 and December 31st, 2014. All 62 nasopharingeal aspirates were analyzed using both assay methods. The main outcome to be measured was the difference in the proportion of test samples returning a positive virus detection result between the IF and the RT-PCR. The McNemar test was used for data analysis and the results showed that the RT-PCR and the IF methods produced 55 (88.7 %) and 17 (27.4 %) virus-positive samples, respectively (p < 0.001). The most prevalent virus was rhinovirus (45.5 % of the RT-PCR positive samples). The RT-PCR method increased the detection rates of human respiratory syncytial virus, influenza A virus and parainfluenza 3 virus. The RT-PCR and IF had concordant results in 19 samples (30.6 %) and discordant results in 43 samples (69.4 %). It is concluded that in comparison to the Oxoid IMAGEN® IF method, the CLART® PneumoVir multiplex RT-PCR method had a greater potential to contribute to the clinical management of hospitalized children due its greater ability in detecting respiratory viruses than the IF method.

Cost minimization analysis of an in-house molecular test for cytomegalovirus in relation to a commercial molecular system

ABSTRACT Introduction: Cytomegalovirus may cause severe disease in immunocompromised patients. Nowadays, quantitative polymerase chain reaction is the gold-standard for both diagnosis and monitoring of cytomegalovirus infection. Most of these assays use cytomegalovirus automated molecular kits which are expensive and therefore not an option for small laboratories, particularly in the developing world. Objective: This study aimed to optimize and validate an in-house cytomegalovirus quantitative polymerase chain reaction test calibrated using the World Health Organization Standards, and to perform a cost-minimization analysis, in comparison to a commercial cytomegalovirus quantitative polymerase chain reaction test. Study design: The methodology consisted of determining: optimization, analytical sensitivity, analytical specificity, precision, curve variability analysis, and inter-laboratorial reproducibility. Patients (n = 30) with known results for cytomegalovirus tested with m2000 RealTime System (Abbott Laboratories, BR) were tested with the in-house assay, as well as patients infected with other human herpes virus, in addition to BK virus. A cost-minimization analysis was performed, from a perspective of the laboratory, assuming diagnostic equivalence of the methodologies applied in the study. Results: The in-house assay had a limit of detection and quantification of 60.3 IU/mL, with no cross-reactivity with the other viral agents tested. Moreover, the test was precise and had a R 2 of 0.954 when compared with the m2000 equipment. The cost analysis showed that the assay was economically advantageous costing a median value of 37.8% and 82.2% in comparison to the molecular test in use at the hospital and the m2000 equipment, respectively. Conclusions: These results demonstrated that in-house quantitative polymerase chain reaction testing is an attractive alternative in comparison to automated molecular platforms, being considerably less expensive and as efficacious as the commercial methods.

Cost minimization analysis of an in-house molecular test for cytomegalovirus in relation to a commercial molecular system.

INTRODUCTION: Cytomegalovirus may cause severe disease in immunocompromised patients. Nowadays, quantitative polymerase chain reaction is the gold-standard for both diagnosis and monitoring of cytomegalovirus infection. Most of these assays use cytomegalovirus automated molecular kits which are expensive and therefore not an option for small laboratories, particularly in the developing world. OBJECTIVE: This study aimed to optimize and validate an in-house cytomegalovirus quantitative polymerase chain reaction test calibrated using the World Health Organization Standards, and to perform a cost-minimization analysis, in comparison to a commercial cytomegalovirus quantitative polymerase chain reaction test. STUDY DESIGN: The methodology consisted of determining: optimization, analytical sensitivity, analytical specificity, precision, curve variability analysis, and inter-laboratorial reproducibility. Patients (n=30) with known results for cytomegalovirus tested with m2000 RealTime System (Abbott Laboratories, BR) were tested with the in-house assay, as well as patients infected with other human herpes virus, in addition to BK virus. A cost-minimization analysis was performed, from a perspective of the laboratory, assuming diagnostic equivalence of the methodologies applied in the study. RESULTS: The in-house assay had a limit of detection and quantification of 60.3IU/mL, with no cross-reactivity with the other viral agents tested. Moreover, the test was precise and had a R2 of 0.954 when compared with the m2000 equipment. The cost analysis showed that the assay was economically advantageous costing a median value of 37.8% and 82.2% in comparison to the molecular test in use at the hospital and the m2000 equipment, respectively. CONCLUSIONS: These results demonstrated that in-house quantitative polymerase chain reaction testing is an attractive alternative in comparison to automated molecular platforms, being considerably less expensive and as efficacious as the commercial methods.

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.