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Summary: 5‐Azacitidine has been used before stem cell transplantation in juvenile myelomonocytic leukaemia (JMML) patients. Recently, we have described immunophenotypic features in JMML at diagnosis. Here, our aim was to examine the changes in the immunophenotypic features during azacitidine treatment, correlating it with clinical response. Patients treated with 5‐azacitidine were evaluated at diagnosis and after three and six cycles of medication. Among 32 patients entering the study, 28 patients were examined after three cycles and 25 patients after six. Patients showed a reduction in CD34/CD117+ cells: median 3.35% at diagnosis, 2.8% after three cycles and 1.63% after six. B‐cell progenitors were decreased at diagnosis and decreased after treatment. Monocytes decreased: 11.91% to 6.4% and 4.18% respectively. Complete response was associated with increase in classical monocytes. T lymphocytes, reduced at diagnosis, increased in patients responding to 5‐azacitidine. Immunophenotypic aberrancies including expression of CD7 in myeloid progenitors remained after treatment. This feature was associated with a worse response to treatment, as well as presence of NF1. Immunophenotyping was feasible in all patients. Clinical response was associated with a decrease of myeloid progenitors and monocytes and a rise in T lymphocytes although phenotypic aberrancies persisted. The largest effect was observed after three cycles. [ABSTRACT FROM AUTHOR]

Fluorescence lifetime imaging (FLIM) has been used in living cells to measure metabolic activity and demonstrate cell differentiation. The aim of this study was to investigate whether the FLIM technique could be able to demonstrate cell maturation during myelopoiesis and erythropoiesis in unlabeled routine bone marrow (BM) preparations. Air‐dried, unstained smears of BM aspiration samples of 32 patients without BM disease and a normal morphology on May–Grünwald–Giemsa (MGG) stained smears entered the study. FLIM images were captured with a Zeiss LSM 780 NLO multiphoton microscope equipped with a Becker & Hickl SPC‐830 TCSPC FLIM module and HPM‐100‐40 hybrid detector. The samples were irradiated by two‐photon excitation at 800 nm with a titanium‐sapphire laser of the LSM 780 NLO. FLIM images were compared with those obtained by autofluorescence high resolution imaging. FLIM images of unstained smears were highly contrasted. Different cell types could be easily recognized as they were similar to those seen in MGG stained preparations. Cytoplasm of cells from the erythroid lineage revealed relatively short fluorescence lifetimes due to the presence of hemoglobin, and therefore could easily be distinguished from granulocytic precursors. Nuclear fluorescence lifetimes of all cell types were higher than those of the corresponding cytoplasm. So, FLIM of unstained BM smears obtained under routine real‐life conditions permits an easy identification of BM cells, by highlighting differences of their physicochemical properties. [ABSTRACT FROM AUTHOR]

The diagnosis of juvenile myelomonocytic leukaemia (JMML) is based on clinical, laboratory and molecular features but immunophenotyping [multiparametric flow cytometry (MFC)] has not been used routinely. In the present study, we describe the flow cytometric features at diagnosis with special attention to the distribution of monocytic subsets and the relation between MFC and molecular subgroups. MFC was performed with an eight‐colour platform based on Euroflow. We studied 33 JMML cases. CD34+/CD117+/CD13+ cells >2% was found in 25 cases, and 51·5% presented an aberrant expression of CD7. A decrease of CD34+/CD19+/CD10+ cells was seen in eight cases and in four they were absent. The granulocytic population had a decreased side scatter in 29 cases. Bone marrow monocytic precursors were increased in 28 patients, with a decrease in classical monocytes (median 80·7%) and increase in CD16+ (intermediate and non‐classical). A more pronounced increase in myeloid CD34+ cells was seen in patients with Neurofibromatosis type 1 (NF1) and tyrosine‐protein phosphatase non‐receptor type 11 (PTPN11), with aberrant CD7 expression in four of six and 10/12 patients respectively. Thus, JMML shows an immunophenotypic profile similar to myelodysplastic syndromes, and a different monocyte subset distribution when compared with chronic MML. MFC proved to be an important diagnostic tool that can help in differential diagnosis with other clonal diseases with monocytosis. [ABSTRACT FROM AUTHOR]

Introduction: Flow cytometry (FC) is a helpful tool for the diagnosis of myelodysplastic syndrome (MDS). Different FC score systems have been developed. The "Ogata score" is a simple diagnostic score that has been validated having a sensitivity of 69% and a specificity of 92% in low‐risk MDS. We aimed to study the feasibility and the utility of the "Ogata score" for the diagnosis of MDS among Latin America (LA) Laboratories. Methods: This is a case and control study conducted in LA institutions members of Grupo Latinoamericano de Mielodisplasia (GLAM). A total of 146 MDS patients and 57 control patients were included. "Ogata score" was calculated. Results: The sensitivity of "Ogata score" was 75.6% (95% CI, 66.8‐81.3), specificity was 91.2% (95% CI, 79.7‐96.7), PPV was 95.6% (95% CI, 88.5‐98.3), and NPV was 65.4% (95% CI, 49.1‐71.9). In low/intermediate‐1 IPSS patients group, the sensitivity was 70.1% (95% CI, 60.2‐78.2), specificity was 91.2% (CI‐95%, 79.7‐96.7), PPV was 94.2% (95% CI, 86.4‐97.8), and NPV was 62.1% (95% CI, 53.0‐78.7). In the group of patients "without MDS specific markers" (patients without ring sideroblasts, blast excess, or chromosomal abnormalities), the sensitivity was 66.7% (CI‐95%, 55.8‐76.0), specificity was 91.2% (95% CI, 79.7‐96.7), PPV was 92.3% (95% CI, 82.2‐97.1), and NPV was 63.5% (95% CI, 51.9‐73.5). Conclusions: The diagnostic power found in this study was similar to the reported by Della‐Porta et al. Also in LA, the analysis was made in modern equipment with acquisition of at least 100 000 events which permits a good reproducibility of the results. [ABSTRACT FROM AUTHOR]

Our aim was to evaluate the cost‐effectiveness of a minimal lymphocyte subset quantification (LSQ) by flow cytometry as the first screening in children with clinically suspected primary immunodeficiency (PID). Two hundred sixty‐eight Brazilian patients (0–21 years old) were studied. They were divided by clinical and phenotypical features into those fulfilling criteria for PID (PID phenotype) according to the 2017 International Union of Immunological Societies (IUIS) classification and those not fulfilling these criteria (non‐PID phenotype). We evaluated how many patients had values below the 10th percentile for five lymphocyte subsets in peripheral blood, (suggestive of PID) according to reference values for Brazil, Italy and USA. Three lymphocyte subsets (T CD3/CD4, B CD19 and NK CD16/CD56) had p‐value < 0.05 and Odds Ratio (OR) indicating a risk at least two times higher for the diagnosis of a PID phenotype. The application of Kappa coefficient (k) on Brazilian vs Italian and Brazilian vs US data sets resulted in k compatible with strong or excellent level of agreement between the three classification systems. The authors conclude that a number of CD3+/CD4+, CD19+ and CD16+/CD56+ (NK) cells in peripheral blood <10th percentile represented a significant risk for the diagnosis of PID in this cohort. Natural killer (NK) deficiency is quite rare and has a very specific clinical profile. So, the analysis of these cells could be requested only in some cases, saving even more costs. The minimal immunophenotyping, with quantification of T CD4+, CD19+ and in some cases CD16+/CD56+ cells, may be a useful tool for the first screening of PID, saving costs, especially in developing countries. [ABSTRACT FROM AUTHOR]

Aims. To develop a fast and robust DNA-based assay to detect insertions and deletions mutations in exon 34 that encodes the PEST domain of NOTCH1 in order to evaluate patients with chronic lymphocytic leukemia (CLL). Methods. We designed a multiplexed allele-specific polymerase chain reaction (PCR) combined with a fragment analysis assay to detect specifically the mutation c.7544_7545delCT and possibly other insertions and deletions in exon 34 of NOTCH1. Results. We evaluated our assay in peripheral blood samples from two cohorts of patients with CLL. The frequency of NOTCH1 mutations was 8.4% in the first cohort of 71 unselected CLL patients. We then evaluated a second cohort of 26 CLL patients with known cytogenetic abnormalities that were enriched for patients with trisomy 12. NOTCH1 mutations were detected in 43.7% of the patients with trisomy 12. Conclusions. We have developed a fast and robust assay combining allele-specific PCR and fragment analysis able to detect NOTCH1 PEST domain insertions and deletions. [ABSTRACT FROM AUTHOR]

TET2, a member of the ten-eleven-translocation (TET) family genes that modify DNA by converting 5-methylcytosine (5- mC) to 5-hydroxymethylcytosine (5-hmC), is located in chromosome 4q24 and is frequently mutated in myeloid malignancies. The impact of TET2 mutation on survival outcomes is still controversial; however, functional studies have proved that it is a loss-of-function mutation that impairs myeloid cell differentiation and contributes to the phenotype of myeloid neoplasia. We, herein, aimed to investigate TET2 expression in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A significantly decreased TET2 expression was observed in bone marrow cells from AML ( n = 53) and patients with MDS ( n = 64), compared to normal donors ( n = 22). In MDS, TET2 expression was significantly reduced in RAEB-1/RAEB-2 compared to other WHO 2008 classifications, and a lower TET2 expression was observed at the time of MDS disease progression in four of five patients. In multivariate analysis, low TET2 expression ( P = 0.03), male gender ( P = 0.02), and WHO 2008 classification ( P < 0.0001) were independent predictors of poorer overall survival. These results suggest that defective TET2 expression plays a role in the MDS pathophysiology and predicts survival outcomes in this disease. [ABSTRACT FROM AUTHOR]

Background Umbilical cord blood ( UCB) is a good source of hematopoietic stem cells for transplantation and cell therapy. In 2006, the Brazilian Public Network of Cord Blood Banks was founded; however, because our country is large, logistic problems could hamper the collection of numerous samples. Our aim was to evaluate the viability of several UCB cell subsets until 96 hours after collection, to examine whether this delay would be acceptable for processing and freezing the samples. Study Design and Methods Two experiments were performed: in the first one, volume reduction of the UCB units was carried out before analysis. In the second one, analysis was carried out with no previous manipulation. Samples were stored at room temperature and one aliquot was taken daily for analysis. We examined CD34+ cell, B-cell precursor, mature B and T lymphocyte, monocyte, granulocyte, and mesenchymal stem cell ( MSCs) concentrations. Results Thirty-six UCB units were analyzed. CD34+ cells and mature T lymphocytes increased (viability 99%). Mature B lymphocytes and MSCs decreased, maintaining viability. Granulocytes decreased with loss of viability. Monocytes and immature B lymphocytes remained stable. Clonogenic assays showed a decrease in colony-forming unit ( CFU) number in UCB units stored for 96 hours. Conclusion UCB manipulation did not influence cell viability. All cell subsets remained viable until 96 hours after collection. CD34+ cells and T lymphocytes increased, probably due to the loss of other subsets. CFU growth during the period analyzed and confirmed stem cell functionality, despite the decrease at 96 hours. Results demonstrated that UCB units could probably be processed up to 96 hours after collection. [ABSTRACT FROM AUTHOR]

Objectives: APAF-1 is a central component of the intrinsic pathway of apoptosis, where APAF-1 dysregulation results in the development of diverse human neoplasms. The aim of this study was to characterize the mRNA expression levels of APAF-1 transcripts in low-risk and high-risk MDS and to elucidate whether the expression levels of APAF-1 transcripts are modulated with increased apoptosis in CD34+ MDS cells undergoing erythroid differentiation. Methods: APAF-1 ( ) expression was verified, by quantitative RT-PCR, in bone marrow aspirates from 33 patients with myelodysplastic syndromes (MDS), at the time of diagnosis, and in erythroid differentiation cultures from CD34+ from normal donors and patients with MDS. Results: APAF-1 expression was significantly higher in low-risk, compared to high-risk MDS, according to IPSS ( P < 0.0001), FAB ( P = 0.0265), and cytogenetic risk ( P = 0.0134). Low-risk MDS-derived differentiated erythroid cells demonstrated an increased expression of APAF-1, compared with normal cells, accompanied by an augmented rate of apoptosis. Conclusions: Increased expression of APAF-1 in low-risk disease and its positive correlation with the apoptotic rate observed during the erythroblast differentiation of low-risk MDS cells may indicate that the modulation of APAF-1, at the transcriptional level, participates in the pathophysiology of MDS. [ABSTRACT FROM AUTHOR]

BACKGROUND: Optimization of peripheral blood stem cell (PBSC) collection for autologous bone marrow transplantation is necessary for a good standard of care and cost-effectiveness. An algorithm was validated for prediction of the day of maximum peripheral CD34+ cell concentration after mobilization chemotherapy (DayCD34peak). STUDY DESIGN AND METHODS: This study compared mobilization and collection variables of a cohort of patients where apheresis was started at the DayCD34peak predicted by the algorithm with a patient group where PBSCs were collected when PB CD34+ cell concentration reached 10 per µL per day (DayCD34threshold). DayCD34peak was calculated according to the equation DayCD34peak = −0.41 × HbD0 + 0.99 × DayCD34threshold + 7.8 (with HbD0 representing the hemoglobin value on Day 0). RESULTS: The mean number of apheresis procedures per patient based on the DayCD34threshold was 1.74, but decreased to 1.35 when applying the new method (DayCD34peak). For lymphomas, the mean number of apheresis procedures decreased from 1.98 to 1.47 (p = 0.03), while in patients with multiple myeloma it did not change significantly (1.23 and 1.26, respectively). Age and primary disease influenced the number of apheresis procedures needed to achieve the collection target. CONCLUSION: The application of our algorithm can lower the number of apheresis procedures by improving the timing, especially in patients suffering from malignant lymphomas with a poor marrow potential after several chemotherapy lines. [ABSTRACT FROM AUTHOR]

The fractal nature of the DNA arrangement has been postulated to be a common feature of all cell nuclei. We investigated the prognostic importance of the fractal dimension (FD) of chromatin in blasts of patients with acute precursor B lymphoblastic leukemia (B-ALL). In 28 patients, gray scale transformed pseudo-3D images of 100 nuclei (May–Grünwald–Giemsa stained bone marrow smears) were analyzed. FD was determined by the Minkowski–Bouligand method extended to three dimensions. Goodness-of-fit of FD was estimated by the R2 values in the log-log plots. Whereas FD presented no prognostic relevance, patients with higher R2 values showed a prolonged survival. White blood cell count (WBC), age and mean fluorescence intensity of CD45 (MFICD45) were all unfavorable prognostic factors in univariate analyses. In a multivariate Cox-regression, R2, WBC, and MFICD45, entered the final model, which showed to be stable in a bootstrap resampling study. Blasts with lower R2 values, equivalent to accentuated “coarseness” of the chromatin pattern, which may reflect profound changes of the DNA methylation, indicated a poor prognosis. In conclusion the goodness-of-fit of the Minkowski–Bouligand dimension of chromatin can be regarded as a new and biologically relevant prognostic factor for patients with B-ALL. [ABSTRACT FROM AUTHOR]

In malignant lymphomas, cell kinetics has shown to be related with histologic type as well as with the clinical behaviour. The aim of our study was to investigate the relevance of cell proliferation parameters on overall survival in non‐Hodgkin's lymphomas as well as their relationship with prognostic factors such as International Prognostic Index (IPI). We performed DNA‐flow‐cytometry (S‐phase fraction and detection of DNA‐aneuploidy) as well as cytologic examination and the AgNOR technique in material obtained by fine needle aspiration of lymph nodes at diagnosis. The majority of the patients were stage IV by Ann Arbor and intermediate risk by IPI (42/55). When analyzing all patients together, histologic type by the WHO classification, IPI and the presence of a DNA‐aneuploid clone could not separate well patients with a different survival. For all patients, univariate Cox analysis revealed S‐phase (SPF) and AgNOR parameters to be of prognostic value. In the multivariate analysis, however, only SPF remained in the final model. Yet, when stratifying for DNA‐ploidy, only the total number of AgNORs/nucleus was an independent parameter. Looking only at the DNA‐diploid cases, the AgNOR pattern remained the most important parameter, whereas for the DNA‐aneuploid cases this was true for SPF. When studying patients with B large cell lymphoma separately, only DNA‐ploidy was a prognostic factor. In summary, cell kinetic parameters reveal important prognostic information in NHL patients. Furthermore, DNA‐aneuploidy seems to interfere with the analysis of the AgNOR pattern. [ABSTRACT FROM AUTHOR]

The frequency of the hybrid Vγ/Jβ trans-rearrangement in peripheral blood lymphocytes (PBLs) was analysed in a transversal study of paediatric patients (n = 210) with acute lymphoblastic leukaemia (ALL) and solid tumours (ST). Different amounts of DNA were used as the template for a nested polymerase chain reaction to evaluate the frequency of hybrid Vγ/Jβ genes, using silver-stained gels. The frequency of the rearrangement was evaluated in groups before, during and after therapy. A greatly increased frequency of Vγ/Jβ trans-rearrangement was found in PBLs of both groups of patients during exposure to chemotherapeutic agents compared with patients before chemotherapy. In patients who had finished treatment, the frequency of the rearrangement fell promptly to the baseline levels in ST but showed a slow decrease in ALL in those in whom increased levels could be found until 4 years after the end of treatment. We hypothesize that the chemotherapeutic agents are able to induce the Vγ/Jβ trans-rearrangement, but this is transient in most cases. The exact relationship between the persistence of the rearrangement and the occurrence of secondary leukaemia remains to be determined. [ABSTRACT FROM AUTHOR]

We analyzed the outcome of a series of 19 newly diagnosed patients with acute promyelocytic leukemia treated with AIDA modified protocol, using mitoxantrone in place of idarubicin. Eleven patients achieved morphologic CR (58%). The remaining 8 patients had induction failure due to death during induction. Ten of eleven patients in CR achieved molecular remission after induction therapy and all the 8 patients had molecular remission after consolidation. Eight patients completed the three consolidation courses as scheduled and then proceeded to maintenance therapy. After a median follow up of 52 months, no molecular or hematological relapse has occurred. The 4-year disease-free survival is 82%. The study showed the antileukemic efficacy of mitoxantrone and that it could be used as a reasonable option in anthracycline-based strategies in APL. [ABSTRACT FROM AUTHOR]

Chédiak-Higashi syndrome approached by several different microscopy imaging technologies A 16-year-old girl with a previous diagnosis of Chédiak-Higashi syndrome was admitted with a high fever. It was not possible to detect any internal heterogeneity either by conventional light microscopy or by Airy-scanned super-resolution microscopy. [Extracted from the article]

Presents a letter to the editor regarding a spontaneous apoptosis in chronic lymphocytic leukemia.

A letter to the editor is presented in response to goodness-of-fit of the fractal dimension as a prognostic factor.

Examines the relationship between histopathological findings and phylogenetic divergence in Trypanosoma cruzi. Variation of the cardiac and brain tropism of T. cruzi; Differences of the clinical features of patients with Chagas' disease; Establishment of histological findings of patients with recrudescence of Chagas' disease.

Abstract: Objectives: Glutathione S-transferases (GST) modulate the effects of exposure to various cytotoxic and genotoxic agents, including those associated with increased risks of the myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML) and aplastic anemia (AA). Both the GST mu 1 (GSTM1) and GST theta 1 (GSTT1) genes have a null variant allele in which the entire gene is absent. In this study, we tested whether null genotypes for the GSTM1 and GSTT1 genes altered the risks for MDS, AML and AA. Methods: Genomic DNA from 49 MDS, 38 AML and 37 AA patients and 276 controls was analysed using the polymerase chain reaction (PCR). Results: The frequencies of GSTM1 (73.6%) and GSTT1 (34.2%) null genotypes were significantly higher in AML patients than in the controls (36.9 and 18.1%, respectively). A higher frequency of the combined null genotype for both genes was also observed in patients with AML (26.3% compared with 5.0% in the controls). In contrast, no differences in the frequencies of the null genotypes were found among MDS patients, AA patients and the controls. Conclusion: Our observation of a 4.7-fold (95% CI: 2.1–11.0) and 2.3-fold (95% CI: 1.0–5.2) increased risk associated with the GSTM1 and GSTT1 null genotypes, respectively, and a 6.6-fold (95% CI: 2.4–7.9) increased risk associated with the combined null genotype presents preliminary evidence that the inherited absence of this carcinogen detoxification pathway may be an important determinant of AML. [ABSTRACT FROM AUTHOR]