Natália Aydos Marcondes

Prevalence of low bone mineral density in adolescents and adults with cystic fibrosis

OBJECTIVE The aim of this cross-sectional study was to evaluate the prevalence of low bone mass density in cystic fibrosis patients as well as to evaluate the factors associated with bone mass in such patients. METHODS Bone mass density was measured by dual-photon X-ray absorptiometry of lumbar spine (L1-L4), in patients ≤19 years old, or lumbar spine and femur (total and neck) in patients ≥20 years old. Evaluations of nutritional status, biochemical parameters, and lung function were performed. Medication data were obtained from medical records. RESULTS Fifty-eight patients were included in the study (25 males/ 33 females), mean age 23.9 years (16-53 years). The prevalence of bone mass below the expected range for age at any site was 20.7%. None of the subjects had history of fracture. Lumbar spine Z-score in cystic fibrosis patients correlated positively with body mass index (r= 0.3, p=0.001), and forced expiratory volume in the first second (% predicted) (r=0.415, p=0.022). Mean lumbar spine Z-score was higher in women (p=0.001), in patients with no pancreatic insufficiency (p=0.032), and in patients with no hospitalization in the last 3 months (p=0.02). After multivariate analysis, body mass index (p= 0.001) and sex (p=0.001) were independently associated with Z-score in lumbar spine. CONCLUSION Low bone mass is a frequent problem in patients with CF, being independently associated with body mass index, and male sex.

Quantitative flow cytometric evaluation of CD200, CD123, CD43 and CD52 as a tool for the differential diagnosis of mature B-cell neoplasms

Background Distinction between mature B-cell neoplasms can be difficult due to overlapping of immunologic features and clinical manifestations. This study investigated whether quantifying mean fluorescence intensity of four monoclonal antibodies in a flow cytometry panel is useful for the differential diagnosis and characterization of these disorders. Methods The expressions of CD52, CD200, CD123 and CD43 were analyzed in samples from 124 patients with mature B-cell neoplasms. The quantitative estimation of these antigens was assessed by mean fluorescence intensity. Results The cases included were 78 chronic lymphocytic leukemias, three atypical chronic lymphocytic leukemias, six marginal zone lymphomas, 11 splenic marginal zone lymphomas, nine lymphoplasmacytic lymphomas, six mantle cell lymphomas, two hairy cell leukemias, two hairy cell leukemias variant, five follicular lymphomas, one Burkitt lymphoma and one diffuse large B-cell lymphoma. The mean fluorescence intensity of CD200 was higher in atypical chronic lymphocytic leukemia, chronic lymphocytic leukemia and hairy cell leukemia cases. CD123 showed higher mean fluorescence intensities in hairy cell leukemia cells. Chronic lymphocytic leukemia, atypical chronic lymphocytic leukemia and mantle cell lymphoma had higher expression of CD43 and all follicular lymphoma cases had very low mean fluorescence intensity values. CD52 expression was consistently positive among all cases. Conclusion Quantitative evaluation of these markers can be a useful additional tool to better identify some types of mature B-cell neoplasms.

Expression of Bruton’s tyrosine kinase in B-cell neoplasms evaluated by flow cytometry

Bruton’s tyrosine kinase (BTK) is a cytoplasmatic protein that is part of the B-cell antigen receptor signaling pathway. Our aim was to evaluate the expression of BTK in B-cell neoplasms and compare it to normal B-cell lymphocytes. After surface staining with CD19 and CD45, flow cytometry staining for intracellular BTK was performed in leukemic or mature B-cells from bone marrow or peripheral blood samples. No differences in BTK expression were identified between groups, or in comparison to control samples, there was no association between BTK expression and the clinical variables evaluated. BTK expression in B-cell neoplasms was similar to that of normal B-cell lymphocytes.

Lineage determination in acute leukemias: Letter to the Editor

We read with great interest the case study interpretation section (CSI) in the last issue of Cytometry Part B. We perform flow cytometry tests and it is always constructive to challenge our analysis and interpretation skills. Case 2 of the CSI, reported by Weina Chen (1), describes a case of an acute leukemia diagnosed as mixed phenotype leukemia T/myeloid. According to the World Health Organization (WHO), myeloperoxidase (MPO) or at least two markers of monocytic differentiation, are required to assign the lineage of a blast population as myeloid, although it does not establishes a minimum percent of myeloid population for the leukemia to be considered of mixed phenotype (2). In the immunophenotypic analysis of the case only 3% of the blasts expressed cytoplasmatic MPO (cMPO). This small subset of blasts could represent normal myeloid progenitor cells since this population was in the compartment where it would be expected to have nonmalignant myeloid blasts (3), and there was no evidence of cytoplasmatic CD3 coexpression in the FCS files. Also, in the lymph node biopsy, the cells largely lacked MPO and it could have been expressed in myeloid cells instead of blasts. As suggested recently, a threshold of 10% for cMPO staining detected by flow cytometry in blast cells would be a secure lower limit for the expression of this marker and subsequent classification of the leukemia as myeloid lineage (3). We believe that a 10% positivity for flow cytometric analysis would be a good choice for the classification of the leukemia as mixed phenotype as well (4). Although mixed phenotype acute leukemias are rare, more studies are needed in order to make possible the definition of a minimum percentage of expression for lineage determination. These objective evidences are important to define more precise criteria in the next edition of WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues.

Hypovitaminosis D in patients with cystic fibrosis: a cross-section study in South Brazil.

Cystic fibrosis (CF) patients have a susceptibility to vitamin D deficiency because of nutrient malabsorption.

Ki-67 expression in mature B-cell neoplasms: a flow cytometry study

OBJECTIVE Ki-67 is a nuclear protein associated with cellular proliferation in normal or leukemic conditions that can help identify more aggressive diseases and is usually evaluated with immunohistochemistry. The aim of this was to assess Ki-67 expression on mature B-cell neoplasms samples with flow cytometry immunophenotyping. METHOD After surface staining with CD19 and CD45, intracellular staining for Ki-67 was performed in leukemic mature B-cells. Ki-67 expression was evaluated with flow cytometry. RESULTS Ki-67 expression was higher in mantle cell lymphoma, Burkitt lymphoma, and diffuse large B-cell lymphoma cases. It was also associated with CD38 mean fluorescence intensity. CONCLUSIONS Ki-67 expression evaluated by flow cytometry can be a useful tool in the diagnosis of mature B-cell neoplasms. More studies are needed to validate Ki-67 assessment with flow cytometry immunophenotyping.

Polyclonal B-cell lymphocytosis: Report of three cases: POLYCLONAL B-CELL LYMPHOCYTOSIS

Persistent polyclonal B‐cell lymphocytosis (PPBL) is a rare benign condition characterized by a polyclonal B‐cell lymphocytosis with binucleated lymphocytes. We hereby report three cases of PPBL.

Expression of neutrophil surface markers in icteric neonates before and after phototherapy: NEUTROPHIL SURFACE MARKERS IN ICTERIC NEONATES

Jaundice due to indirect hyperbilirubinemia affects more than 60% of neonates and phototherapy is the treatment for severe types. There are no previous studies evaluating the effect of phototherapy on the function of neonates neutrophils. The aim of this study was to assess and compare the function of neutrophils by measuring the expression of neutrophils main surface markers in icteric neonates before and after phototherapy.

Flow cytometry assessment of intracellular BTK expression

Dear Editor, We read with great interest the original article “Increased expression of Bruton’s tyrosine kinase in peripheral blood is associated with lupus nephritis” recently published in Clinical Rheumatology (1). In this paper, Kong et al. (2017) evaluated Bruton’s tyrosine kinase (BTK) expression in peripheral blood B-cells from healthy controls and systemic lupus erythematosus (SLE) patients. The authors report an increased BTK expression in Bcells from SLE patients compared to controls, nevertheless only 2–4% of peripheral B-cells showed BTK expression in the study. Nisitani et al. (2000) used flow cytometry to evaluate BTK expression in developing hematopoietic cells, the protein expression was detected in all lineages except for T cells, with higher expression in immature B-cells and a lower expression in B-cells from peripheral sites (2). Wang et al. (2015) assessed BTK expression in B-cells from rheumatoid arthritis patients compared to control subjects with flow cytometry and no differences between groups were identified (3). Most recently, Corneth et al. (2017) evaluated BTK expression in B-cells from healthy controls, rheumatoid arthritis patients, and primary Sj€ ogren’s syndrome patients, where they showed that BTK protein expression correlated with BTK phosphorylation and also that BKT levels were enhanced in most patients with autoimmune diseases diseases (4). We also evaluated BTK expression in B-cells from healthy controls compared to mature B-cell neoplasms (5). In our study, there were no statistical differences among B-cells from evaluated groups and BTK expression in CD19/ CD45 lymphocytes was within the negative control range. In our validation protocol, we tested the same FIX&PERMVR Cell Fixation and Permeabilization Kit (product code GAS-002, Nordic-MUbio, NED) for intracellular staining, but the staining was unsatisfactory and the mean fluorescence intensity (MFI) of BTK in B-cells was too weak. We selected and validated the Transcription Factor Buffer Set (product code 562574, BD Pharmingen, USA), which showed a BTK staining that was not very bright, but allowed an appropriate distinction among positive and negative events. This limitation of the staining kit could be responsible for the very low BTK expressing B-cells identified in both groups, with expression levels near the isotype control in the study by Kong et al. Despite the very low MFI of BTK staining, it is possible to identify a difference in the protein expression as shown in figure 1 (1), but this results should be cautiously interpreted with the understanding of the limitations imposed by the staining technique. Flow cytometry staining protocols should allow the distinction between negative and positive populations, as well as a quantifiable MFI, in order to enable comparison of results among different studies.

Comparison of JC-1 and MitoTracker probes for mitochondrial viability assessment in stored canine platelet concentrates: A flow cytometry study: Mitochondrial Viability in Stored Platelets

Mitochondria perform crucial roles in many biochemical processes, and mitochondrial depolarization is an early sign of platelet apoptosis. The mitochondrial membrane potential is usually evaluated through JC‐1 probe, but it can also be assessed with MitoTracker probes. Our aim was to evaluate mitochondrial viability in stored canine platelet concentrates (PCs) with the fluorescent probes JC‐1 and MitoTracker. Platelets from 22 canine PCs were stained with JC‐1 and MitoTracker probes on days 1, 3, and 5 of storage. Data on metabolic parameters were also collected for correlation studies. Results of JC‐1 and MitoTracker revealed a decrease in mitochondrial membrane potential in day 5 of storage compared to days 1 and 3, providing evidence of mitochondrial depolarization, a finding that was confirmed by the data on metabolic parameters. MitoTracker probes also added information regarding platelet swelling. In conclusion, MitoTracker probes offered a more complete mitochondrial analysis in the evaluation of stored canine PCs.