Mariester Malvezzi

Normal patterns of expression of glycosylphosphatidylinositol-anchored proteins on different subsets of peripheral blood cells: a frame of reference for the diagnosis of paroxysmal nocturnal hemoglobinuria.

Evaluation of the expression of glycosylphosphatidylinositol‐anchored membrane proteins (GPI‐AP) is currently used for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). In this study, we analyzed the amount of expression of a wide variety of GPI‐AP in different subsets of hematopoietic cells present in normal peripheral blood (PB), to establish their normal patterns of expression and provide a frame of reference for the definition of the best combination of GPI‐AP and PB cell subsets to be applied in the diagnosis and monitoring of PNH.

Evaluation of Bone Marrow Mesenchymal Stem Cell Standard Cryopreservation Procedure Efficiency

INTRODUCTION Mesenchymal stem cells are obtained from a variety of sources, particularly bone marrow. These cells have great potential for clinical research due to their potential to regenerate tissue. As is well known, the cryopreservation process can store any cell type, particularly blood cells, for an indeterminate time. OBJECTIVE The aim of this study was to analyze the efficiency of standard cryopreservation procedures for adult mesenchymal stem cells from bone marrow. METHODS Mononuclear stem cells isolated from 10 Wistar male rats were cultivated for 4 weeks to obtain mesenchymal stem cells. The parameters considered in this study were trypan blue exclusion test and annexin V conjugated with 7-amino-actinomycin for flow cytometry before cryopreservation in liquid nitrogen vapor phase for 1 month and after thawing. RESULTS The viabilities determined by the trypan blue exclusion test were 94.76% and 90.58%, and the flow cytometry assay (annexin V conjugated with 7-amino-actinomycin) were 85.52% and 66.25%, before cryopreservation and after thawing, respectively. CONCLUSIONS Standard procedures for cryopreservation were not efficient for those cells. The flow cytometry assay was more sensitive than the trypan blue exclusion test to demonstrate nonviability.

Are the review criteria for automated complete blood counts of the International Society of Laboratory Hematology suitable for all hematology laboratories

Objective to verify whether the review criteria for automated blood counts suggested by the International Consensus Group for Hematology Review of the International Society for Laboratory Hematology are suitable for the Hematology Laboratory of Hospital de Clinicas, Universidade Federal do Paraná. Methods initially, the review criteria of the International Society for Laboratory Hematology were adapted due to limitations in the Institutions electronic hospital records and interfacing systems. The adapted review criteria were tested using 1977 samples. After this first assessment, an additional 180 inpatient samples were analyzed to evaluate the screening criteria of the review criteria in conjunction with positive smear findings established by the institution. The performance of the review criteria was verified by determining false positive, false negative, true positive and true negative rates, sensitivity, specificity, positive predictive value, negative predictive value, microscopic review rate and efficiency. Results initial analysis showed false negatives = 6.73%, false positives = 23.27%, microscopic review rate = 46.03% and efficiency = 70.0%. An evaluation of the screening criteria adapted from the review criteria together with the positive smear findings of the institution showed false negatives = 15.5%, false positives = 10.5%, microscopic review rate = 37.3% and efficiency = 73.8%. In both situations the safety limit (false negative <5%) recommended by the review criteria was exceeded. Conclusions the review criteria adapted from the International Society for Laboratory Hematology are neither suitable nor safe for use in the hematology laboratory of the Hospital de Clinicas. This implies a need to develop and validate institution-specific review criteria in order to decrease false negative results to an acceptable and safe rate for patients.

Pap Test as the First Step in Screening Genetic Stability in Cell-BasedTherapy

The possibility of cell modifications compromises the safety of stem cell therapy under standardized conditions. The aim of this study was to analyze adipose tissue-derived mesenchymal stem cells (AT-MSCs) using the Pap test as a first screening step to evaluate genetic stability. Methods: Human adipose tissue from six healthy female donors was obtained from elective liposuction procedures. The cells were isolated, cultivated at P2/P3, characterized by flow cytometric analysis, and differentiation induced. The AT-MSCs were stained by Papanicolaou (Pap) staining and analyzed according to the Bethesda classification, and viability-apoptosis relationships were evaluated. Results: The Pap test for Sample I indicated high-grade alterations consistent with genetic instability; for Samples II-V, atypical cells of undetermined significance; and for Sample VI, normal cells. Conclusions: These results demonstrate the potential of using the Pap test as an initial screening step to evaluate the genetic stability of cultured AT-MSCs as well for other adherent stem cells.

Immunophenotypic Expression by Flow Cytometric Analysis of Cocultured Skeletal Muscle and Bone Marrow Mesenchymal Stem Cells for Therapy Into Myocardium

The product generated by skeletal muscle and bone marrow mesenchymal stem cell cocultures has been demonstrated to improve the functional outcomes after cell therapy in postinfarction or Chagas myocardiopathy. This coculture method allows cell interactions in vitro, diminishing the operational costs of the culture/expansion as well as leading to angiogenesis and myogenesis for regeneration of the injured heart. Flow cytometric analysis may better characterize the cellular types in this model. Our objective was to use flow cytometry to analyze the immunophenotype expressed in this coculture model. The coculture was performed in accordance with Carvalho for 21 days. Flow cytometry was performed before and after coculture to characterize the immunophenotypic profile of cellular subsets, namely, the surface markers CD31, CD34, CD44H, CD45, CD49d, CD54, CD73, CD90, CD105, CD106, Myo-D, M-cadherin, and Connexin-43. Statistics were performed by the nonparametric Friedman test (P < .05) with post-hoc analysis by the nonparametric Wilcoxon test (P < or = .017, Bonferroni correction). The results demonstrated statistical significance for CD45(+) in 89.49% of mononuclear cells, 3.58% in skeletal muscle cells, and 4.74% among cocultured cells (P = .0094); and CD90(+) in 36.18% of mononuclear cells, 6.01% in skeletal muscle cells, and 48.94% among cocultured cells (P = .0420). The cocultured cells expressed the markers CD73(++), CD90(+++), CD45(-), CD34(+), CD105(-/+), CD106(-/+), M-cadherin(-/+), and Connexin-43(-/+). In conclusion, flow cytometric analysis showed a heterogeneous adherent cell population in this coculture model.

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 GPIbα, 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.

Angiogenesis without functional outcome after mononuclear stem cell transplant in a doxorubicin-induced dilated myocardiopathy murine model

Objectives Cell transplantation is considered a novel approach in the treatment of myocardiopathy. The objective of this study was to evaluate the effects of autologous mononuclear stem cell therapy in doxorubicin-induced dilated myocardiopathy by conducting both functional and histopathologic analysis. Methods Seventy male rats were doxorubicin injected intraperitoneally for 2 weeks. At 1 month, the animals that had demonstrated left ventricular ejection fractions less than 40% were randomly divided into a mononuclear stem cell group and controls. Mononuclear stem cells were isolated. All animals underwent echocardiographic study: baseline, pre-cell therapy, and at 1 month post-cell therapy, and analyzed by the nonparametric Mann-Whitney test. Transplants were performed by subepicardial injections. Standard staining was performed. Results Twenty-three animals were randomly treated: mononuclear stem cell and control groups, with 11 rats completing the study. Cell viability was 85%. Mononuclear stem cells (n=5; 5×106 cells /300 μL medium) and control (n=6; 300 μL medium) were used. The resulting left ventricular ejection fraction in the cell therapy group was not significantly different compared with controls (p=0.54). New vessels were demonstrated in the subepicardial region. Conclusions Autologous mononuclear stem cell therapy was not functionally effective in doxorubicin-induced dilated myocardiopathy in the animal model under study with the experimental conditions, despite occurrence of angiogenic activity.

[Imerslund-Gräsbeck syndrome: report of two cases]

INTRODUCTION: The Imerslund-Gräsbeck syndrome is a rare hereditary autosomal recessive disease, characterized by the onset of megaloblastic anemia and asymptomatic proteinuria during the first 2 years of life. OBJECTIVE: To emphasize the importance of early detection of this disorder, due to high morbidity when not correctly treated, in addition to the necessity of screening and genetic counseling of the asymptomatic family members. METHODS: The authors report two patients, male and female, 8 and 10 years old, respectively. Their past history revealed anemia and multiple blood transfusions since their infancy. They evolved with pancytopenia during childhood and diagnosis of Severe Aplastic Anemia or Fanconi Syndrome was suspected. They were referred to the Bone Marrow Transplantation Section -HC- UFPR. RESULTS: Laboratory investigations revealed pancytopenia in peripheral blood. Bone marrow aspiration showed a marked megaloblastic erythropoiesis. Twenty-four-hour urine collection revealed proteinuria (3.0 and 5.8 g/dl respectively). Cytogenetic analysis was normal. Resolution of symptoms followed replacement therapy with parenteral vitamin B12. CONCLUSIONS: The presence of megaloblastic anemia in children should be followed by investigation of proteinuria, due to the existence of this rare disorder, that has a simple diagnosis and an effective treatment.

Trisomy 4 in acute nonlymphocytic leukemia: The first brazilian case

We report the first South American case of acute nonlymphocytic leukemia, French-American-British (FAB) subtype M1, [1] with trisomy 4 as the sole chromosome abnormality. The patient denied exposure to toxic or carcinogenic substances.

Paroxysmal nocturnal hemoglobinuria clone in 103 Brazilian patients: diagnosis and classification

Background Paroxysmal nocturnal hemoglobinuria is an acquired chronic hemolytic anemia, which often manifests as peripheral blood cytopenias and thrombosis. Objective The aim of this study is to describe a Brazilian population of paroxysmal nocturnal hemoglobinuria patients. Methods One hundred and three paroxysmal nocturnal hemoglobinuria cases were retrospectively reviewed and the clinical presentation, thrombosis, survival, and clone size were assessed. Diagnosis was established by flow cytometry. Results Fifty-two male and 51 female patients with a median age of 24.1 years (5.5–62 years) were studied. Clinical symptoms included hemoglobinuria (18.4%), infection (46.6%) and thrombosis (16.5%), and 80.6% had pancytopenia. Patients were classified as classic paroxysmal nocturnal hemoglobinuria (10), paroxysmal nocturnal hemoglobinuria with aplastic anemia (39), and paroxysmal nocturnal hemoglobinuria with subclinical features and aplastic anemia (54). There were significant differences in terms of median age, size of clone, clinical symptoms, and peripheral blood cell counts between the three subcategories. The clone size in erythrocytes and granulocytes were respectively 0.04% (range: 0–18%) and 7.3% (range: 0.3–68.7%) in patients with subclinical features and aplastic anemia, 15.8% (range: 0–99.7%) and 63.0% (range: 1.7–99.8%) in patients with aplastic anemia alone, and 82.2% (range: 0–99.85%) and 98.0% (81.3–100.0%) in Classic disease. Statistical differences were identified for platelets (p-value = 0.001), lactate dehydrogenase (p-value = 0.002) and the clone size (p-value < 0.001) in patients who suffered thrombotic events compared to those who did not. Overall survival was 81.7%, with patients with subclinical features and aplastic anemia having lower overall survival (76.5%). Conclusion This retrospective review of 103 patients over an 11-year period represents the largest collection of paroxysmal nocturnal hemoglobinuria cases from a single center in Brazil. Flow cytometry showed that a larger clone was associated with classical symptoms and increased risk of thrombosis, even in patients with bone marrow failure, whereas a smaller clone was associated with bone marrow aplasia.