Fernanda G. Pereira

The Fractal Dimension of Nuclear Chromatin as a Prognostic Factor in Acute Precursor B Lymphoblastic Leukemia

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.

Microparticles in deep venous thrombosis, antiphospholipid syndrome and Factor V Leiden

Microparticles (MPs) are blebs released from cellular surfaces during activation/apoptosis. They are procoagulant, pro-inflammatory and could contribute to pathogenesis of deep venous thrombosis (DVT). This study compared the number, cellular origin and procoagulant activity of MPs on DVT patients in different clinical situations: at diagnosis (n = 9, 5F/4M; mean age = 41.11), 1-3 years after warfarin withdrawal (n = 10, 7F/3M; mean age = 32.90), associated to antiphospholipid syndrome (APS; n = 11, 9F/2M; mean age = 33.82), or asymptomatic carriers of Factor V Leiden (FVL; n = 7, 7F/0M; mean age = 34.00) vs healthy controls (CTR). The quantification and characterization were performed by flow cytometry using CD235, CD61, CD45, CD31, CD14, CD45, anti-TF and Annexin V. The plasmatic procoagulant activity was investigated by prothrombin fragment 1 + 2 (F1 + 2) determination. The MPs procoagulant activity was analyzed by D-dimer (DD2) and Thrombin Generation Test (TGT) on a healthy pool of plasmas adjusted or not by their number (10,000 MPs). The MPs percentages were not different between the groups, but absolute number was increased in patients 1-3 years after warfarin withdrawal vs CTR (P = 0.02). There was no difference of the MPs cellular origin comparing patients to controls. TGT using 10,000 MPs was lower on these patients (P = 0.01). APS patients showed a reduction of plasmatic procoagulant activity (P = 0.004), but they were under warfarin therapy. DD2 in the presence of MPs, independently of its number, was higher in patients with DVT at diagnosis (P < 0.0001). MPs of patients with spontaneous DVT at diagnosis can promote coagulation activation demonstrated by increased DD2. Even the increased MPs from patients 1-3 years after thrombotic episode generated lower amount of thrombin, they can have a protective effect by activation of Protein C anticoagulant pathway.

Mechanisms underlying the inhibitory effects of lipopolysaccharide on human platelet adhesion

Alterations in platelet aggregation in septic conditions are well established. However, little is known about the effects of lipopolysaccharide (LPS) on platelet adhesion. We have therefore investigated the effects of LPS in human platelet adhesion, using an in vitro model of platelet adhesion to fibrinogen-coated wells. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 × 108 platelets/ml) were allowed to adhere. Adherent platelets were quantified through measurement of acid phosphatase activity. Calcium mobilization in Fura2-AM-loaded platelets was monitored with a spectrofluorimeter. Platelet flow cytometry in thrombin-stimulated platelets was performed using monoclonal mouse anti-platelet GPIIb/IIIa antibody (PAC-1). Prior incubation of washed platelets with LPS (0.01–300 µg/ml) for 5 to 60 min concentration- and time-dependently inhibited non-activated platelet adhesion. In thrombin-activated (50 mU/ml) platelets, LPS inhibited the adhesion to a significantly lesser extent than non-activated platelets. Cyclohexamide, superoxide dismutase polyethylene glycol (PEG-SOD) or catalase polyethylene glycol did not affect the LPS responses. No alterations in cyclic GMP levels were seen after platelet incubation with LPS, except with the highest concentration employed (300 µg/ml) where an increase of 36% (P < 0.05) was observed. Thrombin increased by 7.5-fold the internal Ca2+ platelet levels, an effect markedly inhibited by LPS. Thrombin induced concentration-dependent platelet GPIIb/IIIa activation, but LPS failed to affect the activation state of this membrane glycoprotein. In conclusion, LPS inhibits human platelet adhesion to fibrinogen by mechanisms involving blockade of external Ca2+, independently of cGMP generation and activation of GPIIb/IIIa complex.

Proliferation in non‐Hodgkin's lymphomas and its prognostic value related to staging parameters

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‐Hodgkins 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.

Spontaneous apoptosis in chronic lymphocytic leukemia is not an independent prognostic factor for stability of disease when compared with combined AgNOR and TTM scores.

It has been shown that AgNOR staining can provide useful information for diagnosis in cytology of the oral cavity [22], body fluids [3,18], hematologic smears [10,11,14–17] or lymph node aspiration cytology [12]. We would like draw attention of the readers towards an easy AgNOR evaluation which has shown to be very useful as prognostic marker in chronic lymphoid leukemia (CLL) patients. This disease shows considerable variability of its clinical presentation and evolution. Patients in stable phase may, after months or years, change to a progressive phase. Whereas initially no treatment is warranted, chemotherapy will be necessary in the latter. In a previous exploratory study [17] we suggested that the percentage of CLL cells in peripheral blood with AgNOR clusters at first diagnosis might be an independent prognostic marker for the duration of the stable phase, besides the total tumor mass (TTM). An index created by summing up both values showed to be a more powerful prognostic factor than other common clinical and laboratory parameters [17]. In order to validate this model, we decided to repeat this study with other CLL patients. Since the apoptosis rate has shown to be of prognostic importance for various neoplasias and since resistance to apoptosis could be involved in the disease progression of B-CLL, [19], we also investigated the spontaneous apoptosis rate of CLL cells in culture. Unselected patients with newly diagnosed B-CLL entered the study. Counting of AgNOR clusters and the determination of TTM were done as previously described [10,16,17]. AgNOR staining of cytological preparations from acute leukaemias allows the differentiation of clusters (aggregations of precipitations within a common matrix in the nucleolus) and dots (small singular precipitations without a matrix). CLL clusters showed a longest chord of 2.07 μm (95% CI 1.85–2.4 μm) and compact nucleoli, 1.07 μm (95% CI 0.95–1.2 μm) (Fig. 1) [16]. Apoptosis rate was measured at diagnosis by flow cytometry as the percentage of annexin V positive cells after a 48 hours culture as described previously [19]. Pearson’s correlations were calculated between the variables. Univariate Cox regression analyses were performed to examine the relationship between the treatment-free period and percentage of annexin V positive cells, TTM, percentage of cells with AgNOR clusters in peripheral lymphocytes and the Index (= percentage of cells with AgNOR clusters + TTM). For all further analyses the Index values were logarithmized, in order to get a good approximation to the normal distribution. Then we tried to find out, whether comparing all these parameters in a multivariate Cox-regression, the Index would again be the strongest variable, as postulated previously [17]. Finally we tested the strongest variable from this regression together with the parameter “annexin V positive cells”. In order to estimate the stability of the models we applied the Cox regression to 200 new data sets created by bootstrap resampling [17]. For all calculations SPSS 8.0 software was used. During the study period 32 patients were analyzed. The mean observation time was 18 months and during this period 22 patients fulfilled the criteria for start of chemotherapy. Median time of the stable phase was 13 months in the Kaplan–Meier curve. Pearson’s correlations demonstrated statistically significant inverse correlations between the apoptosis rate and TTM (r = −0.47), the AgNOR score (r = −0.40) and the (logarithmized) Index (r = −0.52). In univariate Cox analyses TTM (Exp(B) = 1.087; CI 95% 1.027 to 1.15; p = 0.004), percentage of cells with AgNOR clusters (Exp(B) = 1.137; CI 95% 1.014 to 1.274; p = 0.027) and the Index (logIndex: Exp(B) = 5.84; CI 95% 1.83 to 18.6; p = 0.003) were unfavourable predictive variables. In a multivariate Cox regression only the logIndex remained in the final model. In the stability test by bootstrap resampling the variable log Index was included in 69.5% of all models, whereas TTM in only 33% and the AgNOR score in only 29.5% of the cases. Spontaneous apoptosis rate was a favourable prognostic factor for treatment-free period in the uni-

Costimulatory Molecule Expression on Leukocytes from Mice with Experimental Autoimmune Encephalomyelitis Treated with IFN-β

Interferon-beta (IFN-beta) is of benefit in the treatment of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), but the mechanisms by which it exerts this beneficial effect remain uncertain. The present data demonstrate that IFN-beta therapy impairs the proliferative response to concanavalin A (ConA) and myelin basic protein (MBP), decreases expression of the CD80 molecule on leukocytes of treated mice, and may thereby impede the Th1 cell activation-promoting anergy in EAE. Moreover, IFN-beta therapy increases expression of the CTLA4 molecule, which induces a counterregulatory Th2 response. The reduction of CD80 expression with concomitant increase of CTLA4 expression alters the course of EAE and may be useful as a monitor in therapy with IFN-beta.

Diagnosis of Scott syndrome in patient with bleeding disorder of unknown cause.

Scott syndrome is a rare bleeding disorder due to an impaired exposure of phosphatidilserine on the platelet membrane, compromising the platelet procoagulant activity, thrombin generation and, thus, the clot formation. We report a case of a 17-year-old female adolescent with bleeding episodes of unknown cause. She had normal coagulation, but altered platelet aggregation under arteriolar flow, indicating platelet dysfunction. Furthermore, the expression of Annexin V was markedly reduced and the diagnosis of Scott syndrome was established. She was treated with platelet transfusions and demonstrated a clinical improvement. Scott syndrome may be investigated in cases with bleeding history and normal coagulation tests.

A utilidade da citologia por punção com agulha fina aliada a imunofenotipagem no diagnóstico dos linfomas não-Hodgkin

The WHO classification of non-Hodgkins lymphoma stresses the importance of the immunophenotype for diagnosis. The aim of our study was to evaluate the use of cytology together with flow cytometric examination using a panel of monoclonal antibodies including DNA S phase analysis. Material was obtained from lymph node aspiration of 78 patients. The panel for flow cytometric analysis comprised: CD19/CD10, CD20/CD5, CD23, CD3/CD4, CD3/CD8, CD38/CD7, kappa/lambda. The final diagnosis was confirmed by lymph node biopsy. In 85% of cases, cytology combined with immuno-phenotyping and percentage of Phase S cells allowed the correct diagnosis. In the remaining cases it was possible to differentiate T or B lymphomas and estimate their aggressiveness. The panel, although small, was sufficient in all cases except for anaplastic lymphoma. S-phase fraction was important for the diagnosis of large B-cell NHL vs. Follicular NHL. In cases of T-cell lymphomas a reliable diagnosis was only possible for lymphoblastic lymphomas. In conclusions, combined cytology and cytophotometric diagnosis of lymph node aspirations is a good alternative to histologic examination, except for T-cell lymphomas. In contrast to biopsy this method is less invasive and may be repeated if necessary.

The influence of storage and leukocyte depletion on the antigen densities of FY1, FY2, MNS3 and MNS4 measured by flow cytometry

Red blood cell (RBC) antigens may present changes in density during storage and leukocyte reduction. We evaluated the influence of these variables on FY1, FY2, MNS3 and MNS4 antigens using quantitative flow cytometry (FCM). Forty-eight RBC units were divided into two sub-units each immediately after collection. One of them was leukocyte reduced before storage. Antigen expression was analyzed on days 1 and 35 of storage by gel-centrifugation and FCM. Three RBC samples were submitted to papain and bromelin treatment. The gel-centrifugation test could not detect any influence of storage or leukocyte reduction. However, by FCM, a wide variation of antigen density among the donors was found. Leukocyte depletion did not change the antigen density but after storage, expression of FY1 and MNS4 showed a slight decrease. Median antigenic density of FY1 was 11,332 in FY1,2 and 23,436 in FY1,-2 donors. FY2 presented 7204 and 7868, respectively. MNS4 had 100,589 and 214,340 sites in donors MNS3,4 and MNS-3,4, respectively, and MNS3 had 10,389 and 21,122 sites, respectively. After enzyme treatments none of the antigens could be detected. FCM was a reproducible technique, suitable for the quantification of the antigens studied in RBC concentrates stored and leukocyte reduced in conditions normally used for blood transfusion.

O uso de um painel restrito de anticorpos monoclonais no diagnóstico diferencial das síndromes linfoproliferativas

The immunological profile and the morphology of lymphoid cells are the main diagnostic clues for a correct differential diagnosis of chronic lymphoproliferative disorders. In the present study, we used a small panel of monoclonal antibodies for this purpose: CD19/CD10, CD20/CD5, CD23, CD3/CD4, CD3/CD8, kappa chain/lambda chain (for the detection of surface immunoglobulin). This panel was applied in 44 patients seen at our Service, using peripheral blood or lymph node morphology as a confirmatory element. Among them 29 had a typical immunologic profile of B-CLL (16 with kappa light chain and 13 with lamba chain restriction) and 2 were T-CLL. In 8 cases, immunologic study needed peripheral blood morphology for diagnostic confirmation: 6 cases of immunocytoma and 2 prolymphocytic leukemias. The 5 patients with mantle cell lymphoma were diagnosed based on the immunologic profile of peripheral blood or lymph node aspirates. The present observation underlines the importance of the immunophenotype in the correct diagnosis of the chronic lymphoproliferative syndromes. The small panel used was sufficient, when morphological examination of peripheral blood or lymph node was used for diagnostic confirmation if necessary.