T-Large Granular Lymphocytic Leukemia with Hepatosplenic T-Cell Lymphoma? A Rare Case of Simultaneous Neoplastic T-Cell Clones Highlighted by Flow Cytometry and Review of Literature.

Lymphoproliferative diseases are a heterogeneous set of malignant clonal proliferations of lymphocytes. Despite well-established diagnostic criteria, the diagnosis remains difficult due to their variety in clinical presentation and immunophenotypic profile. Lymphoid T-cell disorders are less common than B-cell entities, and the lack of a clear immunophenotypic characteristic makes their identification hard. Flow cytometry turned out to be a useful tool in diagnosing T-cell disorders and to resolve complicated cases, especially if the number of analyzable neoplastic cells is small. We present a case of a 55-year-old man with simultaneous lymphoproliferative neoplastic T-cell clones, one αß and the other γδ, identified and characterized by flow cytometry (FC), exploiting the variable expression intensity of specific markers. However, the patient's rapid decline made it impossible to define a differential diagnosis in order to confirm the identity of the γδ clone, which remains uncertain. This case is added to the few other cases already documented in the literature, characterized by the co-existence of T-large granular lymphocytic leukemia (T-LGLL)-αß and T-LGLL-γδ/Hepatosplenic T-cell lymphoma (HSTCL). Our case underlines the key role of sensitive diagnostic tools in the assessment of potential relationship between the diagnosis, prognosis, and treatment in the two pathologies.

What Does Atypical Chronic Lymphocytic Leukemia Really Mean? A Retrospective Morphological and Immunophenotypic Study.

Atypical chronic lymphocytic leukemia (CLL) is still defined according to morphological criteria. However, deviance from the typical surface immunological profile suggests an atypical immunological-based CLL. A large cohort of patients with CLL was retrospectively evaluated aiming at assessing morphological (FAB criteria), immunophenotypical (two or more discordances from the typical profile), and clinical-biological features of atypical CLL. Compared to typical cases, morphologically atypical CLL showed a greater percentage of unmutated IgVH and CD38 positivity, and a higher expression of CD20. Immunophenotypically atypical CLL was characterized by more advanced clinical stages, higher expression of CD20, higher rate of FMC7, CD79b and CD49d positivity, and by an intermediate-high expression of membrane surface immunoglobulin, compared to typical cases. When patients were categorized based on immunophenotypic and morphologic concordance or discordance, no difference emerged. Finally, morphological features better discriminated patients' prognosis in terms of time-to-first treatment, while concordant atypical cases showed overall a worse prognosis. Discordant cases by immunophenotype and/or morphology did not identify specific prognostic groups. Whether-in the era of molecular markers used as prognostic indicators-it does make sense to focus on morphology and immunophenotype features in CLL is still matter of debate needing further research.

Preliminary analysis of double-negative T, double-positive T, and natural killer T-like cells in B-cell chronic lymphocytic leukemia.

BACKGROUND: B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the expansion of CD5+ malignant B lymphocytes. Recent discoveries have shown that double-negative T (DNT) cells, double-positive T (DPT) cells, and natural killer T (NKT)-cells may be involved in tumor surveillance. METHODS: A detailed immunophenotypic analysis of the peripheral blood T-cell compartment of 50 patients with B-CLL (classified in three prognostic groups) and 38 healthy donors (as controls) matched for age was performed. The samples were analyzed by flow cytometry using a stain-lyse-no wash technique and a comprehensive six-color antibody panels. RESULTS: Our data confirmed a reduction in percentage values and an increase in absolute values of T lymphocytes in patients with B-CLL, as already reported. In particular, DNT, DPT, and NKT-like percentages were significantly lower than in the controls, except for NKT-like in the low-risk prognostic group. Moreover, a significant rise in the absolute counts of DNT cells in each prognostic group and in the low-risk prognostic group of NKT-like cells was found. A significant correlation of the absolute values of NKT-like cells in the intermediate-risk prognostic group versus B cells was observed. Furthermore, we analyzed whether the increase in T cells was related to the subpopulations of interest. Only DNT cells were positively correlated with the increase in CD3+ T lymphocytes, regardless of the stage of the disease, supporting the hypothesis that this T-cell subset plays a key role in the immune T response in B-CLL. CONCLUSION: These early results supported that DNT, DPT, and NKT-like subsets may be related to disease progression and should encourage further studies aimed at identifying the potential immune surveillance role of these minority T subpopulations.

Systematic numerical assessment of occupational exposure to electromagnetic fields of transcranial magnetic stimulation.

PURPOSE: This study aims to perform a classification and rigorous numerical evaluation of the risks of occupational exposure in the health environment related to the administration of transcranial magnetic stimulation (TMS) treatment. The study investigates the numerically estimated induced electric field that occurs in the human tissues of an operator caused by exposure to the variable magnetic field produced by TMS during treatments. This could be a useful starting point for future risk assessment studies and safety indications in this context. METHODS: We performed a review of the actual positions assumed by clinicians during TMS treatments. Three different TMS coils (two circular and one figure-of-eight) were modeled and characterized numerically. Different orientations and positions of each coil with respect to the body of the operator were investigated to evaluate the induced electric (-E) field in the body tissues. The collected data were processed to allow comparison with the safety standards for occupational exposure, as suggested by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) 2010 guidelines. RESULTS: Under the investigated conditions, exposure to TMS shows some criticalities for the operator performing the treatment. Depending on the model of the TMS coil and its relative position with respect to the operator's body, the numerically estimated E-field could exceed the limits suggested by the ICNIRP 2010 guidelines. We established that the worst-case scenario for the three coils occurs when they are placed in correspondence of the abdomen, with the handle oriented parallel to the body (II orientation). Working at a maximum TMS stimulator output (MSO), the induced E-field is up to 7.32 V/m (circular coil) and up to 1.34 V/m (figure-of-eight coil). The induced E-field can be modulated by the TMS percentage of MSO (%MSO) and by the distance between the source and the operator. At %MSO equal to or below 80%, the figure-of-eight coil was compliant with the ICNIRP limit (1.13 V/m). Conversely, the circular coil causes an induced E-field above the limits, even when powered at a %MSO of 30%. Thus, in the investigated worst-case conditions, an operator working with a circular coil should keep a distance from its edge to be compliant with the guidelines limit, which depends on the selected %MSO: 38 cm at 100%, 32 cm at 80%, 26.8 cm at 50%, and 19.8 cm at 30%. Furthermore, attention should be paid to the induced E-field reached in the operator's hand as the operator typically holds the coil by hand. In fact in the hand, we estimated an induced E-field up to 10 times higher than the limits. CONCLUSIONS: Our numerical results indicate that coil positions, orientations, and distances with respect to the operator's body can determine the levels of induced E-field that exceed the ICNIRP limits. The induced E-field is also modulated by the choice of %MSO, which is related to the TMS application. Even under the best exposure conditions, attention should be paid to the exposure of the hand. These findings highlight the need for future risk assessment studies to provide more safety information for the correct and safe use of TMS devices.

Issues in the Implementation of Directive 2013/35/EU Regarding the Protection of Workers against Electromagnetic Fields.

In 2016 the Directive 2013/35/EU regarding the protection of health and safety of workers exposed to electromagnetic fields was transposed in Italy. Since then, the authors of this paper have been faced with several issues related to the implementation of the provisions of the Directive, which pose some interpretative and operative concerns. A primary critical feature of the Directive is that, in some circumstances, conditions of "overexposure", i.e., of exceeding the exposure limits, are allowed. In the case of transient effects, the "flexibility" concerning the compliance with exposure limits is based on the approach introduced by ICNIRP in its guidelines on static magnetic fields and on time-varying electric and magnetic fields. On the contrary, the possibility of exceeding the exposure limits for health effects, formally recognized in the article of the Directive dealing with derogations, is not included in the ICNIRP guidelines. This paper analyzes the main concerns in interpreting and managing some provisions of the Directive with particular reference to the issue of how the employer can manage the situations of overexposure.

Circulating Regulatory T-Cell Number Does Not Predict Prognosis of Monoclonal Gammopathies of Uncertain Significance.

FOXP3-expressing regulatory T-cells (Tregs), which suppress aberrant immune response against self-antigens, also suppress anti-tumor immune response. It has been shown that there is an increased proportion of Tregs in several different human malignancies, although the actual mechanism remains unclear. The research aims to explore the relationship between the number of Tregs and a predict prognosis in particular hematological diseases as monoclonal gammopathies of uncertain significance (MGUS). Tregs were evaluated by means of flow cytometry (CD4+CD25high/+ CD127low/-) in whole peripheral blood of 56 patients with MGUS to predict progression to overt multiple myeloma (MM). In two groups of patients, MGUS versus MGUS evolved to MM, we found a significative difference for the number of white blood cells, but not in terms of clinical and laboratory features evaluated at diagnosis. The study demonstrated the absence of a prognostic relevance of Tregs in MGUS. Nevertheless, their role in these disorders is still to be defined.

CD200 Baseline Serum Levels Predict Prognosis of Chronic Lymphocytic Leukemia.

Membrane-bound CD200 is overexpressed in chronic lymphocytic leukemia (CLL), and there is some evidence that its soluble ectodomain (sCD200) could also be involved in the pathophysiology and the disease. However, very little is known about sCD200's prognostic significance. sCD200 was tested at diagnosis in 272 patients with CLL and in 78 age- and sex-matched healthy subjects using a specific human CD200 (OX-2 membrane glycoprotein) ELISA kit. A significantly higher concentration of sCD200 was found in CLL patients compared to controls. In our cohort, sCD200 was significantly higher in patients who were older than 66 years, with Binet stage C, unmutated IgVH and unfavorable (del11q or del17p) FISH. Time-to-first treatment and overall survival were significantly shorter in patients with higher sCD200 concentration, using as a cut-off 1281 pg/mL, the median value for sCD200 concentration in the whole CLL cohort. However, the prognostic impact of sCD200 was not confirmed in multivariate analysis. Baseline sCD200 values appeared to have an impact on the response to chemotherapy or chemo-immunotherapy, but not to targeted agents. Collectively, our data show that sCD200 serum levels correlate with more aggressive clinical and biological features and are able to predict a worse prognosis. This work supports the relevant role of CD200 not only as a diagnostic tool but also as a prognostic indicator and a potential therapeutic target in CLL.

Hyperandrogenism by Liquid Chromatography Tandem Mass Spectrometry in PCOS: Focus on Testosterone and Androstenedione.

The identification of hyperandrogenism in polycystic ovary syndrome (PCOS) is concerning because of the poor accuracy of the androgen immunoassays (IA) and controversies regarding which androgens should be measured. The aim of our study was to evaluate the impact of the assessment of testosterone (T) and androstenedione (A) by liquid chromatography in tandem with mass spectrometry (LC/MS-MS), in the diagnosis of PCOS. We evaluated 131 patients referred for suspected PCOS. Fourteen patients in total were excluded, some because of other diagnosis (n = 7) or incomplete diagnostic workup (n = 7). We measured T and A both by IA and LC-MS/MS in the 117 subjects included. We calculated free T (fT) by the Vermeulen formula and recorded clinical and metabolic data. 73 healthy females served as controls to derive immunoassays (IA) and LC-MS/MS reference intervals for T, fT and A. PCOS was confirmed in 90 subjects by IA and in 93 (+3.3%) by LC-MS/MS. The prevalence of biochemical hyperandrogenism in PCOS by LC-MS/MS increased from 81.7% to 89.2% if A was also considered. The most frequently elevated androgens were fT (73.1%) and A (64.5%) and they had similar levels of accuracy in differentiating PCOS and controls (0.34 ng/dL, Sn 91% Sp 89%; 1.16 ng/mL, Sn 91% Sp 88%, respectively). Free testosterone correlated with body mass index (BMI), homeostatic model assessment (HOMA)-index, glycated hemoglobin (HbA1c), and sex-binding globulin (SHBG). The results confirm that LC-MS/MS is slightly more sensitive than IA in the diagnosis of PCOS with LC-MS/MS detecting higher levels of fT and A. Moreover, assessment of fT and A by LC-MS/MS had a similar level of accuracy in discriminating between PCOs and control subjects. Lastly, fT by LC-MS/MS correlates with adverse metabolic parameters.

Extremely High Frequency Electromagnetic Fields Facilitate Electrical Signal Propagation by Increasing Transmembrane Potassium Efflux in an Artificial Axon Model.

Among the many biological effects caused by low intensity extremely high frequency electromagnetic fields (EHF-EMF) reported in the literature, those on the nervous system are a promising area for further research. The mechanisms by which these fields alter neural activity are still unclear and thus far there appears to be no frequency dependence regarding neuronal responses. Therefore, proper in vitro models for preliminary screening studies of the interaction between neural cells with EMF are needed. We designed an artificial axon model consisting of a series of parallel RC networks. Each RC network contained an aqueous solution of lipid vesicles with a gradient of potassium (K+) concentration as the functional element. We investigated the effects of EHF-EMF (53.37 GHz-39 mW) on the propagation of the electric impulse. We report that exposure to the EHF-EMF increases the amplitude of electrical signal by inducing a potassium efflux from lipid vesicles. Further, exposure to the EHF-EMF potentiates the action of valinomycin - a K+ carrier - increasing the extent of K+ transport across the lipid membrane. We conclude that exposure to the EHF-EMF facilitates the electrical signal propagation by increasing transmembrane potassium efflux, and that the model presented is promising for future screening studies of different EMF frequency spectrum bands.

Normal reference ranges of antithrombin, protein C and protein S: effect of sex, age and hormonal status.

INTRODUCTION: Antithrombin (AT), protein C (PC) and protein S (PS) deficiencies are risk factors for venous thromboembolism. Overlapping values between heterozygous carriers and normal individuals often make a correct classification of a deficiency difficult. The aim of this study was to investigate the effect of sex, age, menopause and hormone therapy on natural anticoagulant plasma levels in a large group of healthy individuals, and to evaluate the need of separate reference ranges. MATERIALS AND METHODS: AT and PC were measured with a chromogenic assay, antigenic free PS with an ELISA test. To evaluate the effect of sex, age, oral contraception, hormonal status (and their interaction) on AT, PC and PS levels, linear regression models were used. Biological relevance and the value of the normal deviate z were chosen as rules to decide for separate reference ranges. RESULTS: The study population consisted of 1837 healthy adult individuals (741 men, 1096 women), aged 18-85 years (median age: 44 years). In men AT levels decreased after the age of 50 years. Men had higher levels of PS than women, particularly at young ages. In women, after correction for menopause, only PC levels increased with age. Menopause affected AT and PS, but not PC levels. Oral contraceptive intake was associated with a decrease of AT and PS, and an increase of PC levels. CONCLUSIONS: For AT, PC and PS, sex- and age-specific normal reference ranges can be useful, in order to better discriminate true carriers of a natural anticoagulant deficiency.