Alessandra Fanelli

Control of breathing in patients with severe hypothyroidism

PURPOSE Hypothyroid patients have been reported to have a blunted ventilatory response to carbon dioxide stimulation. However, previous data did not clarify the localization of abnormalities responsible for that disorder. The present investigation was aimed at evaluating to what extent central (neural) and/or peripheral (muscular) factors are involved in the abnormalities of the ventilatory control system in hypothyroid patients. PATIENTS AND METHODS We studied 13 patients with severe hypothyroidism before and after 6 to 9 months of replacement therapy; 7 age- and sex-matched normal subjects were also studied as a control. In each subject, we assessed (1) inspiratory muscle strength by measuring maximal inspiratory pressure (MIP), and (2) respiratory control system during a carbon dioxide rebreathing test by measuring minute ventilation (VE), tidal volume (VT), mean inspiratory flow (VT/TI), and electromyographic (EMG) activity of the diaphragm (Edi) and intercostal (Eint) muscles. RESULTS Compared with the normal control group (Group C), patients exhibited similar MIP, and similar VE and EMG response slopes to carbon dioxide. However, evaluating individual VE response slopes, we were able to identify two subsets of patients: Group A (six patients) with low VE response (less than mean -SD.1.65 of Group C) and Group B (seven patients) with normal VE response. Compared with both Groups B and C, Group A exhibited significantly lower VT/TI, Edi, and Eint response slopes; the difference between Groups B and C was not significant. Six patients (two from Group A and four from Group B) exhibited low MIP values compared with that in Group C. After replacement therapy, (1) VE, VT/TI, and Edi response slopes increased significantly in Group A; and (2) MIP increased, but not significantly in patients with low MIP. CONCLUSIONS We conclude that: (1) In patients with severe hypothyroidism, the ventilatory control system may be altered at the neural level, as indicated by a blunted chemosensitivity; (2) Impaired respiratory muscle function does not seem to play a major role in the decreased ventilatory response to carbon dioxide stimulation; (3) Replacement therapy appears to normalize the response to hypercapnic stimulation, but not respiratory muscle strength.

Possible Pathogenetic Role of Activated Platelets in the Primary Antiphospholipid Syndrome Involving the Central Nervous System

Neurological disorders occurring in the primary antiphospholipid syndrome (neuro-PAPS) have not yet been completely understood. Platelet activation has been suggested to play a crucial role in the pathogenesis of hemostatic disorders in the antiphospholipid syndrome, but no association with neuro-PAPS has been investigated so far. Therefore, we investigated 16 patients with PAPS by flow cytometry in the presence of circulating activated platelets as defined by the surface expression of activation-dependent glycoprotein CD62. In addition, the relationship among activated platelets and anticardiolipin antibodies (aCL) was evaluated. Compared to normal subjects CD62 was found significantly increased in these patients. Furthermore, a significantly increased percentage of CD62-positive platelets was found in the neuro-PAPS group (nine patients) compared to the non-neuro-PAPS patients (seven subjects). On the contrary, no significant difference was found between the two groups with regard to aCL IgG and platelet number. Furthermore, within the neuro-PAPS group, no difference was evidenced, in the CD62-positive platelet percentage, between the four subjects with thrombocytopenia and the five with the normal blood platelet count. Similarly, neuro-PAPS subjects with previous peripheral arterial and/or venous thrombosis did not show a significantly more elevated level of CD62-positive platelets. Finally, a linear correlation was found between the aCL IgG level and the CD62-positive platelet percentage in all the patients and, more significantly, in the neuro-PAPS group, but not within the non-neuro-PAPS patients. Our data demonstrate that circulating activated platelets are detectable by flow cytometry in the majority of PAPS patients and suggest the existence of a relationship among activated platelets, aCL, and neurological disease that patients affected by PAPS might undergo.

Flow cytometric measurement of DNA content in human solid tumors: a comparison with cytogenetics.

The aims of this study were: (1) to test the accuracy of flow cytometery (FC) in the measurement of DNA content in human solid tumors, (2) to correlate the FC DNA-index (DI) with the chromosome modal number (CMN) provided by cytogenetic analysis (CG), and (3) to investigate the most frequent pitfalls in FC histograms classification. FC and CG analyses were performed in parallel on 113 samples of human solid tumors of different origin. FC provided an evaluable histogram in 110 out of 113 cases (97%), whereas a successful CG culture was obtained in 79 out of 113 samples (72%). In the 79 cases evaluable by both FC and CG, a concordant ploidy status was found in 66 cases (84%) (47 diploid and 19 aneuploid) (P < 0.001, chi-square test). In the 19 concordant aneuploid tumors a close correlation between the CMN and the DI was found (y = 0.019 x + 0.151; r = 0.860). Concerning the 13 discordant cases, 11 (85%) were classified as aneuploid by FC and as diploid by CG, while 2 cases (15%) were CG aneuploid (1 near-diploid and 1 tetraploid) and FC diploid. The current study suggests that FC is a reliable method for the measurement of tumor DNA content of the studied solid tumors. Special attention should be paid to the improvement of DNA histograms quality, in order to reduce the difficulties in the detection of near-diploid and near-tetraploid cell populations. Multiple sampling should be warranted whenever possible.

Reticulocytes and Reticulated Platelets: Simultaneous Measurement in Whole Blood by Flow Cytometry

Abstract Reticulated platelets are a fraction of newly released circulating elements characterized by a residual amount of RNA. It has been suggested that the reticulated platelet count, providing an estimate of thrombopoiesis in the same way as erythrocyte reticulocyte count is a measure of erythropoiesis, may be useful in the study of thrombocytopenic disorders. Reticulated red cells and platelets can be analyzed by flow cytometry using specific stains for nucleic acids such as Thiazole Orange and Auramine-O. The aim of our work was to perform the simultaneous evaluation of reticulated elements in whole blood using a standard flow cytometer and to correlate the results obtained with a dedicated cytometer. A group of 14 patients with abnormal absolute reticulocyte counts (range 1.1–11%) and a group of 41 patients showing a platelet discrimination error when analyzed with a dedicated flow cytometer (Sysmex R1000) were enrolled. Linear amplification of both scatter and fluorescence was used to perform reticulocyte count. A gate was set on platelet dimensions, and logarithmic amplification of scatter and fluorescence was used to count reticulated platelets. A good correlation was obtained both for results of reticulocyte count (r2 = 0.9825) and for reticulated platelets (r2 = 0.8717) between our method and those using dedicated instruments. These data show that reticulated platelet count may be easily introduced in clinical laboratories that routinely perform reticulocyte count by flow cytometry.

The c-erbB-2 oncogene amplification by competitive PCR in aneuploid cell clones flow sorted from breast cancer samples.

Abstract The amplification of c-erbB-2 oncogene has been reported to have clinical relevance as a prognostic index in breast cancer. However, controversies still remain about its interpretation, mainly due to the inaccuracy of methods used for this purpose and to the unpredictable variability of the ratio between cancer and normal cells. Accurate quantitative assay, combined with strategies for selection or enrichment of tumor cell populations, could shed a new light on the relationships between molecular alterations and their clinical relevance. In this study, amplification of c-erbB-2 was measured by competitive PCR in 21 aneuploid breast cancers using a multiple DNA competitor both in whole homogenized cancer cells and in aneuploid enriched clones obtained after flow cytometry cell sorting. Most breast cancers (10/12) carrying c-erbB-2 oncogene amplification showed a significant increase in copy number in sorted aneuploid clones, and 2/9 apparently not amplified in basal samples were found to be amplified after being sorted for the aneuploid population. A general concordance between amplification and c-erbB-2 overexpression was found. The mean degree of amplification in sorted aneuploid clones is increased in breast cancers with the highest levels of cerbB-2 protein overexpression. These data indicate that in breast cancers the amplification of c-erbB-2 oncogene is mainly associated with aneuploid cells.

Control of Breathing in Patients with Chronic Pulmonary Obstructive Disease: Response to Bamiphylline

In two groups (A and B) of patients with severe chronic obstructive pulmonary disease (COPD), matched for age and routine pulmonary function testing, we evaluated inspiratory muscle strength (MIP), breathing pattern, mouth occlusion pressure (P0.1), inspiratory neural drive, assessed in terms of electromyographic activity of both diaphragm (EMGd) and intercostal (EMGint) muscles, and P0.1/EMGd ratio, an index of inspiratory neuromuscular coupling. Group A (8 patients) was studied before and after a 7-day period of a new oral xanthine derivative (bamiphylline, 1.2 g daily), and group B (7 patients) was given a placebo. Under control conditions, compared with an age-matched normal control group, groups A and B both exhibited a decrease in MIP, more rapid (increase in respiratory frequency RF) and shallower (decrease in tidal volume; VT) breathing (RSB), a marked increase in both EMGd and EMGint and a lower P0.1/EMGd ratio. With bamiphylline FEV1 and PaO2 significantly increased, while a substantial increase in MIP was found in 5 out of the 8 patients. VT and inspiratory time (Ti) also significantly increased, and RF decreased. Both EMGd and EMGint significantly decreased, whereas P0.1/EMGd exhibited a substantial increase in 5 out of the 8 patients. Conversely, no significant changes were observed in group B during the study period. From these data we conclude that in patients with COPD, clinical manifestations, probably associated with inspiratory muscle overloading (decrease in muscle strength, RSB, increase in respiratory neural drive, and derangement in neuromuscular coupling) may benefit from a short-term treatment with bamiphylline.

Comparing the performance of three panels rules of blood smear review criteria on an Italian multicenter evaluation

The aims of this study were to compare the diagnostic accuracy of blood smear review criteria, by means of three different panel rules, those proposed by: the International Consensus Group for Hematology [41‐ICGH rules], the Italian Survey [IS rules] and the Working Group on Hematology‐SIBioC (WGH) consensus rules (WGH rules).

Two-site evaluation of the diagnostic performance of the Sysmex XN Body Fluid (BF) module for cell count and differential in Cerebrospinal Fluid

Cellular analysis in cerebrospinal fluid (CSF) provides important diagnostic information in many pathological settings. The aim of this two‐site study was to evaluate the Sysmex XN Body Fluid mode (XN‐BF) for cell analysis of CSF compared to light microscopy (LM).

Performance evaluation of the automated nucleated red blood cell count of five commercial hematological analyzers

Recent automated hematology analyzers (HAs) can identify and report nucleated red blood cells (NRBC) count as a separate population out of white blood cells (WBC). The aim of this study was to investigate the analytical performances of NRBC enumeration on five top of the range HAs.

Proposal for a New Score-Based Approach To Improve Efficiency of Diagnostic Laboratory Workflow for Acute Bacterial Meningitis in Adults

ABSTRACT Microbiological tests on cerebrospinal fluid (CSF) utilize a common urgent-care procedure that does not take into account the chemical and cytological characteristics of the CSF, resulting sometimes in an unnecessary use of human and diagnostic resources. The aim of this study was to retrospectively validate a simple scoring system (bacterial meningitis-Careggi score [BM-CASCO]) based on blood and CSF sample chemical/cytological parameters for evaluating the probability of acute bacterial meningitis (ABM) in adults. BM-CASCO (range, 0 to 6) was defined by the following parameters: CSF cell count, CSF protein levels, CSF lactate levels, CSF glucose-to-serum glucose ratio, and peripheral neutrophil count. BM-CASCO was retrospectively calculated for 784 cases of suspected ABM in adult subjects observed during a four-and-a-half-year-period (2010 to 2014) at the emergency department (ED) of a large tertiary-care teaching hospital in Italy. Among the 28 confirmed ABM cases (3.5%), Streptococcus pneumoniae was the most frequent cause (16 cases). All ABM cases showed a BM-CASCO value of ≥3. Most negative cases (591/756) exhibited a BM-CASCO value of ≤1, which was adopted in our laboratory as a cutoff to not proceed with urgent microbiological analysis of CSF in cases of suspected ABM in adults. During a subsequent 1-year follow-up, the introduction of the BM-CASCO in the diagnostic workflow of ABM in adults resulted in a significant decrease in unnecessary microbiological analysis, with no false negatives. In conclusion, BM-CASCO appears to be an accurate and simple scoring system for optimization of the microbiological diagnostic workflow of ABM in adults.