Anna Caldini

Multicenter evaluation of hemoglobin A1c assay on capillary electrophoresis.

BACKGROUND Hemoglobin A1c (HbA1c) measurement is currently used for the routine monitoring of long-term glycemic status, thus playing a fundamental role in the management of this disease. Since this marker has recently been recommended as an additional tool for diagnosing diabetes, its of the utmost importance to ensure that the precision and accuracy of HbA1c methods are satisfactory. METHODS We assessed the analytical performances of the Capillarys 2 Flex Piercing® analyzer and compared the results obtained with those from two other widely used HPLC instruments. Furthermore, we evaluated the convenience and ergonomics of the system in authentic routine work conditions in three centers. RESULTS Within-laboratory (n=40) and between-laboratory (n=120) imprecision CV% using four blood samples with different concentrations of HbA1c were <3.4% and <3.1% using IFCC units and <2.1% and <2.0% using NGSP units, respectively. The obtained trueness (<3 mmol/mol, <0.3%) was highly satisfactory, nor was HbA1c measurement compromised by the presence of the commonly present hemoglobinopathies. The comparison made with established methods revealed excellent agreement (r>0.985). CONCLUSIONS The evaluated method is precise, accurate and robust, with a high throughput. It also allows the identification of the most frequent Hb variants and therefore may be a valid alternative to other methods currently proposed for routine use in clinical laboratories.

Plasma adiponectin and hyperglycaemia in diabetic patients

Abstract The insulin-sensitising adipose hormone adiponectin is reduced in type 2 diabetic patients. We assessed the relationships between plasma adiponectin and chronic hyperglycaemia. Adiponectin levels and glycated haemoglobin (HbA1c) were measured at enrolment and after 90 days in 16 patients with type 2 diabetes aged (mean ± SEM) 63.0±0.6 years, with body mass index (BMI) 30.2±0.5 kg/m2 and HbA1c concentration 7.4± 0.1%, who did not modify their hypoglycaemic treatment during the observation period. Furthermore, plasma adiponectin was measured in 29 adult patients with type 1 diabetes and compared with 29 control subjects matched for sex, age, BMI, waist circumference and bioimpedance-estimated fat mass. In type 2 diabetic patients at enrolment, adiponectin concentration correlated with BMI (r = −0.46; p < 0.05), but not with HbA1c. During the prospective observation, variations of adiponectin showed a significant correlation with variations of BMI (r = −0.47; p < 0.01), but not with variations of HbA1c concentration. These results were confirmed by multivariate analysis after adjustment for sex and age. Adiponectin levels in type 1 diabetic patients (380.8±13.7 ng/ml in women, 192.5±13.9 ng/ml in men) were significantly (p < 0.05) higher than in control subjects (277.6±11.0 ng/ml in women, 102.7±5.1 ng/ml in men); plasma adiponectin correlated significantly with BMI and waist circumference, but not with HbA1c. In conclusion, the reduction of plasma adiponectin levels in type 2 diabetic patients does not appear to be determined by chronic hyperglycaemia. Adiponectin levels are increased in type 1 diabetes, but this phenomenon is not attributable to differences in nutritional status or body composition.l21

Diagnosis of Ventilator-Associated Pneumonia: A Pilot, Exploratory Analysis of a New Score Based on Procalcitonin and Chest Echography

BACKGROUND To facilitate the clinical diagnosis of ventilator-associated pneumonia (VAP) in the ICU, the Clinical Pulmonary Infection Score (CPIS) has been proposed but has shown a low diagnostic performance in subsequent studies. We propose a new score based on procalcitonin level and chest echography with the aim of improving VAP diagnosis: the Chest Echography and Procalcitonin Pulmonary Infection Score (CEPPIS). METHODS This retrospective pilot study recruited patients admitted to the Intensive Care Unit of the Emergency Department, Careggi University Hospital (Florence, Italy), from January 2009 to December 2011. Patients were retrospectively divided into a microbiologically confirmed VAP group or a control group based on diagnosis of VAP and positive tracheal aspirate culture. RESULTS A total of 221 patients were included, with 113 in the microbiologically confirmed VAP group and 108 in the control group. A CEPPIS > 5 retrospectively fixed was significantly better in predicting VAP (OR, 23.78; sensitivity, 80.5%; specificity, 85.2%) than a CPIS > 6 (OR, 3.309; sensitivity, 39.8%; specificity, 83.3%). The receiver operating characteristic area under the curve analysis also showed a significantly higher diagnostic value for CEPPIS > 5 than CPIS > 6 (0.829 vs 0.616, respectively; P < .0001). CONCLUSIONS In this pilot, exploratory analysis, CEPPIS is effective in predicting VAP. Prospective validation is needed to confirm the potential value of this score to facilitate VAP diagnosis.

Preparation and evaluation of steroid chemiluminescent tracers

Six different isoluminol derivatives have been synthesized and evaluated as potential labels for conjugation to steroid molecules. The resulting steroid-chemiluminescent tracers have been investigated in terms of detection limit and affinity for the homologous antibody. The conjugation to the steroid molecule did not significantly affect the quantum yield of the chemiluminescent label. Consequently the detection limit was very similar using tracers with the same chemiluminescent label and different steroids and it was always at picomolar levels. The affinity of the chemiluminescent tracer versus the homologous antibody was evaluated using tritiated steroids as reference. The bridge and the chemiluminescent molecule could influence the affinity of the chemiluminescent tracer which could be higher, similar or lower than that of the tritiated steroid. In conclusion some isoluminol derivatives (i.e. ABEI or AEEI) can be considered as universal labelling compounds for steroids and their metabolites, because they possess a high light efficiency (similar to luminal itself) and favourable reaction conditions. Moreover the conjugation to the steroid molecule does not modify their chemiluminescent properties.

Evaluation of the analytic performances of the new HPLC system HLC-723 G7 for the measurement of hemoglobin A1c.

OBJECTIVES The analytical performance of a new automated HPLC system, for the determination of HbA1C in blood (Tosoh HLC-723 G7), was studied. DESIGN AND METHODS The study design included the evaluation of imprecision, linearity, interference and carryover. Comparison study was performed by comparing HbA1C results with those obtained from an established method (Bio-Rad Variant II). RESULTS Total imprecision was less than 1.34% and the results were linear up to 17.2% HbA1C. The method showed a wide analytical range, and no carryover between specimens. Comparison study yielded, r=0.989, Sy.x=0.255, regression equation (y=0.9895x-0.35); Bland-Altman plot showed a mean bias=- 0.43% HbA1C with confidence limits ranging from -0.48% to -0.38% HbA1C. The presence of abnormal hemoglobin was clearly revealed, and no interference from labile HbA1C was apparent. CONCLUSION The HLC-723 G7 instrument seems to be a reliable system for routine assay of HbA1C.

Original Research: Critical CareDiagnosis of Ventilator-Associated Pneumonia: A Pilot, Exploratory Analysis of a New Score Based on Procalcitonin and Chest Echography

BACKGROUND To facilitate the clinical diagnosis of ventilator-associated pneumonia (VAP) in the ICU, the Clinical Pulmonary Infection Score (CPIS) has been proposed but has shown a low diagnostic performance in subsequent studies. We propose a new score based on procalcitonin level and chest echography with the aim of improving VAP diagnosis: the Chest Echography and Procalcitonin Pulmonary Infection Score (CEPPIS). METHODS This retrospective pilot study recruited patients admitted to the Intensive Care Unit of the Emergency Department, Careggi University Hospital (Florence, Italy), from January 2009 to December 2011. Patients were retrospectively divided into a microbiologically confirmed VAP group or a control group based on diagnosis of VAP and positive tracheal aspirate culture. RESULTS A total of 221 patients were included, with 113 in the microbiologically confirmed VAP group and 108 in the control group. A CEPPIS > 5 retrospectively fixed was significantly better in predicting VAP (OR, 23.78; sensitivity, 80.5%; specificity, 85.2%) than a CPIS > 6 (OR, 3.309; sensitivity, 39.8%; specificity, 83.3%). The receiver operating characteristic area under the curve analysis also showed a significantly higher diagnostic value for CEPPIS > 5 than CPIS > 6 (0.829 vs 0.616, respectively; P < .0001). CONCLUSIONS In this pilot, exploratory analysis, CEPPIS is effective in predicting VAP. Prospective validation is needed to confirm the potential value of this score to facilitate VAP diagnosis.

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.

Luminescent immunoassay (LIA) methods for steroid hormones

The application of luminescence to the development of non-isotopic immunoassay methods for steroids and urinary steroid metabolites is reviewed. On-line computer analysis of the light emission is particularly useful, as it reveals the interfering effects of biological compounds on the reaction and this can improve the quality of luminescence immunoassay (LIA) methods. The main characteristics of chemiluminescent tracers are stability, safety, speed and sensitivity of detection, and reliability. Homogeneous methods, not requiring phase separation, have also been reported and validated. Heterogeneous methods which use dextran-coated charcoal or solid-phase techniques can be used for direct determination of urinary steroids in unextracted samples.

CSF proteomic analysis in patients with normal pressure hydrocephalus selected for the shunt: CSF biomarkers of response to surgical treatment

The aim of our pilot study was to investigate, by a proteomic approach, the expressed differences in cerebrospinal fluid (CSF) protein patterns in order to aid in the diagnosis and treatment of normal pressure hydrocephalus (NPH). Seventeen patients with NPH, selected by Intracranial-Pressure monitoring (ICPmo), underwent implantation of a shunt and after 6 months were clinically re-evaluated. Thirteen patients improved, whereas four did not. During ICPmo CSF was collected and its proteoma was analyzed by 2D gel electrophoresis and mass spectrometry. The over-expression of α2HS glycoprotein, α1 antichimotrypsin and α1beta glycoprotein and the under-expression of glial fibrillary acidic protein, apolipoproteins (AIV, J and E), complement C3c, anti-thrombin, α2 antiplasmin and albumin seem to be associated with a positive response to surgery. Most of these proteins have been reported to be altered in Alzheimer disease, supporting the hypothesis of a possible link between these two nosological entities.

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.