Publications by authors named "Alessandra Fanelli"

17 Publications

  • Page 1 of 1

Prognostic Role of Procalcitonin in Patients with Fever and Respiratory Complains During the COVID-19 Pandemic.

SN Compr Clin Med 2021 Mar 23:1-2. Epub 2021 Mar 23.

Department of Emergency Medicine, Careggi University Hospital, largo Brambilla 3, 50134 Firenze, Italy.

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http://dx.doi.org/10.1007/s42399-021-00862-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985222PMC
March 2021

Procalcitonin for the differential diagnosis of COVID-19 in the emergency department. Prospective monocentric study.

Intern Emerg Med 2021 09 2;16(6):1733-1735. Epub 2021 Feb 2.

Department of Emergency Medicine, Careggi University Hospital, largo Brambilla 3, 50134, Firenze, Italy.

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http://dx.doi.org/10.1007/s11739-021-02641-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851503PMC
September 2021

Multicentric evaluation of analytical performances digital morphology with respect to the reference methods by manual optical microscopy.

J Clin Pathol 2020 Sep 14. Epub 2020 Sep 14.

ASST Papa Giovanni XXIII, Bergamo, Italy

Aims: Optical microscopic (OM) evaluation of peripheral blood (PB) cells is still a crucial step of the laboratory haematological workflow. The morphological cell analysis is time-consuming and expensive and it requires skilled operator. To address these challenges, automated image-processing systems, as digital morphology (DM), were developed in the last few years. The aim of this multicentre study, performed according to international guidelines, is to verify the analytical performance of DM compared with manual OM, the reference method.

Methods: Four hundred and ninety PB samples were evaluated. For each sample, two May Grunwald-stained and Giemsa-stained smears were performed and the morphological evaluation of cells was analysed with both DM and OM. In addition, the assessment times of both methods were recorded.

Results: Comparison of DM versus OM methods was assessed with Passing-Bablok and Deming fit regression analysis: slopes ranged between 0.17 for atypical, reactive lymphocytes and plasma cells (LY(AT)) and 1.24 for basophils, and the intercepts ranged between -0.09 for blasts and 0.40 for LY(AT). The Bland-Altman bias ranged between -6.5% for eosinophils and 21.8% for meta-myemielocytes. The diagnostic agreement between the two methods was 0.98. The mean of assessment times were 150 s and 250 s for DM and OM, respectively.

Conclusion: DM shows excellent performance. Approximately only 1.6% of PB smears need the OM revision, giving advantages in terms of efficiency, standardisation and assessment time of morphological analysis of the cells. The findings of this study may provide useful information regarding the use of DM to improve the haematological workflow.
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http://dx.doi.org/10.1136/jclinpath-2020-206857DOI Listing
September 2020

Low bone mass and hypovitaminosis D in haemophilia: A single-centre study in patients with severe and moderate haemophilia A and B.

Haemophilia 2020 Sep 25;26(5):898-906. Epub 2020 Aug 25.

Center for Bleeding Disorders and Coagulation, Careggi University Hospital, Firenze, Italy.

Introduction: Haemophilia (H) is frequently associated with a multifactorial reduction in bone mineral density (BDM), but little is known about possible differences between HA and HB according to their severity.

Aim: To evaluate the association between low bone mineral density (BMD), 25-hydroxyvitamin D [25(OH)D] concentrations and bone turnover markers in patients with HA and HB younger or older than 50 years.

Methods: In 78 patients <50 years and 33 patients >50 years with severe (S) or moderate (M) HA and HB, BMD was measured by dual-energy X-ray absorptiometry at femoral neck (FN) and lumbar spine and then correlated to annual bleeding rate (ABR), World Federation of Haemophilia orthopaedic joint scale (WFH score), 25(OH)D concentrations, parathyroid hormone (PTH), amino-terminal telopeptide of type 1 collagen (NTx), urinary pyridinolines, osteocalcin and bone-specific alkaline phosphatase.

Results: Overall, a high prevalence of hypovitaminosis D was diagnosed. In patients <50 years, low FN-BMD was significantly more frequent in HA than in HB, while PTH, pyridinolines, ABR and WFH score were associated with H type and severity. In patients >50 years, similarly low FN-BMD was observed in HA and HB, while ABR and WFH score were associated with H type and severity, being milder in HB.

Conclusions: Low bone mass is a frequent comorbidity in haemophilic patients of all ages, apart from those with MHB. Clinical and laboratory assessments confirm a higher bone impairment and faster bone resorption in HA compared with HB. Looking at H type and severity, MHB seems to have a normal bone metabolism and a less severe disease.
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http://dx.doi.org/10.1111/hae.14127DOI Listing
September 2020

Predictors of Resumption of Menses in Anorexia Nervosa: A 4-Year Longitudinal Study.

Psychosom Med 2020 10;82(8):782-786

From the Psychiatry Unit, Department of Health Sciences (Castellini, Rossi, Cassioli, Giardinelli, Ricca), University of Florence; Central Laboratory (Fanelli), Azienda Ospedaliero Universitaria Careggi; and Andrology, Women's Endocrinology and Gender Incongruence Unit, Department of Experimental, Clinical, and Biomedical Sciences (Fisher, Vignozzi), University of Florence, Florence, Italy.

Objective: Amenorrhea is a disabling medical consequence of anorexia nervosa (AN); therefore, resumption of menses (ROM) represents an important goal in the treatment for these patients. The aim of the present study was to evaluate possible clinical, psychopathological, and biological predictors of ROM, including age, body mass index (BMI), AN subtype, childhood abuse, duration of illness, general and eating disorder (ED)-specific psychopathology, and sex hormones.

Methods: Fifty amenorrheic patients with AN were enrolled. Baseline clinical data and information on childhood abuse were collected. Questionnaires to evaluate general and ED-specific psychopathology were administered, and blood samples were drawn. All patients received treatment as usual and underwent regular follow-up visits for 4 years or until ROM. Time to ROM, BMI at last evaluation, and data regarding diagnostic crossover into bulimia nervosa were collected.

Results: Twenty-nine (58.0%) patients recovered menses. Diagnostic crossover was associated with a higher probability of ROM (odds ratio = 10.3, p = .030). Time-to-event analysis showed that a shorter duration of illness (χ(1) = 11.00, p = .001), binge-eating/purging subtype (χ(1) = 7.01, p = .008), and history of childhood abuse (χ(1) = 4.03, p = .045) were associated with an earlier ROM. Furthermore, higher baseline ED-specific psychopathology was associated with a reduced likelihood for ROM, whereas higher general psychopathology and follicle-stimulating hormone levels predicted an earlier ROM (all, p < .050). Age, BMI, luteinizing hormone, and estrogen hematic levels had no predictive value with respect to ROM.

Conclusions: The present study provides data in support of an integrated model, emphasizing the importance of duration of illness, childhood abuse, and psychopathological characteristics of amenorrheic patients with AN in predicting ROM.
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http://dx.doi.org/10.1097/PSY.0000000000000849DOI Listing
October 2020

Improvement in estimation of time since death by albumin and potassium concentrations in vitreous humor.

Forensic Sci Int 2020 Sep 27;314:110393. Epub 2020 Jun 27.

Section of Forensic Medical Sciences, Department of Health Sciences, University of Florence, Florence, Italy.

The measurement of the potassium concentration (K) in vitreous humor (VH) has been a well-known adjunct for the estimation of the post-mortem interval (PMI) since the early 1960s. For years, however, many authors have been using other biochemical markers in an attempt to improve predictions. In this paper we confirm the role of K in the determination of the PMI adopting a linear regression model and we investigate whether other biochemical markers could improve the model through a multiple regression analysis. Additionally, the research aims to confirm the data of the analytes of interest among different techniques and instrumentations. We deemed this as an important issue because a primary concern in the literature is that automated analytical methods are often calibrated and, for the most part, validated for serum or urine analysis. Our results confirmed the well-established role of K as well as highlighted Albumin as a novel marker to be considered for further improvement of prediction models, especially since 72 h after death.
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http://dx.doi.org/10.1016/j.forsciint.2020.110393DOI Listing
September 2020

Macro-aspartate aminotransferase in a healthy woman.

Clin Mol Hepatol 2020 07 23;26(3):378-381. Epub 2020 Jun 23.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

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http://dx.doi.org/10.3350/cmh.2020.0069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7364350PMC
July 2020

Multicenter evaluation of analytical performances of platelet counts and platelet parameters: Carryover, precision, and stability.

Int J Lab Hematol 2020 Oct 18;42(5):552-564. Epub 2020 Apr 18.

Clinical Chemistry Laboratory, Papa Giovanni XXIII Hospital, Bergamo, Italy.

Introduction: The correctness of the results of automated platelet analysis is still highly debated. The aim of this multicenter study, conducted according to international guidelines, was to verify the analytical performance of nine different types of hematology analyzers (HAs) in the automated platelet analysis.

Methods: Four hundred eighty-six peripheral blood samples (PB), collected in K EDTA tubes, were analyzed by ABX Pentra, ADVIA2120i, BC-6800, BC-6800 Plus, Cell-DYN Sapphire, DxH800, XE-2100, XE-5000, XN-20 with PLT-F App. Within-run imprecision and between-run imprecision were carried out using PB and material control, respectively. The carryover, low limit of quantification (LoQ), and the PB stability were evaluated.

Results: The carryover was absent for all HAs. The LoQ of PLT ranged between 2.0 (Cell-Dyn Sapphire) and 25.0 × 10 /L (ADVIA 2120i), while immature platelet fraction (IPF) ranged between 1.0 (XN-20) and 12.0 × 10 /L (XE-5000). The imprecision (%CV) increases as the platelet count decreases. No HAs showed desirable CV for PLT counts less than 50.0 × 10 /L, with the exception of Cell-DYN Sapphire (CV 3.0% with PLT-O mean value of 26.7 × 10 /L), XN-20 (CV 2.4% with PLT-F mean value of 21.5 × 10 /L), and BC-6800 Plus (CV 1.9% with PLT-O mean value of 26.5 × 10 /L). The sample stability ranged between under two hours for MPV by ADVIA2120i and 8 hours for other PLT parameters and HAs.

Conclusion: The findings of this study may provide useful information regarding carryover, precision, and stability of platelet counts and parameters, especially in thrombocytopenic samples. Moreover, the stability of sample for platelet analysis is conditioned by the HA and by temperature and storage time.
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http://dx.doi.org/10.1111/ijlh.13204DOI Listing
October 2020

Identification of Amoxicillin Crystals in Urine: a Case Report.

Clin Lab 2020 Mar;66(3)

Background: The case concerns a 30-year-old woman in the 24th week of pregnancy presenting to the medical emergency room with fever and abdominal pain. Urine sediment microscopy revealed the presence of unknown needle-shaped crystals.

Methods: Crystals identification was performed by Fourier-Transform Infrared Spectroscopy coupled to Attenuated Total Reflectance (FTIR-ATR).

Results: Amoxicillin crystals were verified with semiquantitative results of 87.7%.

Conclusions: Drug-induced crystalluria is a frequent finding in urine examination and it may be asymptomatic. FTIR spectroscopy is a rapid and specific tool in identification of crystals and could be useful supporting renal disease diagnosis and monitoring drug therapy.
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http://dx.doi.org/10.7754/Clin.Lab.2019.190606DOI Listing
March 2020

Stem-Cell-Derived Circulating Progenitors Dysfunction in Behçet's Syndrome Patients Correlates With Oxidative Stress.

Front Immunol 2019 13;10:2877. Epub 2019 Dec 13.

Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Firenze, Firenze, Italy.

Behçet's syndrome (BS) is a systemic vasculitis considered as the prototype of a systemic inflammation-induced thrombotic condition whose pathogenesis cannot be explained just by coagulation abnormalities. Circulating hematopoietic progenitor cells (CPC), a population of rare, pre-differentiated adult stem cells originating in the bone marrow and capable of both self-renewal and multi-lineage differentiation, are mobilized in response to vascular injury and play a key role in tissue repair. In cardiovascular and thrombotic diseases, low circulating CPC number and reduced CPC function have been observed. Oxidative stress may be one of the relevant culprits that account for the dysfunctional and numerically reduced CPC in these conditions. However, the detailed mechanisms underlying CPC number reduction are unknown. On this background, the present study was designed to evaluate for the first time the possible relationship between CPC dysfunction and oxidative stress in BS patients. In BS patients, we found signs of plasma oxidative stress and significantly lower CD34+/CD45 and CD34+/CD45/CD133+ CPC levels. Importantly, in all the considered CPC subsets, significantly higher ROS levels with respect to controls were observed. Higher levels of caspase-3 activity in all the considered CPC population and a strong reduction in GSH content in CPC subpopulation from BS patients with respect to controls were also observed. Interestingly, in BS patients, ROS significantly correlated with CPC number and CPC caspase-3 activity and CPC GSH content significantly correlated with CPC number, in all CPC subsets. Collectively, these data demonstrate for the first time that CPC from BS patients show signs of oxidative stress and apoptosis and that a reduced CPC number is present in BS patients with respect to controls. Interestingly, we observed an inverse correlation between circulating CPC number and CPC ROS production, suggesting a possible toxic ROS effect on CPC in BS patients. The significant correlations between ROS production/GSH content and caspase-3 activity point out that oxidative stress can represent a determinant in the onset of apoptosis in CPC. These data support the hypothesis that oxidative-stress-mediated CPC dysfunctioning may counteract their vascular repair actions, thereby contributing to the pathogenesis and the progression of vascular disease in BS.
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http://dx.doi.org/10.3389/fimmu.2019.02877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923242PMC
November 2020

Reperfusion Injury after ischemic Stroke Study (RISKS): single-centre (Florence, Italy), prospective observational protocol study.

BMJ Open 2018 05 24;8(5):e021183. Epub 2018 May 24.

Institute of Neuroscience, Italian National Research Council, Florence, Italy.

Introduction: Treatments aiming at reperfusion of the acutely ischaemic brain tissue may result futile or even detrimental because of the so-called reperfusion injury. The processes contributing to reperfusion injury involve a number of factors, ranging from blood-brain barrier (BBB) disruption to circulating biomarkers. Our aim is to evaluate the relative effect of imaging and circulating biomarkers in relation to reperfusion injury.

Methods And Analysis: Observational hospital-based study that will include 140 patients who had ischaemic stroke, treated with systemic thrombolysis, endovascular treatment or both. BBB disruption will be assessed with CT perfusion (CTP) before treatment, and levels of a large panel of biomarkers will be measured before intervention and after 24 hours. Relevant outcomes will include: (1) reperfusion injury, defined as radiologically relevant haemorrhagic transformation at 24 hours and (2) clinical status 3 months after the index stroke. We will investigate the separate and combined effect of pretreatment BBB disruption and circulating biomarkers on reperfusion injury and clinical status at 3 months. Study protocol is registered at http://www.clinicaltrials.gov (ClinicalTrials.gov ID: NCT03041753).

Ethics And Dissemination: The study protocol has been approved by ethics committee of the Azienda Ospedaliero Universitaria Careggi (Università degli Studi di Firenze). Informed consent is obtained by each patient at time of enrolment or deferred when the participant lacks the capacity to provide consent during the acute phase. Researchers interested in testing hypotheses with the data are encouraged to contact the corresponding author. Results from the study will be disseminated at national and international conferences and in medical thesis.

Trial Registration Number: NCT03041753.
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http://dx.doi.org/10.1136/bmjopen-2017-021183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988101PMC
May 2018

Harmonization of interpretative comments in laboratory hematology reporting: the recommendations of the Working Group on Diagnostic Hematology of the Italian Society of Clinical Chemistry and Clinical Molecular Biology (WGDH-SIBioC).

Clin Chem Lab Med 2018 12;57(1):66-77

Section of Clinical Biochemistry, University Hospital of Verona, Verona, Italy.

The goal of harmonizing laboratory testing is contributing to improving the quality of patient care and ultimately ameliorating patient outcome. The complete blood and leukocyte differential counts are among the most frequently requested clinical laboratory tests. The morphological assessment of peripheral blood cells (PB) through microscopic examination of properly stained blood smears is still considered a hallmark of laboratory hematology. Nevertheless, a variable inter-observer experience and the different terminology used for characterizing cellular abnormalities both contribute to the current lack of harmonization in blood smear revision. In 2014, the Working Group on Diagnostic Hematology of the Italian Society of Clinical Chemistry and Clinical Molecular Biology (WGDH-SIBioC) conducted a national survey, collecting responses from 78 different Italian laboratories. The results of this survey highlighted a lack of harmonization of interpretative comments in hematology, which prompted the WGDH-SIBioC to develop a project on "Harmonization of interpretative comments in the laboratory hematology report", aimed at identifying appropriate comments and proposing a standardized reporting system. The comments were then revised and updated according to the 2016 revision of the World Health Organization classification of hematologic malignancies. In 2016, the Working Group on Diagnostic Hematology of the Italian Society of Clinical Chemistry and Clinical Molecular Biology (WGDH SIBioC) published its first consensus based recommendation for interpretative comments in laboratory hematology reporting whit the purpose of evaluating comments and the aim to (a) reducing their overall number, (b) standardizing the language, (c) providing information that could be easily comprehended by clinicians and patients, (d) increasing the quality of the clinical information, and (e) suggesting additional diagnostic tests when necessary. This paper represents a review of the recommendations of the former document.
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http://dx.doi.org/10.1515/cclm-2017-0972DOI Listing
December 2018

Appropriate use of laboratory test requests in the emergency department: a multilevel intervention.

Eur J Emerg Med 2019 Jun;26(3):205-211

Departments of Emergency Medicine.

Objective: Laboratory test requests in the emergency department (ED) are increasing worldwide. We evaluated whether a multilevel intervention on the basis of the optimization of test profiles and educational meetings with physicians could reduce the number of tests ordered.

Patients And Methods: In a single-center before and after study design, the 8-month intervention period was compared with the 8-month preintervention period. Laboratory test profiles were reduced from 6 to 2 and the number of tests in each profile was reduced by 50%. All physicians received education about the costs and appropriate use of the tests. Primary outcomes were the number of laboratory blood tests and their costs, with a focus on high-cost tests. Secondary outcomes were ED and laboratory performances (patients' waiting time, number of deaths in ED, re-entry, laboratory turn-around time, and add-on tests).

Results: Overall, 61 976 and 61 154 patients were evaluated, respectively, during the intervention and the preintervention period. Laboratory blood test requests were decreased by 207 637 (-36.3%) in the intervention period (P < 0.05), which corresponds to a reduction of 337.3 tests/100 patients. Costs were decreased by 608 079&OV0556; ( - 29.6%, P < 0.05), leading to a cost reduction of 981.2&OV0556;/100 patients. High-cost test requests decreased by 11 457 ( - 27.3%) and contributed toward the overall reduction in costs with 197 206&OV0556; ( - 30.5%). No significant differences were found in ED and laboratory performances between intervention and preintervention periods.

Conclusions: Optimization of test profiles and education on the costs and appropriate use of the tests significantly reduced laboratory test ordering and costs without affecting ED and laboratory performances.
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http://dx.doi.org/10.1097/MEJ.0000000000000518DOI Listing
June 2019

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

J Clin Microbiol 2016 07 11;54(7):1851-1854. Epub 2016 May 11.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

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.
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http://dx.doi.org/10.1128/JCM.00149-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922106PMC
July 2016

Diagnosis of a T-lineage acute lymphoblastic leukemia through digitalized cell analysis of the pleural effusion.

Int Med Case Rep J 2013 1;6:77-80. Epub 2013 Nov 1.

General Laboratory Unit (Microscopy and Clinical Cytometry Unit), Firenze, Italy.

Introduction: Pleural effusion as the first clinical manifestation of acute lymphoblastic leukemia (ALL) is a relatively rare event. An early and accurate diagnosis of this clinical picture is very important for adequate patient management.

Case Presentation: We report the atypical onset of T-lineage ALL in a 31-year-old man. The patient was admitted to the emergency room due to lung failure; at that moment, the patient's initial blood count was normal; the chest X-ray radiography showed a massive pleural effusion and a thoracentesis was carried out. Routine investigations performed on the pleural fluid using a new technology system and digitalized cell analysis demonstrated infiltration by immature cells. Therefore, bone marrow aspirate and flow cytometry analyses were performed, leading to the diagnosis of T-lineage ALL. A cord blood transplantation procedure was performed at the first hematological remission following chemotherapy regimens. The patient died of septic shock.

Conclusion: The case we reported underlines the usefulness of using automated instruments to identify abnormal lymphoid cells in body fluids.
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http://dx.doi.org/10.2147/IMCRJ.S49278DOI Listing
November 2013

High platelet turnover and reactivity in renal transplant recipients patients.

Thromb Haemost 2010 Oct 5;104(4):804-10. Epub 2010 Aug 5.

Department of Medical and Surgical Critical Care, Thrombosis Centre, Center for the Study at Molecular and Clinical Level of Chronic, Degenerative and Neoplastic Diseases to Develop Novel Therapies, University of Florence, Italy.

Renal transplant recipients (RTRs) patients are at increased risk of cardiovascular morbidity and mortality. We aimed this study to assess reticulated platelets (RP), platelet reactivity and von Willebrand factor (vWF) levels in RTRs patients. In 150 RTRs patients [84 (56%) not on acetylsalicylic acid (ASA) treatment, group A; 66 (44%) on ASA 100 mg treatment, group B] and in 60 healthy control subjects, RP were measured by a Sysmex XE-2100 and were expressed as the percentage of RP of the total optical platelet count (immature platelet fraction; IPF), as the percentage of RP highly fluorescent (H-IPF) and as the absolute number of RP (IPF#). Platelet function was assessed by optical aggregometry (PA) induced by 1 mmol arachidonic acid (AA-PA), 2 and 10 μM ADP (ADP2-PA and ADP10-PA) and 2 μg/ml collagen (Coll-PA). vWF levels were measured by using a miniVidas analyser. Group A and group B showed significant higher values of RP than controls. At a multiple linear regression analysis IPF and IPF# were significantly and positively related to collagen-PA. By analysing group B according to residual platelet reactivity (RPR), we observed a significant higher number of RP among patients with RPR by collagen. Moreover at a multiple logistic regression analysis, IPF# significantly affected the risk of having a RPR by collagen. With regard to vWF, RTRs patients showed higher levels than control subjects. We documented a higher platelet turn-over in both groups of RTRs patients and increased platelet reactivity in RTRs patients not on ASA therapy than controls.
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http://dx.doi.org/10.1160/TH10-02-0124DOI Listing
October 2010

Epoetin alpha, epoetin beta and darbepoetin alfa: two-dimensional gel electrophoresis isoforms characterization and mass spectrometry analysis.

Proteomics 2003 Jun;3(6):937-41

Laboratorio Centrale Analisi Biochimico-Cliniche, Azienda Ospedaliera Careggi, Florence, Italy.

Since 1989 recombinant human erythropoietin (rhEPO) has been used as a drug for the correction of anemia, but the misuse of rhEPO as an ergogenic agent among athletes is a widespread doping practice. As a consequence there is a need for developing reference methods for the detection of rhEPO in biological fluids, and to be able to differentiate the recombinant from the natural protein. Recombinant human erythropoietin differs from its natural counterpart in the glycidic part of the molecule. Three different commercial recombinant products Epoetin alpha (Eprex, Janssen Cilag), Epoetin beta (Neorecormon, Roche) and Darbepoetin alfa (Nespo, Dompè) have been used to evaluate the performance of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) for the separation of isoforms and the identification of the proteins respectively. All the compounds studied were well separated by means of 2-DE: Epoetin alpha and beta focused in the same isoelectric point region giving rise to six and eight spots respectively, whereas Darbepoetin alfa was found in a more acidic zone with two spots. Results obtained with micro high-performance liquid chromatography-electrospray ionization-time of flight (TOF) MS and matrix-assisted laser desorption/ionization-time of flight MS for the three rhEPOs are reported. These preliminary results suggest that by means of 2-DE and MS it should be possible to reveal the presence of rhEPOs for antidoping purposes.
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http://dx.doi.org/10.1002/pmic.200300405DOI Listing
June 2003
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