Massimo Locatelli - Academia.edu (original) (raw)

Papers by Massimo Locatelli

Research paper thumbnail of Smart Immunosensors for Point-of-Care Serological Tests Aimed at Assessing Natural or Vaccine-Induced SARS-CoV-2 Immunity

Sensors

Innovative and highly performing smart voltammetric immunosensors for rapid and effective serolog... more Innovative and highly performing smart voltammetric immunosensors for rapid and effective serological tests aimed at the determination of SARS-CoV-2 antibodies were developed and validated in human serum matrix. Two immunosensors were developed for the determination of immunoglobulins directed against either the nucleocapsid or the spike viral antigen proteins. The immunosensors were realized using disposable screen-printed electrodes modified with nanostructured materials for the immobilization of the antigens. Fast quantitative detection was achieved, with analysis duration being around 1 h. Signal readout was carried out through a smart, compact and battery-powered potentiostat, based on a Wi-Fi protocol and devised for the Internet of Things (IoT) paradigm. This device is used for the acquisition, storage and sharing of clinical data. Outstanding immunosensors’ sensitivity, specificity and accuracy (100%) were assessed, according to the diagnostic guidelines for epidemiological ...

Research paper thumbnail of Routine blood tests as an active surveillance to monitor COVID-19 prevalence. A retrospective study

Acta Bio Medica : Atenei Parmensis, 2020

Background: In Italy, one of the country most affected by the COVID-19 pandemic, the first autoch... more Background: In Italy, one of the country most affected by the COVID-19 pandemic, the first autochthonous case appeared in Lombardy on February 20th, 2020. One month later, the number of COVID-19 patients in Lombardy exceeded 17000 and about 3500 had died. Because of this rapid increase in infected people scientists wonder whether SARS-CoV-2 was already highly circulating in Lombardy before such date. Plasma levels of aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were shown to be highly increased in COVID-19 patients. Monitoring their levels in Emergency Room patients during the months preceding February 20th, 2020, might shade light on the prevalence of the disease in the pre-COVID-19 period. Methods: We retrospectively analyzed the AST and LDH levels from more than 30.000 patients admitted to the San Raffaele Hospital Emergency Room (ER) between September 2019 and May 2020 as well as between September 2018 and May 2019. The number of patients diagnosed with respi...

Research paper thumbnail of SARS-CoV-2 infection despite high levels of vaccine-induced anti-Receptor-Binding-Domain antibodies: a study on 1110 health-care professionals from a northern Italian university hospital

Clinical Microbiology and Infection, 2021

Research paper thumbnail of Severely low testosterone in males with COVID‐19: A case‐control study

Andrology, 2021

Background: Circulating androgens could have a relevant pathobiological role in clinical outcomes... more Background: Circulating androgens could have a relevant pathobiological role in clinical outcomes in men with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19). Objectives: We aimed to assess: (a) circulating sex steroids levels in a cohort of 286 symptomatic men with laboratory-confirmed COVID-19 at hospital admission compared to a cohort of 281 healthy men; and (b) the association between serum testosterone levels (tT), COVID-19, and clinical outcomes. Materials and Methods: Demographic, clinical, and hormonal values were collected for all patients. Hypogonadism was defined as tT ≤9.2 nmol/l. The Charlson Comorbidity Index (CCI) was used to score health-significant comorbidities. Severe clinical outcomes were defined as patients either transferred to intensive care unit

Research paper thumbnail of COVID-19 in recipients of allogeneic stem cell transplantation: favorable outcome

Bone Marrow Transplantation, 2021

Research paper thumbnail of MYD88 L265P mutation and interleukin‐10 detection in cerebrospinal fluid are highly specific discriminating markers in patients with primary central nervous system lymphoma: results from a prospective study

British Journal of Haematology, 2021

Reliable biomarkers are needed to avoid diagnostic delay and its devastating effects in patients ... more Reliable biomarkers are needed to avoid diagnostic delay and its devastating effects in patients with primary central nervous system (CNS) lymphoma (PCNSL). We analysed the discriminating sensitivity and specificity of myeloid differentiation primary response (88) (MYD88) L265P mutation (mut‐MYD88) and interleukin‐10 (IL‐10) in cerebrospinal fluid (CSF) of both patients with newly diagnosed (n = 36) and relapsed (n = 27) PCNSL and 162 controls (118 CNS disorders and 44 extra‐CNS lymphomas). The concordance of MYD88 mutational status between tumour tissue and CSF sample and the source of ILs in PCNSL tissues were also investigated. Mut‐MYD88 was assessed by TaqMan‐based polymerase chain reaction. IL‐6 and IL‐10 messenger RNA (mRNA) was assessed on PCNSL biopsies using RNAscope technology. IL levels in CSF were assessed by enzyme‐linked immunosorbent assay. Mut‐MYD88 was detected in 15/17 (88%) PCNSL biopsies, with an 82% concordance in paired tissue‐CSF samples. IL‐10 mRNA was detected in lymphomatous B cells in most PCNSL; expression of IL‐6 transcripts was negligible. In CSF samples, mut‐MYD88 and high IL‐10 levels were detected, respectively, in 72% and 88% of patients with newly diagnosed PCNSL and in 1% of controls; conversely, IL‐6 showed a low discriminating sensitivity and specificity. Combined analysis of MYD88 and IL‐10 exhibits a sensitivity and specificity to distinguish PCNSL of 94% and 98% respectively. Similar figures were recorded in patients with relapsed PCNSL. In conclusion, high detection rates of mut‐MYD88 and IL‐10 in CSF reflect, respectively, the MYD88 mutational status and synthesis of this IL in PCNSL tissue. These biomarkers exhibit a very high sensitivity and specificity in detecting PCNSL both at initial diagnosis and relapse. Implications of these findings in patients with lesions unsuitable for biopsy deserve to be investigated.

Research paper thumbnail of Lower nasopharyngeal viral load during the latest phase of COVID-19 pandemic in a Northern Italy University Hospital

Clinical Chemistry and Laboratory Medicine (CCLM), 2020

Objectives A milder clinical course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-... more Objectives A milder clinical course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been anecdotally reported over the latest phase of COVID-19 pandemic in Italy. Several factors may contribute to this observation, including the effect of lockdown, social distancing, lower humidity, lower air pollution, and potential changes in the intrinsic pathogenicity of the virus. In this regard, the clinical severity of COVID-19 could be attenuated by mutations in SARS-CoV-2 genome that decrease its virulence, as well as by lower virus inocula. Methods In this pilot study, we compared the reverse transcription polymerase chain reaction (RT-PCR) amplification profile of 100 nasopharyngeal swabs consecutively collected in April, during the peak of SARS-CoV-2 epidemic, to that of 100 swabs collected using the same procedure in May. Results The mean Ct value of positive samples collected in May was significantly higher than that of samples collected in the previous pe...

Research paper thumbnail of A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries

Molecular Biotechnology, 2019

Single-domain antigen-binding fragments of camelid antibodies, known as VHHs or nanobodies, are w... more Single-domain antigen-binding fragments of camelid antibodies, known as VHHs or nanobodies, are widely used affinity reagents. However, their production involving animal immunization is time-and resource-intensive. Starting from a sequence dataset of llama VHHs, we designed a novel scaffold, based on conserved framework sequences, suitable for bacterial nanobody expression and synthetic library construction. The consensus scaffold was validated by grafting the CDRs from two known nanobodies. While maintaining their binding properties, the two chimeric nanobodies showed higher levels of expression and solubility in E. coli when compared to the corresponding wild types. A proof-of-concept synthetic combinatorial library, suitable for ribosome display (RD) selection, was obtained by encoding three randomized complementarity determining regions within the consensus framework. The library, made of linear DNA fragments, has an estimated complexity of > 10 12 that is three orders of magnitude higher than common phage display libraries. The bacterial expression of several library clones showed a high production of soluble recombinant proteins. The high complexity of the library, confirmed by sequencing of a subset of clones, as well as a preliminary RD selection of a maltose binding protein binder, indicated this approach as a starting point in the construction of synthetic combinatorial libraries to be used as animal-free tools for the low-cost selection of target-specific nanobodies.

Research paper thumbnail of Association between solar ultraviolet doses and vitamin D clinical routine data in European mid-latitude population between 2006 and 2018

Photochemical & Photobiological Sciences, 2019

The major source of vitamin D in humans is represented by the ultraviolet radiation which induces... more The major source of vitamin D in humans is represented by the ultraviolet radiation which induces dermal synthesis of cholecalciferol, however, low vitamin D status is common in Europe even...

Research paper thumbnail of Biological variation estimates for prostate specific antigen from the European Biological Variation Study; consequences for diagnosis and monitoring of prostate cancer

Clinica Chimica Acta, 2018

Background: Prostate-specific antigen (PSA) is central in the diagnosis of prostate cancer. Howev... more Background: Prostate-specific antigen (PSA) is central in the diagnosis of prostate cancer. However, high-quality biological variation (BV) estimates for PSA are scarce. Here BV estimates from the European Biological Variation Study (EuBIVAS) for total (tPSA), free (fPSA), conjugated PSA (cPSA), and percent free PSA (%fPSA) are provided. Method: EuBIVAS samples were collected weekly from thirty-seven healthy males (22-59 years) for 10 weeks. All samples, stored at −80°C, were measured in duplicate with a Roche Cobas e801. Outlier and homogeneity analysis were performed followed by CV-ANOVA to determine BV, analytical variation, analytical performance specifications (APS), reference change values (RCV) and the number of samples required to estimate the homeostatic set points. Results: Within-subject BV estimates were for tPSA 6.8% (6.1-7.4); fPSA 7.1% (6.5-7.7) cPSA: 8.8% (8.0-9.7) and %fPSA 5.3% (4.8-5.8), delivering RCV for increase of 15-20% and indicating that one sample is sufficient to estimate the homeostatic set points within ± 15%. BV estimates for tPSA were lower than previously published estimates. Estimates for fPSA, cPSA and %fPSA have not previously been reported in healthy subjects. Conclusions: Highly powered EuBIVAS BV estimates of tPSA, fPSA, cPSA and %fPSA provide updated APS and RCV for monitoring for prostate cancer. List of abbreviations APS analytical performance specification B APS analytical performance specification for bias BIVAC Biological Variation Data Critical Appraisal Checklist BV biological variation CV A analytical variation CV APS analytical performance specification for imprecision CV I within-subject biological variation CV G between-subject biological variation EuBIVAS European Biological Variation Study

Research paper thumbnail of A nucleoside-sparing regimen of dolutegravir plus ritonavir-boosted atazanavir in HIV-1-infected patients with virological failure: the DOLATAV study

Drug Design, Development and Therapy, 2019

Research paper thumbnail of Alcohol and illicit drugs in drivers involved in road traffic crashes in the Milan area. A comparison with normal traffic reveals the possible inadequacy of current cut-off limits

Forensic science international, Jan 22, 2017

Driving under the influence of alcohol and/or illicit drugs in Italy is regulated by the articles... more Driving under the influence of alcohol and/or illicit drugs in Italy is regulated by the articles 186 and 187 of the National Street Code. Epidemiological studies on drivers involved in road traffic crashes (RTC) provide useful information about the use/abuse of these substances in the general population. Comparison with case control studies may reveal important information like the cut-off limits adequacy. Data from 1587 blood tests for alcohol and 1258 blood tests for illicit drugs on drivers involved in RTC around Milan between 2012 and 2016, were analyzed and compared with a published random survey (DRUID) from the European Community. Our data from RTC-involved drivers show that alcohol abuse is not age-related whereas illicit drugs are more common in young people. Cannabinoids are frequent among younger drivers (median age 27) whereas cocaine is more often detected in adults (median age 34). The calculated odds ratio after comparison with the DRUID survey shows that a blood alc...

Research paper thumbnail of Vitamina D: valutazione di un metodo cromatografico liquido ultra veloce (UPLC) e confronto con tecnica immunometrica e cromatografia liquida ad alta prestazione (HPLC) tradizionale

La Rivista Italiana della Medicina di Laboratorio - Italian Journal of Laboratory Medicine, 2012

Vitamin D: evaluation of an ultrafast liquid chromatography method (UPLC) and its comparison with... more Vitamin D: evaluation of an ultrafast liquid chromatography method (UPLC) and its comparison with a traditional immunoassay and high-performance liquid chromatography (HPLC) technique

Research paper thumbnail of Determination of Aspirin and Salicylic Acid in Human Plasma by Column-Switching Liquid Chromatography Using On-Line Solid-Phase Extraction

Analytical Chemistry, 1998

ABSTRACT A column-switching liquid chromatographic method is described for the simultaneous deter... more ABSTRACT A column-switching liquid chromatographic method is described for the simultaneous determination of aspirin and salicylic acid in human plasma. Blood samples are taken into chilled tubes containing a fluoride anticoagulant, and the plasma is isolated by centrifugation. Following a simple acidification step, a 200 microL aliquot of the sample is injected directly onto the HPLC system. The C-18 extraction column is washed with acidified water for 2 min, after which time the compounds are removed by back-flushing directly onto the analytical column (C-8 Nucleosil, 5 microns, 250 mm x 4.6 mm). The flow rate through both columns is 1 mL/min, and the analytes are quantified by measurement of their UV absorbance at 225 nm. The mobile phase is a mixture of water-methanol-acetonitrile-orthophosphoric acid (650:200:150:1 v/v/v/v). The method is linear in the concentration ranges 0.10-5.00 micrograms/mL for aspirin and 0.25-15.00 micrograms/mL for salicylic acid. Both compounds have a limit of quantitation of 0.10 microgram/mL and a limit of detection of 0.04 microgram/mL. Extensive stability tests have been carried out, and validation studies reveal the method to be reproducible and repeatable. Excellent recoveries from plasma obviate the need for an internal standard. The procedure is easier to execute and requires less sample handling than methods currently described in the literature. It has been successfully applied to the investigation of the levels of aspirin and salicylic acid in a healthy, nonfasting volunteer following a 600 mg oral dose of aspirin.

Research paper thumbnail of Role of time-normalized laboratory findings in predicting COVID-19 outcome

Diagnosis, 2020

Objectives The pandemic COVID-19 currently reached 213 countries worldwide with nearly 9 million ... more Objectives The pandemic COVID-19 currently reached 213 countries worldwide with nearly 9 million infected people and more than 460,000 deaths. Although several Chinese studies, describing the laboratory findings characteristics of this illness have been reported, European data are still scarce. Furthermore, previous studies often analyzed the averaged laboratory findings collected during the entire hospitalization period, whereas monitoring their time-dependent variations should give more reliable prognostic information. Methods We analyzed the time-dependent variations of 14 laboratory parameters in two groups of COVID-19 patients with, respectively, a positive (40 patients) or a poor (42 patients) outcome, admitted to the San Raffaele Hospital (Milan, Italy). We focused mainly on laboratory parameters that are routinely tested, thus, prognostic information would be readily available even in low-resource settings. Results Statistically significant differences between the two groups...

Research paper thumbnail of Routine blood analysis greatly reduces the false-negative rate of RT-PCR testing for COVID-19

Acta Bio Medica : Atenei Parmensis, 2020

Background: The COVID-19 outbreak is now a pandemic disease reaching as much as 210 countries wor... more Background: The COVID-19 outbreak is now a pandemic disease reaching as much as 210 countries worldwide with more than 2.5 million infected people and nearly 200.000 deaths. Amplification of viral RNA by RT-PCR represents the gold standard for confirmation of infection, yet it showed false-negative rates as large as 15-20% which may jeopardize the effect of the restrictive measures taken by governments. We previously showed that several hematological parameters were significantly different between COVID-19 positive and negative patients. Among them aspartate aminotransferase and lactate dehydrogenase had predictive values as large as 90%. Thus a combination of RT-PCR and blood tests could reduce the false-negative rate of the genetic test. Methods: We retrospectively analyzed 24 patients showing multiple and inconsistent RT-PCR, test during their first hospitalization period, and compared the genetic tests results with their AST and LDH levels. Results: We showed that when consideri...

Research paper thumbnail of Antibody Titer Kinetics and SARS-CoV-2 Infections Six Months after Administration with the BNT162b2 Vaccine

Vaccines, 2021

Background: Studies reporting the long-term humoral response after receiving the BNT162b2 COVID-1... more Background: Studies reporting the long-term humoral response after receiving the BNT162b2 COVID-19 vaccine are important to drive future vaccination strategies. Yet, available literature is scarce. Covidiagnostix is a multicenter study designed to assess the antibody response in >1000 healthcare professionals (HCPs) who received the BNT162b2 vaccine. Methods: Serum was tested at time-0 (T0), before the first dose, T1, T2, and T3, respectively, 21, 42, and 180 days after T0. Antibodies against the SARS-CoV-2 nucleocapsid-protein were measured to assess SARS-CoV-2 infections, whereas antibodies against the receptor-binding domain of the spike protein were measured to assess the vaccine response. Neutralization activity against the D614G, B.1.1.7, and B.1.351 variants were also analyzed. Results: Six months post-vaccination HCPs showed an antibody titer decrease of approximately 70%, yet, the titer was still one order of magnitude higher than that of seropositive individuals before ...

Research paper thumbnail of Characterization of a Lineage C.36 SARS-CoV-2 Isolate with Reduced Susceptibility to Neutralization Circulating in Lombardy, Italy

Viruses, 2021

SARS-CoV-2 spike is evolving to maximize transmissibility and evade the humoral response. The mas... more SARS-CoV-2 spike is evolving to maximize transmissibility and evade the humoral response. The massive genomic sequencing of SARS-CoV-2 isolates has led to the identification of single-point mutations and deletions, often having the recurrence of hotspots, associated with advantageous phenotypes. We report the isolation and molecular characterization of a SARS-CoV-2 strain, belonging to a lineage (C.36) not previously associated with concerning traits, which shows decreased susceptibility to vaccine sera neutralization.

Research paper thumbnail of Changes in 25-(OH) Vitamin D Levels during the SARS-CoV-2 Outbreak: Lockdown-Related Effects and First-to-Second Wave Difference—An Observational Study from Northern Italy

Biology, 2021

Background. We investigated the changes in 25-hydroxy vitamin D [25-(OH)D] concentrations values,... more Background. We investigated the changes in 25-hydroxy vitamin D [25-(OH)D] concentrations values, during the first and the second pandemic waves and the impact of the lockdown periods, with their different approaches to home confinement, among different age groups. Methods. Daily cloud-modified vitamin D UV dose (UVDVC), for the area of interest (Milano, Italy), was obtained. Single-center 25-(OH)D determinations from 40,996 individuals in a 2019 (pre-pandemic), 32,355 individuals from 1 January to 31 August 2020 (containing the first pandemic wave) and 27,684 individuals from 1 June to 30 November 2020 (containing the second wave) were compared to investigate both the effect of the lockdown on vitamin D status and the association between 25-(OH)D and SARS-CoV-2 positivity. Results. No direct association was found between UVDVC, 25-(OH)D status and SARS-CoV-2 infection. The stringent confinement implemented during the first wave lockdown has not had any effect on 25-(OH)D status, al...

Research paper thumbnail of Biological variation of serum insulin: updated estimates from the European Biological Variation Study (EuBIVAS) and meta-analysis

Clinical Chemistry and Laboratory Medicine (CCLM), 2020

Research paper thumbnail of Smart Immunosensors for Point-of-Care Serological Tests Aimed at Assessing Natural or Vaccine-Induced SARS-CoV-2 Immunity

Sensors

Innovative and highly performing smart voltammetric immunosensors for rapid and effective serolog... more Innovative and highly performing smart voltammetric immunosensors for rapid and effective serological tests aimed at the determination of SARS-CoV-2 antibodies were developed and validated in human serum matrix. Two immunosensors were developed for the determination of immunoglobulins directed against either the nucleocapsid or the spike viral antigen proteins. The immunosensors were realized using disposable screen-printed electrodes modified with nanostructured materials for the immobilization of the antigens. Fast quantitative detection was achieved, with analysis duration being around 1 h. Signal readout was carried out through a smart, compact and battery-powered potentiostat, based on a Wi-Fi protocol and devised for the Internet of Things (IoT) paradigm. This device is used for the acquisition, storage and sharing of clinical data. Outstanding immunosensors’ sensitivity, specificity and accuracy (100%) were assessed, according to the diagnostic guidelines for epidemiological ...

Research paper thumbnail of Routine blood tests as an active surveillance to monitor COVID-19 prevalence. A retrospective study

Acta Bio Medica : Atenei Parmensis, 2020

Background: In Italy, one of the country most affected by the COVID-19 pandemic, the first autoch... more Background: In Italy, one of the country most affected by the COVID-19 pandemic, the first autochthonous case appeared in Lombardy on February 20th, 2020. One month later, the number of COVID-19 patients in Lombardy exceeded 17000 and about 3500 had died. Because of this rapid increase in infected people scientists wonder whether SARS-CoV-2 was already highly circulating in Lombardy before such date. Plasma levels of aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were shown to be highly increased in COVID-19 patients. Monitoring their levels in Emergency Room patients during the months preceding February 20th, 2020, might shade light on the prevalence of the disease in the pre-COVID-19 period. Methods: We retrospectively analyzed the AST and LDH levels from more than 30.000 patients admitted to the San Raffaele Hospital Emergency Room (ER) between September 2019 and May 2020 as well as between September 2018 and May 2019. The number of patients diagnosed with respi...

Research paper thumbnail of SARS-CoV-2 infection despite high levels of vaccine-induced anti-Receptor-Binding-Domain antibodies: a study on 1110 health-care professionals from a northern Italian university hospital

Clinical Microbiology and Infection, 2021

Research paper thumbnail of Severely low testosterone in males with COVID‐19: A case‐control study

Andrology, 2021

Background: Circulating androgens could have a relevant pathobiological role in clinical outcomes... more Background: Circulating androgens could have a relevant pathobiological role in clinical outcomes in men with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19). Objectives: We aimed to assess: (a) circulating sex steroids levels in a cohort of 286 symptomatic men with laboratory-confirmed COVID-19 at hospital admission compared to a cohort of 281 healthy men; and (b) the association between serum testosterone levels (tT), COVID-19, and clinical outcomes. Materials and Methods: Demographic, clinical, and hormonal values were collected for all patients. Hypogonadism was defined as tT ≤9.2 nmol/l. The Charlson Comorbidity Index (CCI) was used to score health-significant comorbidities. Severe clinical outcomes were defined as patients either transferred to intensive care unit

Research paper thumbnail of COVID-19 in recipients of allogeneic stem cell transplantation: favorable outcome

Bone Marrow Transplantation, 2021

Research paper thumbnail of MYD88 L265P mutation and interleukin‐10 detection in cerebrospinal fluid are highly specific discriminating markers in patients with primary central nervous system lymphoma: results from a prospective study

British Journal of Haematology, 2021

Reliable biomarkers are needed to avoid diagnostic delay and its devastating effects in patients ... more Reliable biomarkers are needed to avoid diagnostic delay and its devastating effects in patients with primary central nervous system (CNS) lymphoma (PCNSL). We analysed the discriminating sensitivity and specificity of myeloid differentiation primary response (88) (MYD88) L265P mutation (mut‐MYD88) and interleukin‐10 (IL‐10) in cerebrospinal fluid (CSF) of both patients with newly diagnosed (n = 36) and relapsed (n = 27) PCNSL and 162 controls (118 CNS disorders and 44 extra‐CNS lymphomas). The concordance of MYD88 mutational status between tumour tissue and CSF sample and the source of ILs in PCNSL tissues were also investigated. Mut‐MYD88 was assessed by TaqMan‐based polymerase chain reaction. IL‐6 and IL‐10 messenger RNA (mRNA) was assessed on PCNSL biopsies using RNAscope technology. IL levels in CSF were assessed by enzyme‐linked immunosorbent assay. Mut‐MYD88 was detected in 15/17 (88%) PCNSL biopsies, with an 82% concordance in paired tissue‐CSF samples. IL‐10 mRNA was detected in lymphomatous B cells in most PCNSL; expression of IL‐6 transcripts was negligible. In CSF samples, mut‐MYD88 and high IL‐10 levels were detected, respectively, in 72% and 88% of patients with newly diagnosed PCNSL and in 1% of controls; conversely, IL‐6 showed a low discriminating sensitivity and specificity. Combined analysis of MYD88 and IL‐10 exhibits a sensitivity and specificity to distinguish PCNSL of 94% and 98% respectively. Similar figures were recorded in patients with relapsed PCNSL. In conclusion, high detection rates of mut‐MYD88 and IL‐10 in CSF reflect, respectively, the MYD88 mutational status and synthesis of this IL in PCNSL tissue. These biomarkers exhibit a very high sensitivity and specificity in detecting PCNSL both at initial diagnosis and relapse. Implications of these findings in patients with lesions unsuitable for biopsy deserve to be investigated.

Research paper thumbnail of Lower nasopharyngeal viral load during the latest phase of COVID-19 pandemic in a Northern Italy University Hospital

Clinical Chemistry and Laboratory Medicine (CCLM), 2020

Objectives A milder clinical course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-... more Objectives A milder clinical course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been anecdotally reported over the latest phase of COVID-19 pandemic in Italy. Several factors may contribute to this observation, including the effect of lockdown, social distancing, lower humidity, lower air pollution, and potential changes in the intrinsic pathogenicity of the virus. In this regard, the clinical severity of COVID-19 could be attenuated by mutations in SARS-CoV-2 genome that decrease its virulence, as well as by lower virus inocula. Methods In this pilot study, we compared the reverse transcription polymerase chain reaction (RT-PCR) amplification profile of 100 nasopharyngeal swabs consecutively collected in April, during the peak of SARS-CoV-2 epidemic, to that of 100 swabs collected using the same procedure in May. Results The mean Ct value of positive samples collected in May was significantly higher than that of samples collected in the previous pe...

Research paper thumbnail of A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries

Molecular Biotechnology, 2019

Single-domain antigen-binding fragments of camelid antibodies, known as VHHs or nanobodies, are w... more Single-domain antigen-binding fragments of camelid antibodies, known as VHHs or nanobodies, are widely used affinity reagents. However, their production involving animal immunization is time-and resource-intensive. Starting from a sequence dataset of llama VHHs, we designed a novel scaffold, based on conserved framework sequences, suitable for bacterial nanobody expression and synthetic library construction. The consensus scaffold was validated by grafting the CDRs from two known nanobodies. While maintaining their binding properties, the two chimeric nanobodies showed higher levels of expression and solubility in E. coli when compared to the corresponding wild types. A proof-of-concept synthetic combinatorial library, suitable for ribosome display (RD) selection, was obtained by encoding three randomized complementarity determining regions within the consensus framework. The library, made of linear DNA fragments, has an estimated complexity of > 10 12 that is three orders of magnitude higher than common phage display libraries. The bacterial expression of several library clones showed a high production of soluble recombinant proteins. The high complexity of the library, confirmed by sequencing of a subset of clones, as well as a preliminary RD selection of a maltose binding protein binder, indicated this approach as a starting point in the construction of synthetic combinatorial libraries to be used as animal-free tools for the low-cost selection of target-specific nanobodies.

Research paper thumbnail of Association between solar ultraviolet doses and vitamin D clinical routine data in European mid-latitude population between 2006 and 2018

Photochemical & Photobiological Sciences, 2019

The major source of vitamin D in humans is represented by the ultraviolet radiation which induces... more The major source of vitamin D in humans is represented by the ultraviolet radiation which induces dermal synthesis of cholecalciferol, however, low vitamin D status is common in Europe even...

Research paper thumbnail of Biological variation estimates for prostate specific antigen from the European Biological Variation Study; consequences for diagnosis and monitoring of prostate cancer

Clinica Chimica Acta, 2018

Background: Prostate-specific antigen (PSA) is central in the diagnosis of prostate cancer. Howev... more Background: Prostate-specific antigen (PSA) is central in the diagnosis of prostate cancer. However, high-quality biological variation (BV) estimates for PSA are scarce. Here BV estimates from the European Biological Variation Study (EuBIVAS) for total (tPSA), free (fPSA), conjugated PSA (cPSA), and percent free PSA (%fPSA) are provided. Method: EuBIVAS samples were collected weekly from thirty-seven healthy males (22-59 years) for 10 weeks. All samples, stored at −80°C, were measured in duplicate with a Roche Cobas e801. Outlier and homogeneity analysis were performed followed by CV-ANOVA to determine BV, analytical variation, analytical performance specifications (APS), reference change values (RCV) and the number of samples required to estimate the homeostatic set points. Results: Within-subject BV estimates were for tPSA 6.8% (6.1-7.4); fPSA 7.1% (6.5-7.7) cPSA: 8.8% (8.0-9.7) and %fPSA 5.3% (4.8-5.8), delivering RCV for increase of 15-20% and indicating that one sample is sufficient to estimate the homeostatic set points within ± 15%. BV estimates for tPSA were lower than previously published estimates. Estimates for fPSA, cPSA and %fPSA have not previously been reported in healthy subjects. Conclusions: Highly powered EuBIVAS BV estimates of tPSA, fPSA, cPSA and %fPSA provide updated APS and RCV for monitoring for prostate cancer. List of abbreviations APS analytical performance specification B APS analytical performance specification for bias BIVAC Biological Variation Data Critical Appraisal Checklist BV biological variation CV A analytical variation CV APS analytical performance specification for imprecision CV I within-subject biological variation CV G between-subject biological variation EuBIVAS European Biological Variation Study

Research paper thumbnail of A nucleoside-sparing regimen of dolutegravir plus ritonavir-boosted atazanavir in HIV-1-infected patients with virological failure: the DOLATAV study

Drug Design, Development and Therapy, 2019

Research paper thumbnail of Alcohol and illicit drugs in drivers involved in road traffic crashes in the Milan area. A comparison with normal traffic reveals the possible inadequacy of current cut-off limits

Forensic science international, Jan 22, 2017

Driving under the influence of alcohol and/or illicit drugs in Italy is regulated by the articles... more Driving under the influence of alcohol and/or illicit drugs in Italy is regulated by the articles 186 and 187 of the National Street Code. Epidemiological studies on drivers involved in road traffic crashes (RTC) provide useful information about the use/abuse of these substances in the general population. Comparison with case control studies may reveal important information like the cut-off limits adequacy. Data from 1587 blood tests for alcohol and 1258 blood tests for illicit drugs on drivers involved in RTC around Milan between 2012 and 2016, were analyzed and compared with a published random survey (DRUID) from the European Community. Our data from RTC-involved drivers show that alcohol abuse is not age-related whereas illicit drugs are more common in young people. Cannabinoids are frequent among younger drivers (median age 27) whereas cocaine is more often detected in adults (median age 34). The calculated odds ratio after comparison with the DRUID survey shows that a blood alc...

Research paper thumbnail of Vitamina D: valutazione di un metodo cromatografico liquido ultra veloce (UPLC) e confronto con tecnica immunometrica e cromatografia liquida ad alta prestazione (HPLC) tradizionale

La Rivista Italiana della Medicina di Laboratorio - Italian Journal of Laboratory Medicine, 2012

Vitamin D: evaluation of an ultrafast liquid chromatography method (UPLC) and its comparison with... more Vitamin D: evaluation of an ultrafast liquid chromatography method (UPLC) and its comparison with a traditional immunoassay and high-performance liquid chromatography (HPLC) technique

Research paper thumbnail of Determination of Aspirin and Salicylic Acid in Human Plasma by Column-Switching Liquid Chromatography Using On-Line Solid-Phase Extraction

Analytical Chemistry, 1998

ABSTRACT A column-switching liquid chromatographic method is described for the simultaneous deter... more ABSTRACT A column-switching liquid chromatographic method is described for the simultaneous determination of aspirin and salicylic acid in human plasma. Blood samples are taken into chilled tubes containing a fluoride anticoagulant, and the plasma is isolated by centrifugation. Following a simple acidification step, a 200 microL aliquot of the sample is injected directly onto the HPLC system. The C-18 extraction column is washed with acidified water for 2 min, after which time the compounds are removed by back-flushing directly onto the analytical column (C-8 Nucleosil, 5 microns, 250 mm x 4.6 mm). The flow rate through both columns is 1 mL/min, and the analytes are quantified by measurement of their UV absorbance at 225 nm. The mobile phase is a mixture of water-methanol-acetonitrile-orthophosphoric acid (650:200:150:1 v/v/v/v). The method is linear in the concentration ranges 0.10-5.00 micrograms/mL for aspirin and 0.25-15.00 micrograms/mL for salicylic acid. Both compounds have a limit of quantitation of 0.10 microgram/mL and a limit of detection of 0.04 microgram/mL. Extensive stability tests have been carried out, and validation studies reveal the method to be reproducible and repeatable. Excellent recoveries from plasma obviate the need for an internal standard. The procedure is easier to execute and requires less sample handling than methods currently described in the literature. It has been successfully applied to the investigation of the levels of aspirin and salicylic acid in a healthy, nonfasting volunteer following a 600 mg oral dose of aspirin.

Research paper thumbnail of Role of time-normalized laboratory findings in predicting COVID-19 outcome

Diagnosis, 2020

Objectives The pandemic COVID-19 currently reached 213 countries worldwide with nearly 9 million ... more Objectives The pandemic COVID-19 currently reached 213 countries worldwide with nearly 9 million infected people and more than 460,000 deaths. Although several Chinese studies, describing the laboratory findings characteristics of this illness have been reported, European data are still scarce. Furthermore, previous studies often analyzed the averaged laboratory findings collected during the entire hospitalization period, whereas monitoring their time-dependent variations should give more reliable prognostic information. Methods We analyzed the time-dependent variations of 14 laboratory parameters in two groups of COVID-19 patients with, respectively, a positive (40 patients) or a poor (42 patients) outcome, admitted to the San Raffaele Hospital (Milan, Italy). We focused mainly on laboratory parameters that are routinely tested, thus, prognostic information would be readily available even in low-resource settings. Results Statistically significant differences between the two groups...

Research paper thumbnail of Routine blood analysis greatly reduces the false-negative rate of RT-PCR testing for COVID-19

Acta Bio Medica : Atenei Parmensis, 2020

Background: The COVID-19 outbreak is now a pandemic disease reaching as much as 210 countries wor... more Background: The COVID-19 outbreak is now a pandemic disease reaching as much as 210 countries worldwide with more than 2.5 million infected people and nearly 200.000 deaths. Amplification of viral RNA by RT-PCR represents the gold standard for confirmation of infection, yet it showed false-negative rates as large as 15-20% which may jeopardize the effect of the restrictive measures taken by governments. We previously showed that several hematological parameters were significantly different between COVID-19 positive and negative patients. Among them aspartate aminotransferase and lactate dehydrogenase had predictive values as large as 90%. Thus a combination of RT-PCR and blood tests could reduce the false-negative rate of the genetic test. Methods: We retrospectively analyzed 24 patients showing multiple and inconsistent RT-PCR, test during their first hospitalization period, and compared the genetic tests results with their AST and LDH levels. Results: We showed that when consideri...

Research paper thumbnail of Antibody Titer Kinetics and SARS-CoV-2 Infections Six Months after Administration with the BNT162b2 Vaccine

Vaccines, 2021

Background: Studies reporting the long-term humoral response after receiving the BNT162b2 COVID-1... more Background: Studies reporting the long-term humoral response after receiving the BNT162b2 COVID-19 vaccine are important to drive future vaccination strategies. Yet, available literature is scarce. Covidiagnostix is a multicenter study designed to assess the antibody response in >1000 healthcare professionals (HCPs) who received the BNT162b2 vaccine. Methods: Serum was tested at time-0 (T0), before the first dose, T1, T2, and T3, respectively, 21, 42, and 180 days after T0. Antibodies against the SARS-CoV-2 nucleocapsid-protein were measured to assess SARS-CoV-2 infections, whereas antibodies against the receptor-binding domain of the spike protein were measured to assess the vaccine response. Neutralization activity against the D614G, B.1.1.7, and B.1.351 variants were also analyzed. Results: Six months post-vaccination HCPs showed an antibody titer decrease of approximately 70%, yet, the titer was still one order of magnitude higher than that of seropositive individuals before ...

Research paper thumbnail of Characterization of a Lineage C.36 SARS-CoV-2 Isolate with Reduced Susceptibility to Neutralization Circulating in Lombardy, Italy

Viruses, 2021

SARS-CoV-2 spike is evolving to maximize transmissibility and evade the humoral response. The mas... more SARS-CoV-2 spike is evolving to maximize transmissibility and evade the humoral response. The massive genomic sequencing of SARS-CoV-2 isolates has led to the identification of single-point mutations and deletions, often having the recurrence of hotspots, associated with advantageous phenotypes. We report the isolation and molecular characterization of a SARS-CoV-2 strain, belonging to a lineage (C.36) not previously associated with concerning traits, which shows decreased susceptibility to vaccine sera neutralization.

Research paper thumbnail of Changes in 25-(OH) Vitamin D Levels during the SARS-CoV-2 Outbreak: Lockdown-Related Effects and First-to-Second Wave Difference—An Observational Study from Northern Italy

Biology, 2021

Background. We investigated the changes in 25-hydroxy vitamin D [25-(OH)D] concentrations values,... more Background. We investigated the changes in 25-hydroxy vitamin D [25-(OH)D] concentrations values, during the first and the second pandemic waves and the impact of the lockdown periods, with their different approaches to home confinement, among different age groups. Methods. Daily cloud-modified vitamin D UV dose (UVDVC), for the area of interest (Milano, Italy), was obtained. Single-center 25-(OH)D determinations from 40,996 individuals in a 2019 (pre-pandemic), 32,355 individuals from 1 January to 31 August 2020 (containing the first pandemic wave) and 27,684 individuals from 1 June to 30 November 2020 (containing the second wave) were compared to investigate both the effect of the lockdown on vitamin D status and the association between 25-(OH)D and SARS-CoV-2 positivity. Results. No direct association was found between UVDVC, 25-(OH)D status and SARS-CoV-2 infection. The stringent confinement implemented during the first wave lockdown has not had any effect on 25-(OH)D status, al...

Research paper thumbnail of Biological variation of serum insulin: updated estimates from the European Biological Variation Study (EuBIVAS) and meta-analysis

Clinical Chemistry and Laboratory Medicine (CCLM), 2020