Articoli in pubblicazione

Introduction: new equations for the estimation of the glomerular filtration rate (eGFR) have been recently proposed. Equation changes may result in differences regarding the Chronic Kidney Disease (CKD) classification of the studied populations.
Methods: the serum creatinine-based equations elaborated by Modified Diet for Renal Disease Study (MDRD), Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) 2009 and 2021, European Kidney Function Consortium (EKFC) were compared for correlation, concordance and effect on CKD staging. Serum creatinine concentration from 38118 consecutive subjects (18 to 75 years, 51% female) were measured by an IDMS traceable method.
Results: compared to the 2009 CKD-EPI, the mean difference (limits of agreement) of the MDRD, 2021 CKD-EPI and EKFC equations were -5.8 (-19.3 to 7.8), 3.4 (1.0 to 5.9), -4.9 (-12.6 to 2.7) mL/min x 1.73m2 respectively. The concordance, in the same comparisons, was evaluated as excellent (K-Cohen >0.80). The prevalence of CKD [Kidney Disease: Improving Global Outcomes (KDIGO) classes G3a to G5] was 7.6% by MDRD, 5.9% by CKD-EPI 2009, 7.6% by EKFC and 4.8% according to CKD-EPI 2021. The recognized MDRD equation limits regarding age ranges and normal/high eGFR values were confirmed.
Discussion: these data confirm that MDRD equation has less accuracy and limited age-related applicability. The other three studied equations demonstrate high level of correlation and very small quantitative differences. However, these differences significantly affect the staging and prevalence of CKD, especially for mild to moderate classes. Awareness of the problem and appropriate communication are required to be addressed before any change to the eGFR equations can be prosed and used. Greater worldwide harmonization of the eGFR would also be desirable.

Background: Nucleic Acid Amplification Test (NAAT) can detect the SARS-CoV-2 at low viral load, but the risk of isolating unnecessarily people who are no longer infectious may preclude its use as screening test. Immunoassay can be an alternative screening tool to detect viral antigens, thanks to their high throughput, feasibility and practicability.
In this study, we compare the results obtained by a chemiluminescent immunoassay against the molecular test.
Methods: 105 positive NAAT nasopharyngeal swabs (NPS) with threshold cycle (Ct) between 39 and 13, and 15 negative samples were analyzed with two different methods based on chemiluminescent technology (CLIA) targeted to nucleocapsid SARS-Cov-2 antigen: Maglumi (SNIBE diagnostic Shenzen, China), and Liaison XL (DiaSorin s.p.a., Saluggia, Italy).
Results: all 15 negative NPS (Ct >40) were confirmed as negative with both CLIA assays; 31 positive NPS out of 105 positive (Ct ≤40) were confirmed positive on Maglumi and Liaison (29.5%). The agreement between the two immunoassays was 100%.
Conclusions: as expected, CLIA tests demonstrated to be less sensitive to the viral presence than NAAT. In particular, Maglumi and Liaison can detect a positivity when the virus is detected at a Ct <25. The range between 24 and 25 Ct was a grey area, where NAAT and CLIA were not in perfect agreement. It seems that antigen tests reach satisfactory sensitivities when infected people are more likely to be contagious; this characteristic makes them a useful tool for screening the population, especially during the pandemic

Introduction: this single-center retrospective analysis was aimed to investigate the burden of coronavirus disease 2019 (COVID-19) pandemic on utilization of laboratory resources. Methods: a retrospective analysis was conducted in the database of the Management Control Unit of the Pederzoli hospital in Peschiera del Garda (Verona, Italy), to retrieve information on the volume of total and some specific laboratory tests performed in the year range 2018-2022. Results: the mean yearly volume of total tests has increased by 10% during the pandemic compared to the prepandemic period, by 5% without considering SARS-CoV-2 diagnostic tests. The peak over the reference year 2018 was reached in 2021 (+19%, +12% without SARS-CoV-2 tests), but the volume remained high in 2022 (+16%; +11% without SARS-CoV-2 tests). The requests for D-dimer, vitamin D, blood culture, lactate dehydrogenase (LDH) and thyroid stimulating hormone (TSH) tests increased in the pandemic period (between 19-57%), those of white blood cell count (WBC) count, plasma glucose and creatinine remained almost unvaried (between -3% to 2%), whilst those for cardiac troponin T (cTnT) and prostate specific antigen (PSA) declined (-17% and -19%, respectively). Conclusion: the COVID-19 pandemic has had remarkable impact on laboratory activity, by increasing the demand for certain tests whose increment has remained even after the emergency period, and by decreasing the demand for other tests perhaps attributable to social limitations and modified demography (i.e., death of older people)

Introduction: cryoproteinemia is associated with a broad spectrum of clinical symptoms and significant variability among patients; in this context, laboratory diagnostics play a crucial role in diagnosis and treatment. However, diagnosing cryoglobulinemia is difficult from a clinical point of view and in the laboratory. SIBioC (Italian Society of Clinical Biochemistry and Molecular Biology) has proposed a survey to the Italian laboratories to assess the adopted methods.
Methods: the survey consisted of 40 questions: 8 concerning general items, 9 on the pre-analytical phase, 14 on the analytical phase, 4 on the post-analytical phase, and 5 on the cryofibrinogen.
Results: sixty-six laboratories responded to the survey, mainly from Northern Italy. The results show non-homogeneous situation. Preanalytical phase: some laboratories are not equipped with adequate devices for transporting the sample maintaining a temperature-controlled chain. Analytical phase: not all laboratories apply the same procedures to separate the serum, to wash the cryo-precipitate and to measure the cryocrit. Postanalytical phase: almost all laboratories apply the Brouet classification. 11% of laboratories perform cryofibrinogen testing, 20% participate in an external quality assessment, and 62% of participants feel the need for an adequate scientific information and training.
Conclusions: the survey shows that the procedures for cryoprotein testing vary widely among laboratories. The need of recommendations emerges strongly; these could start from the existing literature in the field, the already available procedures, trying make a synthesis where the processes are diversified. It is also crucial to participate to External Quality Assessment and to spread the culture of continuous dialogue with clinicians.

Introduction: point of care testing (POCT) is an alternative near to the patient approach to laboratory testing that should generate results comparable to those obtained in laboratory. In our study, we estimated the concordance of the blood cell count carried out on Norma Icon-5 POCT analyzer with that performed in laboratory on an automated blood cell count analyzer (Sysmex XN1000), to evaluate the possible utilization of the POCT in the Emergency Department (ED).
Methods: Venous blood samples were collected in tubes containing K3EDTA anticoagulant from 40 patients admitted to the ED. The degree of concordance between POCT results of red blood cells (RBC), white blood cells (WBC), platelets (PLT) count and haemoglobin (Hb) concentration were evaluated. 
Results: The POCT versus XN1000 bias was -2.44% (95%CI: -3.45 to -1.43), 0.61% (95%CI: -0.70 to 1.92), 1.22% (95%CI: 0.612 to 1.83) and -10.45% (95%CI: -12.33 to -8.67) for RBC, WBC, Hb and PLT respectively. Passing-Bablok regression equation was y=-0.015 (95%CI: -0.309 to 0.240) +0.976x (95%CI: 0.915 to 1.0435); y=-0.186 (95%CI: -0.404 to 0.012) + 1.038x (95%CI: 1.008 to 1.068); y=-1.889 (95%CI: -7.224 to 1.500) + 1.030x (95%CI: 1.000 to 1.069) and y=-12.098 (95%CI: -21.500 to -3.824) + 0.964x (95%CI: 0.924 to 1.000) for RBC, WBC, Hb and PLT respectively.
Conclusion: The blood cell counter POCT results were statistically concordant with those of the laboratory system only for WBC count, but showed a clinically acceptable bias also for RBC count and Hb concentration values. However, PLT showed an underestimation statistically significant and also exceeding the minimum clinical allowable bias. Therefore, the POCT blood cell counter can be used for clinical purpose only for RBC, WBC and Hb determination. 

Background: sepsis is a serious disease with high mortality rate, threatening human health. Clinicians need to diagnose the condition in time to develop an effective treatment plan; therefore, a quick and early screening to rule in or rule out sepsis has become an urgent problem in clinical laboratories. Different markers are used to diagnose sepsis, but they have limitations and require additional examination. The aim of this study is to use leucocyte count and other haematological parameters obtained by Mindray BC-6800-plus, to identify sepsis biomarkers that are early, quickly, conveniently, and at low cost.

Methods: a total of 479 venous whole blood samples were collected: 63 control samples (blood donors), and 416 samples hospitalized at the emergency department with symptoms attributable to sepsis with a procalcitonin request. Morphometric parameters are reported using Mindray BC-6800 plus analyzer: white blood cell count-related parameters like cell population data named X, Y, Z, neutrophil-lymphocyte ratio, and index of immature granulocytes. Statistical analysis was performed using the Kruskal-Wallis test to evaluate significative differences among the groups. ROC curves were also examined to extrapolate a cut-off of pathogenicity.

Results: we found a significant statistically difference between the control and the sepsis groups for a number of haematological parameters. The ROC curves highlight acceptable sensitivity and specificity values for some parameters, and suggest possible cut-offs.

Discussion: some qualitative and quantitative parameters of total count blood can be of help with the screening of sepsis upon admission to the emergency department.

Introduction: the results of 5 randomized controlled meta-analysis studies showed the association between genetic  deficiency of folate cycle and autistic spectrum disorders in children. The purpose of the present study is to investigate  the biochemical alterations in children with autism spectrum disorders associated with genetic deficiency of the folate  cycle.  Methods: the experimental group involved 225 children diagnosed with autism spectrum disorders (Diagnostic and  Statistical Manual of Mental Disorders IV-TR, International Statistical Classification of Diseases and Related Health  Problems 10), who suffered from a genetic deficiency of the folate cycle (methylenetetrahydrofolate reductase C677T  + methylenetetrahydrofolate reductase A1298C and/or methionine synthase A2756G and/or methionine synthase 
reductase A66G; polymerase chain reaction). The control group included 51 healthy children who followed the same  age and gender distribution pattern of the patients group.  Results: the study revealed that the patients had a specific pattern of biochemical alterations in serum (p˂0.05; Z˂Z0.05).  Mean value and (SD) are reported. Hyperhomocysteinemia: 9.63 (5.36) μmol/L, r.v. ˂5.2); deficiencies of vitamins: B6  [6.32 (3.58) μg/L, r.i. 8.7-27.2], folic acid [2.97 (6.85) pg/mL, r.i. 3.89-26.8], B12 [112.64 (374.2) pg/mL, r.i. 197-771],  and D3 [13.98 (20.41) ng/mL, r.i. 30-60]; hypercreatininemia [69.13 (64.21) μmol/L, r.i. 3 years: 21-36, 3-5 years: 27-42, 5-8 years: 28-50], increased creatine kinase [314.12 (443.42) U/L, r.i. 39-308 U/L]; and lactate dehydrohenase  [378.47 (443.72) U/L, r.i.135-225]. At the time of examination, an increase in the serum concentration of homocysteine  was registered in 88% of the patients. The associations between different folate cycle genes polymorphisms with  certain biochemical abnormalities were shown Discussion: autism spectrum disorders associated with genetic deficiency of the folate cycle in children are  characterized by a specific pattern of biochemical changes that is not found in healthy children and may be involved in  the pathogenesis of immunodeficiency and encephalopathy. These data can be used in clinical practice for diagnostic  purposes to identify a subgroup of children with autism spectrum disorders associated with genetic folate cycle  deficiency and for the selection of biochemical correction during treatment.

The recent position paper by the European Federation of Laboratory Medicine (EFLM) advises against following
the US recommendations on the use of the new race-free version of the Chronic Kidney Disease-Epidemiology
Consortium (CKD-EPI 2021) equation to estimate the glomerular filtration rate (eGFR). The main concern derives
from a possible poor CKD estimation, causing an underestimation of renal impairment for the Caucasian population,
and therefore for all Europeans. Considering the relevance of the EFLM indication, Biochimica Clinica published the
Italian translation of the document and an accompanying editorial suggesting that the European position could be an
end point for a better eGFR harmonization.
However, the use of different equations for eGFR in US and Europe will entail rather a worse harmonisation: the eGFR
values are used for patients enrolment in numerous clinical studies, for the admission to diagnostic procedures or
therapies and for the evaluation of their complications and adverse effects. As a consequence, many clinical trials will
not be comparable, guidelines and recommendations will be less easily transferable from one country to another and
the "regionality" of the equations will not slow down the search for new equations, for different groups and different
countries. With equally shared responsibility between US and Europe, this situation seems to be a paradigmatic
example of how recommendations in laboratory medicine should not be developed.
Nevertheless, the EFLM indication not to adopt the CKD-EPI 2021 race-free equation should be accepted for the
moment in Europe, since the harmonization between neighbouring and regional sanitary structures is a priority in the
patient care. It should be strengthened that the problem has not been solved yet, and that a greater harmonization
action must be rapidly sought, with the aim of a homogeneous calculation and reporting of this widely diffused estimate.

The principal aim of this document is to provide a detailed documentation of the scientific activity carried out by the Italian Study Group on Cardiovascular biomarkers during these last three difficult years characterized by COVID-19 pandemic, that has put in strain the activity of all clinical laboratories (not only in Italy). Another important aim of this document is to discuss some important pathophysiological and clinical issues related to cardiac-specific biomarkers (i.e., cardiac natriuretic peptides and cardiac troponins), that still need not only further theoretical investigation, but also some multicenter clinical trials in order to evaluate and to improve their utilization in diagnosis, prognosis and managing of patients with cardiovascular disease

We report the case of a diabetic woman from west Africa with a presumptive diagnosis of HbSS previously suggested in another laboratory. However, during analysis for HbA1c with a novel instrument for capillary electrophoresis capable of both HbA1c and hemoglobin variants analysis, we suspected and later confirmed (with a centralized molecular study) the presence of a double heterozigosity for a beta gene defect, namely HbS and Hereditary Persistence of Fetal Hemoglobin (HbS/HPFH). Our case highlights the importance of ruling out any possible interference affecting the HbA1c measurement that could compromise its interpretation, posing a risk for erroneous clinical management of diabetes mellitus. The availability of both high resolution HbA1c and hemoglobin variants analysis on the same instrument could lead to a rapid presumptive identification of previously unknown or misdiagnosed hemoglobinopathies. 

Haemoglobin A1c (HbA1c) is a test routinely used to monitor long-term glycemic control in diabetic patients as well as for the diagnosis of diabetes. Capillary electrophoresis (CE) is an accurate and valid method to separate and quantify HbA1c and to detect HbF, HbA2 and haemoglobin variants. The purpose of this study is to report three Hb variants accidentally observed during HbA1c testing. HbA1c tests had been carried out using Capillarys 3 TERA “HbA1c kit” (Sebia, France) in the Hub Laboratory of AUSL Romagna. To screen for Hb variants interfering with HbA1c determination, Capillarys 3 TERA “Hemoglobine kit” was used. Molecular analysis of globin genes was performed by Next Generation Sequencing (NGS) technology. During routine HbA1c testing, we observed three cases of interference with HbA1c assay that led to the suspicion of Hb variants that subsequently were not detectable by the specific “Hemoglobine kit” assay. NGS identified a woman with Hb La Desirade (β 129(H7) Ala-->Val), a man with Hb Bleuland (α 108(G15) Thr-->Asn). Genetic analysis for the third case is still in progress. The two identified Hb variants are clinically asymptomatic; however, if they co-exist with other Hb variants or thalassemia, they could cause clinical symptoms. HbA1c testing performed by CE is an essential part of routine management of diabetes and in addition has proven to be an effective and powerful method to detect rare or silent Hb variants. We suggest laboratory personnel to be aware of the limitations of their method with respect to the identification of Hb variants.

A 82-year-old man suffering from pemphigus and Parkinson’s disease presented at the emergency department of the Misericordia Hospital in Grosseto (Tuscany, Italy) with subconjunctival bleeding. Laboratory blood tests showed a prolonged activated thromboplastin time (aPTT), with normal prothrombin time (PT) and slightly reduced haemoglobin. The negative family and personal history of haemorrhagic disease rose the suspicion of the presence of an acquired inhibitor. The patient was referred for further diagnostics to the University Hospital of Siena where second level tests were performed at the Coagulation Unit. The aPTT mixture test revealed a non-correction both at room temperature and, more markedly, after incubation at 37°C for 2h, confirming the presence of an intrinsic pathway inhibitor. Among the measured factors, only the activity level of factor VIII was extremely low. The titration of FVIII inhibitor confirmed the diagnosis of Acquired Haemophilia A (AHA). 

A new β-globin variant was found in a 68-year-old woman of Sicilian origin living in Alessandria, Italy. This variant was detected by capillary electrophoresis (CE) method during the measurement of HbA1c; other separative methods, particularly those based on high-performance liquid chromatography (HPLC), were inconclusive. Molecular characterization of the defect on the beta globin gene by direct DNA sequencing revealed a G>T transversion at codon 37 and thus the substitution of a tryptophan residue (Trp) for a leucine residue (Leu). The new hemoglobin variant was named Hb Alessandria [β37(C3) Trp>Leu; HBB: c.113G>T]. The oxygen tension at which hemoglobin is 50% saturated (P50) was slightly decreased while the stability test at 37°C in isopropyl alcohol and the main erythrocyte parameters were normal. This finding confirms the importance of electrophoretic or chromatographic methods in the diagnosis and monitoring of diabetes mellitus, as well as the use of alternative confirmatory methods in case of the detection of hemoglobin variants.

Prekallikrein (PK) is a contact factor of the intrinsic pathway of the coagulation cascade. Patients with PK deficiency usually do not show bleeding tendency despite prolonged activated partial thromboplastin time (aPTT). Here we report two cases of male patients in their seventies, both with a prolonged aPTT detected at a pre-operative screening. The aPTT correction after 1:1 mixing with normal pool plasma (NPP) indicated a coagulation factor deficiency; however,
factors XII, XI, IX and VIII activities were normal. The aPTT test was then performed increasing the pre-incubation time of the patient’s plasma. This resulted in a significant shortening of the aPTT. The PK deficiency was ultimately confirmed by direct measurement of PK plasma activity. This experience shows that PK deficiency should be considered for asymptomatic patients with prolonged aPTT and that, whenever a PK deficient plasma is not available, the diagnosis
can be correctly obtained through a simple modification of the procedure of the aPTT test.