Evaluation of Stability and Accuracy Compared to the Westergren Method of ESR Samples Analyzed at VES-MATIC 5.

The Erythrocyte Sedimentation Rate (ESR) is a diagnostic estimator of systemic inflammation as a reflection of acute phase proteins circulating in the blood. The purpose of this manuscript is to evaluate the blood stability at room temperature (RT) and at 4 °C to avoid ESR diagnostic errors, as well as the accuracy of the VES-MATIC 5 analyzer. The ESR stability evaluation at RT for 24 h (4 h "T1", 6 h "T2", 8 h "T3", 10 h "T4", 24 h "T5") and at 4 °C (24 h, 36 h, 48 h) was carried out using 635 total samples, starting with T0 (2 h of venipuncture). For method comparison, 164 patients were analyzed using VES-MATIC 5 and then the Westergren reference method. The sample at RT is established by a significant gradual decrease in correlation R = 0.99 (T0 vs. T1), R = 0.97 (T0 vs. T2), R = 0.92 (T0 vs. T3), R = 0.87 (T0 vs. T4), and R = 0.40 (T0 vs. T5). The stability at 4 °C after 24 h, 36 h, and 48 h showed a regression of R = 0.99, R = 0.97, and R = 0.95, respectively. Therefore, ESR measurements on RT samples beyond 6 h after collection cannot be carried out, but the ESR can be measured until 36 h for samples stored at 4 °C. Moreover, the VES-MATIC 5 accuracy performance compared to the Westergren method (R = 0.96) is confirmed.

Identification of a Novel Non-V600E BRAF Mutation in Papillary Thyroid Cancer.

Papillary thyroid cancer (PTC) is a common endocrine malignancy, and its incidence is reported to be constantly increasing. BRAF mutation is detected in approximately 44% of PTCs, and the most common BRAF mutation is thymine (T) to adenine (A) missense mutation in nucleotide 1796 (T1796A, V600E). Although BRAFV600E represents 95% of all BRAF mutations, uncommon BRAF mutations have been reported in thyroid carcinomas and represent an alternative mechanism of BRAF activation with unclear clinical significance. We report a novel non-V600E mutation (c.1799_1812delinsAT, p.V600_W604delinsD), identified preoperatively with next-generation sequencing (NGS) on the material obtained with fine-needle aspiration cytology (FNAC) performed on a thyroid nodule cytologically suspicious for malignancy in a 35-year-old male patient. The presence of this new variant of BRAF mutation was subsequently confirmed in the postoperative phase by direct Sanger sequencing. In conclusion, we report a new non-V600E variant previously undetected in papillary thyroid cancer. In addition, this case report shows that the NGS technique on cytological tissue allows to detect the presence of rare mutations, thus increasing the diagnostic specificity of molecular analysis.

BL-MOL-AR Project, Preliminary Results about Liquid Biopsy: Molecular Approach Experience and Research Activity in Oncological Settings.

Background Liquid biopsy is mainly used to identify tumor cells in pulmonary neoplasms. It is more often used in research than in clinical practice. The BL-MOL-AR study aims to investigate the efficacy of next-generation sequencing (NGS) and clinical interpretation of the circulating free DNA (cfDNA) levels. This study reports the preliminary results from the first samples analyzed from patients affected by various neoplasms: lung, intestinal, mammary, gastric, biliary, and cutaneous. Methods The Biopsia Liquida-Molecolare-Arezzo study aims to enroll cancer patients affected by various malignancies, including pulmonary, intestinal, advanced urothelial, biliary, breast, cutaneous, and gastric malignancies. Thirty-nine patients were included in this preliminary report. At time zero, a liquid biopsy is executed, and two types of NGS panels are performed, comprising 17 genes in panel 1, which is already used in the routine tissue setting, and 52 genes in panel 2. From the 7th month after enrollment, 10 sequential liquid biopsies are performed up to the 17th month. The variant allele frequency (%) and cfDNA levels (ng/mL) are measured in every plasmatic sample. Results The NGS results obtained by different panels are similar even though the number of mutations is more concordant for lung pathologies. There are no significant differences in the actionability levels of the identified variants. Most of the molecular profiles of liquid biopsies reflect tissue data. Conclusions Preliminary data from this study confirm the need to clarify the limitations and potential of liquid biopsy beyond the lung setting. Overall, parameters related to cfDNA levels and variant allele frequency could provide important indications for prognosis and disease monitoring.

Novel Biomarkers for Early Detection of Acute Kidney Injury and Prediction of Long-Term Kidney Function Decline after Partial Nephrectomy.

BACKGROUND: Identifying acute kidney injury (AKI) within few hours of onset is certainly helpful. However, early prediction of a long-term eGFR decline may be an even more important goal. Our aim was to identify and compare serum [creatinine, kineticGFR, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL)] and urinary (NephroCheck, NGAL, proteinuria, albuminuria, acantocytes at urinary sediment) predictors of AKI that might efficiently predict long-term GFR decline after robotic Nephron-Spearing Surgery (rNSS). METHODS: Monocentric prospective observational study. Patients scheduled for rNSS for suspected localized Renal Cell Carcinoma from May 2017 to October 2017 were enrolled. Samples were collected preoperatively and postoperatively (timepoints: 4 h, 10 h, 24 h, 48 h), while kidney function was re-assessed up to 24 months. RESULTS: 38 patients were included; 16 (42%) developed clinical AKI. The eGFR decline at 24 months was more pronounced after postoperative AKI (-20.75 vs. -7.20, p < 0.0001). KineticGFR at 4 h (p = 0.008) and NephroCheck at 10 h (p = 0.001) were, at multivariable linear regression analysis, efficient predictors of post-operative AKI and long-term eGFR decline if compared to creatinine (R2 0.33 vs. 0.04). CONCLUSIONS: NephroCheck and kineticGFR have emerged as promising noninvasive, accurate, and early biomarkers of postoperative AKI and long-term GFR decline after rNSS. Combining NephroCheck and kineticGFR in clinical practice would allow to identify high risk of postoperative AKI and long-term GFR decline as early as 10 h after surgery.

Monocyte distribution width and the fighting action to neutralize sepsis (FANS) score for sepsis prediction in emergency department.

BACKGROUND AND AIMS: The aim of this study was to evaluate the diagnostic accuracy of sepsis markers and to develop a multiparametric score, using demographic and clinical variables as well as laboratory parameters to predict sepsis in patients admitted in the ED with suspected symptoms. MATERIALS AND METHODS: Patients with clinical presentation of suspected sepsis were enrolled in the ED of San Donato Hospital in Arezzo between September 2019 and May 2020. Anagraphic, anamnestic, clinical and laboratory data were collected for all subjects. PCT, MDW, WBC, MPV and BT were utilised to formulate FANS score. RESULTS: The AUC of the FANS score, PCT, MDW and CRP was 0.87, 0.80, 0.77 and 0.71, respectively, when used to predict sepsis in all 308 subjects. Instead, the AUC of the FANS (Fighting Action To Neutralize Sepsis) score, PCT, MDW and CRP was 0.93, 0.84, 0.83 and 0.77, respectively, when used to predict sepsis excluding subjects with infection (clinically classified as the Infections group). CONCLUSIONS: The results obtained with PCT, PCR and MDW confirm the results of these markers for the identification of sepsis obtained from other studies. The multiparametric approach, obtained from the statistical study of the parameters using binary logistic regression, identified those PCT, WBC, MPV, BT and MDW as the most significant and effective clinical classifiers for diagnosing sepsis.

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19.

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage.

The role of Monocyte Distribution Width (MDW) in the prognosis and monitoring of COVID-19 patients.

The new parameter derived from the standard deviation of the monocyte distribution width (MDW) has shown a good diagnostic efficacy in COVID-19 patients. In this study, we propose MDW as a prognostic and monitoring parameter in patients with severe forms of COVID-19. Sixty SARS-CoV-2-positive patients admitted to the San Donato Hospital in Arezzo were enrolled. A blood sample taken to measure the complete blood count was used for the determination of MDW using a UniCel DxH 900 instrument (Beckman Coulter). For each patient, a mean of 6 ± 2 measurements of MDW were taken. The difference between the last and first MDW results was reported as the ΔMDW variable. The ΔMDW and age were significantly correlated to the outcome. In non-survivors patients, the difference in the mean of the MDW between the first and other points was not significant, while in survivors, the first point was higher than the other points (p < 0.005), with the exception of the mean of the second point (p-value = NS). The ΔMDW area under the curve (AUC) was 0.84, and with a cut-off lower than 0.00 the sensitivity and specificity were 88% and 81%, respectively. The most important result of this study is the ΔMDW calculated on the basis of the difference between the first and third measurement, after approximately the 5-7th day of hospitalisation. A ΔMDW less than one was indicative of an unfavourable prognosis. The data reported suggest that MDW could be used to support monitoring and surveillance, alongside other tests such as procalcitonin, in critically ill patients in the ICU.

Isolated case of platelet satellitism around white blood cells and phagocytosis by neutrophils and monocytes.

INTRODUCTION: Rarely, platelets can interact with other blood elements, forming platelet aggregates. This paper presents an isolated case of platelet satellitism around neutrophils, lymphocytes and monocytes with platelet phagocytosis by both neutrophils and monocytes. CASE PRESENTATION: The subject was an 89-year-old woman with breast cancer on anti-estrogenic hormone cancer therapy. Whole blood sample collected in a tube with EDTA (Ethylenediaminetetra-acetic acid) anticoagulant was analysed within 4 hours, using an XN haematology analyser (Sysmex). The CBC (complete blood count) presented the following results: WBC (White blood cell) 4.0 x 109/L, RBC (Red blood cell) 3.58 x 1012/L, haemoglobin 116 g/L, haematocrit 34.9%, MCV (Mean corpuscular volume) 97.5 fL, MCH (Mean corpuscular haemoglobin) 32.5 pg, MCHC (Mean corpuscular haemoglobin concentration) 33.2 g /dL, RDW (Red blood cell distribution width) 14.6% and PLT (Platelet) 136. CONCLUSION: This manuscript describes the platelet satellitism around neutrophils, lymphocytes and monocytes and the interesting, very rare and singular phenomenon of platelet phagocytosis by not only neutrophils but also monocytes.

Experience of Uncommon EGFR Mutation in Lung Scheme Quality Program: Discussing Risks and Opportunities for the Improvement of Laboratory Response.

Background The quality programs can be considered to be a valuable tool for global and individual growth. Each result, obtained by a single laboratory, contributes to define the standardization of the response. In the case of the uncommon epidermal growth factor receptor (EGFR) mutations, the molecular result is sometimes difficult to interpret in terms of biological significance and therapy choosing. The standardization effort in the diagnostic lung setting also consists of active quality program participation. Materials and Methods The quality control analysis, which is defined as a clinical case, was performed by the extraction of DNA from FFPE sections and by RT-PCR on the EGFR (exons 19, 20, 21), BRAF, and KRAS genes. The laboratory performed a validation sequencing of EGFR exon 20 with the help of the Sanger method. Results The laboratory reported positivity for EGFR exon 20 insertions and negative results for BRAF and KRAS. The quality test finished with the redaction of a report containing the recommendation to consider the efficacy of therapy with tyrosine kinase inhibitors (TKI). This specific interpretation has determined poor performance judgment by the quality provider, which explained why most of these mutations are TKI-resistant. Conclusions This experience provides an opportunity to reflect on the critical aspects of this diagnostic setting. The detection of some uncommon EGFR mutations should entail the mutation characterization, especially for the rare exon 20 insertions, of which are not classifiable as "resistant." Moreover, this experience allows reflecting on the quality program design, mandatory actions for the laboratory, and routine activity in the oncologic multidisciplinary team.

Comparison of serologic and molecular SARS-CoV 2 results in a large cohort in Southern Tuscany demonstrates a role for serologic testing to increase diagnostic sensitivity.

BACKGROUND: Since February 2020, Italian hospitals registered COVID-19 (COronaVIrus Disease 19) cases more often than the rest of the Europe. During this epidemic, health authorities requested swab tests, while seeking new patient paths. METHODS: A dual laboratory approach was evaluated, consisting of patient care reports for viral RNA detection on swabs and rapid serological tests in 516 patients (192 symptomatic or paucisymptomatic and 324 asymptomatic). RESULTS: We found the molecular positive fraction equal to 12% (23/192) among symptomatic/paucisymptomatic (S/P) and 15.4% (50/324) in asymptomatic (As) sets. Among subsets, we observed serologically positive results, corresponding to 35% (8/23) for S/P and 38% (19/50) for As. Among molecular negative cases, we detected specific Immunoglobulin G or M (Ig G or Ig M) positivity in the S/P cohort equal to 6.6% (11/167) and 6% (15/246) in As cases. For indeterminate molecular results, we found S/P serological positivity equal to 100% (1/1) and 54% (13/24) in As patients. We found higher (p < 0.05) seropositivity in older patients (n = 8) among symptomatic and positives for viral RNA (n.23). CONCLUSIONS: It has been observed that a dual approach of serological and molecular tests detects a higher absolute number of disease cases in a pandemic context,which could improve monitoring and health surveillance efficacy. The age-related seropositivity frequency in this study, if confirmed, could enhance the validity of serological tests, especially in older patients.In these subjects, molecular positivity accompanied by serological positivity (distinct for M and G immunoglobulins) should help determine disease status and support decisions related to patient management.

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

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.

Elevated monocyte distribution width in COVID-19 patients: The contribution of the novel sepsis indicator.

INTRODUCTION: Interesting results regarding the contribution of MDW (Monocyte Distribution Width) in the Infectious Disease Unit have been reported. An observational study is ongoing at San Donato Hospital with the aim to evaluate the contribution of MDW in the diagnostic pathway in adult patients entering in the ED setting and tested for SARS-CoV-2. MATERIAL AND METHOD: COVID-19 symptomatic and paucisymptomatic patients presenting to ED (Emergency Department), have been enrolled consecutively. Whole blood venous samples have been collected on K2 EDTA for MDW determination, at the same time a nasopharyngeal swab for SARS-CoV-2 RNA detection have been collected. RESULTS: One hundred six patients were negative for SARS-CoV-2 with MDW mean value of 20.3 ± 3.3, while forty-one were positive for SARS-CoV-2 with higher MDW mean value of 27.3 ± 4.9 (P < 0.005). The ROC curve analysis has been evaluated showing MDW AUC of 0.91. Finally twenty-three patients hospitalized in high-intensity care unit showed an MDW value higher than the eighteen patients presenting few symptoms [28.8 ± 5.3 vs 25.4 ± 3.6 respectively, P < 0.05]. DISCUSSION: Monocytic population, in Covid19 disease, are the first elements of innate immunity to be involved, these changes are the basis of the modification of the MDW, with evident efficacy in term of sensitivity, particularly in the studied Covid19 patients. Moreover the patients hospitalized in high-intensity care unit showed significantly elevated MDW respects to middle or low symptomatic one, suggest including this parameter as prognostic marker or of therapy efficacy, integrated with other laboratory findings.

Early diagnosis of gestational diabetes mellitus using circulating microRNAs.

DESIGN: Gestational diabetes mellitus (GDM) is one of the most common pregnancy complications and its prevalence is constantly rising worldwide. Diagnosis is commonly in the late second or early third trimester of pregnancy, though the development of GDM starts early; hence, first-trimester diagnosis is feasible. OBJECTIVE: Our objective was to identify microRNAs that best distinguish GDM samples from those of healthy pregnant women and to evaluate the predictive value of microRNAs for GDM detection in the first trimester. METHODS: We investigated the abundance of circulating microRNAs in the plasma of pregnant women in their first trimester. Two populations were included in the study to enable population-specific as well as cross-population inspection of expression profiles. Each microRNA was tested for differential expression in GDM vs control samples, and their efficiency for GDM detection was evaluated using machine-learning models. RESULTS: Two upregulated microRNAs (miR-223 and miR-23a) were identified in GDM vs the control set, and validated on a new cohort of women. Using both microRNAs in a logistic-regression model, we achieved an AUC value of 0.91. We further demonstrated the overall predictive value of microRNAs using several types of multivariable machine-learning models that included the entire set of expressed microRNAs. All models achieved high accuracy when applied on the dataset (mean AUC = 0.77). The significance of the classification results was established via permutation tests. CONCLUSIONS: Our findings suggest that circulating microRNAs are potential biomarkers for GDM in the first trimester. This warrants further examination and lays the foundation for producing a novel early non-invasive diagnostic tool for GDM.

Seminal but not Serum Levels of Holotranscobalamin are Altered in Morbid Obesity and Correlate with Semen Quality: A Pilot Single Centre Study.

Vitamin B12 (cobalamin) is an essential cofactor in the one-carbon metabolism. One-carbon metabolism is a set of complex biochemical reactions, through which methyl groups are utilised or generated, and thus plays a vital role to many cellular functions in humans. Low levels of cobalamin have been associated to metabolic/reproductive pathologies. However, cobalamin status has never been investigated in morbid obesity in relation with the reduced semen quality. We analysed the cross-sectional data of 47-morbidly-obese and 21 lean men at Careggi University Hospital and evaluated total cobalamin (CBL) and holotranscobalamin (the active form of B12; holoTC) levels in serum and semen. Both seminal and serum concentrations of holoTC and CBL were lower in morbidly obese compared to lean men, although the difference did not reach any statistical significance for serum holoTC. Seminal CBL and holoTC were significantly higher than serum levels in both groups. Significant positive correlations were observed between seminal holoTC and total sperm motility (r = 0.394, p = 0.012), sperm concentration (r = 0.401, p = 0.009), total sperm number (r = 0.343, p = 0.028), and negative correlation with semen pH (r = -0.535, p = 0.0001). ROC analysis supported seminal holoTC as the best predictor of sperm number (AUC = 0.769 ± 0.08, p = 0.006). Our findings suggest that seminal rather than serum levels of holoTC may represent a good marker of semen quality in morbidly obese subjects.

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

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.

Mid regional pro-adrenomedullin for the prediction of organ failure in infection. Results from a single centre study.

Biomarkers are widely used to confirm the presence of infection. However, it would be of the greatest importance to predict in advance the occurrence or worsening of organ dysfunction in infected patients allowing timely antibiotic escalation. This study investigates the ability of procalcitonin (PCT) and MR-proADM to predict the transition to sepsis in infected patients. The study was conducted in a neurointensive care unit over a three-month period. We included both patients with and without infection to investigate the specificity of organ dysfunction prediction in infected patients. Daily measurement of PCT and MR-proADM, SOFA, Pitt, and CPIS were performed. To measure the correlation between each biomarker and each severity score, linear mixed-effects models were developed. For each biomarker-score combination we tested the correlation of the score with the biomarker measured one and two days before, the same day, and the day after. Sixty-four critically ill patients, 31 with infection, were enrolled. The statistically significant biomarker-score combinations were PCT-SOFA, MR-proADM-SOFA, MR-proADM-Pitt, and MR-proADM-CPIS. The MR-proADM models predicting Pitt and CPIS variations with 24-hour anticipation showed the best fit. The scores increased by 0.6 ± 0.3 and 0.4 ± 0.2 for each unitary biomarker increase, respectively. The MR-proADM-SOFA combinations were equivalent when the biomarker was measured the day before or the same day (score increases were 1.5 ± 0.4 and 1.9 ± 0.4, respectively). The PCT-SOFA model had the best fit when PCT was measured the same day of the score. There was no difference in the predictive ability of the biomarker in infected and non-infected patients. This was a pivotal study conducted in a single neurointensive centre on a limited number of patients, and as such it does not provide definitive conclusions. PR-proADM predicted occurrence and worsening of organ failure in critically ill patients with and without infection. The combination with infection diagnostic biomarkers such as PCT would allow predicting evolution to sepsis in infected patients.

Effects of fecal sampling on preanalytical and analytical phases in quantitative fecal immunochemical tests for hemoglobin.

BACKGROUND: Information on preanalytical variability is mandatory to bring laboratories up to ISO 15189 requirements. Fecal sampling is greatly affected by lack of harmonization in laboratory medicine. The aims of this study were to obtain information on the devices used for fecal sampling and to explore the effect of different amounts of feces on the results from the fecal immunochemical test for hemoglobin (FIT-Hb). METHODS: Four commercial sample collection devices for quantitative FIT-Hb measurements were investigated. The volume of interest (VOI) of the probes was measured from diameter and geometry. Quantitative measurements of the mass of feces were carried out by gravimetry. The effects of an increased amount of feces on the analytical environment were investigated measuring the Hb values with a single analytical method. RESULTS: VOI was 8.22, 7.1 and 9.44 mm3 for probes that collected a target of 10 mg of feces, and 3.08 mm3 for one probe that targeted 2 mg of feces. The ratio between recovered and target amounts of devices ranged from 56% to 121%. Different changes in the measured Hb values were observed, in adding increasing amounts of feces in commercial buffers. CONCLUSIONS: The amounts of collected materials are related to the design of probes. Three out 4 manufacturers declare the same target amount using different sampling volumes and obtaining different amounts of collected materials. The introduction of a standard probes to reduce preanalytical variability could be an useful step for fecal test harmonization and to fulfill the ISO 15189 requirements.