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Digital droplet PCR (ddPCR) is a recent version of quantitative PCR (QT-PCR), useful for measuring gene expression, doing clonality assays and detecting hot spot mutations. In respect of QT-PCR, ddPCR is more sensitive, does not need any reference curve and can quantify one quarter of samples already defined as “positive but not quantifiable”. In the IgH and TCR clonality assessment, ddPCR recapitulates the allele-specific oligonucleotide PCR (ASO-PCR), being not adapt for detecting clonal evolution, that, on the contrary, does not represent a pitfall for the next generation sequencing (NGS) technique. Differently from NGS, ddPCR is not able to sequence the whole gene, but it is useful, cheaper, and less time-consuming when hot spot mutations are the targets, such as occurs with IDH1, IDH2, NPM1 in acute leukemias or T315I mutation in Philadelphia-positive leukemias or JAK2 in chronic myeloproliferative neoplasms. Further versions of ddPCR, that combine different primers/probes fluorescences and concentrations, allow measuring up to four targets in the same PCR reaction, sparing material, time, and money. ddPCR is also useful for quantitating BCR-ABL1 fusion gene, WT1 expression, donor chimerism, and minimal residual disease, so helping physicians to realize that “patient-tailored therapy” that is the aim of the modern hematology.

Polycythemia vera (PV) causes thrombosis. Erythrocytosis and cell adhesiveness are responsible for thrombosis. JAK2V617F causes inflammation and autoimmunity; however, whether or not autoimmunity or inflammation causes thrombosis has yet to be proven. In 60 PV patients, we analyzed JAK2V671F and its allele burden, autoimmune Th17 cells, interleukin-17 (IL-17), anti-endothelial cell antibodies (AECAs), endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and von Willebrand factor antigen (VWF: Ag). Fifty blood donors were used as the controls. All patients were on phlebotomy-maintaining hematocrit

COVID-19-associated coagulopathy (CAC) identifies the coagulation changes in coronavirus disease 2019 (COVID-19) and is responsible for thrombosis. CAC has been studied in critical and severe stage COVID-19 disease through tests including the D-Dimer (DD), prothrombin time (PT), thromboplastin partial time (PTT), platelet count, fibrinogen (Fib), and platelet factor 4 (PF4) tests. However, these tests have some limitations. The aim of this study was to identify more accurate warning tests for early recognition of CAC and to prevent its deterioration to disseminated intravascular coagulation (DIC). First, we measured Interleukin-1α (IL-1α) and IL-8, and tissue factor pathway inhibitor (TFPI) as inflammation and endothelial damage markers, respectively. Second, we measured thrombin antithrombin complex (TAT), β-Thromboglobulin (β-TG), and thromboelastometric parameters including clotting time (CT), clot formation time (CFT), clot firmness (MCF), and clot lysis at 30 min (LY-30), as markers of coagulation and platelet activation. This study included 100 non-severe patients with COVID-19 that developed pulmonary embolism (PE) compared to 80 healthy patients. IL-1α and IL-8, and TFPI were higher as well as TAT and β-TG and thromboelastometric parameters, indicating hypercoagulability. If confirmed in other studies, these results could help in predicting the deterioration of non-severe COVID-19 disease, thereby reducing hospitalizations and health costs.

Background: Detection of BCR-ABL1 transcript level via real-time quantitative-polymerase-chain reaction (Q-PCR) is a clinical routine for disease monitoring, assessing Tyrosine Kinase Inhibitor therapy efficacy and predicting long-term response in chronic myeloid leukemia (CML) patients. For valid Q-PCR results, each stage of the laboratory procedures need be optimized, including the cell-counting method that represents a critical step in obtaining g an appropriate amount of RNA and reliable Q-PCR results. Traditionally, manual or automated methods are used for the detection and enumeration of white blood cells (WBCs). Here, we compared the performance of the manual counting measurement to the flow cytometry (FC)-based automatic counting assay employing CytoFLEX platform. Methods: We tested five different types of measurements: one manual hemocytometer-based count and four FC-based automatic cell-counting methods, including absolute, based on beads, based on 7-amino actinomycin D, combining and associating beads and 7AAD. The recovery efficiency for each counting method was established considering the quality and quantity of total RNA isolated and the Q-PCR results in matched samples from 90 adults with CML. Results: Our analyses showed no consistent bias between the different types of measurements, with comparable number of WBCs counted for each type of measurement. Similarly, we observed a 100% concordance in the amount of RNA extracted and in the Q-PCR cycle threshold values for both BCR-ABL1 and ABL1 gene transcripts in matched counted specimens from all the investigated groups. Overall, we show that FC-based automatic absolute cell counting has comparable performance to manual measurements and allows accurate cell counts without the use of expensive beads or the addition of the time-consuming intercalator 7AAD. Conclusions: This automatic method can replace the more laborious manual workflow, especially when high-throughput isolations from blood of CML patients are needed.

Background Patients with Philadelphia-negative myeloproliferative neoplasms (MPN) can develop venous thrombosis and MPN are the leading cause of splanchnic vein thrombosis. Cerebral vein thrombosis (CVT) is a rare life-threatening disease that in approximately 3% of cases encounters MPN among risk factors and tends to recur in 2-4% of patients as CVT and in 4-7% as venous thrombosis at other sites. Little or no information is available on patients with MPN who develop CVT. Objective and design To investigate the characteristics and clinical course of CVT in patients with MPN we carried out a multicenter (n=11), observational, retrospective cohort study. Patients Centers were asked to provide information on index patients with MPN who developed CVT (group MPN-CVT). For each of them, 2 patients with MPN and venous thrombosis other than CVT (group MPN-VT) and 4 patients with MPN and no venous thrombosis (group MPN-NoVT) were provided, matched by sex, age at diagnosis of MPN (+/-5 years) and type of MPN (polycytemia vera, essential thrombocytemia, myelofibrosis) with index patients. Results From January 1982 to June 2013, 48 MPN-CVT, 87 MPN-VT and 178 MPN-NoVT patients were identified in a population of 5,500 patients with MPN. Diagnosis of MPN and thrombosis coincided in 46% of MPN-CVT and 29% of MPN-VT patients (p=0.046). Compared to MPN-NoVT, MPN-CVT and MPN-VT patients had a higher prevalence of thrombophilia abnormalities (40% and 35% vs 21%, p=0.015) and, among those with essential thrombocytemia, of the JAK2 V617F mutation (76% and 78% vs 55%, p=0.059). Compared to MPN-VT, MPN-CVT patients had a higher rate of recurrent thrombosis (42% vs 25%, p=0.049) that in two-third of patients in both groups was venous, with a similar site distribution. This difference occurred despite a shorter median follow-up period (6.1 vs 10.3 years, p=0.019), a higher proportion of patients on long-term antithrombotic treatment (94% vs 84%, p=0.099) and a similar proportion of patients on cytoreductive treatment (75% vs 72%, p=0.745) among MPN-CVT than MPN-VT patients. The incidence of recurrent thrombosis was 8.8% patients/year in MPN-CVT and 4.2% patients/year in MPN-VT patients (log-rank test, p=0.022) and CVT was the only variable in a multivariate model including blood counts, thrombophilia, cytoreductive and antithrombotic treatment, that was predictive of recurrent thrombosis (HR 1.86, 95%CI 1.00-3.58). Conclusions Patients with MPN develop recurrent thrombosis in a much higher proportion than those without, particularly if they had a CVT. Patients with MPN and CVT have an approximately 2-fold increased probability to develop recurrent thrombosis than those with MPN and venous thrombosis at other sites, independently of other risk factors. Disclosures: No relevant conflicts of interest to declare.