Inherited Thrombophilia Associated With Ischemic Pediatric Stroke in Parent-Child Pairs.

BACKGROUND: We aimed to examine inherited thrombophilia frequencies by extending genetic profile to previously rarely or not investigated polymorphisms in children with ischemic pediatric stroke (IPS) and their parents. METHODS: The study included 33 children: 23 with perinatal arterial ischemic stroke (PAIS), eight with childhood arterial ischemic stroke (CAIS), and two with sinovenous thrombosis and their parents (33 mother-child, 12 father-child, and 12 mother-father-child pairs). Genotyping of FV-Leiden, FV-H1299R, FII-G20210A, ß-fibrinogen-455G>A, FXIII-A-Val34Leu, PAI-1(4G/5G), HPA-1, MTHFR-C677T, MTHFR-A1298C, ACE(I/D), and APOE(ε2-4) was performed using CVD Strip assay (ViennaLab, Austria). RESULTS: At least one and up to seven simultaneously present polymorphisms were observed in all children with IPS, mothers, and fathers. More than five simultaneously present polymorphisms were identified threefold more frequently in children with IPS (10 of 33; 30%) compared with the child control group (17 of 150; 11%), yielding a statistically significant difference between the two groups (odds ratio [OR] = 3.40; 95% confidence interval [CI] = 1.39 to 8.35; P = 0.012). Stronger association was revealed for PAIS (OR = 4.17; 95% CI = 1.55 to 11.29; P = 0.008) and CAIS subgroups (OR = 7.82; 95% CI = 1.79 to 34.20; P = 0.012). Complete match of polymorphisms was not identified in any parent-child pair. A partial match (one to four mutual polymorphisms) was found in 11 of 12 parent-child pairs where until three mutual polymorphisms was present in 11 of 12 (91.7%) father-child compared with 21 of 33 (63.6%) mother-child pairs. CONCLUSIONS: According to obtained results the simultaneous presence of more than five polymorphisms is associated with a higher risk for IPS occurrence, suggesting the risk enhancement for PAIS in the presence of pregnancy complications or for CAIS in conjunction with maternal comorbidity and positive family history. The presence of up to three mutual polymorphisms more frequently in father-child than mother-child pairs suggests significant paternal contribution of inherited thrombophilia to increased risk of IPS.

Importance of Cellular Immunity and IFN-γ Concentration in Preventing SARS-CoV-2 Infection and Reinfection: A Cohort Study.

Recent studies have highlighted the underestimated importance of the cellular immune response after the emergence of variants of concern (VOCs) of SARS-CoV-2, and the significantly reduced neutralizing power of antibody titers in individuals with previous SARS-CoV-2 infection or vaccination. Our study included 303 participants who were tested at St. Catherine Specialty Hospital using the Quan-T-Cell SARS-CoV-2 in combination with the Quan-T-Cell ELISA (Euroimmun Medizinische Labordiagnostika, Lübeck, Germany) for the analysis of IFN-γ concentration, and with Anti-SARS-CoV-2 QuantiVac ELISA IgG (Euroimmun Medizinische Labordiagnostika, Lübeck, Germany) for the detection of human antibodies of the immunoglobulin class IgG against the S1 domain of the SARS-CoV-2 spike protein. The statistical analysis showed a significant difference in the concentration of IFN-γ between reinfected participants and those without infection (p = 0.012). Participants who were not infected or reinfected with SARS-CoV-2 after vaccination and/or previous SARS-CoV-2 infection had a significantly higher level of cellular immunity. Furthermore, in individuals without additional vaccination, those who experienced infection/reinfection had significantly lower levels of IFN-γ compared to uninfected participants (p = 0.016). Our findings suggest a long-lasting effect of cellular immunity, measured by IFN-γ concentrations, which plays a key role in preventing infections and reinfections after the emergence of SARS-CoV-2 variants of concern.

Type 1 von Willebrand Disease in a Pediatric Patient Caused by a Novel Heterozygous Deletion of Exons 1 to 6 of the von Willebrand Factor Gene: A Case Report.

A 6-year-old boy was referred to a hematologist due to excessive mucocutaneous bleeding. Diagnostic assessment for von Willebrand disease (VWD) was indicated and included both coagulation and genetic testing. Laboratory testing revealed proportionally decreased von Willebrand factor (VWF) glycoprotein Ib-binding activity (23.6%) compared to VWF antigen (24.7%), similarly decreased VWF collagen-binding activity (24.2%), and normally distributed VWF multimers, with decreased intensity of all fractions. Diagnosis of type 1 VWD was established. Genetic analysis by means of next-generation sequencing (NGS) of VWF and coagulation factor VIII genes did not identify any causative mutations. Additionally, multiplex ligation-dependent probe amplification (MLPA) of VWF gene exons revealed a heterozygous deletion of exons 1 to 6, which is reported in type 1 VWD for the first time. Application of MLPA was crucial for revealing the genetic basis of type 1 VWD in this case, which would have remained undetected if only NGS was used.

Unusual total anti-SARS-CoV-2 antibody kinetics observed during longitudinal monitoring after BNT162b2 vaccination.

The present study aimed to clarify unusual total antibody kinetics in three female individuals observed during longitudinal monitoring of antibody response to BNT162b2 COVID-19 vaccine in 54 healthy volunteers. Total and IgG antibodies against the SARS-CoV-2 spike glycoprotein were measured using Roche and Abbott quantitative assays, respectively, a day before and 8, 71, 135 and 217 days after the second dose. Samples showing unusual kinetics were additionally tested with Beckman Coulter and Euroimmun IgG assays, as well as IgA assay. Antibody levels peaked 8 days after the second dose (total:2769 U/mL; IgG:20022 AU/mL) and declined to 611 U/mL (total) and 783 AU/mL (IgG), after 217 days. A delayed increase of total but not IgG antibodies evidenced in three females, was in two cases coupled with an increase in IgA antibodies. This study identified a previously unknown contribution of anti-SARS-CoV-2 IgA antibodies to a delayed total antibody increase in a subgroup of vaccinated individuals. It also emphasizes that different commercially available serological assays do not provide uniform information about the post-vaccination immune status and that thorough understanding the assays' features is crucial for the proper interpretation of antibody response monitoring.

Next-generation sequencing of von Willebrand factor and coagulation factor VIII genes: a cross-sectional study in Croatian adult patients diagnosed with von Willebrand disease.

AIM: To identify the von Willebrand factor (VWF) gene variant status in Croatian adult patients diagnosed with von Willebrand disease (VWD), provide differential diagnosis of VWD subtypes, and identify patients with mild hemophilia A (HA) who were earlier misdiagnosed as VWD. METHODS: Coagulation testing included determination of VWF gain-of-function mutant glycoprotein Ib binding activity (VWF:GPIbM), VWF antigen, VWF collagen-binding activity, and multimeric analysis. Genetic analysis of VWF and FVIII genes was performed with next-generation sequencing (NGS). RESULTS: The study enrolled 50 patients (72% women; median age 37 years, range 18-75) from 44 unrelated families. Fourteen patients were heterozygous for VWF gene variants compatible with type-1 VWD. Twelve had variants associated with type 2, of whom seven were classified as type 2A, four as type 2B, and one as type 2N. Six type-3 VWD patients were either homozygotes for null variants or combined heterozygotes. Eleven variants within the VWF gene were novel. Three female patients had variants within the FVIII gene, and were re-classified as mild-HA carriers, of whom one had causative novel variants both within VWF and FVIII genes. Fifteen patients remained without a defined genetic cause of their disorder, of whom five had VWF:GPIbM levels below 50%. CONCLUSION: Croatian adult patients with VWD have considerable genetic heterogeneity. NGS of both VWF and FVIII genes provided accurate differential diagnosis of VWD subtypes and distinction of VWD from mild HA.

Association of polymorphisms in genes encoding prothrombotic and cardiovascular risk factors with disease severity in COVID-19 patients: A pilot study.

The present study aimed to assess the association of 16 polymorphisms in genes encoding prothrombotic and cardiovascular risk factors with COVID-19 disease severity: FV G1691A, FV H1299R, FII G20210A, MTHFR C677T, MTHFR A1298, factor XIII V34L, PAI-1 4G/5G, EPCR haplotypes (A1/A2/A3), eNOS -786 T > C, eNOS G894T, LTA C804A, ACE I/D, ITGB3 PIA1/A2, ITGA2B Baka/b, ß-Fbg -455 G > A and ApoB R3500Q. The study included 30 patients with severe COVID-19 and 49 non-severe COVID-19 patients. All studied polymorphisms except ITGA2B Baka/b were determined using multilocus genotyping assays CVD StripAssays (ViennaLab Diagnostics), while ITGA2B was genotyped using a real-time PCR method based on TaqMan technology. A higher frequency of carriers of at least one ITGB3 PIA2 allele was found in severe COVID-19 patients (p = 0.009). The distribution of genotypes was significantly different for ß-Fbg -455 G > A (p = 0.042), with only three homozygous AA genotypes found among severe COVID-19 patients. The association with an increased risk for severe COVID-19 was found for ITGB3, with carriers of at least one ITGB3 PIA2 allele having a 3.5-fold greater risk of severe COVID-19 (p = 0.011). Genotype distribution differences were obtained for the combinations of FV H1299R and FXIII V34L (p = 0.026), ITGB3 PIA1/A2 and ITGA2B Baka/b (p = 0.024), and ACE I/D and PAI-1 4G/5G (p = 0.046). ITGB3 polymorphism emerged as an independent risk factor for severe COVID-19 and homozygosity for ß-Fbg -455 G > A mutation could contribute to disease severity. The combined effect of polymorphisms in genes encoding prothrombotic and cardiovascular risk factors could further contribute to disease severity.

Cellular Immunity-The Key to Long-Term Protection in Individuals Recovered from SARS-CoV-2 and after Vaccination.

Previous clinical and epidemiological studies have shown that over time antibody titers decrease, and they do not provide long-term mucosa protection against SARS-CoV-2 infection. Additionally, the increase in breakthrough infections that occur more frequently in the vaccinated than in the study participants with previous SARS-CoV-2 infection has recently become a priority public health concern. We measured the amount of interferon-gamma (Quan-T-Cell ELISA) and the level of antibodies (Anti-SARS-CoV-2 QuantiVac ELISA IgG) in the blood of the same patients simultaneously to compare cellular and humoral immunity. A total of 200 study participants (before Omicron variant appearance) were divided into four groups whose levels of cellular and humoral immunity we compared: study participants previously infected with SARS-CoV-2 (group 1); study participants vaccinated with EMA-approved vaccines (group 2); study participants previously infected with SARS-CoV-2, and vaccination history (group 3); and study participants without a history of SARS-CoV-2 infection or vaccination (group 4). Our results showed that study participants who received one of the EMA-approved vaccines and who recovered from COVID-19 (group 3) had significantly higher levels of cellular immunity and antibody titers in comparison with groups 1 and 2. Additionally, we have noticed that the study participants previously infected with SARS-CoV-2 and the study participants vaccinated with EMA-approved vaccines had a long-lasting cellular immunity. Furthermore, antibody levels showed a negative correlation with time since the last contact with a viral antigen, while cellular immunity within 20 months showed as long-term protection. Moreover, out of 200 study participants, only 1 study participant who recovered from COVID-19 (0.5%) was re-infected, while a total of 6 study participants (3%) were infected with SARS-CoV-2 after receiving the vaccine. This study suggests that cellular immunity-unlike humoral immunity, thanks to memory T cells-represents long-term protection in individuals recovered from SARS-CoV-2 and after vaccination.

Reevaluation of von Willebrand disease diagnosis in a Croatian paediatric cohort combining bleeding scores, phenotypic laboratory assays and next generation sequencing: a pilot study.

INTRODUCTION: This study reevaluated von Willebrand disease (vWD) diagnosis in a Croatian paediatric cohort by combining bleeding scores (BS), phenotypic laboratory testing, and next-generation sequencing (NGS). MATERIALS AND METHODS: A total of 25 children (11 males and 14 females, median age 10 years, from 2 to 17) previously diagnosed with vWD were included. BS were calculated using an online bleeding assessment tool. Phenotypic laboratory analyses included platelet count, platelet function analyser closure times, prothrombin time, activated partial thromboplastin time, von Willebrand factor antigen (vWF:Ag), vWF gain-of-function mutant glycoprotein Ib binding activity (vWF:GPIbM), vWF collagen binding activity (vWF:CBA), factor VIII activity (FVIII:C) and multimeric analysis. Next-generation sequencing covered regions of both vWF and FVIII genes and was performed on MiSeq (Illumina, San Diego, USA). RESULTS: Disease-associated variants identified in 15 patients comprised 11 distinct heterozygous vWF gene variants in 13 patients and one novel FVIII gene variant (p.Glu2085Lys) in two male siblings. Four vWF variants were novel (p.Gln499Pro, p.Asp1277Tyr, p.Asp1277His, p.Lys1491Glu). Three patients without distinctive variants had vWF:GPIbM between 30 and 50%. Patients with identified vWF gene variants had statistically significant lower values of vWF:GPIbM (P = 0.002), vWF:Ag (P = 0.007), vWF:CBA (P < 0.001) and FVIII:C (P = 0.002), compared to those without. Correlations between BS and phenotypic laboratory test results were not statistically significant for either of the tests. CONCLUSION: The applied diagnostic approach confirmed the diagnosis of vWD in 13 patients and mild haemophilia A in two. Limited utility of BS in the paediatric population was evidenced.

Rapid COVID-19 Antigen Testing in Croatia: Risk Perception Plays an Important Role in the Epidemic Control.

Aim: To explore the clinical presentation and epidemiological history of the subjects who underwent SARS-CoV-2 antigen testing. Methods: We included 1,000 consecutive subjects who presented themselves at the diagnostic clinic in Croatia and analyzed their symptoms and epidemiological history. All subjects were classified into three groups, according to their reason of arrival; symptomatic, contacts of confirmed patients, and those who were tested due to administrative reasons. Results: On average, there were 24% of positive antigen results; the positivity rate was 51% among symptomatic, 16% in contacts, and 5% of administrative patients. The commonest symptoms of the disease included febrility and anosmia. We developed a clinical score to predict SARS-CoV-2 positivity, which had an area under the curve of 79.3 [95% confidence intervals (CI) 75.8-82.8]. Contact with the isolated person [odds ratio 0.54 (95% CI 0.31-0.94)] and international travel had a protective effect [0.20 (0.09-0.43)], suggesting that risk perception and mandatory pretravel measures had a key role in the determination of the infection risk. Conclusions: A combination of clinical symptoms can have reasonable predictive power for an antigen-positive test result. Risk perception seems to have a role in the epidemic spread, probably via stricter adherence to personal preventative measures.

B regulatory cells and monocyte subpopulations in patients with chronic graft-vs-host disease.

AIM: To assess the correlations of B regulatory cells (Bregs) and monocyte subsets in peripheral blood with the National Institutes of Health (NIH)-consensus-defined clinical manifestations of chronic graft-vs-host disease (cGvHD), in an attempt to establish their role as cellular biomarkers. METHODS: This multidisciplinary prospective study enrolled adult cGVHD patients treated in the University Hospital Center Zagreb and University of Zagreb School of Medicine. Immunophenotypic subpopulations of CD24highCD38high Bregs (CD27-, CD27+, and total) and monocyte (classical, intermediate, and non-classical) counts were correlated with demographic, transplant, and cGVHD-related data. Bivariate correlation analysis was performed to evaluate the correlations between Bregs and monocytes subsets and cGVHD organ involvement, as well as cGVHD severity and immunosuppression intensity. RESULTS: Twenty-two adult patients (54.5% female) with cGVHD were enrolled. The median (range) age was 44.5 years (24-65). All patients were transplanted for hematologic malignancies and 40.9% had severe NIH cGVHD global score. The median time from cGVHD diagnosis to the analysis was 16.6 months (0-176). The organ most frequently affected with cGVHD were the eyes (68.2%), skin (45.5%), lungs (45.5%), and liver (40.9%). Lower total and CD27-Bregs counts were correlated with worse cGVHD severity, higher immunosuppression intensity, and lung cGVHD, in terms of cell count, but also with skin cGVHD, in terms of percentages. Patients with liver and joint/fascia cGVHD had a lower percentage of non-classical monocytes and patients with more severe global NIH score had a higher classical monocytes count. CONCLUSION: Different organs affected by cGVHD are differently associated with different subpopulations of Bregs and monocytes.

Overall hemostasis potential and aPTT-clot waveform analysis as powerful laboratory diagnostic tools for identification of hemophilia A patients with unexpected bleeding phenotype.

INTRODUCTION: Traditionally used laboratory methods do not always accurately reflect bleeding severity in hemophilia A (HA) patients. The ability of three global assays for identifying patients with unexpected bleeding phenotype was investigated. METHODS: Overall hemostasis potential (OHP), aPTT-clot waveform analysis (aPTT-CWA), endogenous thrombin potential (ETP), FVIII activities, and prothrombin fragment 1 + 2 concentrations were measured in 62 HA patients (30 severe and 32 non-severe) and 27 male controls. Bleeding phenotype was determined using our proposed scoring system including age at first joint bleed, number of target joints, and number of joint/muscle bleeds per year. Bleeding score ≤ 4 defined patients with mild bleeding phenotype (N = 27); score ≥ 5 defined severe bleeding phenotype (N = 35). RESULTS: The receiver operating characteristic analysis performed for distinguishing patients with severe and mild bleeding phenotype yielded following values of area under the curve: 0.910 (FVIII); 0.891 (aPTT-CWA parameter DELTA); 0.769 (OHP); and 0.634 (ETP). Unexpected bleeding phenotype was identified in 11/62 HA patients: 8/32 (25%) non-severe HA patients had severe, while 3/30 (10%) severe HA patients had mild bleeding phenotype, and global assays enabled the identification of all these patients. OHP and DELTA were revealed as the most reliable parameters for bleeding phenotype determination (10/11 and 9/11 unexpected results, respectively). CONCLUSION: This study emphasizes OHP and aPTT-CWA as a powerful laboratory diagnostic tool in identifying HA patients with unexpected bleeding presentations, with the best results achieved by combining both assays. Global assays should not completely replace FVIII activity measurement but should be a part of the HA diagnostic algorithm.

Verification of automated latex-enhanced particle immunoturbidimetric D-Dimer assays on different analytical platforms and comparability of test results.

INTRODUCTION: The aim of the study was the analytical verification of automated latex-enhanced particle immunoturbidimetric (LPIA) D-Dimer assay INNOVANCE D-dimer on Sysmex CS-5100 and Atellica COAG 360 analysers, and HemosIL D-dimer HS500 on ACL TOP 550, as well as the comparison with the enzyme-linked immunofluorescent assay (ELFA) on the miniVidas analyser. MATERIALS AND METHODS: Verification included assessment of within-run and between-run precision, bias, measurement uncertainty (MU), verification of the cut-off, method comparison between all assessed assays, and the reference commercial ELFA VIDAS D-Dimer Exclusion II. RESULTS: Within-run coefficients of variations (CVs) ranged from 1.6% (Atellica COAG 360) to 7.9% (ACL TOP 550), while between-run CVs ranged from 1.7% (Sysmex CS-5100) to 6.9% (Atellica COAG 360). Spearman's rank correlation coefficients were > 0.99 between LPIAs and ≥ 0.93 when comparing ELFA with LPIA. Passing-Bablok regression analysis yielded constant and proportional difference for comparison of ACL TOP 550 with both Sysmex CS-5100 and Atellica COAG360, and for miniVidas with Atellica COAG360. Small proportional difference was found between miniVidas and both Sysmex CS-5100 and ACL TOP 550. Calculated MUs using D-dimer HS 500 calibrator were 12.6% (Sysmex CS-5100) and 15.6% (Atellica COAG 360), while with INNOVANCE D-dimer calibrator 12.0% (Sysmex CS-5100), 10.0% (Atellica COAG 360) and 28.1% (ACL TOP 550). Excellent agreement of results was obtained, with occasional discrepancies near the cut-off. The cut-off (0.5 mg/L FEU) was confirmed. CONCLUSIONS: The obtained results prove satisfactory analytical performance of LPIAs, their high comparability and almost equal discriminatory characteristics, suggesting them as a valid alternative to ELFA.

Role of platelet gene polymorphisms in ischemic pediatric stroke subtypes: a case-control study.

AIM: To assess the role of human platelet antigens (HPA), P-selectin gene (SELP) polymorphisms, and HPA and SELP haplotypes with factor V (FV) R506Q in ischemic pediatric stroke (IPS) subtypes: cerebral sinovenous thrombosis (CSVT), perinatal (PAIS), and childhood (CAIS) arterial ischemic stroke. METHODS: This case-control study enrolled 150 children with confirmed IPS and 150 age- and sex-matched controls. FV R506Q and HPA-1 were genotyped with CVD StripAssay®, HPA-2 and HPA-3 with real-time polymerase chain reaction, SELP S290N, V599L, and T715P with high resolution melting analysis, and SELP N562D with sequence-specific polymerase chain reaction. RESULTS: HPA-1b allele (odds ratio [OR] 2.75, 95% confidence interval [CI] 1.02-7.42, P=0.048) and HPA-1a2a3b (OR 5.46, 95% CI 1.51-19.76, P=0.011), HPA-1b2a3a (OR 7.00, 95% CI 1.25-39.13, P=0.028), and HPA-1b2b3a (OR 11.39, 95% CI 1.39-92.95, P=0.024) haplotypes increased the risk for CSVT. HPA-3b allele was significantly associated with 2-fold lower risk for PAIS (OR 0.49, 95% CI 0.26-0.89, P=0.020) and CAIS (OR 0.47, 95% CI 0.26-0.86, P=0.014) and non-significantly associated with increased risk for CSVT (OR 6.43, 95% CI 0.83-50.00, P=0.022). HPA-1a2b3a haplotype was significantly associated with CAIS (OR 6.76, 95% CI 2.13-21.44, P=0.001). The inclusion of FV R506Q in SELP haplotype analysis increased the risk for PAIS 4-fold in QNDVT carriers (OR 8.14, 95% CI 0.93-71.33, P=0.060) compared with NDVT haplotype (OR 2.45, 95% CI 0.98-6.18, P=0.058), but the result was not significant. CONCLUSION: Individual HPAs, and particularly HPA haplotypes, are involved in IPS subtypes pathogenesis. A possible risk-inducing synergistic effect of SELP haplotypes with FV R506Q is restricted to PAIS only.

Analytical validation of the iSED automated analyzer for erythrocyte sedimentation rate.

INTRODUCTION: iSED is an alternate automated analyzer for erythrocyte sedimentation rate (ESR) based on photometric rheology technology that estimates ESR by measuring rouleaux formation. The aim was to evaluate the analytical performance of the iSED analyzer and compare the results with the Westergren method and another alternate ESR analyzer, TEST1. METHODS: Validation was performed at two study sites according to the recommendations by the International Council for Standardization in Haematology and included determination of intrarun precision and inter-run precision, bias, carryover, and method comparison, which was further assessed for samples with normal and low hematocrit, as well as per low, middle, and upper third of the analytical range. RESULTS: Intrarun coefficients of variation (CVs) with commercial controls were 4.0% and 1.8%, while inter-run CVs 7.5% and 0.7%, for the normal and pathological range, respectively. Intrarun CVs obtained with patient samples were 19.9%, 9.9%, 10.3%, and 9.4%, the highest being for the lowest ESR value. Correlation coefficients for the comparison between iSED and Westergren were 0.862 (Site-1) and 0.916 (Site-2). While proportional difference with a positive bias was revealed at Site-1, comparison at Site-2 showed both constant and proportional difference and a negligible negative bias. Higher correlation was obtained for samples with low than normal hematocrit. Comparison between iSED and TEST1 yielded a correlation coefficient of 0.986, constant and proportional difference, and positive bias. Carryover was 3.2%. CONCLUSION: This study proved the analytical validity of the iSED analyzer, despite minor discrepancies to the Westergren method that can be attributed to methodological differences.

Influence of flavonoids' lipophilicity on platelet aggregation.

Flavonoids are natural polyphenolic compounds present in a wide spectrum of plants that have a beneficial effect on human health. In the context of cardiovascular diseases related to plaque and thrombus formation, flavonoids exhibit an anti-aggregatory effect. Previously, it has been reported that all tested flavonoids exhibit an antiaggregatory effect on platelet aggregation when measured by impedance aggregometry on whole blood, in the test of aggregation induced by adenosine diphosphate (ADP). As not all flavonoids have the same targets within signaling pathways, an assumption of a common non-specific mechanism related to lipophilicity is to be considered. To test this hypothesis, reverse-phase thin layer chromatography was used to assess the lipophilicity of flavonoids; impedance aggregometry was used for testing of platelet aggregation and flow cytometry to monitor the influence of flavonoids on platelet activation. Lipophilicity analysis showed a highly negative correlation of logP and MINaAC for groups of flavones and flavanones. As determined by flow cytometry, the exposition of receptors necessary for the promotion of platelet activation and primary clot formation was diminished, i.e., lowered expression of the activated form of integrin αIIbß3 was observed in the presence of flavanone. Platelet membrane stabilization by flavonoids as a mechanism of antiaggregatory effect has been supported by impedance aggregometry experiments when specific inhibitors of platelet aggregation signaling pathways (U73122, indomethacin, verapamil) were used in the presence of a weak (ADP) and a strong (TRAP-6) agonist of aggregation. While individual flavonoids can have specific targets within aggregation signaling pathways, all flavonoids share a common non-specific mechanism of platelet aggregation inhibition related to their lipophilicity and membrane stabilization that, to some extent, contributes to their antiaggregatory effect.

Results of the European survey on the assessment of deep molecular response in chronic phase CML patients during tyrosine kinase inhibitor therapy (EUREKA registry).

PURPOSE: The advent of tyrosine kinase inhibitor (TKI) therapies has revolutionized the treatment of chronic myeloid leukemia (CML). The European LeukemiaNet (ELN) recommends quantification of BCR-ABL1 transcripts by real-time quantitative PCR every 3 months during TKI treatment. Since a proportion of patients in deep molecular response (DMR: MR4, MR4.5, MR5) maintain remission after treatment stop, assessment of DMR is crucial. However, systematically collected molecular data, monitored with sensitive standardized assays, are not available outside clinical trials. METHODS: Data were collected on the standardized assessment of molecular response in the context of real-life practice. BCR-ABL1 transcript levels after > 2 years of TKI therapy were evaluated for DMR by local laboratories as well as standardized EUTOS laboratories. Since standardized molecular monitoring is a prerequisite for treatment discontinuation, central surveillance of the performance of the participating laboratories was carried out. RESULTS: Between 2014 and 2017, 3377 peripheral blood samples from 1117 CML patients were shipped to 11 standardized reference laboratories in six European countries. BCR-ABL1 transcript types were b3a2 (41.63%), b2a2 (29.99%), b2a2/b3a2 (3.58%) and atypical (0.54%). For 23.72% of the patients, the initial transcript type had not been reported. Response levels (EUTOS laboratory) were: no MMR, n = 197 (6.51%); MMR, n = 496 (16.40%); MR4, n = 685 (22.64%); MR4.5, n = 937 (30.98%); MR5, n = 710 (23.47%). With a Cohen's kappa coefficient of 0.708, a substantial agreement between EUTOS-certified and local laboratories was shown. CONCLUSIONS: Multicenter DMR assessment is feasible in the context of real-life clinical practice in Europe. Information on the BCR-ABL1 transcript type at diagnosis is crucial to accurately monitor patients' molecular response during or after TKI therapy.

Influence of Blood Count, Cardiovascular Risks, Inherited Thrombophilia, and JAK2 V617F Burden Allele on Type of Thrombosis in Patients With Philadelphia Chromosome Negative Myeloproliferative Neoplasms.

INTRODUCTION: Thrombosis is the most common complication in Philadelphia chromosome negative (Ph-) myeloproliferative neoplasms patients. PATIENTS AND METHODS: In a cohort of 258 Ph- myeloproliferative neoplasm patients, the difference between patients with and without thrombosis was analyzed according to genetic thrombophilia factors, JAK2 V617F status and burden allele, blood count, cardiovascular risk factors and age. Patients were also divided in polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) subgroups as well as by the type of thrombosis. RESULTS: Analysis of cardiovascular risk factors regarding arterial thrombosis showed that PV patients with thrombosis had higher incidence of diabetes (P = .030), ET patients more often had hypertension (P = .003) and hyperlipidemia (P = .005), while PMF patients had hyperlipidemia (P = .046) and at least one cardiovascular risk factor (P = .044). Moreover, leukocytes > 18 × 109/L and V617F burden allele > 25.7% were statistically significantly different in PV patients (P = .019 and borderline significant at P = .055, respectively), while in ET patients leukocytes > 9.2 × 109/L (P < .001) and age at diagnosis of > 55 years were statistically significantly different (P = .002). PMF patients with V617F burden allele ≤ 34.8% were more prone to thrombosis (P = .032). When comparing patients with and without venous thrombosis, cutoff value of V617F burden allele > 90.4% was significant for PV patients with thrombosis (P = .036), as was > 56.7% for PMF patients with thrombosis (P = .046). Platelets ≤ 536 × 109/L and age at diagnosis > 54 years showed statistically significant difference for ET patients with thrombosis (P = .015 and P = .041, respectively). CONCLUSION: On the basis of our results, a new scoring system for thrombosis risk in PV could be made, while PMF prognostic model may be expanded for better recognition of potential thrombotic risk factors.

Development and validation of a rapid method for genotyping three P-selectin gene polymorphisms based on high resolution melting analysis.

BACKGROUND: High resolution melting (HRM) analysis is one of the newer, reliable, and sensitive genotyping techniques, which offers considerable time and cost savings. P-selectin is an adhesion molecule that has a role in the initial phases of leukocyte adhesion to stimulated platelets and endothelial cells in inflammation. Multiple polymorphisms in P-selectin gene (SELP) that affect the protein sequence have been described. The aim of this study was to design, optimize, and validate a simple and rapid in-house HRM-based method for genotyping the NM_003005.3:c.992G>A (c.992G>A), NM_003005.3:c.1918G>T (c.1918G>T), and NM_003005.3:c.2266A>C (c.2266A>C) SELP polymorphisms. METHODS: Initial genotyping of three SELP polymorphisms was performed by applying polymerase chain reaction (PCR) with sequence-specific primers (SSP), which was used as a reference method for determination of analytical sensitivity. PCR-HRM was performed with primers for c.2266A>C reported in the literature. Primers for the remaining two polymorphisms were designed using Primer-BLAST. Precision testing was performed using three samples with different genotypes. For accuracy, analytical sensitivity and specificity testing, 20 wild type, 10 heterozygous, and 10 homozygous samples were chosen per polymorphism. Results were expressed as percentage of concordance with the acceptability criterion ≥95%. RESULTS: Agreement of results was 100% for all validation parameters except for analytical sensitivity for c.1918G>T and c.2266A>C, with agreement of 90%. Repeated analysis using both methods revealed an error in initial genotyping and correct genotyping by PCR-HRM, which was confirmed by Sanger sequencing. CONCLUSION: The validation confirmed PCR-HRM as a precise, accurate, and specific method for genotyping the c.992G>A, c.1918G>T, and c.2266A>C SELP polymorphisms.

The incidence of atypical patterns of BCR-ABL1 rearrangement and molecular-cytogenetic response to tyrosine kinase inhibitor therapy in newly diagnosed cases with chronic myeloid leukemia (CML).

BACKGROUND: To analyze the frequency of atypical fluorescence in situ hybridization signal patterns and estimate the complete cytogenetic response (CCyR) and major molecular response (MMR) during 12 months of tyrosine kinase inhibitor therapy in patients with newly diagnosed chronic myeloid leukemia. METHODS: The study included bone marrow and peripheral blood samples from 122 patients with newly diagnosed chronic myeloid leukemia. Detection of the breakpoint cluster region-Abelson fusion gene (BCR-ABL1) was performed using fluorescence in situ hybridization with a dual-color dual-fusion translocation probe, and MMR analysis was performed using the real-time quantitative polymerase chain reaction method. RESULTS: Variant translocation was determined in 10 samples and a deletion on the derivative chromosome 9 (del/der(9)) was found in 20 samples. The rates of CCyR and MMR were similar between patients with reciprocal translocation, variant translocation, deletion of derivative BCR, or ABL1-BCR fusion gene. The Kaplan-Meier test did not show any significant differences in the rates of CCyR and MMR among those groups of patients. CONCLUSION: The frequencies of variant translocation and del/der(9) in the present study agree with the results of other studies performed worldwide. No differences were observed in the rates of CCyR and MMR between patients with atypical patterns and reciprocal translocation.