Thrombin Generation Profile Using ST-Genesia after PEG-asparaginase in Pediatric Patients with Acute Lymphoblastic Leukemia.

BACKGROUND: Venous thromboembolism (VTE) etiology in children with acute lymphoblastic leukemia (ALL) is multifactorial. The use of global assays of hemostasis as a thrombin generation test (TGT) is useful to individualize VTE risk in adult patients. This prospective cohort study aimed to evaluate the usefulness of an automated TGT to evaluate VTE risk during ALL treatment in children. METHODS: TGT (automated analyzer ST Genesia; ThromboScreen) and pro- and anticoagulant plasma proteins were analyzed during ALL treatment in pediatric patients following LAL-SEHOP-PETHEMA-2013 guidelines. Results were compared with a series of pediatric normal controls and evaluated according to pegylated asparaginase PEG-ASP administration and to VTE risk factors. RESULTS: The study included 67 patients: males n = 35, B-ALL (n = 60). None had a VTE during the evaluated period. Compared to healthy controls, the normalized endogenous thrombin potential (N-ETP) ratio in patients was higher and ETP inhibition (ETP-inh) was lower, especially after PEG-ASP administration. Plasmatic protein C and protein S levels decreased after PEG-ASP administration, but antithrombin mean level did not. A bivariant analysis showed that ETP-inh was lower in patients >10 years old (p = 0.05) and in those with non-O blood type (p = 0.005). A linear mixed model also showed a higher TGT prothrombotic profile in patients with inherited thrombophilia. CONCLUSION: TGT could be a biomarker of a high VTE risk in ALL pediatric patients. Non-O blood group and inherited thrombophilia were associated with a significantly higher thrombotic profile, and an increased profile was also observed after administration of PEG-ASP.

Gaining Insights into Inherited Bleeding Disorders of Complex Etiology in Pediatric Patients: Whole-Exome Sequencing as First-Line Investigation Tool.

INTRODUCTION: Investigation of the molecular basis of inherited bleeding disorders (IBD) is mostly performed with gene panel sequencing. However, the continuous discovery of new related genes underlies the limitation of this approach. This study aimed to identify genetic variants responsible for IBD in pediatric patients using whole-exome sequencing (WES), and to provide a detailed description and reclassification of candidate variants. MATERIAL AND METHODS: WES was performed for 18 pediatric patients, and variants were filtered using a first-line list of 290 genes. Variant prioritization was discussed in a multidisciplinary team based on genotype-phenotype correlation, and segregation studies were performed with available family members. RESULTS: The study identified 22 candidate variants in 17 out of 18 patients (94%). Eleven patients had complete genotype-phenotype correlation, resulting in a diagnostic yield of 61%, 5 (28%) were classified as partially solved, and 2 (11%) remained unsolved. Variants were identified in platelet (ACTN1, ANKRD26, CYCS, GATA1, GFI1B, ITGA2, NBEAL2, RUNX1, SRC, TUBB1), bleeding (APOLD1), and coagulation (F7, F8, F11, VWF) genes. Notably, 9 out of 22 (41%) variants were previously unreported. Variant pathogenicity was assessed according to the American College of Medical Genetics and Genomics guidelines and reclassification of three variants based on family segregation evidence, resulting in the identification of 10 pathogenic or likely pathogenic variants, 6 variants of uncertain significance, and 6 benign or likely benign variants. CONCLUSION: This study demonstrated the high potential of WES in identifying rare molecular defects causing IBD in pediatric patients, improving their management, prognosis, and treatment, particularly for patients at risk of malignancy and/or bleeding due to invasive procedures.

Membrane Protein Detection and Morphological Analysis of Red Blood Cells in Hereditary Spherocytosis by Confocal Laser Scanning Microscopy.

In hereditary spherocytosis (HS), genetic mutations in the cell membrane and cytoskeleton proteins cause structural defects in red blood cells (RBCs). As a result, cells are rigid and misshapen, usually with a characteristic spherical form (spherocytes), too stiff to circulate through microcirculation regions, so they are prone to undergo hemolysis and phagocytosis by splenic macrophages. Mild to severe anemia arises in HS, and other derived symptoms like splenomegaly, jaundice, and cholelithiasis. Although abnormally shaped RBCs can be identified under conventional light microscopy, HS diagnosis relies on several clinical factors and sometimes on the results of complex molecular testing. It is specially challenging when other causes of anemia coexist or after recent blood transfusions. We propose two different approaches to characterize RBCs in HS: (i) an immunofluorescence assay targeting protein band 3, which is affected in most HS cases and (ii) a three-dimensional morphology assay, with living cells, staining the membrane with fluorescent dyes. Confocal laser scanning microscopy (CLSM) was used to carry out both assays, and in order to complement the latter, a software was developed for the automated detection of spherocytes in blood samples. CLSM allowed the precise and unambiguous assessment of cell shape and protein expression.

Venous thromboembolism in pediatric patients with acute lymphoblastic leukemia under chemotherapy treatment. Risk factors and usefulness of thromboprophylaxis. Results of LAL-SEHOP-PETHEMA-2013.

INTRODUCTION: Symptomatic venous thromboembolism (VTE) is diagnosed in 3%-14% of patients during pediatric acute lymphoblastic leukemia (ALL) therapy. There are well-known risk factors, but the role of others as inherited thrombophilia is still controversial. Prophylaxis with low molecular weight heparin (LMWH) has been described, but its use is not globally accepted. METHODS: A retrospective multicentric study in ALL patients 1-18 years old following SEHOP-PETHEMA-2013 treatment guideline was performed to evaluate VTE rate, anticoagulant treatment, outcome, risk factors, and safety and usefulness of LMWH administration as primary thromboprophylaxis in children with inherited thrombophilia. RESULTS: A total of 652 patients were included in the study. VTE incidence was 8.7%. Most of the cases occurred during induction therapy associated with central venous catheter. Univariant analysis showed that family history of thrombosis, presence of mediastinal mass, high-risk treatment group, and inherited thrombophilia were statistically significant risk factors. LMWH administration seemed to decrease VTE rate in patients with inherited thrombophilia and those with T-cell ALL phenotype. CONCLUSION: Most of the VTE cases occurred in patients without inherited thrombophilia, but when it is present, the VTE risk is higher. LMWH administration was useful to decrease VTE in these patients.

Thrombin generation in children using ThromboScreen reagent kit with ST Genesia-A pilot study.

INTRODUCTION: Thrombin generation assays assess overall coagulation system and are widely used in research; however, they still need standardization and clinical validation. The new ST Genesia is a benchtop, automated analyzer that normalizes each thrombin generation parameter using a reference plasma. The ThromboScreen reagent kit has two triggers, one of which contains thrombomodulin to assess the effect of the protein C pathway. This study aimed to make a pilot approach to the ThromboScreen reference range in children and evaluate the impact of sex, age, and pro- and anticoagulant plasma proteins on thrombin generation parameters. METHODS: This study included 55 healthy children from the following age groups: 1-6 years (n = 14), 7-11 years (n = 15), and 12-17 years (n = 26). Children younger than 1 year were excluded from the study. We measured thrombin generation using ThromboScreen, coagulation routine and test, pro- and anticoagulant proteins. RESULTS: Age did not influence ThromboScreen results. Males showed significantly lower endogenous thrombin potential and peak height values than females. The strongest determinants of endogenous thrombin potential were von Willebrand factor parameters, whereas for endogenous thrombin potential inhibition, the strongest determinants were protein C and protein S. No statistically significant differences were found between groups on temporal parameters. CONCLUSIONS: For the ThromboScreen reagent kit, it may not be necessary to subdivide reference ranges according to age for children (>1 year).

Impact of previous admission to an intensive care unit on stem cell transplantation outcome / Impacto de un ingreso previo en una unidad de medicina intensiva sobre los resultados del trasplante hematopoyético

INTRODUCTION: The impact of an admission to ICU before stem cell transplantation (SCT) on post-SCT outcome is not well established. PATIENTS AND METHODS: We reviewed the medical records of patients who had received a first SCT between 2000 and 2016 in our institution. The outcome of 22 patients who required ICU admission during chemotherapy prior to SCT (ICU group) was compared with 44 matched patients (1:2) who did not need it (NO-ICU group). RESULTS: There were no differences in transplant complications, in time to neutrophil and platelet recovery or in the length of hospital stay during SCT between the ICU and NO-ICU groups. However, microbiologically documented infections were more common in the ICU group (16/20) than in the NO-ICU group (18/39) (p=.027). The 5-yr overall survival probability (CI 95%) was 49% (28-70%) in the ICU vs. 45% (29-61%) in the NO-ICU group (p=.353), while the 5-yr incidence of non-relapse mortality was 32% (14-52%) and 24% (12-38%) (p=.333), respectively. Six patients (27%) in the ICU group and 8 (18%) in the NO-ICU group required admission to the ICU during or after the SCT procedure (p=.293). Twelve (54%) patients in the ICU and 22 (50%) in the NO-ICU group died, the causes of death were similar in both groups. CONCLUSION: Our results show that admission to the ICU prior to SCT does not have a negative impact on patient outcomes following SCT and should not be considered as an exclusion criterion for SCT

INTRODUCCIÓN: No se conoce con exactitud el impacto de la necesidad de ingreso previo en una unidad de cuidados intensivos (UCI) en la supervivencia postrasplante de progenitores hematopoyéticos (TPH). PACIENTES Y MÉTODOS: Se revisaron los archivos de pacientes que habían recibido un TPH entre el 2000 y 2016 en una única institución. El resultado del TPH en 22 pacientes que habían precisado de ingreso en una UCI durante las quimioterapias administradas previas al TPH (grupo UCI) se comparó con el de 44 pacientes controles (1:2) trasplantados que no habían precisado ingreso previo en UCI (grupo NO-UCI). RESULTADOS: No hallamos diferencias en las complicaciones post-TPH, en el tiempo de injerto de neutrófilos o de plaquetas, ni tampoco en la duración del ingreso hospitalario entre el grupo UCI y el grupo NO-UCI (p = 0,353). Sin embargo, la incidencia de infecciones documentadas microbiológicamente fue mayor en el grupo UCI (16/20) que en el NO-UCI. La probabilidad de supervivencia a 5 años (IC95%) fue del 49% (28-70%) para el grupo UCI vs. el 45% (29-61%) para el grupo NO-UCI (p = 0,353), mientras que la mortalidad relacionada con el TPH a los 5 años fue del 32% (14-52%) y 24% (12-38%) (p = 0,333), respectivamente. Seis pacientes (27%) en el grupo UCI y 8 (18%) en el grupo NO-UCI precisaron ingreso en UCI durante o después del proceso de TPH (p = 0,293). Doce pacientes (54%) en el grupo UCI y 22 (50%) en el NO-UCI fallecieron, y las causas de muerte fueron similares en ambos grupos. CONCLUSIÓN: El ingreso en UCI no tiene necesariamente un impacto negativo en los resultados de un TPH posterior en pacientes hematológicos y no debería ser criterio de exclusión para dicho procedimiento

Impact of previous admission to an intensive care unit on stem cell transplantation outcome.

INTRODUCTION: The impact of an admission to ICU before stem cell transplantation (SCT) on post-SCT outcome is not well established. PATIENTS AND METHODS: We reviewed the medical records of patients who had received a first SCT between 2000 and 2016 in our institution. The outcome of 22 patients who required ICU admission during chemotherapy prior to SCT (ICU group) was compared with 44 matched patients (1:2) who did not need it (NO-ICU group). RESULTS: There were no differences in transplant complications, in time to neutrophil and platelet recovery or in the length of hospital stay during SCT between the ICU and NO-ICU groups. However, microbiologically documented infections were more common in the ICU group (16/20) than in the NO-ICU group (18/39) (p=.027). The 5-yr overall survival probability (CI 95%) was 49% (28-70%) in the ICU vs. 45% (29-61%) in the NO-ICU group (p=.353), while the 5-yr incidence of non-relapse mortality was 32% (14-52%) and 24% (12-38%) (p=.333), respectively. Six patients (27%) in the ICU group and 8 (18%) in the NO-ICU group required admission to the ICU during or after the SCT procedure (p=.293). Twelve (54%) patients in the ICU and 22 (50%) in the NO-ICU group died, the causes of death were similar in both groups. CONCLUSION: Our results show that admission to the ICU prior to SCT does not have a negative impact on patient outcomes following SCT and should not be considered as an exclusion criterion for SCT.

Dabigatran for catastrophic antiphospholipid syndrome.

: Vitamin K antagonists (VKA) remain the treatment of choice for catastrophic antiphosphilipid syndrome (CAPS). However, when VKAs do not work for a specific patient, direct oral anticoagulants (DOAC) may be a valid therapeutic alternative. We present a patient with a psychiatric disorder and CAPS who was noncompliant to VKA and low-molecular-weight heparin. He was started on dabigatran and has remained thrombosis-free for 8 years. Due to CAPS he has developed progressive renal failure but dabigatran levels were within the expected range. In conclusion, this case report provides anecdotic evidence that dabigatran may be of use in patients with high-risk APS in whom VKA are not an option.

Thromboembolism and bleeding in patients with cancer and mechanical heart valves.

Mechanical heart valves (MHV) require life-long anticoagulation with vitamin K antagonists (VKA), but anticoagulation management is complex in patients with cancer due to a high risk of thrombosis and bleeding. This is a retrospective, single-center study to assess anticoagulation management and thrombotic (stroke/valve thrombosis) and bleeding events in patients with active cancer and MHV. The incidence of thrombotic complications was compared to a control group (matched 1:1) of patients with MHV but without cancer. We included 48 patients, 60% of whom had aortic prostheses, 23% mitral prostheses and 17% both types. All patients received VKA as anticoagulant. With a median follow-up of 5.12 years, we observed two arterial thrombotic events (two strokes and no heart valve thrombosis). The 5-year incidence (95% confidence interval [CI]) of stroke/valve thrombosis was 5.7% (0.9-17.9%). The control group had a similar incidence of stroke/valve thrombosis (5-year incidence 7.9% [95%CI 2-19.8], p = 0.16). There were also 15 major bleeding episodes in the cancer group, 11 of which were related to a surgical procedure. The 5-year incidence (95% CI) of major bleeding was 32.9% (18.5-48%), and that of major bleeding unrelated to any procedure was 10.3% (3-23%). We found a low incidence of thrombotic events in this series of patients with active cancer and MHV who were anticoagulated with VKA. However, the incidence of bleeding was high, particularly in relation to invasive procedures.

Refining the Limits of Borderline Lymphoproliferative Disorders.

BACKGROUND: The concept of borderline lymphoproliferative disorder (LPD) has not been clearly defined. METHODS: This study aimed to classify patients with leukemic LPD (n = 597, excluding hairy cell leukemia, mantle cell lymphomas, and CD10-positive LPDs) into CLL or non-CLL applying three diagnostic strategies (the D'Arena and CLLflow scores and CD43 expression) and to better characterize unclassified patients. RESULTS: Patients with concurring CLL-like (n = 441) or non-CLL like (n = 99) results with the three diagnostic strategies were determined to have CLL and non-CLL, respectively. Patients with discordant results (n = 57) were analyzed taking into consideration each individual cytometric marker and cytogenetic data: 41 were classified (11 CLL, 30 non-CLL) and 16 (2.7% of the entire series) could not and were considered borderline LPD. Excluding borderline LPD, the CLLflow score had the highest accuracy of the three strategies. With the addition of CD43 no patient was misclassified. With the aid of hierarchical clustering, 12 of the 16 borderline patients seemed to fall into two well-defined antigenic groups. None of the diagnostic strategies could reliably pick out borderline LPD. CONCLUSION: The combination of the CLLflow score and CD43 generally has a high diagnostic accuracy for leukemic LPD but it is not reliable to identify or diagnose borderline LPD. This latter group needs further study to determine its underlying biology. © 2018 International Clinical Cytometry Society.

Importancia de la dosis de anticoagulantes orales directos / Importance of dosing direct oral anticoagulants appropriately

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Indications and use of, and incidence of major bleeding with, antithrombotic agents in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) and antithrombotic medication both increase the risk of bleeding. We set out to analyze the prevalence of use, indications and bleeding risk of antithrombotic therapy in patients with MDS in a retrospective, single-center study including all patients with MDS with >20 × 109/L platelets. 193 patients (59% male, median age 75 years) were included; 122 did not receive antithrombotic treatment, 51 received antiplatelet agents and 20 received anticoagulants. The cumulative incidence of major bleeding was higher in both the antiplatelet group (11.8% at 4 years, 95% confidence interval [95%CI]: 4.7-22.3%) and the anticoagulation group (21.2% at 4 years, 95%CI 6-42.5%) than in the control group (2.8% at 4 years 95%CI: 0.7-7.3%). The prevalence of use of antithrombotic medication in this cohort of patients with MDS was high and bleeding risk was increased in these patients.

Incidence, predictive factors, management, and survival impact of atrial fibrillation in non-Hodgkin lymphoma.

Atrial fibrillation (AF) and cancer are common disorders in the general population but there are few studies in patients with both diseases. More specifically, there are scarce data on AF in patients with non-Hodgkin lymphoma (NHL). We assessed the incidence, predictive factors, management, and survival impact of AF in a cohort of patients with NHL from a single institution between 2002 and 2016 (n = 747). Twenty-three patients were diagnosed with AF before and 40 after the diagnosis of NHL (of the later, 16 were secondary to an extracardiac comorbidity and 24 unrelated to any triggering event [primary AF]). The 5-year cumulative incidence of new-onset AF was 4% (95% confidence interval [CI] 3-6%). Age and hypertension were the only predictive factors for the development of AF. Management of AF was heterogeneous, primarily with anti-vitamin K agents but also antiplatelet therapy in a significant proportion of patients. Among the 63 patients, there were six episodes of ischemic stroke/transient ischemic attack and four venous thromboembolic events, with four major bleeding episodes. Overall survival (OS) was inferior in patients with AF (HR 0.1, 95% CI 0.01-0.7, p = 0.02), largely due to secondary AF. We conclude that the incidence of new-onset AF in NHL patients seemed somewhat higher than in the general population, although with similar predictive factors. The management was heterogeneous, and the risk of ischemic and hemorrhagic events did not seem higher than in cancer-free patients. Survival was particularly poor for patients with secondary AF.

Usefulness of the CLLflow score.

BACKGROUND: The CLLflow score was recently suggested as an improvement over the Moreau score (MS) for the diagnosis and classification of B-cell lymphoproliferative disorders (B-LPD). METHODS: We determined the CLLflow score in peripheral blood or bone marrow of a series of cases with an inconclusive immunophenotype, including samples with a MS of 3 (n = 52) and CD5-positive with a score of 2 (n = 38). As controls, B-LPD with a MS of 0-1 (n = 95), CD5-negative score 2 (n = 24), and score 4-5 (i.e., chronic lymphocytic leukemia [CLL], n = 166) were included. RESULTS: The CLLflow score was positive (suggestive of CLL) in all CLL cases and negative in all MS <2, regardless of CD200-positivity, which occurred in 31% (29/95) of cases. The CLLflow score was positive in 71%, 29%, and 8% of samples with a MS 3, CD5-positive score 2, and CD5-negative score 2, respectively. DISCUSSION: Our results suggest that the CLLflow is useful in the differential diagnosis of cases with inconclusive immunophenotype. © 2018 International Clinical Cytometry Society.