Inherited deficiency of stress granule ZNFX1 in patients with monocytosis and mycobacterial disease.

Human inborn errors of IFN-γ underlie mycobacterial disease, due to insufficient IFN-γ production by lymphoid cells, impaired myeloid cell responses to this cytokine, or both. We report four patients from two unrelated kindreds with intermittent monocytosis and mycobacterial disease, including bacillus Calmette-Guérin-osis and disseminated tuberculosis, and without any known inborn error of IFN-γ. The patients are homozygous for ZNFX1 variants (p.S959* and p.E1606Rfs*10) predicted to be loss of function (pLOF). There are no subjects homozygous for pLOF variants in public databases. ZNFX1 is a conserved and broadly expressed helicase, but its biology remains largely unknown. It is thought to act as a viral double-stranded RNA sensor in mice, but these patients do not suffer from severe viral illnesses. We analyze its subcellular localization upon overexpression in A549 and HeLa cell lines and upon stimulation of THP1 and fibroblastic cell lines. We find that this cytoplasmic protein can be recruited to or even induce stress granules. The endogenous ZNFX1 protein in cell lines of the patient homozygous for the p.E1606Rfs*10 variant is truncated, whereas ZNFX1 expression is abolished in cell lines from the patients with the p.S959* variant. Lymphocyte subsets are present at normal frequencies in these patients and produce IFN-γ normally. The hematopoietic and nonhematopoietic cells of the patients tested respond normally to IFN-γ. Our results indicate that human ZNFX1 is associated with stress granules and essential for both monocyte homeostasis and protective immunity to mycobacteria.

Different profile of thrombin generation in children with acute lymphoblastic leukaemia treated with native or pegylated asparaginase: A cohort study.

BACKGROUND: Asparaginase (Asp) and corticosteroid (CS) treatment in patients with acute lymphoblastic leukaemia (ALL) is associated with an increased risk of thrombotic events. OBJECTIVE: Characterization of global haemostatic phenotypes of patients with ALL during Asp therapy. PROCEDURE: Thrombin generation (TG) was monitored in platelet-poor plasma of 56 children treated for a B lineage ALL (36 with native, 20 with PEG Asp) using 1 pM tissue factor and 4 µM phospholipids, with and without thrombomodulin. Protein C activity (PC), free protein S (PS), antithrombin (AT) and fibrinogen levels were also measured. RESULTS: Elevated endogenous thrombin potential (ETP) and peak of TG were noted at diagnosis, throughout the Induction phase and Late Intensification but was significantly less for PEG than for native Asp (P < 0.001), while age, sex, type of corticosteroid during Induction and molecular response had no significant effect. The reduction of ETP after addition of thrombomodulin was significantly lower in ALL children compared with that in controls, suggesting impairment in PS/PC pathway. Three patients experienced thrombosis: two treated with native and one with PEG Asp. The two patients with native Asp had, at the time of thrombosis, a prothrombotic profile. CONCLUSIONS: Treatment with Asp, in combination with CS, enhances TG in children with ALL, more significantly with native than PEG Asp, which is present early at diagnosis, persists during Induction and reappears during Late Intensification. This is consistent with the high incidence of thrombotic events described during these phases of therapy. The less pronounced effect of PEG Asp remains to be elucidated.

HIL Interferences on Three Hemostasis Analyzers and Contribution of a Preanalytical Module for Routine Coagulation Assays.

BACKGROUND: Preanalytical issues are a major part of routine coagulation laboratory errors. Automation in detection of preanalytical problems, including hemolysis, icterus and lipemia (HIL), improper tube fill volume, and undue clotting, has recently been implemented on specific hemostasis instruments. The aim of this study was to assess the added value of a new preanalytical module integrated into hemostasis analyzers compared to visual inspection of samples. METHODS: The detection of preanalytical issues was performed by visual or manual inspection of the samples and by the new preanalytical module integrated on the ACL TOP 550. Additional tests were done to evaluate the interference of hemoglobin (Hb), bilirubin or triglycerides (TG). Plasma pools containing the interference substance were tested for routine coagulation assays on the STA-R Evolution, CS-5100, and ACL TOP 550. RESULTS: Visual or manual inspection detected statistically less samples with preanalytical issues than the new preanalytical module integrated on the ACL TOP 550 (3.5% vs. 6.6%, p < 0.001). The majority of the samples were rejected for poor filling. HIL interferences appeared on assays when the concentration of Hb, bilirubin or TG exceeded a certain threshold that was analyzer and reagent dependent. CONCLUSIONS: Automatic and standardized check of routine coagulation samples by ACL TOP 550 increased the accuracy and consistency in detection of preanalytical issues as compared to visual inspection only. The main advantages were the detection of insufficient filled tubes and icteric samples that are not detected visually.

Heparin Reversal After Cardiopulmonary Bypass: Are Point-of-Care Coagulation Tests Interchangeable?

OBJECTIVE: Protamine is used to neutralize heparin after patient separation from cardiopulmonary bypass (CPB). Different bedside tests are used to monitor the adequacy of heparin neutralization. For this study, the interchangeability of the activated coagulation time (ACT) and thromboelastometry (ROTEM; Tem Innovations GmbH, Basel, Switzerland) clotting time (CT) ratios in children undergoing cardiac surgery was assessed. DESIGN: Single-center, retrospective, cohort study between September 2010 and January 2012. SETTING: University children's hospital. PARTICIPANTS: The study comprised children 0 to 16 years old undergoing elective cardiac surgery with CPB. Exclusion criteria were preoperative coagulopathy, Jehovah's witnesses, and children in a moribund condition (American Society of Anesthesiologists score 5). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: After heparin neutralization with protamine, the ratio between ACT, with and without heparinase, and the CT measured with INTEM/HEPTEM (intrinsic test activated with ellagic acid was performed without heparinase [INTEM] and with heparinase [HEPTEM]) using tests of ROTEM were calculated. Agreement was evaluated using Cohen's kappa statistics, Passing-Bablok regression, and Bland-Altman analysis. Among the 173 patients included for analysis, agreement between both tests showed a Cohen's kappa statistic of 0.06 (95% CI: -0.02 to 0.14; p = 0.22). Bland-Altman analysis showed a bias of 0.01, with a standard deviation of 0.13, and limits of agreement between -0.24 and 0.26. Passing-Bablok regression showed a systematic difference of 0.40 (95% CI: 0.16-0.59) and a proportional difference of 0.61 (95% CI: 0.42-0.86). The residual standard deviation was 0.11 (95% CI: -0.22 to 0.22), and the test for linearity showed p = 0.10. CONCLUSION: ACT, with or without heparinase, and the INTEM/HEPTEM CT ratios are not interchangeable to evaluate heparin reversal after pediatric patient separation from CPB. Therefore, the results of these tests should be corroborated with the absence/presence of bleeding and integrated into center-specific treatment algorithms.

Effective tranexamic acid concentration for 95% inhibition of tissue-type plasminogen activator induced hyperfibrinolysis in children with congenital heart disease: A prospective, controlled, in-vitro study.

BACKGROUND: Although recent studies have assessed tranexamic acid (TXA) pharmacokinetics in different subgroups, the effective concentration of TXA required to completely inhibit fibrinolysis remains to be determined. OBJECTIVE: An in-vitro determination of the effective TXA concentration needed for 95% inhibition (EC95) of tissue-type plasminogen activator (t-PA) activated fibrinolysis, using an experimental model designed for thromboelastometry (ROTEM). DESIGN: A prospective interventional study. SETTING: Department of Anaesthesiology, Queen Fabiola Children's University Hospital and Laboratory of Haematology and Haemostasis, Brugmann University Hospital. Patients were enrolled between June 2013 and October 2014. PATIENTS AND VOLUNTEERS: Twenty children, aged between 1 and 10 years, undergoing elective cardiac catheterisation were included (10 with cyanotic and 10 with noncyanotic diseases). Exclusion criteria were child requiring a procedure in a moribund state. Ten adult volunteers were also included as controls. INTERVENTION: Citrated whole blood samples were obtained from children and volunteers. MAIN OUTCOMES MEASURES: The extrinsic coagulation pathway was activated by tissue factor using the EXTEM test on ROTEM. The degree of lysis measured 30 min (LI30) after the clotting time (CT), and clot amplitudes measured at different times were recorded at baseline, after addition of 1535 units t-PA ml(-1), and following the addition of increasing TXA concentrations in t-PA activated samples. RESULTS: The concentration-effect analysis performed with lysis index after 30 min (LI30) allowed the determination of TXA efficacy concentration 50% (EC50), and calculation of the EC95, which was significantly lower in cardiac surgery children than in adults [8.6 µg ml(-1); 95% confidence interval (95% CI) 6.9 to 14.9 versus 11.3 µg ml(-1); 95% CI 10.6 to 12.9, P < 0.001]. CONCLUSION: In this in-vitro study, we observed that the EC95 TXA concentration that completely inhibited t-PA induced hyperfibrinolysis in children with congenital heart was significantly lower than the concentration required in healthy adult volunteers. Further studies are needed to confirm that this plasma concentration can effectively inhibit fibrinolysis activation in children undergoing cardiac surgery.

Transient leukemia in a newborn without Down syndrome: case report and review of the literature.

UNLABELLED: Transient neonatal leukemia occurs almost exclusively in Down syndrome babies. We report here the unusual case of a newborn without Down syndrome who presented neonatal transient leukemia and who achieved spontaneously complete remission. Trisomy 21 and GATA1 mutation were both present in leukemic cells. While close follow-up is advised since true leukemia may develop later, the patient is still in remission for 2.5 years. We performed a literature review of 15 other similar cases. CONCLUSION: Our case of transient leukemia without Down syndrome and the literature review highlight the important role of trisomy 21 and GATA1 mutation in the development of transient neonatal leukemia.

Antiphospholipid Antibodies Associated with Native Arteriovenous Fistula Complications in Hemodialysis Patients: A Comprehensive Review of the Literature.

Antiphospholipid antibody (aPL)-persistent positivity is frequent in hemodialysis (HD) patients. Native arteriovenous fistula (AVF) complications such as stenosis and thrombosis are among the most important causes of morbidity and mortality in hemodialysis patients. The association between aPL positivity and AVF thrombosis seems to now be well established. However, whether aPL positivity is associated with other AVF complications, such as maturation failure or stenosis, is not well known. Given the significant impact of AVF failure on patient's prognosis, it is of interest to further investigate this particular point in order to improve prevention, surveillance and treatment, and, ultimately, the patient's outcome. This literature review aims to report the recent literature on aPL-associated native AVF complications.

Thrombin generation in children with sickle cell disease: relationship with age, hemolysis, transcranial Doppler velocity, and hydroxyurea treatment.

INTRODUCTION: Increased thrombin generation (TG) was described in sickle cell disease (SCD) children. The aim of this study was to characterize TG at the individual level and assess its relationship with age, hemolysis, transcranial Doppler velocity (TCD), and hydroxyurea treatment. PATIENTS AND METHODS: TG was triggered in the platelet-poor plasma using tissue factor and phospholipids with addition of thrombomodulin in 97 SCD at steady state and 80 control children. Patients and controls were aged from 2 to 20 years, and they were distributed in four categories of age: [2-5], [6-10], [11-15], and [16-20] years. For each subject, ratio of endogenous thrombin potential (rETP) and peak height (rPeak) was calculated as subject's value divided by the mean value of controls of the same age range. rETP and rPeak of patients were considered abnormal when > mean + 2SD of controls. LDH, total hemoglobin, and reticulocyte count were measured as markers of hemolysis. Data on hydroxyurea treatment and TCD were collected from medical records. RESULTS: Overall, 38.1% and 44.3% of patients showed elevated rETP and rPeak, respectively. rETP and rPeak decreased significantly with increasing age. In homozygous (SS) patients, TCD velocities and all markers of hemolysis correlated significantly with both rETp and rPeak. Negative correlations were observed between these ratios and the duration of hydroxyurea treatment. CONCLUSION: Elevated TG in SCD children is mainly related to younger age and to the intensity of hemolysis. There probably a link between TG and cerebral vasculopathy in these patients. Hydroxyurea may have a beneficial effect, which could be related to the duration of treatment.

Evaluation of three APTT reagents in a routine laboratory: toward a compromise.

BACKGROUND: Our current activated partial thromboplastin time (APTT) reagent (PTT-A) is often prolonged for unexplained reasons. METHODS: We decided to compare this reagent with an alternative reagent (Cephascreen) and with our second line APTT (Actin FS) in terms of cut-off values, sensitivity to in-vitro coagulation factor deficiencies, sensitivity to lupus anticoagulant (LA), and in vivo sensitivity to unfractionated heparin (UFH). RESULTS: Actin FS, PTT-A, and Cephascreen were prolonged for FVIII level at 60%, 40%, and 40% respectively, FIX at 50%, 25%, and 35%, and FXI at 60%, 20%, and 50%. PTT-A showed the same sensitivity and specificity as Cephascreen to LA. Actin FS and PTT-A appeared less suitable to monitor UFH regarding the CLSI criteria. CONCLUSIONS: Cephascreen fulfilled the CLSI performance criteria, with a good compromise in terms of sensitivity to factor deficiency and with a substantial reduction of complementary analysis in our routine practice.

Usefullness of Heparin Calibrated Anti-Xa Activity to Assess Anticoagulant Activity of Apixaban and Rivaroxaban in Emergency Patients Scheduled for Acute Interventions.

(1) Background: Direct oral anticoagulants (DOACs) require monitoring in some critical clinical situations. The specific tests for DOAC monitoring are not yet available in all labs. The aim of this study was to evaluate if a unique, more widespread heparin-calibrated anti-Xa assay could be suitable to estimate the concentrations of apixaban and rivaroxaban in order to establish an algorithm helping our clinicians in their therapeutic decision for patients treated with DOACs in emergencies. (2) Methods: A first retrospective part allowed us to determine of a conversion factor between the measured DOAC concentration and the deducted anti-Xa heparin activity based on optic density. During the second prospective part, both DOAC concentration (ng/mL) and anti-Xa activity heparin (UI/mL) were measured on the same sample, and the previously determined conversion factor was applied to each UI/mL value. We then compared the calculated and measured DOAC concentration values. (3) Results: The analysis of the derivation cohort confirmed a good correlation, especially between the anti-Xa heparin activity and the apixaban concentrations (r = 0.97). Additionally, we determined heparin-calibrated anti-Xa assay cut-offs for invasive procedures at 0.3 UI/mL and for intravenous thrombolysis at 0.51 UI/mL using ROC curves with a sensitivity at 98% and specificity at 95% for 0.3 UI/mL and a sensitivity at 97.7% and specificity at 88.2% for the cut-off of 0.51 UI/mL. In the validation cohort, we confirmed the agreement between measured and calculated DOAC concentrations for the low values, especially around cut-offs with an excellent negative predictive value for 0.51 UI/mL (94% for apixaban and 100% for rivaroxaban) and a good negative predictive value for 0.3 UI/mL (83.3% for apixaban and 85.7% for rivaroxaban). (4) Conclusions: Our results confirm that it is possible to correctly predict or exclude the presence of apixaban/rivaroxaban in emergency situations when specific tests are not readily available.

Thrombin generation reveals high procoagulant potential in the plasma of sickle cell disease children.

Changes in several components of the clotting system are well documented in sickle cell disease (SCD) patients. However, whether the global hemostatic potential of these patients is altered is still unclear. Calibrated automated thrombogram(®) method of thrombin generation (TG) was used to characterize the hemostatic potential of 83 SCD children (75 SS, 6 SC, and 2 Sß (thal)) at steady-state as compared with 50 controls of the same range of age. TG was triggered using 1 pM tissue factor and 4 µM phospholipids with and without thrombomodulin. Thirteen SCD children were also evaluated during vaso-occlusive crisis. Protein C activity, free protein S and D-dimers levels were measured in parallel. SCD patients showed higher rates of thrombin formation, higher thrombin peak height (with and without thrombomodulin), and higher endogenous thrombin potential (ETP) than controls in the presence of thrombomodulin. Reduction of ETP (RETP) in the presence of thrombomodulin was lower in SCD group compared with controls and correlated both with protein C and protein S levels. ETP, RETP, peak height, and velocity index of TG correlated with D-dimers. Compound heterozygous patients showed an intermediate hemostatic phenotype at steady-state. No significant difference was observed when comparing TG parameters during vaso-occlusive crisis to those obtained at steady-state in the same patients. The global hemostatic potential is increased and reflects the hypercoagulable state of SCD patients even at steady-state. The relevance of this finding with respect to the risk of thrombotic complications of the disease needs further investigation.

Vitamin B12 deficiency in a 9-month-old boy.

UNLABELLED: We present a 9-month-old boy with megaloblastic anaemia, neutropenia and hypogammaglobulinaemia due to vitamin B12 deficiency. The deficiency was secondary to prolonged exclusive breastfeeding with inadequate nutritional amounts of vitamin B12 from the mother. There were no clinical or biological signs of maternal anaemia or macrocytosis. Treatment with oral vitamin B12 rapidly improved the biological findings of the child. Vitamin B12 deficiency should be considered in infants older than 2 months presenting with failure to thrive, neurocognitive retardation or even pancytopenia and hypogammaglobulinaemia, even in the absence of any signs of maternal anaemia or macrocytosis. Therefore, evaluation of vitamin B12 status during pregnancy and lactation is necessary in order to prevent B12 deficiency and its possible long-term effects in infants. CONCLUSION: Further studies should be conducted to evaluate the optimal oral dosage of vitamin B12 in children since limited data on the use of oral B12 substitution are available.

Evaluation of the analyzer ST Genesia.

BACKGROUND: Numerous studies have shown that thrombin generation test (TG) allows a better evaluation of the hemorrhagic and the thrombotic risks. The ST Genesia® is a benchtop, fully automated TG device. However, standardization of the technique and establishment of usual values are essential for its implementation in the routine laboratory. OBJECTIVES: We evaluated the fully automated TG analyzer ST Genesia ® and we aimed to determine usual values with special attention to the pediatric population. METHODS: Two ST Genesia® reagents were evaluated (BleedscreenTM and ThromboscreenTM). Precision, stability, practicability and usual values according to age were established as well as the impact of freezing samples. The comparison between calibrated automated thrombogram (CAT) method and ST Genesia® was performed. TG parameters (ETP, peak, velocity, lag time, time to peak) were analyzed on both instruments. All the results were normalized toward a reference plasma. RESULTS: Coefficient of variation associated with the repeatability and reproducibility of the QC norm low, and norm were less than 5% with both Thromboscreen and Bleedscreen reagents. We did not observe any statistical difference between results obtained on fresh or frozen samples. Usual values according to age and sex were established with special attention to the pediatric population. CONCLUSION: This technique provides a fully automated system, a strict control of temperature and the use of a reference plasma to obtain normalized results. This study is a prerequisite to future use of ST Genesia® in clinical application.

Global Hemostasis Potential in COVID-19 Positive Patients Performed on St-Genesia Show Hypercoagulable State.

BACKGROUND: At the dawn of the pandemic, severe forms of COVID-19 were often complicated by thromboembolisms. However, routine laboratory tests cannot be used to predict thromboembolic events. The objective of this study was to investigate the potential value of the thrombin generation test (TGT) in predicting hypercoagulability and thrombotic risk in the aforementioned set of patients. METHODS: The study panel comprised 52 patients divided into two groups (26 COVID-19 positive and 26 COVID-19 negative); COVID-19-positive patients were further grouped in "severe" (n = 11) and "non-severe" (n = 15) categories based on clinical criteria. The routine blood tests and TGT of these patients were retrospectively analyzed. RESULTS: All 26 COVID-19-positive patients showed decreased lymphocyte, monocyte and basophil counts and increased lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine transaminase (ALT) compared with control patients. Conversely, we did not observe statistically significant differences between severe and non-severe patients despite anecdotal variations in the distribution patterns. TGT without thrombomodulin (TM) addition showed statistically significant differences in the thrombin peak heights between COVID-19-positive and negative patients. After addition of TM, peak height, Endogenous Thrombin Potential (ETP) and velocity index were increased in all COVID-19-positive patients while the percentage of inhibition of ETP was reduced. These trends correlated with the severity of disease, showing a greater increase in peak height, ETP, velocity index and a drastic reduction in the percentage of ETP inhibition in more severely affected patients. CONCLUSIONS: Our data suggest that all COVID-19 patients harbor a hypercoagulable TGT profile and that this is further pronounced in severely affected patients.

Acquired hemophilia A revealed by spontaneous bleeding in an 80-year-old man: a marginal diagnosis?

This case report illustrates the difficulty associated with diagnosing acquired hemophilia A by reviewing the case of an 80-year-old man admitted to the hospital for anemia. A prolonged activated partial thromboplastin time (aPTT) was not noticed until the patient developed a severe hemorrhagic syndrome.

Is There Any Improvement of the Coagulation Imbalance in Sickle Cell Disease after Hematopoietic Stem Cell Transplantation?

Several components of the clotting system are modified towards hypercoagulability in sickle cell disease (SCD). To date, hematopoietic stem cell transplantation (HSCT) is the only validated curative treatment of SCD. Here, we investigated the changes in the hemostatic potential of SCD children who've received a successful HSCT. Seventeen children with severe SCD were enrolled in the study. Thrombin generation (TG) was performed on citrated platelet-poor plasma, obtained before and 3, 6, 9, 12 and 15 months after HSCT. TG was triggered using 1 pM tissue factor and 4 µM phospholipids with or without thrombomodulin (TM). Before the HSCT, SCD children showed a higher endogenous thrombin potential (ETP), higher peak, higher velocity and shorter time-to-peak of TG than the normal controls (NC). ETP did not significantly change following the HSCT. However, the peak, velocity and time-to-peak of TG reversed to normal ranges from 3 months post-HSCT and remained so up to 15 months post-HSCT. The reduction of ETP after the addition of thrombomodulin (RETP) was dramatically reduced in SCD children before HSCT as compared with the NC. A partial reversal of RETP was observed from 3 months through 15 months post-HSCT. No statistical difference was observed for patient age or donor hemoglobinopathy status. In summary, successful HSCT improves the kinetics of TG but not the total thrombin capacity in SCD children.

OPG, RANK-L, bone metabolism, and BMD in patients on peritoneal dialysis and hemodialysis.

OBJECTIVES: Bone turnover is regulated locally by osteoprotegerin (OPG) and receptor activator of NFkappaB ligand (RANK-L); it is not known how the circulating concentrations of these cytokines reflect renal osteodystrophy. METHODS: We measured serum OPG, RANK-L, parathyroid hormone (iPTH), collagen C-terminal cross-linked telopeptide (betaCrossLaps), and bone densitometry (BMD) in 79 patients with end-stage renal disease (ESRF) undergoing dialysis. A hand X-ray of these patients was also analyzed. Controls were 65 healthy subjects. RESULTS: ESRF patients had high OPG and RANK-L levels; RANK-L was higher in hemodialysis than in peritoneal dialysis. OPG and RANK-L did not depend on iPTH. The bone markers were significantly increased and correlated with serum iPTH, but not with OPG or RANK-L; neither OPG nor RANK-L correlated significantly with BMD. OPG was significantly higher in patients with acro-osteolysis. CONCLUSIONS: OPG and RANK-L serum concentrations do not strongly reflect bone status in ESRF. However, OPG was significantly higher in patients with acro-osteolysis.