Aberrant MNX1 expression associated with t(7;12)(q36;p13) pediatric acute myeloid leukemia induces the disease through altering histone methylation.

Certain subtypes of acute myeloid leukemia (AML) in children have inferior outcome, such as AML with translocation t(7;12)(q36;p13) leading to an MNX1::ETV6 fusion along with high expression of MNX1. We have identified the transforming event in this AML and possible ways of treatment. Retroviral expression of MNX1 was able to induce AML in mice, with similar gene expression and pathway enrichment to t(7;12) AML patient data. Importantly, this leukemia was only induced in immune incompetent mice using fetal but not adult hematopoietic stem and progenitor cells. The restriction in transforming capacity to cells from fetal liver is in alignment with t(7;12)(q36;p13) AML being mostly seen in infants. Expression of MNX1 led to increased histone 3 lysine 4 mono-, di- and trimethylation, reduction in H3K27me3, accompanied with changes in genome-wide chromatin accessibility and genome expression, likely mediated through MNX1 interaction with the methionine cycle and methyltransferases. MNX1 expression increased DNA damage, depletion of the Lin-/Sca1+/c-Kit+ population and skewing toward the myeloid lineage. These effects, together with leukemia development, were prevented by pre-treatment with the S-adenosylmethionine analog Sinefungin. In conclusion, we have shown the importance of MNX1 in development of AML with t(7;12), supporting a rationale for targeting MNX1 and downstream pathways.

An induced pluripotent stem cell t(7;12)(q36;p13) acute myeloid leukemia model shows high expression of MNX1 and a block in differentiation of the erythroid and megakaryocytic lineages.

Acute myeloid leukemia (AML) results from aberrant hematopoietic processes and these changes are frequently initiated by chromosomal translocations. One particular subtype, AML with translocation t(7;12)(q36;p13), is found in children diagnosed before 2 years of age. The mechanisms for leukemogenesis induced by t(7;12) is not understood, in part because of the lack of efficient methods to reconstruct the leukemia-associated genetic aberration with correct genomic architecture and regulatory elements. We therefore created induced pluripotent stem cell (iPSC) lines that carry the translocation t(7;12) using CRISPR/Cas9. These t(7;12) iPSC showed propensity to differentiate into all three germ layers, confirming retained stem cell properties. The potential for differentiation into hematopoietic stem and progenitor cells (HSPC) was shown by expression of CD34, CD43 and CD45. Compared with the parental iPSC line, a significant decrease in cells expressing CD235a and CD41a was seen in the t(7;12) iPSC-derived HSPC (iHSPC), suggesting a block in differentiation. Moreover, colony formation assay showed an accumulation of cells at the erythroid and myeloid progenitor stages. Gene expression analysis revealed significant down-regulation of genes associated with megakaryocyte differentiation and up-regulation of genes associated with myeloid pathways but also genes typically seen in AML cases with t(7;12). Thus, this iPSC t(7;12) leukemia model of the t(7;12) AML subtype constitutes a valuable tool for further studies of the mechanisms for leukemia development and to find new treatment options.

Relative effects of plasma, fibrinogen concentrate, and factor XIII on ROTEM coagulation profiles in an in vitro model of massive transfusion in trauma.

Massive traumatic haemorrhage is aggravated through the development of trauma-induced coagulopathy, which is managed by plasma transfusion and/or fibrinogen concentrate administration. It is yet unclear whether these treatments are equally potent in ensuring adequate haemostasis, and whether additional factor XIII (FXIII) administration provides further benefits. In this study, we compared ROTEM whole blood coagulation profiles after experimental massive transfusion with different transfusion regimens in an in vitro model of dilution- and transfusion-related coagulopathy. Healthy donor blood was mixed 1 + 1 with six different transfusion regimens. Each regimen contained RBC, platelet concentrate, and either fresh frozen plasma (FFP) or Ringer's acetate (RA). The regimens were further augmented through addition of a low- or medium-dose fibrinogen concentrate and FXIII. Transfusion with FFP alone was insufficient to maintain tissue-factor activated clot strength, coincidental with a deficiency in fibrin-based clot strength. Fibrinogen concentrate conserved, but did not improve coagulation kinetics and overall clot strength. Only combination therapy with FFP and low-dose fibrinogen concentrate improved both coagulation kinetics and fibrin-based clot strength. Administration of FXIII did not result in an improvement of clot strength. In conclusion, combination therapy with both FFP and low-dose fibrinogen concentrate improved clotting time and produced firm clots, representing a possible preferred first-line regimen to manage trauma-induced coagulopathy when RBC and platelets are also transfused. Further research is required to identify optimal first-line transfusion fluids for massive traumatic haemorrhage.

TEG® functional fibrinogen analysis may overestimate fibrinogen levels.

Fibrinogen is of crucial importance in patients with ongoing bleeding. In this study, we compared fibrinogen concentration measured by thrombelastography (TEG®) with fibrinogen plasma concentration determined by Clauss. Sixty-three surgical patients and 38 healthy controls were included. For the whole group (patients and controls, n = 101), TEG® functional fibrinogen was on average 1.0 g/L higher than the plasma fibrinogen concentration (3.5 vs 2.5 g/L, 95% confidence interval for difference 0.8 to 1.2 g/L, P < 0.0001). Similar patterns were observed when patients and healthy controls were analysed separately. The fibrinogen level may be overestimated when assessed using TEG® compared with the fibrinogen plasma concentration measured by the conventional method.

Effect of plasma-to-RBC ratios in trauma patients: a cohort study with time-dependent data*.

BACKGROUND: A widespread approach today is to transfuse bleeding trauma patients with RBC concentrates and plasma at a 1:1 ratio. This regime is supported by a range of observational studies showing lower mortality in bleeding patients receiving equal volumes of plasma and RBCs. The rationale for this practice is still unclear with several studies failing to show any survival benefits of increased plasma use, perhaps due to a failure to account for the timing of transfused units. OBJECTIVE: To study the association between plasma-to-RBC ratios and risk of death in trauma patients, using appropriate methods. DESIGN, SETTINGS, AND PARTICIPANTS: In a retrospective cohort study, we assembled data on 741 transfused trauma patients at a large trauma center. Measures of transfusion therapy were assessed entirely time dependently, and relative risk of death was compared between patients receiving low to high plasma-to-RBC ratio (< 0.85 vs > 0.85). MEASUREMENTS AND RESULTS: In the time-dependent analyses, we saw no significant association between a low plasma ratio and the risk of death. However, age more than 75 years, injury severity score greater than 33, Glasgow Coma Scale less than 8, and systolic blood pressure lower than 90 mm Hg were all significantly associated with increased risk of death. Conversely, when the analyses were conducted with conventional methods, a strong protective effect of high plasma ratios was seen. CONCLUSIONS: The key finding in our study is the strikingly different results produced by time-dependent analyses and the conventional analyses when studying survival and plasma-to-RBC ratio, supporting recent claims that prior studies showing benefit of high plasma ratios might have suffered from survival bias. There is a great need for further studies on the subject to enable improvements in treatment of massively bleeding trauma patients.

Thromboelastography (TEG®) compared to conventional coagulation tests in surgical patients--a laboratory evaluation.

BACKGROUND: Several methods exist for evaluation of hypocoagulation in patients with perioperative bleeding, e.g. thromboelastography (TEG(®)) and conventional methods (platelet count, aPTT, INR and fibrinogen). Considering the vast experience of conventional methods it is important to investigate how well the methods correspond. METHODS: Sixty surgical patients were included prospectively and blood samples were taken perioperatively. TEG(®) and conventional parameters were analyzed simultaneously. An assessment of coagulopathy, based on a synthesis of the conventional methods, was done by two experienced coagulation specialists, blinded from the results of TEG(®) and from the results of each other. Hypocoagulation, defined by TEG(®) parameters; reaction time (R-time), angle, maximal amplitude (MA) and fibrinolysis, was evaluated according to a commonly used algorithm. RESULTS: To detect a platelet count below 150 × 10(9) L(-1), the sensitivity of TEG was 17% (95% CI, 7-36%) with angle and 25% (95% CI, 11-45%) with MA. The sensitivity to detect fibrinogen below 2 g/L was 11% (95% CI, 3-29%) with angle and 21% with MA (95% CI, 8-43%). To detect aPTT more than 40 s and INR more than 1.2 with R-time, the sensitivity was 19% (95% CI, 8-37%) and 0% (95% CI, 0-69%) respectively. The agreement of the evaluator's assessments of hypocoagulation was 100%, but the agreement with the overall TEG(®) analysis was poor with a sensitivity of 33% and a specificity of 95%. CONCLUSION: The agreement between conventional laboratory tests and TEG is poor, but it remains uncertain which type of coagulation tests that best reflects the actual bleeding risk.

Variations and obstacles in the use of coagulation factor concentrates for major trauma bleeding across Europe: outcomes from a European expert meeting.

PURPOSE: Trauma is a leading cause of mortality, with major bleeding and trauma-induced coagulopathy (TIC) contributing to negative patient outcomes. Treatments for TIC include tranexamic acid (TXA), fresh frozen plasma (FFP), and coagulation factor concentrates (CFCs, e.g. prothrombin complex concentrates [PCCs] and fibrinogen concentrate [FCH]). Guidelines for TIC management vary across Europe and a clear definition of TIC is still lacking. METHODS: An advisory board involving European trauma experts was held on 02 February 2019, to discuss clinical experience in the management of trauma-related bleeding and recommendations from European guidelines, focusing on CFC use (mainly FCH). This review summarises the discussions, including TIC definitions, gaps in the guidelines that affect their implementation, and barriers to use of CFCs, with suggested solutions. RESULTS: A definition of TIC, which incorporates clinical (e.g. severe bleeding) and laboratory parameters (e.g. low fibrinogen) is suggested. TIC should be treated immediately with TXA and FCH/red blood cells; subsequently, if fibrinogen ≤ 1.5 g/L (or equivalent by viscoelastic testing), treatment with FCH, then PCC (if bleeding continues) is suggested. Fibrinogen concentrate, and not FFP, should be administered as first-line therapy for TIC. Several initiatives may improve TIC management, with improved medical education of major importance; generation of new and stronger data, simplified clinical practice guidance, and improved access to viscoelastic testing are also critical factors. CONCLUSIONS: Management of TIC is challenging. A standard definition of TIC, together with initiatives to facilitate effective CFC administration, may contribute to improved patient care and outcomes.

A retrospective register study comparing fibrinogen treated trauma patients with an injury severity score matched control group.

BACKGROUND: Fibrinogen concentrate (FC) is frequently used to treat bleeding trauma patients, although the clinical effects are not well known. In this study we describe demographic and clinical outcome data in a cohort of trauma patients receiving FC, compared to a matched control group, who did not receive FC. METHODS: This retrospective, single-center, observational study included adult trauma patients admitted to a level 1-trauma center in Sweden between January 2013 and June 2015. The study population consisted of patients to whom FC was administrated within 24 h (n = 138, "Fib+"). Patients with Injury Severity Score (ISS) > 49 and/or deceased within 1 h from arrival were excluded (n = 30). Controls (n = 108) were matched for age, gender and ISS ("Fib-"). Primary outcome was mortality (24 h-/30 days-/1 year-), and secondary outcomes were blood transfusions, thromboembolic events and organ failure. RESULTS: The Fib+ group, despite having similar ISS as Fib-, had higher prevalence of penetrating trauma and lower Glasgow Coma Scale (GCS), indicating more severe injuries. Patients receiving FC had a higher mortality after 24 h/ 30 days/ 1 year compared to controls (Fib-). However, in a propensity score matched model, the differences in mortality between Fib+ and Fib- were no longer significant. Blood transfusions were more common in the Fib+ group, but no difference was observed in thromboembolic events or organ failure. In both groups, low as well as high P-fibrinogen levels at arrival were associated with increased mortality, with the lowest mortality observed at P-fibrinogen values of 2-3 g/l. CONCLUSIONS: Despite equal ISS, patients receiving FC had a higher mortality compared to the control group, presumably associated to the fact that these patients were bleeding and physiologically deranged on arrival. When applying a propensity score matching approach, the difference in mortality between the groups was no longer significant. No differences were observed between the groups regarding thromboembolic events or organ failure, despite higher transfusion volumes in patients receiving FC.

A criteria-directed protocol for in-hospital triage of trauma patients.

OBJECTIVE: To better match hospital resources to patients' needs of trauma care, a protocol for facilitating in-hospital triage decisions was implemented at a Swedish level I trauma centre. In the protocol, physiological parameters, anatomical injuries and mechanism of injury were documented, and used to activate full or limited trauma team response. The aim of this study was to evaluate the efficacy of the criteria-directed protocol to determine in-hospital trauma triage in an emergency department. METHODS: Level of triage and triage rates were compared before and after implementation of the protocol. Overtriage and undertriage were assessed with injury severity score higher than 15 as the cutoff for defining major trauma. Medical records for undertriaged patients were retrospectively reviewed. RESULTS: In 2011, 78% of 1408 trauma team activations required full trauma response, with an overtriage rate of 74% and an undertriage rate of 7%. In 2013, after protocol implementation, 58% of 1466 trauma team activations required full trauma response. Overtriage was reduced to 52% and undertriage was increased to 10%. However, there were no preventable deaths in the undertriaged patients. CONCLUSION: A criteria-directed protocol for use in the emergency department was efficient in reducing overtriage rates without risking undertriaged patients' safety.

Haemostasis monitored in stored red blood cells, plasma and platelet concentrates in the proportion of 4 :  4 :  1 diluted with crystalloids and colloids.

The aim of this in-vitro study was to evaluate haemostasis analysed with thromboelastometry and blood gas and blood count variables, in stored blood components and the effects after dilution with Ringer[Combining Acute Accent]s acetate, albumin and hydroxyethyl starch (HES). Aliquots from stored red blood cells, plasma and platelet concentrates were mixed in the proportion of 4 : 4 : 1 and analysed with rotational thromboelastometry (ROTEM), blood count [haemoglobin (Hb), haematocrit, platelet count] and blood gas (pH, calcium, sodium, potassium, glucose levels). The blood mix was thereafter diluted 20 and 33% with Ringer's acetate, albumin or HES. The stored blood component mix in a ratio of 4 : 4 : 1 had a low pH (7.11 ±â€Š0.03, mean ±â€Šstandard deviation), nonmeasurable calcium level, and high concentrations of sodium, potassium and glucose but ROTEM curves within normal range after recalcification. With Ringer's acetate dilution, the ROTEM variables changed almost linearly with increasing dilution volume. When albumin was used in the 33% dilution, the clot firmness of the fibrin clot (FibTEM) was further reduced, and with HES dilution, there was a pronounced impairment. The stored blood mix had a low pH and calcium level, both of which might have a significant influence on the coagulation process but normal ROTEM curves after recalcification. Dilution with Ringer's acetate and albumin resulted in moderate deterioration, while dilution with HES showed severely impaired haemostasis.

Suspected Transfusion Related Acute Lung Injury Improving following Administration of Tranexamic Acid: A Case Report.

A 16-year-old woman with craniofacial injury developed severe acute respiratory failure under the primary reconstructive surgical procedure requiring several units of blood and plasma. A transfusion related acute lung injury (TRALI) was suspected and supportive treatment was initiated. Because of the severity of symptoms, acute extracorporeal membrane oxygenation (ECMO) was planned. During preparation for ECMO, a single intravenous dose, 1 g of tranexamic acid, was administered and a remarkable improvement was observed shortly thereafter. The patient was placed on ECMO for 16 hours. The further course was uncomplicated and the patient was discharged from ICU on the 6th day after admission fully and she recovered. A clinical improvement was observed in a timely fashion following the administration of tranexamic acid. The handling of a suspected TRALI and potential benefit from administration of tranexamic acid are discussed in this case report.

In vitro combinations of red blood cell, plasma and platelet components evaluated by thromboelastography.

BACKGROUND: Thromboelastography is increasingly used to evaluate coagulation in massively bleeding patients. The aim of this study was to investigate how different combinations of blood components affect in vitro whole blood clotting measured by thromboelastography. MATERIALS AND METHODS: Packed red blood cells, plasma and platelets from fresh and old blood components were mixed in vitro, in proportions of 4:4:1, 5:5:2, 8:4:1 and 2:1:0, and analysed with thromboelastography. For the ratio 4:4:1 the experiment was done at both 37 °C and 32 °C. RESULTS: Thromboelastography curves were within normal reference values for the blood component proportions of 4:4:1 and 5:5:2. For 8:4:1, the angle and maximal amplitude were reduced below normal values, indicating low levels of fibrinogen and/or platelets. For the 2:1:0 proportion, all parameters were affected resulting in severely impaired in vitro clot formation. The reaction-time, reflecting the coagulation factor-dependent, initial clot formation, was slightly increased at a low temperature. Prolonged storage of the components did not affect the curve. DISCUSSION: With the introduction of guidelines on the management of massive bleeding it is important to have tools for the assessment of the new protocols. In vitro evaluation of mixtures of packed red blood cells, plasma and platelets by thromboelastography may be relevant in the prediction of in vivo clot formation and haemostasis.